Protein Kinase C-β as a Therapeutic Target in Breast Cancer

Protein Kinase C-␤ as a Therapeutic Target in Breast Cancer George W. Sledge, Jr and Yesim Gökmen-Polar Combining existing breast cancer therapies with novel agents that interfere with major signaling pathways is a promising approach. Targeting protein kinase C (PKC)-␤ may serve as an attractive candidate in this regard for the following reasons: first, PKC-␤ II (a splice variant of PKC-␤) has been implicated in tumorigenesis in human and rodent models. Second, PKC-␤, mainly PKC-␤II, is the predominant mediator of vascular endothelial growth factor-induced endothelial cell proliferation, which is a well-known stimulator of tumor angiogenesis and growth in breast cancer. There is increasing evidence that PKC␤-selective inhibitors are effective in both preclinical and clinical trials. Enzastaurin, a potent inhibitor of PKC-␤, suppresses both tumor growth and tumor-induced angiogenesis in human tumor xenografts. Phase II trials of enzastaurin in recurrent high-grade gliomas and lymphomas have shown promising results. A similar compound, ruboxistaurin, is also under investigation in clinical trials for diabetic complications. This review focuses on the rationale for using PKC-␤ as a therapeutic target at both the preclinical and clinical levels in breast cancer. Semin Oncol 33(suppl 9):S15-S18 © 2006 Elsevier Inc. All rights reserved.

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rotein kinase C (PKC) is a family of at least 12 distinct serine/threonine kinases that are involved in diverse cellular functions including cell proliferation, differentiation, apoptosis, tumorigenesis, angiogenesis, and drug resistance.1-3 Individual PKC isozymes have distinct cellular functions based on their differences in tissue expression, subcellular localization, and activator/substrate specificity.4-7 The role of PKC isozymes in cancer has been reviewed in depth by Fields and Gustafson.1 Therefore, it is logical to develop isozyme-specific inhibitors, with the potential for targeting specific intracellular pathways, rather than broadrange PKC inhibitors. This review discusses the rationale underlying therapeutic targeting of PKC-␤ for cancer in general and for breast cancer in particular.

The Role of PKC-␤ in Cancer The PKC-␤ gene codes for two distinct proteins generated by alternative splicing: PKC-␤I and PKC-␤II, which differ in the last 50 amino acids.8 Several studies indicate that these two

Departments of Medicine and Pathology, Indiana University Cancer Center, Indianapolis, IN. Dr Sledge’s work is supported in part by a grant from the Breast Cancer Research Foundation and the Walther Medical Foundation. Address reprint requests to George W. Sledge, Jr, MD, Departments of Medicine and Pathology, Indiana University Cancer Center, RT-473, Indianapolis, IN 46220. E-mail: [email protected]

0093-7754/06/$-see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2006.03.019

splice variants participate in opposing cellular functions: PKC-␤II is involved in cellular proliferation, whereas PKC-␤I is associated with differentiation.7,9,10 The role of PKC-␤II has been well documented in proliferation and tumorigenesis of colorectal carcinomas.9,11 Murray et al9 showed that overexpression of PKC-␤II in the colonic epithelium resulted in hyperproliferation and increased susceptibility to carcinogen-induced colon carcinogenesis. Expression of PKC-␤II increases dramatically early in the colon carcinogenic process.11 Furthermore, PKC-␤II also induced an invasive phenotype in rat intestinal epithelial cells.12 PKC-␤I, on the other hand, has been shown to be down-regulated in colonic neoplasms.13,14 A decrease in PKC-␤I levels has also been observed in the adenomatous polyposis coli multiple intestinal neoplasia (APCmin) mouse colon carcinogenesis model.15 Other studies have also emphasized the contribution of PKC-␤ to the malignant progression of diffuse, large, B-cell lymphomas (DLBCLs)16 and gliomas.17 In DLBCL especially, patient survival was inversely correlated with high levels of PKC-␤ expression.16 Treatment of DLBCL cells with the PKC-␤-specific inhibitor LY379196 induced apoptosis in PKC-␤ overexpressing DLBCL cells.18

PKC-␤ in Breast Cancer Several studies have investigated the expression of PKC isozymes and activity in breast cancer. PKC activity was elevated in malignant breast tumors relative to surrounding norS15

S16 mal tissue,19,20 and was correlated with the more aggressive estrogen receptor-negative phenotype.21,22 However, these early studies failed to differentiate between PKC isozymes and their subtypes. Among the PKC isozymes, PKC-␦ has been shown to be involved in mammary tumor metastasis,23,24 and PKC-␣ has been implicated in malignant transformation and tumor cell proliferation of the breast.25 Despite these results, breast cancer trials on the role of PKC-␤ or any other PKC isozymes in relation to PKC-␤ are not well addressed. Most of the available studies of PKC-␤ in breast cancer relate to in vitro cell line studies, typically with a limited number of human breast cancer cell lines. In one in vitro study, stable overexpression of PKC-␣ significantly increased the endogenous expression of PKC-␤, accompanied by decreases in levels of PKC-␩ and -␦. This model also showed increased proliferation and anchorage-independent growth, alterations in cellular morphology characterized by loss of epithelial appearance with a marked increase in vimentin expression, decreased estrogen receptor messenger RNA and estrogen-dependent gene expression, and enhanced tumorigenesis and metastasis when injected into nude mice.26 Another study by Morse-Gaudio et al27 confirmed the association of PKC-␤ with MCF-7 cells overexpressing PKC-␣ and showed that conventional PKC isozymes are associated with the metastatic phenotype in breast cancer cells. In contrast, a separate preclinical study showed that overexpression of PKC-␣ and -␤ (PKC-␤I) induced a less-aggressive phenotype, which was characterized by reduced in vitro invasiveness and markedly diminished tumor formation and growth.28 Improved anti-estrogen resistance in mammary tumor cells has also been associated with MCF-7 cells overexpressing active PKC-␦.29 These discrepancies can be explained in two ways. First, it is important to analyze the expression of each PKC-␤ splice variant to assess its potential role in breast cancer, because the expression of PKC-␤I and PKC-␤II may be differentially regulated.30 Second, overexpression of one PKC isozyme may alter expression of other PKC isozymes. Therefore, it is necessary to redefine the role of PKC-␤ and its associated PKC isozymes in breast cancer. To date, no large-scale studies of the relationship between PKC isozymes and clinical outcome have been performed. Further studies are underway to analyze human breast cancer specimens for PKC-␤II expression.

PKC-␤ and Angiogenesis Vascular endothelial growth factor (VEGF) is one of a number of genes associated with angiogenesis, which is a process necessary for tumor growth. VEGF acts via kinase insert domain-containing receptor/VEGF receptor (VEGFR)-2 and subsequent phospholipase C (PLC)-␥ tyrosine phosphorylation to promote activation of PKC-␤ in endothelial cells.31 PKC-␤ activation, in turn, induces the activation of the Raf/ mitogen-activated protein kinase (MAPK) and extracellularsignal-regulated kinase (ERK) cascade and subsequent endothelial cell proliferation (Fig 1).32,33 Yoshiji et al34 reported that PKC-␤ inhibition reduced tumor growth by decreasing

G.W. Sledge, Jr and Y. Gökmen-Polar

Figure 1 PKC-␤ in VEGF signaling. Binding of VEGF to its receptor (KDR/VEGFR-2) initiates the tyrosine phosphorylation and activation of phospholipase-␥ resulting in the generation of DAG, IP3, and Ca2⫹ mobilization, and subsequent activation of PKC-␤. Further activation of Raf, MEK, and ERK leads to both mitogenesis and cPLA2 activation, and generation of PGs. Signaling by Ca2⫹ also results in the production of long-term PGs through the PP2B, the transcription NFAT, and COX-2 induction. Abbreviations: COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; IP3, inositol 1,4,5-trisphosphate; KDR, kinase insert domain-containing receptor; MEK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T-cells; P, phosphate; PGs, prostanoids; PKC, protein kinase C; PLC-␥, phospholipase C-␥; PP2B, serine/threonine phosphatase; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

VEGF-dependent MAPK activation, suggesting the involvement of PKC-␤ in VEGF-induced tumor development and angiogenesis. The role of PKC-␤ in the VEGF-induced Akt pathway is still not clear. VEGF may activate phosphatidylinositol-3-kinase and PLC-␥ via different pathways.31,32 The PLC-␥/PKC/ERK pathway also mediates VEGF-induced activation of cytosolic phospholipase A2 and results in generation of cyclooxygenase-derived prostanoids.33 Ca2⫹ also contributes to the activation of the protein serine/threonine phosphatase 2B, which activates transcription factor nuclear factor of activated T cells, and leads to the induction of cyclooxygenase-2, which further activates long-term prostanoid production.33 Recent data from the E2100 first-line metastatic breast cancer trial35 studying the combination of paclitaxel plus bevacizumab showed that the addition of a VEGF-targeted agent to a standard chemotherapeutic agent could result in a significant improvement in progression-free and overall survival. This proof-of-concept trial may provide a foundation for therapeutic targeting of angiogenesis in breast cancer, and

PKC-␤ as a target in breast cancer raises the question of whether other therapeutic approaches to targeting angiogenesis might be beneficial in breast cancer. To date, there are no preclinical or clinical data examining the combination of VEGF and PKC-␤ targeting. It seems reasonable to examine such an “upstream/downstream” approach, both in the laboratory and the clinic. Plans for both areas are being investigated.

Therapeutic Targeting of PKC-␤ PKC-␤ clearly represents a potential angiogenesis therapeutic target. Indeed, the PKC-␤-specific inhibitor LY379196 has been shown to induce apoptosis in DLBCL cells overexpressing PKC-␤.18 Studies of LY379196, as well as LY333531, another PKC-␤ inhibitor, have been reviewed by Goekjian and Jirousek.2 One of the most promising PKC-␤ inhibitors, enzastaurin, is an acyclic bisindolylmaleimide that competes with the adenosine triphosphate binding site of PKC, preventing substrate phosphorylation. While it is capable of inhibiting several members of the PKC family, it is most potent against PKC-␤ (inhibitor concentration 90 ⫽ .069 ␮mol/L).36 It is relatively inactive against other kinases, including tyrosine kinases. Enzastaurin is a more potent inhibitor of endothelial cell proliferation than of tumor cell proliferation, at least for some cancer cell lines in vitro.37 This, of course, raises the possibility that its effects will be mediated predominantly via an anti-angiogenic mechanism, although this remains to be demonstrated in the clinic. In preclinical models, enzastaurin inhibits tumor growth in vitro and in vivo in numerous human tumor models, and has significant anti-angiogenic activity in vitro and in vivo. In vivo, PKC-␤ inhibition with enzastaurin has been shown to inhibit VEGF and basic fibroblast growth factor-stimulated angiogenesis in the rat corneal micropocket assay, a standard angiogenesis assay.37 In some, but not all, tumor-bearing mouse models, PKC-␤ inhibition decreases plasma VEGF levels after 5 to 7 days of treatment.38 In several murine model systems, including the MX-1 human breast cancer xenograft model system, PKC-␤ inhibition results in decreased intratumoral angiogenesis as measured by decreased microvessel density.39 Enzastaurin has entered phase I trials, both as monotherapy40 and in combination with gemcitabine and cisplatin.41 In both trials the agent was well tolerated, with promising initial evidence of activity. More recently, enzastaurin has entered phase II trials for treatment of glioma and lymphoma, and colon, prostate, non–small cell lung, ovarian, and pancreatic cancers, as well as chronic lymphocytic leukemia. In gliomas, the agent showed promising early evidence of activity, with responses observed in heavily pretreated patients.42 In a group of 55 patients with heavily pretreated DLBCL, enzastaurin therapy produced prolonged disease stabilization and occasional remissions.43 To date, there have been no trials of enzastaurin in metastatic breast cancer. In preclinical models, enzastaurin is growth inhibitory in vitro against some (MCF-7, NCI/ADR-RES, and MDA-MB-

S17 435), but not all (HS-578T, BT-549, and T-47D), breast cancer cell lines in the NCI 60 cell-line panel.39 In a preclinical MX-1 xenograft model of human breast cancer, Teicher et al39 showed that the combination of enzastaurin with either paclitaxel or carboplatin resulted in significantly greater anti-tumor activity than either agent alone.38 At present, no preclinical research has been conducted combining this agent with either human epidermal growth factor receptor-2or estrogen-receptor-targeted therapies. A phase II clinical trial of enzastaurin as monotherapy for advanced breast cancer will begin in the near future.

Conclusion PKC-␤ represents an under-explored therapeutic target in breast cancer. The growing evidence for the importance of angiogenesis as a therapeutic target in breast cancer and its modulation via PKC-␤ suggest the promise of targeting this molecule. New data should emerge in the near future showing the biologic importance of PKC-␤ in clinical breast cancer, and will help to determine whether specific agents targeting PKC-␤ (such as enzastaurin) will offer benefit to patients with this disease.

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