- Email: [email protected]
A decade of tyrosine kinases: from gene discovery to therapeutics
Surgical Oncology 12 (2003) 39–50
A decade of tyrosine kinases: from gene discovery to therapeutics Rolf J. Craven*, Harry Lightfoot, William G. Cance Department of Surgery, University of North Carolina at Chapel Hill, 21-237 Lineberger, Comprehensive Cancer Centre, Camous Box 7295, Chapel Hill, NC 27599, USA
Abstract Tyrosine kinases are key regulators of breast cancer cell survival and proliferation. Ten years ago, we conducted a screen for protein kinases expressed in primary human breast tumors and cultured cancer cells. Here, we review the progress from the last ten years in understanding the functions of these kinases with a focus on breast cancer. Three themes emerge: (1) tyrosine kinases regulate proliferation through the MAP Kinase pathway, (2) tyrosine kinases regulate cellular survival through the PI3 Kinase-Akt pathway, and (3) the cell cycle is regulated through a complex series of serine-threonine kinases. Our improved understanding of these signaling cascades has led to novel strategies for therapeutic intervention in breast cancer. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cancer; Tyrosine kinase; Apoptosis; Signaling; Review
1. Overview of tyrosine kinases in breast cancer Breast cancer will ultimately strike one of nine women in the United States during the course of their lives. Risk factors for breast cancer recurrence after surgery are generally related to the rates of cellular proliferation and apoptosis within the tumor. Both proliferation and suppression of apoptosis are associated with overexpression of a family of proteins called tyrosine kinases. Tyrosine kinases phosphorylate other proteins on tyrosine residues, generally activating the target protein or providing binding sites for protein–protein interactions. Tyrosine kinases include transmembrane receptors and intracellular proteins that signal adhesion, proliferation, DNA damage repair, hormonal responses, and specific alterations in gene expression. Several tyrosine kinases are key indicators of breast cancer progression. The HER-2/neu protein encodes a receptor for Heregulin and is amplified or overexpressed in approximately 20% of breast cancers. Because of the importance of HER-2/neu as a prognostic marker in breast cancer, we thought that other tyrosine kinases might also contribute to breast cancer progression. In 1993, we published the results of a screen in which we uncovered 26 protein kinases expressed in cultured breast cancer cell lines and primary breast tumors [1]. The protein kinases identified in our screen consist of several distinct groups: transmembrane receptor tyro*Corresponding author. E-mail address: [email protected] (R.J. Craven).
sine kinases, intracellular tyrosine kinases, and serine/ threonine kinases. The transmembrane receptor tyrosine kinases included HER-2/neu, the Insulin-like Growth factor I Receptor, the Epidermal Growth Factor Receptor, the Fibroblast Growth Factor Receptor-4, a-Platelet-derived Growth Factor Receptor, b-Plateletderived Growth Factor Receptor, Colony Stimulating Factor-1 Receptor, Met, Tyro10, Eph-A7, and Tie-1. The intracellular tyrosine kinases included Abl, Fer, CSK (carboxy-terminal Src kinase), JAK1 (janus kinase—it has two kinase domains), JAK2, JAK3, FAK (focal adhesion kinase), and Rak. Finally, we identified the serine/threonine kinases B-Raf and CDK7 (cyclin-dependent kinase 7), and six of the serine/ threonine kinases identified in breast cancer cells were novel and remain to be characterized. In the years since the original screen, work from a number of labs has demonstrated that tyrosine kinases play important roles in signaling survival and proliferation, pathways that are crucial for tumor formation. This review will focus on kinases from our screen that regulate survival and proliferation in cancer, then describe early attempts to utilize this knowledge to develop therapeutic strategies for breast cancer.
2. Survival signaling in cancer Normal human cells can be transformed minimally by four genetic alterations: inactivation of the tumor
0960-7404/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-7404(03)00004-5
40
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
suppressors Rb and p53, stimulation of the Ras GTPase signaling pathway, and activation of telomerase [2]. The Rb and p53 tumor suppressors regulate the cell cycle and apoptosis [3]. Ras directs responses to proliferation and suppresses apoptosis (reviewed below). Telomerase activation is required for continued cellular proliferative lifespan and maintenance of genetic stability [4]. Thus, tumor growth requires pathways with very different types of signaling. Normal tissues direct an apoptotic (programmed cell death) response to prevent the onset of malignancy, and tumor cells suppress this response through a number of mechanisms. Various tumor types suppress apoptosis by activating Bcl-2 or altering the tumor suppressor protein p53 [5]. Breast cancer cells suppress the apoptotic response by activating tyrosine kinases (Fig. 1). The best characterized tyrosine kinases that are overexpressed in breast cancer are the EGFR and HER-2/neu tyrosine kinases, which were detected in our screen, and these kinases are described in greater detail in the following section. Overexpression of both kinases suppresses the apoptotic response, making them attractive targets for therapeutic intervention. Epithelial cells are stimulated to undergo apoptosis when they are deprived of cell–cell contact (Fig. 1). Invading tumor cells must overcome this apoptotic signal in order to leave their native environment and migrate to distant sites. Cells in culture have a similar response in which they depend on adhesion to a plastic coated dish for survival. Loss of this type of adhesion leads to a specific type of apoptosis called anoikis [6,7]. The suppression of apoptosis due to aberrant cell–cell contact and adhesion signaling are regulated by the FAK, and FAK will be reviewed in a following section.
Fig. 1. Survival signaling through tyrosine kinases in cancer. In the example shown, breast epithelial cells proliferate and become invasive. During this process, a number of apoptotic signals associated with proliferation and loss of cell–cell contact are suppressed for the tumor cells to survive. This signaling pattern is dependent on the action of multiple tyrosine kinases.
3. Survival signaling through EGFR and HER-2/neu Both EGFR and HER-2/neu are overexpressed in breast tumors [8,9]. In a large number of studies, EGFR overexpression was detected reproducably in tumor samples from breast and other tissues, although the prognostic significance of EGFR overexpression with regard to survival in breast cancer was limited (reviewed in Ref. [10]). The overexpression of HER-2/neu is more clearly associated with decreased disease-free and overall survival, and an assessment of the relationship between HER-2/neu expression and response to therapy is ongoing (reviewed in Ref. [11]). The forced overexpression of either EGFR or HER-2/ neu can transform murine fibroblasts into a deregulated, tumorigenic cell type [12,13]. HER-2/neu can function as a heterodimer with EGFR and HER3, and these heterodimeric complexes evade inactivation by decreasing ligand dissociation, receptor internalization, and receptor degradation (reviewed in Ref. [14]). EGFR and HER-2/neu activation leads to elevated MAP kinase and PI3 Kinase recruitment (Fig. 2). PI3 Kinase suppresses apoptosis by activating the Akt anti-apoptotic protein kinase. The ERK kinases deregulate the cell cycle machinery through phosphorylation of a variety of kinases and transcription factors (reviewed in Ref. [14]). Signaling through MAP kinases and PI3 kinases is a common feature of tyrosine kinases, and we will review these pathways briefly in the next sections.
Fig. 2. Survival signaling through receptor tyrosine kinases. Several receptor tyrosine kinases transmit anti-apoptotic signals through a common pathway. The PI3-Kinase is activated by direct binding to the various receptors, leading to the activation of the Akt protein kinase and suppression of apoptosis through the phosphorylation of Akt target proteins.
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
4. Downstream regulators survival signaling: PI3 Kinase and Akt protein kinase PI3 kinase is a key regulator of survival signaling. The activated PI3 Kinase phosphorylates the membranebound lipid phosphotidylinositol-4,5-bisphosphate, converting it to phosphotidylinositol-3,4,5-bisphosphate (recently reviewed in Ref. [15]). The phosphorylated lipid then binds to a number of proteins that contain pleckstrin homology domains, including the serine threonine kinase Akt. Membrane localized Akt is readily phosphorylated, triggering the activation of Akt kinase activity. Akt then phosphorylates the proteins FKHR-L1 (Forkhead-related transcription factor 1), Bad (Bcl-2-associated death protein), and GSK3 (glycogen synthase kinase 3). Bad phosphorylation blocks its association with the Bcl-2 and Bcl-XL proteins, allowing them to perform anti-apoptotic survival functions (reviewed in Ref. [15]). Several of the proteins detected in our original screen affect Akt activity (Fig. 2). EGF stimulation activates Akt via PI3 K [16] as does HER-2/neu [17–19]. FAK binds directly to PI3 K through its amino-terminal domain [20], and inactivation of FAK causes a decrease in Akt expression (see below). Thus, Akt activation directs potent survival signals, and overexpression of tyrosine kinases can initiate this response in breast cancer cells.
41
mulating Factor-1 Receptor, Met, Tyro 10, Eph-A7, and Tie-1. MAP kinases are also regulated by the intracellular FAK [22]. MAP kinase/ERK1 is phosphorylated by the MEK kinase, which is phosphorylated in turn by MEK kinase, also called Raf. We identified the Raf kinase as a gene expressed in breast cancer cell lines [1]. Taken together, the number of genes regulating MAP kinase signaling that were detected in breast cancer underscores the importance of this pathway in breast cancer. MAP kinases are elevated in breast cancer (reviewed by Santen [23]). In an initial study, MAP kinase activity was increased in 11 primary breast tumors [24], and similar findings were reached by two different groups [25,26]. In the latter study, activated MAP kinase paradoxically correlated with both node positivity and disease free survival [26]. While MAP kinases are activated by Ras, and Ras is activated by mutation in cancer, the mechanism of MAP kinase activation in breast tumors is probably not due to mutations of Ras exclusively. Instead, MAP kinase activation appears to depend on overexpression of the EGFR and or HER-2/ neu [27]. Thus, MAP kinase activation is important in the progression of clinical breast tumor progression, and a number of proteins identified in our screen affect MAP kinase activation.
6. A cornucopia of survival signals: additional receptor tyrosine kinases implicated in survival signaling 5. Regulating proliferation: the MAP kinases While PI3 kinase and Akt primarily regulate survival signaling, proliferation is associated with activation of MAP kinases. The majority of the kinases detected in this screen regulate the activation of the MAP kinase pathway. MAP kinases are relatively low molecular weight serine/threonine protein kinases that relay signals from the cell membrane to transcription factors in the nucleus (recently reviewed by Hazzalin and Mahadevan, Ref. [21]). MAP kinases are regulated by a complex cascade that includes binding of adaptor proteins and a series of phosphorylation events. Through this cascade, growth factor binding at the cell membrane can regulate the phosphorylation of transcription factors that direct the onset of proliferation or apoptosis. Growth factor receptors regulate MAP kinase activation by activating the membrane associated Ras protein, which in turn activates protein kinases that eventually direct the phosphorylation of MAP kinases. As stated above, our screen identified a number of receptor tyrosine kinases, including HER-2/neu, the Insulin-like Growth factor I Receptor, the Epidermal Growth Factor Receptor, the Fibroblast Growth Factor Receptor-4, a-Platelet-derived Growth Factor Receptor, bPlatelet-derived Growth Factor Receptor, Colony Sti-
Although we have studied only a few of the kinases identified in our original screens, tyrosine kinases have been studied intensively in cancer over the last 10 years. In addition to the kinases described above, several other tyrosine kinases also have roles in survival signaling. Investigators have also developed a greater appreciation for the complexity of kinase-mediated signaling. For instance, HER-2/neu mediates functions in growth and survival signaling described earlier, but also contributes to regulation of the estrogen receptor [28] and angiogenesis [29]. Thus, it is likely that many of the receptors described in the following section will have diverse, interacting roles in breast cancer progression. Met is the receptor for hepatocyte growth factor/ scatter factor (HGF/SF). Met is overexpressed in breast tumors, and its expression correlates with relapse and poor overall survival [30]. Additional studies substantiate Met as a strong, independent prognostic marker for breast cancer [31]. Met appears to function through a combination of survival signaling and the activation of DNA repair. Treatment of cells with hepatocyte growth factor/scatter factor protects cells against damage by activating a pathway that includes c-Met, PI3-kinase, and Akt [32]. Some of this protective effect is due to the stimulation of DNA repair by an unknown mechanism
42
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
that probably includes widespread alterations in gene expression [33]. Some of the genes induced by HGF/SF include the human checkpoint protein Atm and the DNA repair protein FEN-1, which works as a flap endonuclease in the base excision repair pathway [33]. The IGF1-R also mediates proliferation and survival signaling. IGF1 (insulin-like growth factor 1) is a potent mitogen for breast cancer cell lines [34,35], and its receptor, IGF1-R, is overexpressed in breast tumors ([36], reviewed in Ref. [37]). This suggests that the IGF1 may regulate breast cancer signaling through a paracrine mechanism. Like several other receptor tyrosine kinases, IGF1-R mediates a survival signaling pathway through the activation of the PI3-kinase and Akt, resulting in the suppression of apoptosis ([38,39]; Fig. 2). Some of the other tyrosine kinases identified in our screen suppress apoptosis in various cell types, but their roles in survival signaling in breast cancer have not been characterized. For example, the JAK1 tyrosine kinase is involved in survival signaling in transformed hematopoietic cells [40], but its role in breast cancer growth is unclear. In the next section, we will review recent findings from our lab about a tyrosine kinase with potential survival signaling function that is overexpressed and cleaved in breast epithelial cells. This kinase, called Tie-1, is of particular interest because its expression is generally associated with the growth of blood vessels (angiogenesis). We have found that Tie-1 is overexpressed in breast cancer cells, and review data linking Tie-1 to survival signaling.
7. An angiogenic tyrosine kinase is overexpressed and cleaved in breast cancer: potential roles and regulation of Tie-1 At least three of the protein kinases discovered in our screen, FGFR-4, ephA7, and Tie-1, are directly implicated in angiogenesis, the growth of blood vessels within a tumor. Tie-1 acts as a survival signal in endothelial cells, and we have found that epithelial breast cancer cells overexpress Tie-1. This raises the possibility that Tie-1 may act as a survival signal in tumors. Tie-1 (41, tyrosine kinase with immunoglobulin and Epidermal Growth Factor homology domains) is a receptor tyrosine kinase with an unknown ligand. Tie-1 and the related Tek receptor are transmembrane tyrosine kinases that contain one complete and one incomplete immunoglobulin domain, three epidermal growth factor homology domains, and three fibronectin type III homology domains [41]. Knock-out mice for Tek and Tie-1 have identified distinct functions for the two receptors. Tek knock-out mice die at embryonic day 9.5–10.5, while mice deleted for Tie-1 die at day 14.5 to birth [42,43]. This is a very
different phenotype from mice containing deletions of genes that are required for the early stages of blood vessel formation, such as the VEGFR. VEGFR knockout mice die by embryonic day 8.5 with profound defects in the earliest stages of blood vessel formation [44,45]. In contrast, mice lacking either Tek or Tie-1 form endothelial cells in normal numbers, but Tek-null mice form vessels that lack branching networks and organization into large and small vessels [42,43]. Tie-1null mice die from edema and hemorrhage, suggesting that Tie-1 normally regulates fluid exchange across capillaries and directs responses to hemodynamic stress [43,46]. Tie-1 is expressed predominantly in endothelial cells of adult tissues, in proliferating ovarian capillaries, and during wound healing, suggesting a role in neovascularization [47]. While Tie-1 expression is generally associated with endothelial cells, there is a growing body of evidence linking Tie-1 function to epithelial cell regulation. We originally identified Tie-1 in a breast tumor sample and detected the TIE-1 transcript in 8/9 breast tumor samples, with one tumor expressing high levels of the transcript. More recently, we have used an antibody to the Tie-1 carboxy-terminus, and have found that Tie-1 was expressed in 15/24 breast tumor samples. Surprisingly, immunohistochemistry with the same antibody revealed that Tie-1 was not expressed exclusively in blood vessels within the tumor, but is also expressed by epithelial tumor cells. Tsengi et al. used a similar approach and also found that Tie-1 was expressed in epithelial breast tumor cells [48]. Furthermore, Tie-1-positivity correlated with worse overall survival and disease-free survival. Both reports contradict earlier work by Salven et al. in which an antibody to the Tie-1 amino-terminus indicated that Tie-1 is expressed only in the blood vessels of breast tumors [49]. We believe that these two reports can be reconciled by a recent analysis from our lab. We have found that Tie-1 is overexpressed in breast cancer cells and cleaved near the cell membrane. Thus, the Tie-1 carboxy-terminus would be detected in breast epithelial cells, while the amino-terminal extracellular domain is released. Tie-1 is cleaved in response to phorbol ester stimulation when overexpressed in Chinese hamster ovary cells [50]. This cleavage is dependent on protein kinase C and resembles the processing of a number of other receptor tyrosine kinases, including Axl [51], Kit [52], and colony stimulating factor 1 receptor [53]. It is unclear whether Tie-1 is activated by cleavage in breast cancer cells, and experiments are under way to address the regulation of Tie-1 in cancer cells. If our model is correct, Tie-1 is expressed in epithelial breast cancer cells and cleaved, releasing the extracellular domain and leaving the intracellular domain within the cell. The function of the cleaved intracellular domain
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
Fig. 3. The Tie-1 tyrosine kinase intracellular domain is expressed in breast cancer. By this model, Tie-1 is expressed as a full-length protein in epithelial breast cancer cells and is cleaved, leaving an activated, truncated form of the kinase. Because Tie-1 is capable of survival signaling in other cell types (see text), it is likely that Tie-1 performs an analogous function in breast cancer cells.
is not known, but recent studies indicate a role for the Tie-1 intracellular domain in survival signaling. Overexpression of a chimeric receptor that included the Tie-1 intracellular domain inhibited apoptosis in fibroblasts [54]. These cells contained activated phosphatidylinositol 3-kinase and Akt, key mediators of anti-apoptotic survival signaling (Fig. 2; Ref. [54]). Thus, the Tie-1 intracellular domain functions in anti-apoptotic survival signaling, and future work will determine whether Tie-1 functions as a survival signal in breast cancer (Fig. 3).
8. Downstream effectors of survival signaling: FAK and apoptosis Binding of a ligand to a transmembrane receptor tyrosine kinase activates a complex network of signaling pathways, and one of these responses is the activation of the focal adhesion kinase (FAK, [55]). This section will describe the role of FAK in survival signaling, and the following section will describe the interaction of FAK
43
with the epidermal growth factor receptor. FAK is a tyrosine kinase that localizes to sites of focal contacts between cultured cells and their substratum. FAK is phosphorylated following activation of a number of transmembrane receptors (reviewed in Ref. [56]), suggesting that FAK acts to transmit signals from the membrane to intracellular molecules. We identified FAK in separate PCR-based screens for protein kinases expressed in high-grade sarcomas and breast cancer [1,57]. We subsequently developed a polyclonal antibody to the amino-terminus of FAK, and found that FAK is overexpressed in breast, colon, and thyroid tumors, as well as sarcomas [57–60]. More recently, we developed a monoclonal antibody to the amino-terminal region of FAK, and have found that FAK is overexpressed in invasive breast tumors as well as non-invasive ductal carcinoma in situ (DCIS, Fig. 4, [61]). In contrast to earlier reports that FAK was decreased in metastatic colon cancers, we have recently shown that FAK is expressed in liver metastases at similar or higher levels than the primary tumor (Lark et al., in press). Structurally, FAK contains a kinase domain flanked by extensive amino- and carboxy-terminal domains that direct physical associations with other adhesion and signaling proteins. Little is known about the function of the kinase domain, and no direct substrates of FAK have been characterized. Instead, FAK is thought to act as an adapter molecule, bringing together proteins that regulate adhesion and cell survival. Our original model was that FAK contributed primarily to adhesion in tumors, and as cells became invasive and metastatic, FAK overexpression increased their migration and allowed them to adhere to new substrates. However, detection of FAK in DCIS suggested that FAK activation is not simply a consequence of increased migration, but suggested that FAK might be required for an earlier event in carcinogenesis. We propose that FAK functions in two separate pathways in cancer: regulating adhesion and activation of survival signaling. We will briefly review the data supporting a role for FAK in survival signaling. Our group was the first to provide a link between FAK and apoptosis by showing that disruption of FAK expression in tumor cell lines with antisense oligonucleotides caused loss of adhesion and apoptosis [62]. In subsequent work, Ilic et al. deleted the FAK gene in mice and demonstrated that FAK-deficient cells underwent apoptosis when deprived of serum, suggesting that FAK normally directs survival in these cells [63]. Notably, these FAK-deficient cells only survived following loss of the p53 gene, a situation that is similar to cancer cells. FAK contains a carboxy-terminal domain which directs protein–protein associations with a number of molecules, and overexpression of the FAK
44
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
(A)
(B)
Fig. 4. Overexpression of the FAK in breast tumors. (A) FAK was detected in two separate samples of DCIS by immunohistochemistry using the anti-FAK 4.47 monoclonal antibody. (B) Diagram of potential FAK functions in survival signaling, including Akt regulation via activation of PI3K and a more direct regulation of Akt stability.
carboxy-terminal domain, called FRNK (FAK-related non-kinase [64]) or FAK-CD (FAK carboxy-terminal domain), inhibits FAK function, probably by competing for proteins normally associating with the FAK protein [65]. When FAK-CD is overexpressed in breast cancer cells, the cells lose adhesion [66] and undergo apoptosis [67]. FAK is important as a therapeutic target because FAK inhibition appears to have minimal effects on nontransformed cells. Antisense inhibition of FAK does not induce loss of adhesion or apoptosis in normal fibroblasts [62], and FAK-CD/FRNK overexpression does not induce apoptosis in a broad array of normal cells, including Rat-1 cells [68], rabbit synovial fibroblasts [63], and HME cells [67]. These results suggest that the pathways directed by FAK in non-transformed cells are distinct from those in tumor cells, perhaps due to the presence of redundant signaling pathways in normal cells.
9. Where pathways intersect: EGFR and FAK FAK phosphorylation is elevated in response to a number of extracellular stimuli, suggesting that FAK activation might contribute to these pathways by regulating adhesion and survival signaling. For example, FAK phosphorylation is elevated following EGF stimulation in breast cancer cells, and FAK and EGFR associate physically. Because EGF signals both survival and proliferation, we and other groups have examined the relationship between FAK and EGFR signaling. Initial studies in lung cancer cell lines gave conflicting results and suggested that FAK is dephosphorylated in response to EGF [69], leading to a loss of adhesion in stimulated cells. A second paper using a different lung cancer cell line did not detect changes in FAK phosphorylation following EGF treatment, and found that the role of FAK in these cells was primarily related to migration [70]. However, these lung cancer cell lines express extraordinarily high levels of EGFR, and inhibition of FAK in this tumor cell line does not efficiently induce apoptosis. We utilized a BT474 isolate engineered to express EGFR. In breast cancer cells, inhibition of FAK caused detachment, and this detachment was not affected by EGFR. However, detached cells underwent apoptosis at a much lower rate in EGFR-expressing cells, and the suppression of apoptosis was reversed by small molecule inhibitors of EGFR. We conclude that in breast cancer cells, FAK and EGFR cooperate in survival signaling, with FAK performing a separate function in adhesion. These results confirm that FAK interacts directly with growth factor receptors, and this joint signaling suppresses apoptosis, perhaps by amplifying the survival signal. This is an exciting area of research, and the phenotypic and physical interactions of FAK with other growth factor receptors remain to be elucidated. In particular, it will be important to determine the interactions between FAK, HER-2/neu, and the receptors for CSF-1, PDGF, and ephrins, which were detected in our screen as proteins expressed in breast cancer. In a broader sense, the interactions between FAK and EGFR illustrate the importance of therapeutic strategies targeting multiple pathways.
10. Other tyrosine kinases identified in breast cancer: growth inhibition by the Rak tyrosine kinase While the EGFR, HER-2/neu, FAK, and Tie-1 tyrosine kinases correlate with advanced cancer, other kinases identified in the original screen do not. The Rak tyrosine kinase is highly homologous to the c-Src protooncogene, with single SH2 and SH3 (SH=Src homology) domains and a kinase domain containing a tyrosine residue adjacent to the carboxy-terminal end of the
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
protein [71]. Rak was simultaneously named Frk in a separate study. The activity of the c-Src protein is regulated by phosphorylation of its carboxy-terminal tyrosine Y527. Following phosphorylation, Y527 binds to the amino-terminal SH2 domain, folding the protein in an inactive conformation. The c-Src protein is frequently activated in human tumors, including tumors of the colon and breast, and therapeutic strategies targeting the activity of c-Src in cancer cell lines are capable of inducing apoptosis. The structural similarity of Rak to c-Src suggested that Rak might also be elevated in tumors and contribute to tumorigenesis. However, overexpression of Rak in murine fibroblasts inhibits colony formation [72]. Furthermore, we have recently shown that overexpression of a Rak construct fused to the green fluorescent protein causes human breast cancer cells to arrest the cell cycle in the G1 phase [83]. Thus, the activity of Rak in cultured cells differs markedly from that of Src, which causes oncogenic transformation of murine fibroblasts. The mechanism of Rak function is not known. Rak expression varies in different cell lines, with a nuclear localization in some cell lines [71] and a perinuclear localization in others [83]. Rak is capable of binding to the pRb retinoblastoma tumor suppressor protein in vitro and in vivo [72]. Recent work with Rb+ and Rb osteosarcoma cell lines suggests that the ability of Rak to induce growth arrest is not mediated by binding to pRb. Instead, Rb+ cell lines undergo G1 arrest less efficiently than Rb cell lines following Rak expression, suggesting that Rak is in an inactive conformation when bound to pRb [83]. Our model is that Rak functions in the endoplasmic reticulum to phosphorylate target proteins and trigger growth arrest (Fig. 5). Targets include proteins that directly or indirectly regulate cell cycle entry. In addition, other cell types have nuclear targets of Rak that also regulate the cell cycle. The regulation of Rak in tumors is largely unknown. The RAK transcript is expressed only in epithelial tissues and is highest in liver and kidney [71]. In a panel of nine breast tumors, RAK was expressed weakly in four [1]. A larger western blot analysis of 50 breast tumors suggested that RAK expression was lost in approximately 1/3 of tumors (unpublished observations). The loss of RAK expression in breast cancer is consistent with the chromosomal localization of the RAK gene. RAK is localized to 6q21, a region that is frequently lost in both breast and ovarian cancer. This suggests that loss of RAK expression might be a cause of breast cancer. However, systematic deletion of the murine homologue of RAK, called IYK, did not result in an increase in breast tumor formation [73]. Because of the complexity of tumor suppressive signaling, this result is not entirely surprising. Furthermore, Rak is closely related to eight Src family kinases, and redundant
45
Fig. 5. Growth inhibition through the Rak tyrosine kinase. Rak localization is either nuclear or perinuclear, with localization being cell-type dependent. Rak is thought to transmit an unknown signal from an upstream protein, whereupon Rak phosphorylates a target protein within the endoplasmic reticulum.
functions between the kinases may have suppressed any Rak-associated phenotypes. Thus, Rak expression correlates with growth suppression in breast cancer, but the mechanism of Rak function in breast cancer remains to be elucidated.
11. Cell cycle regulators in breast cancer: the serine threonine kinases CAK/CDK7/STK1 and STK2 In addition to tyrosine kinases, our PCR-based screen identified eight serine/threonine kinases expressed in human breast cancer. The majority have not been characterized, but two have a role in cell cycle regulation. We cloned and sequenced a low molecular weight serine/threonine kinase which we called STK1 (serine-threonine kinase 1 [74]). The STK1 kinase was subsequently cloned as CDK7 (cyclin-dependent kinase 7) or CAK (cyclin-dependent kinase activating kinase), a key regulator of cyclin-dependent kinases (reviewed in Ref. [75]). CAK phosphorylates a threonine residue on the activation loop of CDC2 (cell division cycle 2) and analogous residues on other cyclin-dependent kinases, changing the conformation of the proteins to an active state. CAK also phosphorylates the carboxy-terminal domain of RNA polymerase II, regulating gene transcription (reviewed by [76]). We found the expression of STK1 high in all tissue types examined and in 9/9
46
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
breast tumors [1], although STK1 expression varied somewhat during the cell cycle [74]. In addition to STK1/CDK7/CAK, we identified a second cell cycle regulatory kinase called STK2 in breast cancer cells. The STK2 (serine threonine kinase 2) kinase is part of an evolutionarily conserved family of proteins related to the Apergillus NimA (never in mitosis) protein [77]. Overexpression of STK2 is toxic to most cell types (Yarbrough, unpublished observations), as is the overexpression of proteins related to STK2 [78]. The expression of STK2 is epithelial cellspecific [1], suggesting a role in growth regulation only in certain cell types, unlike the STK1/CDK7/CAK protein, which is expressed ubiquitously. Interestingly, we detected expression of STK2 in only 1/9 breast tumor samples, suggesting that STK2 is not a positive regulator of breast cell proliferation [1]. Recent work has also shown that STK2 is down-regulated following treatment of MDA-MB-453 breast cancer cells with HGF/SF (hepatocyte growth factor/scatter factor) and adriamycin compared to cells treated with adriamycin alone [33]. It is conceivable that STK2 is a pro-apoptotic protein and is down-regulated by HGF/SF survival signaling, and it is notable that STK2 cannot be stably expressed in breast cancer cell lines using standard overexpression systems. Thus, cell cycle regulatory kinases were detected in our previous screen, and they are implicated in cell cycle regulation, control of gene expression, and damage responses.
12. Therapeutics: attacking the tumor survival signals Once a protein is found to be overexpressed in tumors, the next step is to develop a method for inhibiting its function. In the case of the HER-2/neu receptor tyrosine kinase, antibody fragments targeting the extracellular domain of the receptor inhibits its function, presumably by competing for its association with its cognate ligand. As a result, the receptor cannot be activated and its function is nullified. The limitation of HER-2/neu as a therapeutic target is that only 20% of breast tumors overexpress HER-2/neu. Furthermore, the HER-2/neu extracellular domain is overexpressed as a soluble spliced variant, potentially competing for binding to antibodies targeting the membrane-associated receptor. FAK and Tie-1 are expressed in large percentages of breast tumors, and FAK is highly expressed in 80% of breast tumors. However, FAK will be difficult to target with a monoclonal antibody because FAK is an intracellular protein, and antibody fragments will probably not traverse the cell membrane in an active form. Thus, the ideal anti-FAK therapeutic would be a small molecule that can inhibit FAK function or expression after entering the cell.
We have targeted FAK function through two approaches. First, we have attenuated FAK expression with the use of antisense oligonucleotides. These are short oligomers that bind to the coding sequence of the FAK transcript. The DNA–RNA hybrid probably signals degradation of the transcript by RNAses. As described briefly above, FAK attenuation leads to apoptosis in tumor cells in culture, but not in normal fibroblasts. In order to be useful clinically, the main limitation of this approach will be the stability of the oligonucleotides in an organism and the potential side effects of introducing a chemically modified DNA fragment into cells. In spite of all of the concerns regarding antisense oligonucleotides, this approach remains promising for the treatment of cancer. Several of the proteins described in this review have been targetted by antisense oligonucleotides. Inhibition of c-Raf inhibited ovarian cancer cell proliferation and increased levels of apoptosis both in vitro and in xenograph models [79]. However, a Phase I trial of this oligonucleotide revealed serious adverse effects in two out of four patients, including acute hemolytic anemia and acute renal failure and anasarca, but a failure to deplete c-Raf expression [80]. Earlier work indicated that the oligonucleotide was effective in depleting c-Raf [81], suggesting that the timing and dose of treatment are crucial. It is also unclear whether the c-Raf antisense oligonucleotides diminish tumor burden at physiologically relevant doses [82]. Thus, antisense oligonucleotide therapy is still a fledgling technique, and it remains to be seen whether this technique can be used as a therapeutic strategy. In experimental in vitro systems, we have inhibited FAK function by overexpressing a large fragment of the FAK carboxy-terminal region by adenoviral transduction. Even though this virus directed high levels of expression of the FAK carboxy-terminus in normal cells, there was no increase in loss of adhesion or apoptosis in those cells. Nonetheless, in the present climate, it is unlikely that adenoviruses will gain widespread use in the treatment of cancer because of questions about their potential side effects. It is also unclear whether a small molecule inhibitor of FAK kinase activity would be useful as an anticancer therapeutic. FAK appears to function as an adaptor molecule, bringing together proteins that bind to FAK, or perhaps anchoring bound proteins to focal adhesions. The ideal therapeutic would be a small molecule that would disrupt these interactions. While our discussion regards therapeutic approaches for HER2/neu and FAK, the same principles apply for Tie-1. As interest in angiogenesis increases, inhibitors of Tie-1 and Tek will undoubtedly emerge. Our results suggest that novel Tie-1 inhibitors could be useful in preventing the growth of breast cancer cells, and could provide new insights about signaling pathways used in breast cancer.
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
Acknowledgements R.J.C. is a scholar of the Building Interdisciplinary Research Careers in Women’s Health (BIRCWH) program through the NIH (K12HD001441). This work was funded in part by the University of North Carolina University Research Council. W.G.C. is funded by NIH grants CA69510 and CA83895.
References [1] Cance WG, Craven RJ, Weiner TM, Liu ET. Novel protein kinases expressed in human breast cancer. International Journal of Cancer 1993;54:571–7. [2] Hahn WC, Counter CC, Lundberg AS, Beijersberger RL, Brooks MW, Weinberg RA. Creation of human tumour cells with defined genetic elements. Nature 1999;400:464–8. [3] Wang JY, Ki SW. Choosing between growth arrest and apoptosis through the retinoblastoma tumour suppressor protein, Abl and p73. Biochemical Society Transactions 2001;29:666–73. [4] Hackett JA, Greider CW. Balancing instability: dual roles for telomerase and telomere dysfunction in tumorigenesis. Oncogene 2002;21:619–26. [5] Ferreira CG, Epping M, Kruyt FA, Giaccone G. Apoptosis: target of cancer therapy. Clinical Cancer Research 2002;8: 2024–34. [6] Rouslahti E, Reed JC. Anchorage dependence, integrins, and apoptosis. Cell 1994;77:477–8. [7] Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. Journal of Cell Biology 1994;124: 619–26. [8] Sainsbury JRC, Farndon JR, Needham GK, Malcolm AJ, Harris AL. Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet 1987;1: 1398–402. [9] Slamon DJ, Clark GM, Wong SJ, Levin LJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235: 177–82. [10] Nicholson RI, Ge JM, Harper ME. EGFR and cancer prognosis. European Journal of Cancer 2001;4(Suppl):9–15. [11] Lohrisch C, Piccart M. HER2/neu as a predictive factor in breast cancer. Clinical Breast Cancer 2001;2:129–35. [12] Di Fiore PP, Pierce JH, Fleming TP, Hazan R, Ullrich A, King CR, Schlessinger J, Aaronson SA. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 1987;51:1063–70. [13] Hudziak RM, Schlessinger J, Ullrich A. Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis in NIH3T3 cells. Proceedings of the National Academy of Science 1987;84:7159–63. [14] Harari D, Yarden Y. Molecular mechanisms underlying ErbB2/ HER2 action in breast cancer. Oncogene 2000;19:6102–14. [15] Cantley LC. The phosphoinositide 3-kinase family. Science 2002;296:1655–7. [16] Lin J, Adam RM, Santiestevan E, Freeman MR. The phosphotidylinositol 30 -kinase pathway is a dominant growth factoractivated cell survival pathway in LNCaP human prostate carcinoma cells. Cancer Research 1999;59:2891–7. [17] Wen Y, Hu MC-T, Makino K, Spohn B, Bartolomeusz G, Yan D-H, Hung M-C. HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. Cancer Research 2000;60:6841–945.
47
[18] Lim SJ, Lopez-Berestein G, Hung MC, Lupu R, Tari AM. Grb2 downregulation leads to Akt inactivation in heregulin-stimulated and ErbB2-overexpressing breast cancer cells. Oncogene 2000;19:6271–6. [19] Zhou BP, Hu MC, Miller SA, Yu Z, Xia W, Lin SY, Hung MC. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway. Journal of Biological Chemistry 2000;275:8027–31. [20] Chen HC, Guan JL. Association of focal adhesion kinase with its potential substrate phosphotidylinositol 3-kinase. Proceedings of the National Academy of Science 1994;91:10148–52. [21] Hazzalin CA, Mahadevan LC. MAPK-regulated transcription: a continuously variable gene switch? Nature Reviews 2002;3: 30–40. [22] Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrinmediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 1994;372:786–91. [23] Santen RJ, Song RX, McPherson R, Kumar R, Adam L, Jeng M-H, Yue W. The role of mitogen-activated protein (MAP) kinase in breast cancer. Journal of Steroid Biochemistry and Molecular Biology 2002;80:239–56. [24] Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer. Journal of Clinical Investigation 1997;99:1478–83. [25] Salh B, Marotta A, Matthewson C, Ahluwalia M, Flint J, Owen D, Pelech S. Investigation of the MEK-MAP kinase-Rsk pathway in human breast cancer. Anticancer Research 1999;19: 731–40. [26] Mueller H, Flury N, Eppenberger-Castori S, Kueng W, David F, Eppenberger U. Potential prognostic value of mitogen-activated protein kinase activity for disease-free survival of primary breast cancer patients. International Journal of Cancer 2000;89:384–8. [27] Von Lintig FC, Dreilinger AD, Varki NM, Wallace AM, Casteel DE, Boss GR. Ras activation in human breast cancer. Breast Cancer Research Treatment 2000;62:51–62. [28] Kumar R, Yarmand-Bagheri R. The role of HER2 in angiogenesis. Seminars in Oncology 2001;28:27–32. [29] Mazumdar A, Wang RA, Mishra SK, Adam L, BagheriYarmand R, Mandal M, Vadlamudi RK, Kumar R. Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor. Nature Cell Biology 2001;3:30–7. [30] Nagy J, Curry GW, Hillan KJ, McKay IC, Mallon E, Purushotham AD, George WD. Hepatocyte growth factor/scatter factor expression and c-met in primary breast cancer. Surgical Oncology 1996;5(l):15–21. [31] Ghoussoub RA, Dillon DA, D’Aquila T, Rimm EB, Fearon ER, Rimm DL. Expression of c-met is a strong independent prognostic factor in breast carcinoma. Cancer 1998;82:1513–20. [32] Fan S, Ma YX, Wang JA, Yuan RQ, Meng Q, Cao Y, Laterra JJ, Goldberg ID, Rosen EM. The cytokine hepatocyte growth factor/ scatter factor inhibits apoptosis and enhances DNA repair by a common mechanism involving signaling through phosphatidyl inositol 3’ kinase. Oncogene 2000;19:2212–23. [33] Yuan RQ, Fan S, Achary M, Stewart DM, Goldberg ID, Rosen EM. Altered gene expression pattern in cultured human breast cancer cells treated with hepatocyte growth factor/scatter factor in the setting of DNA damage. Cancer Research 2001;61: 8022–31. [34] Yee D, Cullen KJ, Paik KJ, Perdue JF, Hampton B, Schwartz A, Lippman ME, Rosen N. Insulin-like growth factor II mRNA expression in human breast cancer. Cancer Research 1988;48:6691–6. [35] Karey KP, Sirbasku DA. Differential responsiveness of human breast cancer cell lines MCF-7 and T47-D to growth factors and 17-b estradiol. Cancer Research 1988;48:4083–92.
48
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
[36] Cullen KJ, Yee D, Sly WS, Perdue J, Hampton B, Lippman ME, Rosen N. Insulin-like Growth Factor Receptor expression and function in human breast cancer. Cancer Research 1990;50: 48–53. [37] Ellis MJ, Jenkins S, Hanfelt J, Redington ME, Taylor M, Leek R, Siddle K, Harris A. Insulin-like growth factors in human breast cancer. Breast Cancer Research Treatment 1998;52:175–84. [38] Myers Jr MG, Grammer TC, Wang LM, Sun XJ, Pierce JH, Blenis J, White MF. Insulin receptor substrate-1 mediates phosphatidylinositol 30 -kinase and p70S6 K signaling during insulin, insulin-like growth factor-1, and interleukin-4 stimulation. Journal of Biological Chemistry 1994;269:28783–9. [39] Kulik G, Klippel A, Weber MA. Antiapoptotic signaling by the insulin-like growth factor I receptor, phosphatidylinositol 3kinase, and Akt. Molecular Cell Biology 1997;17:1595–606. [40] Oki S, Limnander A, Danial NN, Rothman PB. Functional involvement of Akt signaling downstream of Jak1 in v-Ablinduced activation of hematopoietic cells. Blood 2002;100: 966–73. [41] Partanen J, Armstrong E, Makela TP, Korhonen J, Sandberg M, Renkonen R, Knuutila S, Huebner K, Alitalo K. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Molecular Cell Biology 1992;12:1698–707. [42] Dumont DJ, Gradwohl G, Fong GH, Puri MC, Gertsenstein M, Auerbach A, Breitman ML. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes and Development 1994;8:1897–909. [43] Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maquire M, Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 1995;376:70–4. [44] Carmeliet P, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–9. [45] Farrera N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shae K, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–42. [46] Puri MC, Rossant J, Alitalo K, Bernstein A, Partanen J. The receptor tyrosine kinase TIE is required for the integrity and survival of vascular endothelial cells. EMBO Journal 1995;14:5884–91. [47] Korhonen J, Partanen J, Armstrong E, Vaahtokari A, Elenius K, Jalkanen M, Alitalo K. Enhanced expression of the Tie-1 receptor tyrosine kinase in endothelial cells during neovascularization. Blood 1992;80:2548–55. [48] Tseng L-M, Hsu C-Y, Wang H-C, Liu JM, Chang H-M, Lo S-S, Wu C-W, Lui W-Y, Chi C-W. Tie-1 tyrosine kinase is an independent prognostic indicator for invasive breast cancer. Anticancer Research 2001;21:2163–70. [49] Salven P, Joensuu H, Heikkila P, Matikainen MT, Wasenius VM, Alanko A, Alitalo K. Endothelial Tie-1 growth factor receptor provides antigenic marker for assessment of breast cancer angiogenesis. British Journal of Cancer 1996;74:69–72. [50] Yabkowitz R, Meyer S, Yanagihara D, Brankow D, Staley T, Elliot G, Hu S, Ratzkin B. Regulation of Tie-1 receptor expression on human endothelial cells by protein kinase Cmediated release of soluble Tie-1. Blood 1997;90:706–15. [51] O’Bryan JP, Fridell YW, Koski R, Varnum B, Liu ET. The transforming receptor tyrosine kinase, Axl, is post-translationally regulated by proteolytic cleavage. Journal of Biological Chemistry 1995;270:551–7. [52] Yee NS, Hsiau C-WM, Serve H, Vosseller K, Besmer P. Mechanism of down-regulation of c-kit receptor. Roles of
[53]
[54]
[55]
[56] [57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
receptor tyrosine kinase, phosphotidylinositol 30 -kinase, and protein kinase C. Journal of Biological Chemistry 1994;269: 31991–8. Downing JR, Roussel MF, Sherr CJ. Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Molecular Cell Biology 1989;9:2890–6. Kontos CD, Cha EH, York JD, Peters KG. The endothelial receptor tyrosine kinase Tie1 activates Phosphotidylinositol 3Kinase and Akt to inhibit apoptosis. Molecular Cell Biology 2002;22:1704–13. Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. p125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proceedings of the National Academy of Science 1992;89:5192–6. Cary LA, Guan JL. Focal adhesion kinase in integrin-mediated signaling. Frontiers in Bioscience 1999;4:102–13. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of growth factor receptors, the focal adhesion kinase, and other tyrosine kinases in human soft tissue tumors. Annals of Surgical Oncology 1994;1:18–27. Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, Liu ET, Cance WG. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Research 1995;55:2752–5. Owens LV, Xu L, Dent GA, Yang X, Sturge GC, Craven RJ, Cance WG. The focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer. Annals of Surgical Oncology 1996;3:100–5. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of focal adhesion kinase gene and invasive cancer. Lancet 1993;342: 1024–5. Cance WG, Harris JE, Iacocca MV, Roche E, Yang X, Chang J, Simkins S, Xu L. Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clinical Cancer Research 2000;6: 2417–23. Xu L, Owens LV, Sturge GC, Yang X, Liu ET, Craven RJ, Cance WG. Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth and Differentiation 1996;7:413–8. Ilic D, Almeida EAC, Schlaepfer DD, Dazin P, Aizawa S, Damsky CH. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. Journal of Cell Biology 1998;143:547–60. Schaller MD, Borgman CA, Parsons JT. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase p125FAK. Molecular Cell Biology 1993;13: 785–91. Richardson A, Parsons T. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 1996;380:538–40. Xu LH, Yang X, Craven RJ, Cance WG. The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth and Differentiation 1998;9:999–1005. Xu LH, Yang X, Bradham CA, Brenner DA, Baldwin AS, Craven RJ, Cance WG. The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Journal of Biological Chemistry 2000;275:30597–604. Xiong WC, Macklem M, Parsons JT. Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein. Journal of Cell Science 1998;111:1981–91. Lu Z, Jiang G, Blume-Jensen P, Hunter T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
[70]
[71]
[72]
[73]
[74]
[75] [76]
dephosphorylation and downregulation of the focal adhesion kinase. Molecular Cell Biology 2001;21:4016–31. Hauck CR, Sieg DJ, Hsia DJ, Loftus JC, Gaarde WA, Monia BP, Schlaepfer DD. Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Research 2001;61:7079–90. Cance WG, Craven RJ, Bergman M, Xu L, Alitalo K, Liu ET. Rak, a novel nuclear tyrosine kinase expressed in epithelial cells. Cell Growth and Differentiation 1994;5:1347–55. Craven RJ, Cance WG, Liu ET. The nuclear tyrosine kinase Rak associates with the product of the Retinoblastoma tumor susceptibility gene, pRb. Cancer Research 1995;55:3969–72. Chandrasekharan S, Qiu TH, Alkharouf N, Brantley K, Mitchell JB, Liu ET. Characterization of mice deficient in the Src family nonreceptor tyrosine kinase Frk/rak. Molecular Cell Biology 2002;22:5235–47. Levedakou EN, He M, Baptist EW, Craven RJ, Cance WG, Welcsh PL, Simmons A, Naylor SL, Leach RJ, Lewis TB, Bowcock A, Liu ET. Two novel human serine/threonine kinases with homologies to the cell cycle regulating Xenopus MO15, and NimA kinases: cloning and characterization of their expression pattern. Oncogene 1994;9:1977–88. Kaldis P. The CDK-activating kinase (CAK): from yeast to mammals. Cell Molecular Life Science 1999;55:284–96. Riedl T, Egly JM. Phosphorylation in transcription: the CTD and more. Gene Experiment 2000;9:3–13.
Craven Craven received his Ph.D. in Genetics and Molecular Biology from the University of North Carolina at Chapel Hill with Edison T. Liu, M.D., former director of Clinical Sciences at the N.C.I. Dr. Craven did his Post-doctoral fellowship with Dr. Thomas Petes, member of the National Academy of Sciences. Dr. Craven is an Assistant Professor at UNC in the Surgical Oncology Division of the Surgery Department and is a Scholar of the B.I.R.C.W.H. (Building Interdisciplinary Research Careers in Women’s Health) program.
49
[77] Osmani SA, Pu RT, Morris NR. Mitotic induction and maintenance by over-expression of a G2-specific gene that encodes a potential protein kinase. Cell 1988;53:237–44. [78] Lu KP, Hunter T. Evidence for a NIMA-like mitotic pathway in vertebrate cells. Cell 1995;81:413–24. [79] McPhillips F, Mullen P, Monia BP, Ritchie AA, Dorr FA, Smyth JF, Langdon SP. Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer. British Journal of Cancer 2001;85:1753–8. [80] Rudin CM, Holmlund J, Fleming GF, Mani S, Stadler WM, Schumm P, Monia BP, Johnston JF, Geary R, Yu RZ, Kwoh TJ, Dorr FA, Ratain MJ. Phase I Trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer. Clinical Cancer Research 2001;7:1214–20. [81] O’Dwyer PJ, Stevenson JP, Gallagher M, Cassella A, Vasilevskaya I, Monia BP, Holmlund J, Dorr FA, Yao KS. c-raf-1 depletion and tumor responses in patients treated with the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A). Clinical Cancer Research 1999;5:3977–82. [82] Monia BP. Anti-tumor activity of C-raf antisense–correction. Nature Med 1999;5:127. [83] Meyer S, et al. The analysis of the Tie-1 intracellular domain expression in breast cancer is in press at Tumor Biology. International Journal of Cancer 2003;104:139–46.
Lightfoot Lightfoot received his M.D. from the University of North Carolina at Chapel Hill. He is presently a Senior Resident in the Surgery Department at the University of North Carolina at Chapel Hill.
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
50
Cance Cance earned his B.S. and M.D. degrees from Duke University and did his residency at Washington University in St. Louis. Since his fellowship at SloanKettering, Dr. Cance has become Professor and Chief of Surgical Oncology at the University of North Carolina at Chapel Hill, with an adjunct appointment in the Department of Cell and Developmental Biology. In January 2003, Dr. Cance began as Chair of the Department of Surgery at the University of Florida.
A decade of tyrosine kinases: from gene discovery to therapeutics Rolf J. Craven*, Harry Lightfoot, William G. Cance Department of Surgery, University of North Carolina at Chapel Hill, 21-237 Lineberger, Comprehensive Cancer Centre, Camous Box 7295, Chapel Hill, NC 27599, USA
Abstract Tyrosine kinases are key regulators of breast cancer cell survival and proliferation. Ten years ago, we conducted a screen for protein kinases expressed in primary human breast tumors and cultured cancer cells. Here, we review the progress from the last ten years in understanding the functions of these kinases with a focus on breast cancer. Three themes emerge: (1) tyrosine kinases regulate proliferation through the MAP Kinase pathway, (2) tyrosine kinases regulate cellular survival through the PI3 Kinase-Akt pathway, and (3) the cell cycle is regulated through a complex series of serine-threonine kinases. Our improved understanding of these signaling cascades has led to novel strategies for therapeutic intervention in breast cancer. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cancer; Tyrosine kinase; Apoptosis; Signaling; Review
1. Overview of tyrosine kinases in breast cancer Breast cancer will ultimately strike one of nine women in the United States during the course of their lives. Risk factors for breast cancer recurrence after surgery are generally related to the rates of cellular proliferation and apoptosis within the tumor. Both proliferation and suppression of apoptosis are associated with overexpression of a family of proteins called tyrosine kinases. Tyrosine kinases phosphorylate other proteins on tyrosine residues, generally activating the target protein or providing binding sites for protein–protein interactions. Tyrosine kinases include transmembrane receptors and intracellular proteins that signal adhesion, proliferation, DNA damage repair, hormonal responses, and specific alterations in gene expression. Several tyrosine kinases are key indicators of breast cancer progression. The HER-2/neu protein encodes a receptor for Heregulin and is amplified or overexpressed in approximately 20% of breast cancers. Because of the importance of HER-2/neu as a prognostic marker in breast cancer, we thought that other tyrosine kinases might also contribute to breast cancer progression. In 1993, we published the results of a screen in which we uncovered 26 protein kinases expressed in cultured breast cancer cell lines and primary breast tumors [1]. The protein kinases identified in our screen consist of several distinct groups: transmembrane receptor tyro*Corresponding author. E-mail address: [email protected] (R.J. Craven).
sine kinases, intracellular tyrosine kinases, and serine/ threonine kinases. The transmembrane receptor tyrosine kinases included HER-2/neu, the Insulin-like Growth factor I Receptor, the Epidermal Growth Factor Receptor, the Fibroblast Growth Factor Receptor-4, a-Platelet-derived Growth Factor Receptor, b-Plateletderived Growth Factor Receptor, Colony Stimulating Factor-1 Receptor, Met, Tyro10, Eph-A7, and Tie-1. The intracellular tyrosine kinases included Abl, Fer, CSK (carboxy-terminal Src kinase), JAK1 (janus kinase—it has two kinase domains), JAK2, JAK3, FAK (focal adhesion kinase), and Rak. Finally, we identified the serine/threonine kinases B-Raf and CDK7 (cyclin-dependent kinase 7), and six of the serine/ threonine kinases identified in breast cancer cells were novel and remain to be characterized. In the years since the original screen, work from a number of labs has demonstrated that tyrosine kinases play important roles in signaling survival and proliferation, pathways that are crucial for tumor formation. This review will focus on kinases from our screen that regulate survival and proliferation in cancer, then describe early attempts to utilize this knowledge to develop therapeutic strategies for breast cancer.
2. Survival signaling in cancer Normal human cells can be transformed minimally by four genetic alterations: inactivation of the tumor
0960-7404/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-7404(03)00004-5
40
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
suppressors Rb and p53, stimulation of the Ras GTPase signaling pathway, and activation of telomerase [2]. The Rb and p53 tumor suppressors regulate the cell cycle and apoptosis [3]. Ras directs responses to proliferation and suppresses apoptosis (reviewed below). Telomerase activation is required for continued cellular proliferative lifespan and maintenance of genetic stability [4]. Thus, tumor growth requires pathways with very different types of signaling. Normal tissues direct an apoptotic (programmed cell death) response to prevent the onset of malignancy, and tumor cells suppress this response through a number of mechanisms. Various tumor types suppress apoptosis by activating Bcl-2 or altering the tumor suppressor protein p53 [5]. Breast cancer cells suppress the apoptotic response by activating tyrosine kinases (Fig. 1). The best characterized tyrosine kinases that are overexpressed in breast cancer are the EGFR and HER-2/neu tyrosine kinases, which were detected in our screen, and these kinases are described in greater detail in the following section. Overexpression of both kinases suppresses the apoptotic response, making them attractive targets for therapeutic intervention. Epithelial cells are stimulated to undergo apoptosis when they are deprived of cell–cell contact (Fig. 1). Invading tumor cells must overcome this apoptotic signal in order to leave their native environment and migrate to distant sites. Cells in culture have a similar response in which they depend on adhesion to a plastic coated dish for survival. Loss of this type of adhesion leads to a specific type of apoptosis called anoikis [6,7]. The suppression of apoptosis due to aberrant cell–cell contact and adhesion signaling are regulated by the FAK, and FAK will be reviewed in a following section.
Fig. 1. Survival signaling through tyrosine kinases in cancer. In the example shown, breast epithelial cells proliferate and become invasive. During this process, a number of apoptotic signals associated with proliferation and loss of cell–cell contact are suppressed for the tumor cells to survive. This signaling pattern is dependent on the action of multiple tyrosine kinases.
3. Survival signaling through EGFR and HER-2/neu Both EGFR and HER-2/neu are overexpressed in breast tumors [8,9]. In a large number of studies, EGFR overexpression was detected reproducably in tumor samples from breast and other tissues, although the prognostic significance of EGFR overexpression with regard to survival in breast cancer was limited (reviewed in Ref. [10]). The overexpression of HER-2/neu is more clearly associated with decreased disease-free and overall survival, and an assessment of the relationship between HER-2/neu expression and response to therapy is ongoing (reviewed in Ref. [11]). The forced overexpression of either EGFR or HER-2/ neu can transform murine fibroblasts into a deregulated, tumorigenic cell type [12,13]. HER-2/neu can function as a heterodimer with EGFR and HER3, and these heterodimeric complexes evade inactivation by decreasing ligand dissociation, receptor internalization, and receptor degradation (reviewed in Ref. [14]). EGFR and HER-2/neu activation leads to elevated MAP kinase and PI3 Kinase recruitment (Fig. 2). PI3 Kinase suppresses apoptosis by activating the Akt anti-apoptotic protein kinase. The ERK kinases deregulate the cell cycle machinery through phosphorylation of a variety of kinases and transcription factors (reviewed in Ref. [14]). Signaling through MAP kinases and PI3 kinases is a common feature of tyrosine kinases, and we will review these pathways briefly in the next sections.
Fig. 2. Survival signaling through receptor tyrosine kinases. Several receptor tyrosine kinases transmit anti-apoptotic signals through a common pathway. The PI3-Kinase is activated by direct binding to the various receptors, leading to the activation of the Akt protein kinase and suppression of apoptosis through the phosphorylation of Akt target proteins.
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
4. Downstream regulators survival signaling: PI3 Kinase and Akt protein kinase PI3 kinase is a key regulator of survival signaling. The activated PI3 Kinase phosphorylates the membranebound lipid phosphotidylinositol-4,5-bisphosphate, converting it to phosphotidylinositol-3,4,5-bisphosphate (recently reviewed in Ref. [15]). The phosphorylated lipid then binds to a number of proteins that contain pleckstrin homology domains, including the serine threonine kinase Akt. Membrane localized Akt is readily phosphorylated, triggering the activation of Akt kinase activity. Akt then phosphorylates the proteins FKHR-L1 (Forkhead-related transcription factor 1), Bad (Bcl-2-associated death protein), and GSK3 (glycogen synthase kinase 3). Bad phosphorylation blocks its association with the Bcl-2 and Bcl-XL proteins, allowing them to perform anti-apoptotic survival functions (reviewed in Ref. [15]). Several of the proteins detected in our original screen affect Akt activity (Fig. 2). EGF stimulation activates Akt via PI3 K [16] as does HER-2/neu [17–19]. FAK binds directly to PI3 K through its amino-terminal domain [20], and inactivation of FAK causes a decrease in Akt expression (see below). Thus, Akt activation directs potent survival signals, and overexpression of tyrosine kinases can initiate this response in breast cancer cells.
41
mulating Factor-1 Receptor, Met, Tyro 10, Eph-A7, and Tie-1. MAP kinases are also regulated by the intracellular FAK [22]. MAP kinase/ERK1 is phosphorylated by the MEK kinase, which is phosphorylated in turn by MEK kinase, also called Raf. We identified the Raf kinase as a gene expressed in breast cancer cell lines [1]. Taken together, the number of genes regulating MAP kinase signaling that were detected in breast cancer underscores the importance of this pathway in breast cancer. MAP kinases are elevated in breast cancer (reviewed by Santen [23]). In an initial study, MAP kinase activity was increased in 11 primary breast tumors [24], and similar findings were reached by two different groups [25,26]. In the latter study, activated MAP kinase paradoxically correlated with both node positivity and disease free survival [26]. While MAP kinases are activated by Ras, and Ras is activated by mutation in cancer, the mechanism of MAP kinase activation in breast tumors is probably not due to mutations of Ras exclusively. Instead, MAP kinase activation appears to depend on overexpression of the EGFR and or HER-2/ neu [27]. Thus, MAP kinase activation is important in the progression of clinical breast tumor progression, and a number of proteins identified in our screen affect MAP kinase activation.
6. A cornucopia of survival signals: additional receptor tyrosine kinases implicated in survival signaling 5. Regulating proliferation: the MAP kinases While PI3 kinase and Akt primarily regulate survival signaling, proliferation is associated with activation of MAP kinases. The majority of the kinases detected in this screen regulate the activation of the MAP kinase pathway. MAP kinases are relatively low molecular weight serine/threonine protein kinases that relay signals from the cell membrane to transcription factors in the nucleus (recently reviewed by Hazzalin and Mahadevan, Ref. [21]). MAP kinases are regulated by a complex cascade that includes binding of adaptor proteins and a series of phosphorylation events. Through this cascade, growth factor binding at the cell membrane can regulate the phosphorylation of transcription factors that direct the onset of proliferation or apoptosis. Growth factor receptors regulate MAP kinase activation by activating the membrane associated Ras protein, which in turn activates protein kinases that eventually direct the phosphorylation of MAP kinases. As stated above, our screen identified a number of receptor tyrosine kinases, including HER-2/neu, the Insulin-like Growth factor I Receptor, the Epidermal Growth Factor Receptor, the Fibroblast Growth Factor Receptor-4, a-Platelet-derived Growth Factor Receptor, bPlatelet-derived Growth Factor Receptor, Colony Sti-
Although we have studied only a few of the kinases identified in our original screens, tyrosine kinases have been studied intensively in cancer over the last 10 years. In addition to the kinases described above, several other tyrosine kinases also have roles in survival signaling. Investigators have also developed a greater appreciation for the complexity of kinase-mediated signaling. For instance, HER-2/neu mediates functions in growth and survival signaling described earlier, but also contributes to regulation of the estrogen receptor [28] and angiogenesis [29]. Thus, it is likely that many of the receptors described in the following section will have diverse, interacting roles in breast cancer progression. Met is the receptor for hepatocyte growth factor/ scatter factor (HGF/SF). Met is overexpressed in breast tumors, and its expression correlates with relapse and poor overall survival [30]. Additional studies substantiate Met as a strong, independent prognostic marker for breast cancer [31]. Met appears to function through a combination of survival signaling and the activation of DNA repair. Treatment of cells with hepatocyte growth factor/scatter factor protects cells against damage by activating a pathway that includes c-Met, PI3-kinase, and Akt [32]. Some of this protective effect is due to the stimulation of DNA repair by an unknown mechanism
42
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
that probably includes widespread alterations in gene expression [33]. Some of the genes induced by HGF/SF include the human checkpoint protein Atm and the DNA repair protein FEN-1, which works as a flap endonuclease in the base excision repair pathway [33]. The IGF1-R also mediates proliferation and survival signaling. IGF1 (insulin-like growth factor 1) is a potent mitogen for breast cancer cell lines [34,35], and its receptor, IGF1-R, is overexpressed in breast tumors ([36], reviewed in Ref. [37]). This suggests that the IGF1 may regulate breast cancer signaling through a paracrine mechanism. Like several other receptor tyrosine kinases, IGF1-R mediates a survival signaling pathway through the activation of the PI3-kinase and Akt, resulting in the suppression of apoptosis ([38,39]; Fig. 2). Some of the other tyrosine kinases identified in our screen suppress apoptosis in various cell types, but their roles in survival signaling in breast cancer have not been characterized. For example, the JAK1 tyrosine kinase is involved in survival signaling in transformed hematopoietic cells [40], but its role in breast cancer growth is unclear. In the next section, we will review recent findings from our lab about a tyrosine kinase with potential survival signaling function that is overexpressed and cleaved in breast epithelial cells. This kinase, called Tie-1, is of particular interest because its expression is generally associated with the growth of blood vessels (angiogenesis). We have found that Tie-1 is overexpressed in breast cancer cells, and review data linking Tie-1 to survival signaling.
7. An angiogenic tyrosine kinase is overexpressed and cleaved in breast cancer: potential roles and regulation of Tie-1 At least three of the protein kinases discovered in our screen, FGFR-4, ephA7, and Tie-1, are directly implicated in angiogenesis, the growth of blood vessels within a tumor. Tie-1 acts as a survival signal in endothelial cells, and we have found that epithelial breast cancer cells overexpress Tie-1. This raises the possibility that Tie-1 may act as a survival signal in tumors. Tie-1 (41, tyrosine kinase with immunoglobulin and Epidermal Growth Factor homology domains) is a receptor tyrosine kinase with an unknown ligand. Tie-1 and the related Tek receptor are transmembrane tyrosine kinases that contain one complete and one incomplete immunoglobulin domain, three epidermal growth factor homology domains, and three fibronectin type III homology domains [41]. Knock-out mice for Tek and Tie-1 have identified distinct functions for the two receptors. Tek knock-out mice die at embryonic day 9.5–10.5, while mice deleted for Tie-1 die at day 14.5 to birth [42,43]. This is a very
different phenotype from mice containing deletions of genes that are required for the early stages of blood vessel formation, such as the VEGFR. VEGFR knockout mice die by embryonic day 8.5 with profound defects in the earliest stages of blood vessel formation [44,45]. In contrast, mice lacking either Tek or Tie-1 form endothelial cells in normal numbers, but Tek-null mice form vessels that lack branching networks and organization into large and small vessels [42,43]. Tie-1null mice die from edema and hemorrhage, suggesting that Tie-1 normally regulates fluid exchange across capillaries and directs responses to hemodynamic stress [43,46]. Tie-1 is expressed predominantly in endothelial cells of adult tissues, in proliferating ovarian capillaries, and during wound healing, suggesting a role in neovascularization [47]. While Tie-1 expression is generally associated with endothelial cells, there is a growing body of evidence linking Tie-1 function to epithelial cell regulation. We originally identified Tie-1 in a breast tumor sample and detected the TIE-1 transcript in 8/9 breast tumor samples, with one tumor expressing high levels of the transcript. More recently, we have used an antibody to the Tie-1 carboxy-terminus, and have found that Tie-1 was expressed in 15/24 breast tumor samples. Surprisingly, immunohistochemistry with the same antibody revealed that Tie-1 was not expressed exclusively in blood vessels within the tumor, but is also expressed by epithelial tumor cells. Tsengi et al. used a similar approach and also found that Tie-1 was expressed in epithelial breast tumor cells [48]. Furthermore, Tie-1-positivity correlated with worse overall survival and disease-free survival. Both reports contradict earlier work by Salven et al. in which an antibody to the Tie-1 amino-terminus indicated that Tie-1 is expressed only in the blood vessels of breast tumors [49]. We believe that these two reports can be reconciled by a recent analysis from our lab. We have found that Tie-1 is overexpressed in breast cancer cells and cleaved near the cell membrane. Thus, the Tie-1 carboxy-terminus would be detected in breast epithelial cells, while the amino-terminal extracellular domain is released. Tie-1 is cleaved in response to phorbol ester stimulation when overexpressed in Chinese hamster ovary cells [50]. This cleavage is dependent on protein kinase C and resembles the processing of a number of other receptor tyrosine kinases, including Axl [51], Kit [52], and colony stimulating factor 1 receptor [53]. It is unclear whether Tie-1 is activated by cleavage in breast cancer cells, and experiments are under way to address the regulation of Tie-1 in cancer cells. If our model is correct, Tie-1 is expressed in epithelial breast cancer cells and cleaved, releasing the extracellular domain and leaving the intracellular domain within the cell. The function of the cleaved intracellular domain
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
Fig. 3. The Tie-1 tyrosine kinase intracellular domain is expressed in breast cancer. By this model, Tie-1 is expressed as a full-length protein in epithelial breast cancer cells and is cleaved, leaving an activated, truncated form of the kinase. Because Tie-1 is capable of survival signaling in other cell types (see text), it is likely that Tie-1 performs an analogous function in breast cancer cells.
is not known, but recent studies indicate a role for the Tie-1 intracellular domain in survival signaling. Overexpression of a chimeric receptor that included the Tie-1 intracellular domain inhibited apoptosis in fibroblasts [54]. These cells contained activated phosphatidylinositol 3-kinase and Akt, key mediators of anti-apoptotic survival signaling (Fig. 2; Ref. [54]). Thus, the Tie-1 intracellular domain functions in anti-apoptotic survival signaling, and future work will determine whether Tie-1 functions as a survival signal in breast cancer (Fig. 3).
8. Downstream effectors of survival signaling: FAK and apoptosis Binding of a ligand to a transmembrane receptor tyrosine kinase activates a complex network of signaling pathways, and one of these responses is the activation of the focal adhesion kinase (FAK, [55]). This section will describe the role of FAK in survival signaling, and the following section will describe the interaction of FAK
43
with the epidermal growth factor receptor. FAK is a tyrosine kinase that localizes to sites of focal contacts between cultured cells and their substratum. FAK is phosphorylated following activation of a number of transmembrane receptors (reviewed in Ref. [56]), suggesting that FAK acts to transmit signals from the membrane to intracellular molecules. We identified FAK in separate PCR-based screens for protein kinases expressed in high-grade sarcomas and breast cancer [1,57]. We subsequently developed a polyclonal antibody to the amino-terminus of FAK, and found that FAK is overexpressed in breast, colon, and thyroid tumors, as well as sarcomas [57–60]. More recently, we developed a monoclonal antibody to the amino-terminal region of FAK, and have found that FAK is overexpressed in invasive breast tumors as well as non-invasive ductal carcinoma in situ (DCIS, Fig. 4, [61]). In contrast to earlier reports that FAK was decreased in metastatic colon cancers, we have recently shown that FAK is expressed in liver metastases at similar or higher levels than the primary tumor (Lark et al., in press). Structurally, FAK contains a kinase domain flanked by extensive amino- and carboxy-terminal domains that direct physical associations with other adhesion and signaling proteins. Little is known about the function of the kinase domain, and no direct substrates of FAK have been characterized. Instead, FAK is thought to act as an adapter molecule, bringing together proteins that regulate adhesion and cell survival. Our original model was that FAK contributed primarily to adhesion in tumors, and as cells became invasive and metastatic, FAK overexpression increased their migration and allowed them to adhere to new substrates. However, detection of FAK in DCIS suggested that FAK activation is not simply a consequence of increased migration, but suggested that FAK might be required for an earlier event in carcinogenesis. We propose that FAK functions in two separate pathways in cancer: regulating adhesion and activation of survival signaling. We will briefly review the data supporting a role for FAK in survival signaling. Our group was the first to provide a link between FAK and apoptosis by showing that disruption of FAK expression in tumor cell lines with antisense oligonucleotides caused loss of adhesion and apoptosis [62]. In subsequent work, Ilic et al. deleted the FAK gene in mice and demonstrated that FAK-deficient cells underwent apoptosis when deprived of serum, suggesting that FAK normally directs survival in these cells [63]. Notably, these FAK-deficient cells only survived following loss of the p53 gene, a situation that is similar to cancer cells. FAK contains a carboxy-terminal domain which directs protein–protein associations with a number of molecules, and overexpression of the FAK
44
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
(A)
(B)
Fig. 4. Overexpression of the FAK in breast tumors. (A) FAK was detected in two separate samples of DCIS by immunohistochemistry using the anti-FAK 4.47 monoclonal antibody. (B) Diagram of potential FAK functions in survival signaling, including Akt regulation via activation of PI3K and a more direct regulation of Akt stability.
carboxy-terminal domain, called FRNK (FAK-related non-kinase [64]) or FAK-CD (FAK carboxy-terminal domain), inhibits FAK function, probably by competing for proteins normally associating with the FAK protein [65]. When FAK-CD is overexpressed in breast cancer cells, the cells lose adhesion [66] and undergo apoptosis [67]. FAK is important as a therapeutic target because FAK inhibition appears to have minimal effects on nontransformed cells. Antisense inhibition of FAK does not induce loss of adhesion or apoptosis in normal fibroblasts [62], and FAK-CD/FRNK overexpression does not induce apoptosis in a broad array of normal cells, including Rat-1 cells [68], rabbit synovial fibroblasts [63], and HME cells [67]. These results suggest that the pathways directed by FAK in non-transformed cells are distinct from those in tumor cells, perhaps due to the presence of redundant signaling pathways in normal cells.
9. Where pathways intersect: EGFR and FAK FAK phosphorylation is elevated in response to a number of extracellular stimuli, suggesting that FAK activation might contribute to these pathways by regulating adhesion and survival signaling. For example, FAK phosphorylation is elevated following EGF stimulation in breast cancer cells, and FAK and EGFR associate physically. Because EGF signals both survival and proliferation, we and other groups have examined the relationship between FAK and EGFR signaling. Initial studies in lung cancer cell lines gave conflicting results and suggested that FAK is dephosphorylated in response to EGF [69], leading to a loss of adhesion in stimulated cells. A second paper using a different lung cancer cell line did not detect changes in FAK phosphorylation following EGF treatment, and found that the role of FAK in these cells was primarily related to migration [70]. However, these lung cancer cell lines express extraordinarily high levels of EGFR, and inhibition of FAK in this tumor cell line does not efficiently induce apoptosis. We utilized a BT474 isolate engineered to express EGFR. In breast cancer cells, inhibition of FAK caused detachment, and this detachment was not affected by EGFR. However, detached cells underwent apoptosis at a much lower rate in EGFR-expressing cells, and the suppression of apoptosis was reversed by small molecule inhibitors of EGFR. We conclude that in breast cancer cells, FAK and EGFR cooperate in survival signaling, with FAK performing a separate function in adhesion. These results confirm that FAK interacts directly with growth factor receptors, and this joint signaling suppresses apoptosis, perhaps by amplifying the survival signal. This is an exciting area of research, and the phenotypic and physical interactions of FAK with other growth factor receptors remain to be elucidated. In particular, it will be important to determine the interactions between FAK, HER-2/neu, and the receptors for CSF-1, PDGF, and ephrins, which were detected in our screen as proteins expressed in breast cancer. In a broader sense, the interactions between FAK and EGFR illustrate the importance of therapeutic strategies targeting multiple pathways.
10. Other tyrosine kinases identified in breast cancer: growth inhibition by the Rak tyrosine kinase While the EGFR, HER-2/neu, FAK, and Tie-1 tyrosine kinases correlate with advanced cancer, other kinases identified in the original screen do not. The Rak tyrosine kinase is highly homologous to the c-Src protooncogene, with single SH2 and SH3 (SH=Src homology) domains and a kinase domain containing a tyrosine residue adjacent to the carboxy-terminal end of the
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
protein [71]. Rak was simultaneously named Frk in a separate study. The activity of the c-Src protein is regulated by phosphorylation of its carboxy-terminal tyrosine Y527. Following phosphorylation, Y527 binds to the amino-terminal SH2 domain, folding the protein in an inactive conformation. The c-Src protein is frequently activated in human tumors, including tumors of the colon and breast, and therapeutic strategies targeting the activity of c-Src in cancer cell lines are capable of inducing apoptosis. The structural similarity of Rak to c-Src suggested that Rak might also be elevated in tumors and contribute to tumorigenesis. However, overexpression of Rak in murine fibroblasts inhibits colony formation [72]. Furthermore, we have recently shown that overexpression of a Rak construct fused to the green fluorescent protein causes human breast cancer cells to arrest the cell cycle in the G1 phase [83]. Thus, the activity of Rak in cultured cells differs markedly from that of Src, which causes oncogenic transformation of murine fibroblasts. The mechanism of Rak function is not known. Rak expression varies in different cell lines, with a nuclear localization in some cell lines [71] and a perinuclear localization in others [83]. Rak is capable of binding to the pRb retinoblastoma tumor suppressor protein in vitro and in vivo [72]. Recent work with Rb+ and Rb osteosarcoma cell lines suggests that the ability of Rak to induce growth arrest is not mediated by binding to pRb. Instead, Rb+ cell lines undergo G1 arrest less efficiently than Rb cell lines following Rak expression, suggesting that Rak is in an inactive conformation when bound to pRb [83]. Our model is that Rak functions in the endoplasmic reticulum to phosphorylate target proteins and trigger growth arrest (Fig. 5). Targets include proteins that directly or indirectly regulate cell cycle entry. In addition, other cell types have nuclear targets of Rak that also regulate the cell cycle. The regulation of Rak in tumors is largely unknown. The RAK transcript is expressed only in epithelial tissues and is highest in liver and kidney [71]. In a panel of nine breast tumors, RAK was expressed weakly in four [1]. A larger western blot analysis of 50 breast tumors suggested that RAK expression was lost in approximately 1/3 of tumors (unpublished observations). The loss of RAK expression in breast cancer is consistent with the chromosomal localization of the RAK gene. RAK is localized to 6q21, a region that is frequently lost in both breast and ovarian cancer. This suggests that loss of RAK expression might be a cause of breast cancer. However, systematic deletion of the murine homologue of RAK, called IYK, did not result in an increase in breast tumor formation [73]. Because of the complexity of tumor suppressive signaling, this result is not entirely surprising. Furthermore, Rak is closely related to eight Src family kinases, and redundant
45
Fig. 5. Growth inhibition through the Rak tyrosine kinase. Rak localization is either nuclear or perinuclear, with localization being cell-type dependent. Rak is thought to transmit an unknown signal from an upstream protein, whereupon Rak phosphorylates a target protein within the endoplasmic reticulum.
functions between the kinases may have suppressed any Rak-associated phenotypes. Thus, Rak expression correlates with growth suppression in breast cancer, but the mechanism of Rak function in breast cancer remains to be elucidated.
11. Cell cycle regulators in breast cancer: the serine threonine kinases CAK/CDK7/STK1 and STK2 In addition to tyrosine kinases, our PCR-based screen identified eight serine/threonine kinases expressed in human breast cancer. The majority have not been characterized, but two have a role in cell cycle regulation. We cloned and sequenced a low molecular weight serine/threonine kinase which we called STK1 (serine-threonine kinase 1 [74]). The STK1 kinase was subsequently cloned as CDK7 (cyclin-dependent kinase 7) or CAK (cyclin-dependent kinase activating kinase), a key regulator of cyclin-dependent kinases (reviewed in Ref. [75]). CAK phosphorylates a threonine residue on the activation loop of CDC2 (cell division cycle 2) and analogous residues on other cyclin-dependent kinases, changing the conformation of the proteins to an active state. CAK also phosphorylates the carboxy-terminal domain of RNA polymerase II, regulating gene transcription (reviewed by [76]). We found the expression of STK1 high in all tissue types examined and in 9/9
46
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
breast tumors [1], although STK1 expression varied somewhat during the cell cycle [74]. In addition to STK1/CDK7/CAK, we identified a second cell cycle regulatory kinase called STK2 in breast cancer cells. The STK2 (serine threonine kinase 2) kinase is part of an evolutionarily conserved family of proteins related to the Apergillus NimA (never in mitosis) protein [77]. Overexpression of STK2 is toxic to most cell types (Yarbrough, unpublished observations), as is the overexpression of proteins related to STK2 [78]. The expression of STK2 is epithelial cellspecific [1], suggesting a role in growth regulation only in certain cell types, unlike the STK1/CDK7/CAK protein, which is expressed ubiquitously. Interestingly, we detected expression of STK2 in only 1/9 breast tumor samples, suggesting that STK2 is not a positive regulator of breast cell proliferation [1]. Recent work has also shown that STK2 is down-regulated following treatment of MDA-MB-453 breast cancer cells with HGF/SF (hepatocyte growth factor/scatter factor) and adriamycin compared to cells treated with adriamycin alone [33]. It is conceivable that STK2 is a pro-apoptotic protein and is down-regulated by HGF/SF survival signaling, and it is notable that STK2 cannot be stably expressed in breast cancer cell lines using standard overexpression systems. Thus, cell cycle regulatory kinases were detected in our previous screen, and they are implicated in cell cycle regulation, control of gene expression, and damage responses.
12. Therapeutics: attacking the tumor survival signals Once a protein is found to be overexpressed in tumors, the next step is to develop a method for inhibiting its function. In the case of the HER-2/neu receptor tyrosine kinase, antibody fragments targeting the extracellular domain of the receptor inhibits its function, presumably by competing for its association with its cognate ligand. As a result, the receptor cannot be activated and its function is nullified. The limitation of HER-2/neu as a therapeutic target is that only 20% of breast tumors overexpress HER-2/neu. Furthermore, the HER-2/neu extracellular domain is overexpressed as a soluble spliced variant, potentially competing for binding to antibodies targeting the membrane-associated receptor. FAK and Tie-1 are expressed in large percentages of breast tumors, and FAK is highly expressed in 80% of breast tumors. However, FAK will be difficult to target with a monoclonal antibody because FAK is an intracellular protein, and antibody fragments will probably not traverse the cell membrane in an active form. Thus, the ideal anti-FAK therapeutic would be a small molecule that can inhibit FAK function or expression after entering the cell.
We have targeted FAK function through two approaches. First, we have attenuated FAK expression with the use of antisense oligonucleotides. These are short oligomers that bind to the coding sequence of the FAK transcript. The DNA–RNA hybrid probably signals degradation of the transcript by RNAses. As described briefly above, FAK attenuation leads to apoptosis in tumor cells in culture, but not in normal fibroblasts. In order to be useful clinically, the main limitation of this approach will be the stability of the oligonucleotides in an organism and the potential side effects of introducing a chemically modified DNA fragment into cells. In spite of all of the concerns regarding antisense oligonucleotides, this approach remains promising for the treatment of cancer. Several of the proteins described in this review have been targetted by antisense oligonucleotides. Inhibition of c-Raf inhibited ovarian cancer cell proliferation and increased levels of apoptosis both in vitro and in xenograph models [79]. However, a Phase I trial of this oligonucleotide revealed serious adverse effects in two out of four patients, including acute hemolytic anemia and acute renal failure and anasarca, but a failure to deplete c-Raf expression [80]. Earlier work indicated that the oligonucleotide was effective in depleting c-Raf [81], suggesting that the timing and dose of treatment are crucial. It is also unclear whether the c-Raf antisense oligonucleotides diminish tumor burden at physiologically relevant doses [82]. Thus, antisense oligonucleotide therapy is still a fledgling technique, and it remains to be seen whether this technique can be used as a therapeutic strategy. In experimental in vitro systems, we have inhibited FAK function by overexpressing a large fragment of the FAK carboxy-terminal region by adenoviral transduction. Even though this virus directed high levels of expression of the FAK carboxy-terminus in normal cells, there was no increase in loss of adhesion or apoptosis in those cells. Nonetheless, in the present climate, it is unlikely that adenoviruses will gain widespread use in the treatment of cancer because of questions about their potential side effects. It is also unclear whether a small molecule inhibitor of FAK kinase activity would be useful as an anticancer therapeutic. FAK appears to function as an adaptor molecule, bringing together proteins that bind to FAK, or perhaps anchoring bound proteins to focal adhesions. The ideal therapeutic would be a small molecule that would disrupt these interactions. While our discussion regards therapeutic approaches for HER2/neu and FAK, the same principles apply for Tie-1. As interest in angiogenesis increases, inhibitors of Tie-1 and Tek will undoubtedly emerge. Our results suggest that novel Tie-1 inhibitors could be useful in preventing the growth of breast cancer cells, and could provide new insights about signaling pathways used in breast cancer.
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
Acknowledgements R.J.C. is a scholar of the Building Interdisciplinary Research Careers in Women’s Health (BIRCWH) program through the NIH (K12HD001441). This work was funded in part by the University of North Carolina University Research Council. W.G.C. is funded by NIH grants CA69510 and CA83895.
References [1] Cance WG, Craven RJ, Weiner TM, Liu ET. Novel protein kinases expressed in human breast cancer. International Journal of Cancer 1993;54:571–7. [2] Hahn WC, Counter CC, Lundberg AS, Beijersberger RL, Brooks MW, Weinberg RA. Creation of human tumour cells with defined genetic elements. Nature 1999;400:464–8. [3] Wang JY, Ki SW. Choosing between growth arrest and apoptosis through the retinoblastoma tumour suppressor protein, Abl and p73. Biochemical Society Transactions 2001;29:666–73. [4] Hackett JA, Greider CW. Balancing instability: dual roles for telomerase and telomere dysfunction in tumorigenesis. Oncogene 2002;21:619–26. [5] Ferreira CG, Epping M, Kruyt FA, Giaccone G. Apoptosis: target of cancer therapy. Clinical Cancer Research 2002;8: 2024–34. [6] Rouslahti E, Reed JC. Anchorage dependence, integrins, and apoptosis. Cell 1994;77:477–8. [7] Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. Journal of Cell Biology 1994;124: 619–26. [8] Sainsbury JRC, Farndon JR, Needham GK, Malcolm AJ, Harris AL. Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet 1987;1: 1398–402. [9] Slamon DJ, Clark GM, Wong SJ, Levin LJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235: 177–82. [10] Nicholson RI, Ge JM, Harper ME. EGFR and cancer prognosis. European Journal of Cancer 2001;4(Suppl):9–15. [11] Lohrisch C, Piccart M. HER2/neu as a predictive factor in breast cancer. Clinical Breast Cancer 2001;2:129–35. [12] Di Fiore PP, Pierce JH, Fleming TP, Hazan R, Ullrich A, King CR, Schlessinger J, Aaronson SA. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 1987;51:1063–70. [13] Hudziak RM, Schlessinger J, Ullrich A. Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis in NIH3T3 cells. Proceedings of the National Academy of Science 1987;84:7159–63. [14] Harari D, Yarden Y. Molecular mechanisms underlying ErbB2/ HER2 action in breast cancer. Oncogene 2000;19:6102–14. [15] Cantley LC. The phosphoinositide 3-kinase family. Science 2002;296:1655–7. [16] Lin J, Adam RM, Santiestevan E, Freeman MR. The phosphotidylinositol 30 -kinase pathway is a dominant growth factoractivated cell survival pathway in LNCaP human prostate carcinoma cells. Cancer Research 1999;59:2891–7. [17] Wen Y, Hu MC-T, Makino K, Spohn B, Bartolomeusz G, Yan D-H, Hung M-C. HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. Cancer Research 2000;60:6841–945.
47
[18] Lim SJ, Lopez-Berestein G, Hung MC, Lupu R, Tari AM. Grb2 downregulation leads to Akt inactivation in heregulin-stimulated and ErbB2-overexpressing breast cancer cells. Oncogene 2000;19:6271–6. [19] Zhou BP, Hu MC, Miller SA, Yu Z, Xia W, Lin SY, Hung MC. HER-2/neu blocks tumor necrosis factor-induced apoptosis via the Akt/NF-kappaB pathway. Journal of Biological Chemistry 2000;275:8027–31. [20] Chen HC, Guan JL. Association of focal adhesion kinase with its potential substrate phosphotidylinositol 3-kinase. Proceedings of the National Academy of Science 1994;91:10148–52. [21] Hazzalin CA, Mahadevan LC. MAPK-regulated transcription: a continuously variable gene switch? Nature Reviews 2002;3: 30–40. [22] Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrinmediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 1994;372:786–91. [23] Santen RJ, Song RX, McPherson R, Kumar R, Adam L, Jeng M-H, Yue W. The role of mitogen-activated protein (MAP) kinase in breast cancer. Journal of Steroid Biochemistry and Molecular Biology 2002;80:239–56. [24] Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer. Journal of Clinical Investigation 1997;99:1478–83. [25] Salh B, Marotta A, Matthewson C, Ahluwalia M, Flint J, Owen D, Pelech S. Investigation of the MEK-MAP kinase-Rsk pathway in human breast cancer. Anticancer Research 1999;19: 731–40. [26] Mueller H, Flury N, Eppenberger-Castori S, Kueng W, David F, Eppenberger U. Potential prognostic value of mitogen-activated protein kinase activity for disease-free survival of primary breast cancer patients. International Journal of Cancer 2000;89:384–8. [27] Von Lintig FC, Dreilinger AD, Varki NM, Wallace AM, Casteel DE, Boss GR. Ras activation in human breast cancer. Breast Cancer Research Treatment 2000;62:51–62. [28] Kumar R, Yarmand-Bagheri R. The role of HER2 in angiogenesis. Seminars in Oncology 2001;28:27–32. [29] Mazumdar A, Wang RA, Mishra SK, Adam L, BagheriYarmand R, Mandal M, Vadlamudi RK, Kumar R. Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor. Nature Cell Biology 2001;3:30–7. [30] Nagy J, Curry GW, Hillan KJ, McKay IC, Mallon E, Purushotham AD, George WD. Hepatocyte growth factor/scatter factor expression and c-met in primary breast cancer. Surgical Oncology 1996;5(l):15–21. [31] Ghoussoub RA, Dillon DA, D’Aquila T, Rimm EB, Fearon ER, Rimm DL. Expression of c-met is a strong independent prognostic factor in breast carcinoma. Cancer 1998;82:1513–20. [32] Fan S, Ma YX, Wang JA, Yuan RQ, Meng Q, Cao Y, Laterra JJ, Goldberg ID, Rosen EM. The cytokine hepatocyte growth factor/ scatter factor inhibits apoptosis and enhances DNA repair by a common mechanism involving signaling through phosphatidyl inositol 3’ kinase. Oncogene 2000;19:2212–23. [33] Yuan RQ, Fan S, Achary M, Stewart DM, Goldberg ID, Rosen EM. Altered gene expression pattern in cultured human breast cancer cells treated with hepatocyte growth factor/scatter factor in the setting of DNA damage. Cancer Research 2001;61: 8022–31. [34] Yee D, Cullen KJ, Paik KJ, Perdue JF, Hampton B, Schwartz A, Lippman ME, Rosen N. Insulin-like growth factor II mRNA expression in human breast cancer. Cancer Research 1988;48:6691–6. [35] Karey KP, Sirbasku DA. Differential responsiveness of human breast cancer cell lines MCF-7 and T47-D to growth factors and 17-b estradiol. Cancer Research 1988;48:4083–92.
48
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
[36] Cullen KJ, Yee D, Sly WS, Perdue J, Hampton B, Lippman ME, Rosen N. Insulin-like Growth Factor Receptor expression and function in human breast cancer. Cancer Research 1990;50: 48–53. [37] Ellis MJ, Jenkins S, Hanfelt J, Redington ME, Taylor M, Leek R, Siddle K, Harris A. Insulin-like growth factors in human breast cancer. Breast Cancer Research Treatment 1998;52:175–84. [38] Myers Jr MG, Grammer TC, Wang LM, Sun XJ, Pierce JH, Blenis J, White MF. Insulin receptor substrate-1 mediates phosphatidylinositol 30 -kinase and p70S6 K signaling during insulin, insulin-like growth factor-1, and interleukin-4 stimulation. Journal of Biological Chemistry 1994;269:28783–9. [39] Kulik G, Klippel A, Weber MA. Antiapoptotic signaling by the insulin-like growth factor I receptor, phosphatidylinositol 3kinase, and Akt. Molecular Cell Biology 1997;17:1595–606. [40] Oki S, Limnander A, Danial NN, Rothman PB. Functional involvement of Akt signaling downstream of Jak1 in v-Ablinduced activation of hematopoietic cells. Blood 2002;100: 966–73. [41] Partanen J, Armstrong E, Makela TP, Korhonen J, Sandberg M, Renkonen R, Knuutila S, Huebner K, Alitalo K. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Molecular Cell Biology 1992;12:1698–707. [42] Dumont DJ, Gradwohl G, Fong GH, Puri MC, Gertsenstein M, Auerbach A, Breitman ML. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes and Development 1994;8:1897–909. [43] Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maquire M, Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 1995;376:70–4. [44] Carmeliet P, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–9. [45] Farrera N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shae K, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–42. [46] Puri MC, Rossant J, Alitalo K, Bernstein A, Partanen J. The receptor tyrosine kinase TIE is required for the integrity and survival of vascular endothelial cells. EMBO Journal 1995;14:5884–91. [47] Korhonen J, Partanen J, Armstrong E, Vaahtokari A, Elenius K, Jalkanen M, Alitalo K. Enhanced expression of the Tie-1 receptor tyrosine kinase in endothelial cells during neovascularization. Blood 1992;80:2548–55. [48] Tseng L-M, Hsu C-Y, Wang H-C, Liu JM, Chang H-M, Lo S-S, Wu C-W, Lui W-Y, Chi C-W. Tie-1 tyrosine kinase is an independent prognostic indicator for invasive breast cancer. Anticancer Research 2001;21:2163–70. [49] Salven P, Joensuu H, Heikkila P, Matikainen MT, Wasenius VM, Alanko A, Alitalo K. Endothelial Tie-1 growth factor receptor provides antigenic marker for assessment of breast cancer angiogenesis. British Journal of Cancer 1996;74:69–72. [50] Yabkowitz R, Meyer S, Yanagihara D, Brankow D, Staley T, Elliot G, Hu S, Ratzkin B. Regulation of Tie-1 receptor expression on human endothelial cells by protein kinase Cmediated release of soluble Tie-1. Blood 1997;90:706–15. [51] O’Bryan JP, Fridell YW, Koski R, Varnum B, Liu ET. The transforming receptor tyrosine kinase, Axl, is post-translationally regulated by proteolytic cleavage. Journal of Biological Chemistry 1995;270:551–7. [52] Yee NS, Hsiau C-WM, Serve H, Vosseller K, Besmer P. Mechanism of down-regulation of c-kit receptor. Roles of
[53]
[54]
[55]
[56] [57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
receptor tyrosine kinase, phosphotidylinositol 30 -kinase, and protein kinase C. Journal of Biological Chemistry 1994;269: 31991–8. Downing JR, Roussel MF, Sherr CJ. Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Molecular Cell Biology 1989;9:2890–6. Kontos CD, Cha EH, York JD, Peters KG. The endothelial receptor tyrosine kinase Tie1 activates Phosphotidylinositol 3Kinase and Akt to inhibit apoptosis. Molecular Cell Biology 2002;22:1704–13. Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. p125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proceedings of the National Academy of Science 1992;89:5192–6. Cary LA, Guan JL. Focal adhesion kinase in integrin-mediated signaling. Frontiers in Bioscience 1999;4:102–13. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of growth factor receptors, the focal adhesion kinase, and other tyrosine kinases in human soft tissue tumors. Annals of Surgical Oncology 1994;1:18–27. Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, Liu ET, Cance WG. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Research 1995;55:2752–5. Owens LV, Xu L, Dent GA, Yang X, Sturge GC, Craven RJ, Cance WG. The focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer. Annals of Surgical Oncology 1996;3:100–5. Weiner TM, Liu ET, Craven RJ, Cance WG. Expression of focal adhesion kinase gene and invasive cancer. Lancet 1993;342: 1024–5. Cance WG, Harris JE, Iacocca MV, Roche E, Yang X, Chang J, Simkins S, Xu L. Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clinical Cancer Research 2000;6: 2417–23. Xu L, Owens LV, Sturge GC, Yang X, Liu ET, Craven RJ, Cance WG. Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth and Differentiation 1996;7:413–8. Ilic D, Almeida EAC, Schlaepfer DD, Dazin P, Aizawa S, Damsky CH. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. Journal of Cell Biology 1998;143:547–60. Schaller MD, Borgman CA, Parsons JT. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase p125FAK. Molecular Cell Biology 1993;13: 785–91. Richardson A, Parsons T. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 1996;380:538–40. Xu LH, Yang X, Craven RJ, Cance WG. The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth and Differentiation 1998;9:999–1005. Xu LH, Yang X, Bradham CA, Brenner DA, Baldwin AS, Craven RJ, Cance WG. The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Journal of Biological Chemistry 2000;275:30597–604. Xiong WC, Macklem M, Parsons JT. Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein. Journal of Cell Science 1998;111:1981–91. Lu Z, Jiang G, Blume-Jensen P, Hunter T. Epidermal growth factor-induced tumor cell invasion and metastasis initiated by
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
[70]
[71]
[72]
[73]
[74]
[75] [76]
dephosphorylation and downregulation of the focal adhesion kinase. Molecular Cell Biology 2001;21:4016–31. Hauck CR, Sieg DJ, Hsia DJ, Loftus JC, Gaarde WA, Monia BP, Schlaepfer DD. Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Research 2001;61:7079–90. Cance WG, Craven RJ, Bergman M, Xu L, Alitalo K, Liu ET. Rak, a novel nuclear tyrosine kinase expressed in epithelial cells. Cell Growth and Differentiation 1994;5:1347–55. Craven RJ, Cance WG, Liu ET. The nuclear tyrosine kinase Rak associates with the product of the Retinoblastoma tumor susceptibility gene, pRb. Cancer Research 1995;55:3969–72. Chandrasekharan S, Qiu TH, Alkharouf N, Brantley K, Mitchell JB, Liu ET. Characterization of mice deficient in the Src family nonreceptor tyrosine kinase Frk/rak. Molecular Cell Biology 2002;22:5235–47. Levedakou EN, He M, Baptist EW, Craven RJ, Cance WG, Welcsh PL, Simmons A, Naylor SL, Leach RJ, Lewis TB, Bowcock A, Liu ET. Two novel human serine/threonine kinases with homologies to the cell cycle regulating Xenopus MO15, and NimA kinases: cloning and characterization of their expression pattern. Oncogene 1994;9:1977–88. Kaldis P. The CDK-activating kinase (CAK): from yeast to mammals. Cell Molecular Life Science 1999;55:284–96. Riedl T, Egly JM. Phosphorylation in transcription: the CTD and more. Gene Experiment 2000;9:3–13.
Craven Craven received his Ph.D. in Genetics and Molecular Biology from the University of North Carolina at Chapel Hill with Edison T. Liu, M.D., former director of Clinical Sciences at the N.C.I. Dr. Craven did his Post-doctoral fellowship with Dr. Thomas Petes, member of the National Academy of Sciences. Dr. Craven is an Assistant Professor at UNC in the Surgical Oncology Division of the Surgery Department and is a Scholar of the B.I.R.C.W.H. (Building Interdisciplinary Research Careers in Women’s Health) program.
49
[77] Osmani SA, Pu RT, Morris NR. Mitotic induction and maintenance by over-expression of a G2-specific gene that encodes a potential protein kinase. Cell 1988;53:237–44. [78] Lu KP, Hunter T. Evidence for a NIMA-like mitotic pathway in vertebrate cells. Cell 1995;81:413–24. [79] McPhillips F, Mullen P, Monia BP, Ritchie AA, Dorr FA, Smyth JF, Langdon SP. Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer. British Journal of Cancer 2001;85:1753–8. [80] Rudin CM, Holmlund J, Fleming GF, Mani S, Stadler WM, Schumm P, Monia BP, Johnston JF, Geary R, Yu RZ, Kwoh TJ, Dorr FA, Ratain MJ. Phase I Trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer. Clinical Cancer Research 2001;7:1214–20. [81] O’Dwyer PJ, Stevenson JP, Gallagher M, Cassella A, Vasilevskaya I, Monia BP, Holmlund J, Dorr FA, Yao KS. c-raf-1 depletion and tumor responses in patients treated with the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A). Clinical Cancer Research 1999;5:3977–82. [82] Monia BP. Anti-tumor activity of C-raf antisense–correction. Nature Med 1999;5:127. [83] Meyer S, et al. The analysis of the Tie-1 intracellular domain expression in breast cancer is in press at Tumor Biology. International Journal of Cancer 2003;104:139–46.
Lightfoot Lightfoot received his M.D. from the University of North Carolina at Chapel Hill. He is presently a Senior Resident in the Surgery Department at the University of North Carolina at Chapel Hill.
R.J. Craven et al. / Surgical Oncology 12 (2003) 39–50
50
Cance Cance earned his B.S. and M.D. degrees from Duke University and did his residency at Washington University in St. Louis. Since his fellowship at SloanKettering, Dr. Cance has become Professor and Chief of Surgical Oncology at the University of North Carolina at Chapel Hill, with an adjunct appointment in the Department of Cell and Developmental Biology. In January 2003, Dr. Cance began as Chair of the Department of Surgery at the University of Florida.