Molecular pathways of pancreatic carcinogenesis

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Drug Discovery Today: Disease Mechanisms

DRUG DISCOVERY

TODAY

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA

DISEASE Cancer MECHANISMS

Molecular pathways of pancreatic carcinogenesis Martin E. Fernandez-Zapico1,2, Raul Urrutia1,2,3,* 1

Gastroenterology Research Unit, Mayo Clinic College of Medicine, Alfred 2-445A, Saint Mary’s Hospital, Rochester, MN 55905, USA Tumor Biology Program, Mayo Clinic College of Medicine, Alfred 2-445A, Saint Mary’s Hospital, Rochester, MN 55905, USA 3 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Alfred 2-445A, Saint Mary’s Hospital, Rochester, MN 55905, USA 2

Pancreatic cancer has one of the poorest prognoses among human neoplasms, with an overall five-year survival rate of 3%. Thus, there are significant efforts to understand the molecular mechanisms underlying the development pancreatic cancer that might lead to effective diagnostic and treatment strategies that improve the prognosis of the disease. In this review, we discuss signaling pathways that control cell growth, differentiation and migration that are dysregulated in this cancer. Particular emphasis is given to the potential there is to manipulate these pathways for thera-

Section Editor: Silvio Gutkind – National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA Pancreatic cancer has one of the poorest prognoses among human neoplasms, with an overall five-year survival rate of 3%. It usually occurs without appreciable symptoms and can remain undetected until the tumor is at an advanced stage. Pancreatic cancer tumors are highly resistant to conventional chemotherapy and radiation treatments. Although the use of combined modality therapy has produced modest increases in survival, significant advances in the treatment of this cancer have not been made. In this article, Martin E. Fernandez-Zapico and Raul Urrutia discuss the signaling pathways that control cell growth, differentiation and migration, which are dysregulated in this cancer, and the potential there is to manipulate these pathways for therapeutic purposes.

peutic purposes. Introduction Pancreatic cancer (PC) is the fourth leading cause of cancer deaths of adults in the United States with a one-year survival rate of <20% and a five-year survival rate of 3% [1]. PC generally occurs without appreciable symptoms and thus can go undetected until tumor development is in an advanced stage. Surgical resection has been considered the only curative modality for this disease, but only 10% of patients are candidates for surgery and even then the fiveyear survival rate does not increase much above 3% [2,3]. In addition, these tumors are highly resistant to conventional chemotherapy and radiation treatments [2]. Although the use of combined modality therapy has produced modest increases in survival, significant advances in the treatment of PC have not been made [3]. Thus, further understanding of *Corresponding author: (R. Urrutia) [email protected] 1740-6765/$ ß 2004 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmec.2004.09.003

the molecular cascades involved in the tumor biology as well as the resistance of these neoplasias to therapy is needed to establish a basis for development of more effective treatments of PC. For an overview of these pathways, see Fig. 1.

Tumor suppressor pathways p16INK4a, cyclin-dependent kinase 4 and retinoblastoma protein This pathway is a component of global cell cycle regulation and inactivation might occur through alterations of retinoblastoma gene (Rb) or p16 in PC [4–6]. The p16 gene product inhibits the interaction of cyclin D with cyclin-dependent kinase 4 (CDK4). This cyclin D–CDK4 complex phosphorylates Rb preventing the formation of the E2F–RB complex, which drives cell cycle to S phase [7–8]. Abrogation of p16 occurs through several mechanisms in PC, namely inactivation by homozygous deletion or mutation in addition to www.drugdiscoverytoday.com

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Figure 1. Representative schematic of the molecular pathways involved pancreatic carcinogenesis. As shown in the cartoon the end point of these cascades in the activation of several transcription factors that regulate the expression of genes involved in cell growth and survival in pancreatic cancer cells. Oncogenic pathways involving receptor tyrosine kinases (RTKs) activate Ras and PI3K cascades that lead to modulation of the expression of growth-related genes. Similarly, p16INK4a and TP53 tumor suppressor pathways trigger a signaling cascade that activates downstream effectors such as CDKs, which oppose to the proliferation-driven forces of the oncogenic pathways. Abbreviations: CDK, cyclin-dependent kinase; COX-2, cyclooxygenase 2; ERK extracellular regulated kinase; GEF, guanine–nucleotide exchange factor; Hsp90, heat shock protein 90; PI3K, phosphatidylinositol-3-kinase; TGF-bR, transforming growth factor-b receptor.

hypermethylation in 27–98% of PC [5,6]. This is significant because tumor suppressor gene alterations appear to be associated with the prognosis PC. For instance, Gerdes et al. [9] reported a greater frequency of p16INK4a alterations in shortterm compared with long-term survivors. p19ARF is another tumor suppressor gene that is encoded by the p16INK4a locus and generated by an alternate reading frame using the same second exon as p16INK4a but a separate first exon and promoter. Therefore, p19ARF is inactivated in all tumors bearing p16INK4a deletions and in those with a considerable fraction of tumors within this gene. The p19ARF protein binds to murine double minute 2 gene (mdm2), thereby inhibiting its interaction with tumor suppressor protein 53 (TP53). Normally, mdm2 regulates TP53 by accelerating its degradation and blocking its transcriptional regulatory properties. Consequently, loss of p19ARF mimics mdm2 overexpression by permitting this protein to freely interact with TP53. Therefore, most homozygous deletions of this locus result in the coordi248

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nate loss of three gene products involved in regulating the G1/ S checkpoint with synergistic facilitation of cell-cycle progression [7]. Rb, another component of this pathway, is altered in approximately 5–10% of PC [5,6]. Tumors with p16INK4a alterations do not usually have coexistent Rb1 mutations since these would tend to mitigate the effects of p16INK4a loss. Reconstitution of p16INK4a–Rb pathway blocks cell proliferation, invasion, tumor angiogenesis and cell spreading in xenografts models [10,11]. Hosotani et al. [12] have reported the use of a synthetic peptide that suppresses pancreatic cell growth by blocking CDK4 and CDK6 activity.

Pathways involving transforming growth factor-b Transforming growth factor-b (TGF-b) signaling is associated with tumor suppression by blocking the activation of G1 cycling-dependent kinase leading to suppression of Rb phosphorylation and downregulation of mitogen-induced c-Myc expression [13]. Through these mechanisms, TGF-b signaling

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interferes with the progression of the cell cycle and induces apoptosis. Thus, interference of TGF-b signaling often contributes to a loss of cell growth control. Several different mechanisms can account for loss of TGF-b signaling in PC [14]. These include mutations or transcriptional repression of TGF-b receptors and mutations from the key downstream molecules, for example, similar to mothers against decapentaplegic (Smad4) also known as DPC4 [5,6]. Cells displaying alterations in this pathway do not remain quiescent and are primed to rapidly reenter the cell cycle. Therefore, this pathway is a bona fide therapeutic target for PC. In fact, inhibition of the type II TGF-b receptor attenuates expression of metastasis-associated genes and suppresses PC cell invasion [15]. Restoration of wildtype Smad4 by gene therapy also reverses the invasive phenotype in pancreatic adenocarcinoma cells [16]. Paradoxically, although the tumor suppressive effects of TGF-b signaling are well documented, this pathway contributes to tumor progression. On the basis of this knowledge, the emerging paradigm indicates that TGF-b signaling acts as tumor suppressor in normal cells and at early stages of carcinogenesis, whereas it switches into a tumor promoter during tumor progression.

The TP53 pathways The TP53 family of proteins that has three members, TP53, p63, and p73, which participate in DNA repair, cell cycle arrest, senescence and apoptosis. Mutations in the TP53 gene abrogate its function leading to genetic instability and tumor progression [4,17]. The TP53 gene, which is located on chromosome 17p13, is inactivated in 40–75% of PC. The dominant mechanism of TP53 inactivation is a missense mutation within the DNA-binding domain. In addition, mutations in TP53 result in an abnormal protein that is longer-lived than the wild-type counterpart. Because TP53 normally functions as a dimer, the mutant protein sequesters the wild-type form in nonfunctional heterodimers. These types of alterations arise at later stages of pancreatic carcinogenesis. However, germline mutation of TP53 underlie genetic defect in the LiFraumeni syndrome, in which, PC appears as one of the most commonly associated malignancies. TP53 inhibits the cell cycle by inactivating CDK4 directly, and inactivating p21 (also know as Cip1 and WAF1) indirectly. Thus, TP53 functions at the G1/S transition by blocking entry into S phase caused by DNA damage. TP53 also mediates DNA damageinduced apoptosis in sensitive cells. Apoptosis is the major mechanism by which ionizing radiation and many chemotherapeutic agents cause tumor cell death. Reintroduction of wild-type TP53 in mutant pancreatic tumor cells activates apoptosis. Consequently, TP53 mutations are associated with decreased response to chemotherapy and a decreased survival in some tumors. Furthermore, Hecht et al. used an engineered replication-selective virus that selectively replicates and lyses the tumor cells of PC patient that are deficient in TP53 function but not cells with functional TP53 [18,19].

Drug Discovery Today: Disease Mechanisms | Cancer

The STK11 pathway The Peutz-Jeghers syndrome, an autosomal dominant syndrome, is caused by germline mutation in the tumor suppressor gene Serine/threonine kinase 11 (STK11) also known as LKB1. Affected individuals manifest hamartomatous gastrointestinal polyps and a predisposition to other gastrointestinal tumors, including PC. STK11 inactivation has been identified in 5% of sporadic PC [20,21]. The STK11 protein is involved in two biologically important pathways that lead to cancer. First, STK11 helps to maintain a polarized epithelium and second, STK11 activates the AMP-dependent kinase (AMPK), which controls the cellular energy balance. Activation of AMPK redirects cell metabolism towards the generation of ATP and away from energy expenditure, such as the synthesis of proteins required for cell division. Therefore, cells lacking STK11 display a proliferative advantage, by activating catabolic pathways to fuel cell division. These insights into STK11 function suggest future new therapeutic strategies, by regulation of AMPK activity. For instance, metformin, the widely prescribed oral hypoglycemic for diabetes, activates AMPK. Thus, metformin-related drugs might arrest cell proliferation in PC leading to the development of novel treatments for this disease [22].

Fanconi–BRCA2 pathways This cascade is involved in the cell-cycle checkpoint and in DNA-repair. Several Fanconi proteins, including A, C, E, F and G form a constitutive complex in the nucleus of normal human cells. In response to DNA damage, or during the S phase of the cell cycle, this complex mediates the monoubiquitination (mUb) of other members of the Fanconi family of FANCD2. Activated FANCD2 translocates to DNA-repair chromatin foci. These foci contain BRCA1 and a BRCA2– FANCD1 complex. Mutations in this pathway occur in familial PC [23]. For instance, BRCA2 mutations are also found in sporadic PC, indicating that both germline and sporadic events can have a role in PC tumorigenesis [24]. Similarly, Hahn et al. [25] determined mutations of BRCA2 in 19% of the 26 European families in which at least two first-degree relatives had a confirmed diagnosis of PC. Rogers et al. [26] reported inherited and somatic mutations of FANCC and FANCG in young-onset PC. Thus, these findings indicate a general involvement of Fanconi pathways with an inherited risk of cancer.

DNA mismatch repair pathways Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is caused by germline mutations in DNA mismatch repair genes MutL homolog 1 (MLH1), mutS homolog 2 (MSH2) and mutS homolog 2 (MSH6). HNPCC affected patients develop colorectal cancers and show an increased incidence of other tumor types, including PC. The pancreatic tumors associated with HNPCC show a different mutation www.drugdiscoverytoday.com

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profile, such as lower mutations of K-ras or TP53, and a lessaggressive clinical course compared with the sporadic PC [5,6,23].

Oncogenic pathways Receptor tyrosine kinase pathway The expression of receptor tyrosine kinases (RTKs) and their ligands is an early event in the development of PC [5,6]. The ErbB family of RTKs includes epidermal growth factor receptor 1 (ErbB1), ErbB2 (HER2/neu), ErbB3 and ErbB4, which share sequence similarity and are widely expressed in human tissues. After the binding of the ligand, these receptors both homo- and heterodimerize and become active. Activation involves transphosphorylation of tyrosine residues by their intrinsic TK activity. Phosphotyrosine residues generate docking sites for other signaling molecules harboring either Src homology 2- or phosphotyrosine-interacting domains. The activation of these pathways regulates proliferation thus contributing to cellular transformation [27–30]. Overexpression of ErbB1 and its downstream molecules transform normal cells and frequently occurs in PC. Overexpression of ErbB1 and its ligands occurs in 95% of PC patients and worsens their prognosis [3–6]. Currently, therapeutic strategies targeting ErbB1 consist of inhibitory antibodies and tyrosine kinase inhibitor drugs [31]. The overexpression of ErbB2 in PC correlates with a more glandular, well-differentiated tumor histology and noninvasive intraductal lesions of the pancreas. Other pre-neoplastic intraductal lesions also express ErbB2, suggesting the participation of this receptor during steps of PC development [5,6]. Insulin growth factor receptor 1 (IGF-1R) is another RTK that is involved in cell growth, transformation and resistance to apoptosis. The IGF1R, which binds to IGF-1, IGF-2 and insulin, is also overexpressed in PC [5,32]. The activation of IGF-1R triggers a signaling cascade, which involves IRS-1, IRS-2, Shc and growth factor receptor-bound protein 2 (Grb2), and activation mitogen-activated protein kinase (MAPK), ultimately resulting in a cellular proliferative response. In cancer, IGF1R shows aberrant activation that facilitates rapid entry into the cell cycle promoting cell survival.

The Ras–Raf–MEK–ERK pathway K-ras mutations occur in preneoplastic PC and in >90% of PC. Thus, K-ras gene mutations are an early and essential step in the development of this tumor [32,33]. K-ras encodes a GTPbinding protein involved in signal transduction. Constitutive activation of K-ras occurs by point mutations at codons 12, 13 or 61, locking the protein in a GTP-bound state. K-ras activation requires farnesylation by a farnesyl transferase to attach to the membrane and become active. Thus, blockage of farnesylation inhibits the normal signaling as well as cell transformation by mutant K-ras [34,35]. Unfortunately, however, the results of the farnesyl transferase inhibitors trials 250

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have been disappointing. This lack of clinical efficacy occurs by an alternative geranylgeranylation of K-ras, rather than farnesylation [19,31]. In addition, as PC progress develops additional survival pathways that might compensate for K-ras inhibition such as Vav1 activation (M. FernandezZapico and D.D. Billadeau, unpublished). Interestingly, an antisense approach to PC therapy has been developed. Liposome-mediated gene transfer of this antisense has been used in xenografts of human PC, where it inhibited tumor progression [19]. Other alterations of the K-ras pathway can occur via different mechanisms. For instance, K-ras activates the Raf–MEK–ERK pathway by first localizing Raf to the plasma membrane, initiating a mitogenic cascade [36,37]. Studies with dominant-negative mutants and antisense molecules suggest that inhibition of Raf kinase is an important target for cancer therapy. Preclinical studies indicate that Bay 43-9006 is a potent inhibitor of Raf kinase in vitro and in vivo, and has significant dose-dependent anti-tumor activity [31].

Phosphatidylinositol 3-kinase-AKT-nuclear factor-kB pathway This pathway inhibits apoptosis that is linked to chemoresistance in PC by inactivating pro-apoptotic proteins such as BAD, caspase-9, Forkhead transcription factors and nuclear factor-kB (NF-kB) [38]. NF-kB exists as a homo- or heterodimer of five members of the mammalian RelNF-kB family. These proteins are retained in the cytoplasm by IkB. Several stimuli activate a kinase complex that phosphorylates IkB, triggering its polyubiquitination and subsequent degradation. NF-kB then enters the nuclei, thus, promoting survival and resistance to chemotherapy [39]. Therefore, NF-kB is an important target for therapeutic intervention in PC. However, no NF-kB-specific agents are being tested in clinical trials (Table 1). However, inhibition of protein degradation by blocking the ubiquitin–proteasome pathway that degrades IkB is a novel approach to target NF-kB. PS-341 is the first proteosome inhibitor to have progressed to clinical trials. In pre-clinical studies, this drug showed anti-tumor activity as a single agent and additive effects when combined with standard chemotherapy. Ryan et al. demonstrated that PS-341 has good tolerability and anti-tumor activity in patients with metastatic PC [31]. Finally, another promising approach is based on Akt inactivation, which can be inhibited by interfering with an Akt–Hsp90 complex with ansamycin. Ansamycin reduces the half-life of Akt by inducing its ubiquitination and proteasomal degradation [31].

Cyclooxygenase-lipooxygenase pathway Cyclooxygenase-1 (COX-1) and COX-2 catalyze the conversion of arachidonic acid into prostaglandin H2, which is then further metabolized to form various prostaglandins and other eicosanoids. Despite the structural similarity between the two isoforms, COX-1 and COX-2 differ substantially in the reg-

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Drug Discovery Today: Disease Mechanisms | Cancer

Table 1. Potential target for therapy in pancreatic cancer Target

Therapy

Stage of development

Advantages and disadvantages

Refs

ErbB1 (EGFR)

MAbs RTKIs

Phase III Phase I–III

Increased survival Well tolerated, Phase III is ongoing

[19,31]

ErbB2 (Her2/Neu)

MAbs

Phase II

Partial tumor regression and increased survival

[19,31]

VEGFR

MAbs RTKIs

Phase II Phase I

Partial response Increased survival

[19]

TP53

Gene therapy

Phase I and II

Low Toxicity Partial anti-tumor activity

[18]

K-ras

Gene therapy

Phase II

[19,31,34,35]

FTIs

Phase II and III

Low toxicity Increased survival in combination with Gemcitabine No differences in the rate of progression of PC

Raf

RIs

Phase II

Ongoing

[31]

MEK

MEK-I

Phase II

Limited anti-tumor activity

[31]

Akt

Akt-I

Phase I

Ongoing

[31]

mTOR

mTOR-I

Phase I

Well tolerated

[31]

NF-kB

NFkB (PS-1)

Phase I

Well tolerated Combination with Gemcitabine shows good tolerability and anti-neoplastic activity in metatstatic PC Non-specific

[31]

COX-2

COX2-I

Phase I

Well tolerated No interaction with Gemcitabine

[19,31]

LOX

LOX-I

Phase I

Well tolerated No interaction with Gemcitabine

[31]

Abbreviations: Akt-I, Akt inihibitor; COX2-I, ciclooxygenase 2 inhibitor; FTI, farnesyl transferase inhibitor; LOX-I, lipooxygenase inhibitor, Mabs, monoclonal antibodies; MEK-I, MAP kinase inhibitor; mTOR-I, mTOR inhibitor; NFkB-I, nuclear factor kB inhibitor; PS-I, proteosome inhibitors; Ris, Raf inhibitors; RTKIs, receptor tyrosine kinase inhibitors; VEGFR, vascular endothelial growth factor receptor.

ulation of their expression and in their roles in tissue biology and disease. COX-1 is constitutively expressed in many tissues which provide the prostaglandins required to maintain their homeostasis. By contrast, COX-2 is transcriptionally regulated as an early gene in response to inflammation, growth factors, cytokines and tumor promoters. COX-2 is upregulated in many cancers including in 75% of PC patients. COX-2-specific inhibitors prevent PC cell growth by regulating cell-cycle progression. Consequently, COX-2 inhibitors mediate an accumulation of proteins that are involved in G1 arrest, including p27 and p21 [40]. Thus, inhibiting COX enzymes can be beneficial to control PC development. The lipooxygenase (LOX) pathway converts arachidonic acid into potent signaling mediators, such as leukotriene B4 (LTB4), facilitating the development and progression of human cancers. Overexpression of LOX and the LTB4 receptor in human PC form an autocrine loop that stimulates cell proliferation, which is inactivated by LOX inhibitors [2,19,31].

Summary and conclusions The results described in this article suggests that these pathways have a role in pancreatic tumorigenesis and, therefore, might serve as a foundation for the developing novel thera-

pies to treat PC. For example, the highest frequency alterations in the p16INK4a/Rb genes or constitutively activation of the Ras–Raf–MEK–ERK cascade makes these signaling pathways good targets for therapy in these tumors. Similarly, the effect of proteosomal inhibitors can be beneficial in treating PC by having a pleotrophic effect on several members of the PI3K-Akt-NF-kB pathway. Furthermore, the known hypersensitivity of Fanconi–BRCA pathway deficient cells to mitomycin suggests a therapeutic utility for this drug in PC. Thus, future efforts involving the development of small drugs that target the DNA-repair machinery might also reduce the incidence of familial PC. PC is a major cause of cancer death in the United States and Europe. Most cases are sporadic and are discovered at an incurable stage. Tools from genetics and molecular biology are enabling the characterization of alterations that disturb cell proliferation, survival and apoptosis regulation in pancreatic tumor cells, thereby affecting their response to conventional therapies. Testing for these genetic alterations in PC might enable the selection of optimal therapeutic strategies for individual patients. Furthermore, combined therapies can, in theory, provide higher efficacy with reduced side effects and might offer the best promise in the future. www.drugdiscoverytoday.com

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Acknowledgments This work was supported by the Mayo Foundation, Lustgarten Foundation for Pancreatic Cancer Research and by grants, DK52913 and DK56620, from the National Institutes of Health to R.U.

Outstanding issues    

Effective and early diagnosis. New molecular targets for more specific and effective treatments. New palliative regimens for the treatment of caquexia and pain. Development of chemopreventive strategies.

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