Ann Surg Oncol DOI 10.1245/s10434-015-4463-x

ORIGINAL ARTICLE – PANCREATIC TUMORS

Molecular Pathogenesis and Targeted Therapy of Pancreatic Cancer Shinji Tanaka, MD, PhD, FACS Department of Molecular Oncology, Tokyo Medical and Dental University, Tokyo, Japan

ABSTRACT Accumulation of multiple genetic and/or epigenetic abnormalities is required for generation and progression of cancers, and the survival of cancer cells might depend on addiction to these abnormalities. Because disruption of such dependency on the abnormal molecules should cause the cancer cell death, so-called oncogene addiction is the rationale for molecular targeted therapy. Pancreatic cancer, especially pancreatic ductal adenocarcinoma, is one of the most lethal malignancies in humans, and remains a challenging problem in targeted therapy compared to other malignancies such as pancreatic neuroendocrine tumor. This review summarizes the molecular pathogenesis of pancreatic cancer on the basis of the recent studies of driver mutations including chromatin remodeling factors, and promising concepts ‘‘cancer stemness’’ and ‘‘stromal niche’’ for the strategy of novel targeted therapy.

Pancreatic cancer is one of the leading causes of cancer deaths and the incidence is still increasing in the developed world. Five-year survival is approximately 5 % in patients diagnosed with pancreatic ductal adenocarcinoma, and such poor prognosis has remained virtually unaltered in the past 30 years.1 Pancreatic ductal adenocarcinoma is the most common malignancy of the pancreas ([95 %) and usually referred to as ‘‘pancreatic cancer’’ in general. Because numerous studies on molecular abnormalities have been reported in pancreatic cancer, targeted therapy which specifically inhibits molecular abnormalities emerged to be a novel approach for the innovative and effective medical treatment.2 In order to fulfill this promise, there is an urgent

Ó Society of Surgical Oncology 2015 First Received: 23 October 2014 S. Tanaka, MD, PhD, FACS e-mail: [email protected]

need to identify the optimal targets for the treatment of pancreatic cancer. The sharpshooting strategy for the addictive molecule or molecules has led to understating pancreatic carcinogenesis and progression, leading to novel development of molecular targeted therapy.3 DRIVER MUTATIONS IN PANCREATIC DUCTAL ADENOCARCINOMA: THE FOUR MAJORS PLUS ONE Pancreatic ductal carcinomas arise from noninvasive precursor lesions; mainly pancreatic intraepithelial neoplasms (PanIN).1 Accumulation of genetic alterations is associated with the histologic progression from PanIN-1 (hyperplasia) through PanIN-2 (atypia) and PanIN-3 (carcinoma-in situ) to invasive ductal adenocarcinoma (Fig. 1). Genetic sequencing of numerous pancreatic tumors revealed mutations in KRAS (95 %), CDKN2A/P16 ([90 %), TP53 (75 %), and SMAD4 (55 %) are commonly seen in ductal adenocarcinoma (Fig. 1).4 KRAS mutations happen as the earliest genetic abnormalities, even in PanIN-1 lesions, whereas inactivation of TP53 and SMAD4 are detected in PanIN-3 lesions and invasive carcinomas. Pancreatic duct adenocarcinomas can also evolve from intraductal papillary mucinous neoplasms (IPMN) or mucinous cystic neoplasms. Frequent mutations of KRAS were detected also in IPMN and mucinous cystic neoplasm lesions (70 %). Genetically engineered mouse model expressing an endogenous mutant KRAS gene under a pancreas-specific promoter PDX1 can develop PanINmimic lesions.2 Combining analysis of KRAS with CDKN2A or TP53 mutations in mouse models led to the frequent development of pancreatic ductal carcinoma.2 Recently, further whole genome/exome sequencing revealed the SWI/SNF chromatin-remodeling complex as the fifth candidate of the major driver mutations in pancreatic cancer (Fig. 2).5 Approximately 34 % of pancreatic ductal adenocarcinoma carries genomic aberrations in the

S. Tanaka FIG. 1 Schematic of pancreatic carcinogenesis and genetic alterations. PanIN pancreatic intraepithelial neoplasm, PanIN-1 hyperplasia, PanIN-2 atypia, PanIN-3 carcinoma-in situ

FIG. 2 SWI/SNF chromatin remodeling complexes BAF and PBAF

SWI/SNF subunits such as ARID1A, ARID1B, ARID2, PBRM1, and BRG1.5,6 Loss of BRG1 cooperated with oncogenic KRAS formed IPMN and then progressed to pancreatic ductal adenocarcinoma in the genetically engineered mouse model, indicating the tumor suppressor function of BRG1, one of the SWI/SNF core subunits of ATP-dependent helicase.7 Further analysis should focus on the role of chromatin regulators in epigenetic carcinogenesis and progression of pancreatic cancer. EGFR: THE ORTHODOX TARGET IN PANCREATIC DUCTAL ADENOCARCINOMA EGFR, a homologue of erbB oncogene product, belongs to a receptor tyrosine kinase family including ErbB2/Her2/ Neu, ErbB3/Her3, and ErbB4/Her4.8 Overexpression and constitutive activation of EGFR family members are recognized in various cancers, including pancreatic ductal adenocarcinoma.1 The ligand of EGFR/ErbB1 is not only EGF but also TGF-a, amphiregulin, HB-EGF, b-cellulin, or epiregulin. The binding of the ligand to EGFR/ErbB1 leads to the homodimerization or heterodimerization with ErbB2/Her2/Neu or ErbB3/Her3, inducing tyrosine kinase activation, self-phosphorylation (not in ErbB2/Her3), and intracellular signal transduction (Fig. 3).

Cetuximab (Erbitux, IMC-C225), a chimeric monoclonal antibody against EGFR, has attracted attention in colorectal cancer for KRAS biomarker.9 Cetuximab combined with a conventional anticancer drug gemcitabine was tested in a phase 3 randomized controlled trial (RCT) of patients with advanced pancreatic cancer.10 In 675 patients who were enrolled, median survival time (MST) was 6.3 months for the combination therapy, compared to 5.9 months for the gemcitabine monotherapy. Hazard ratio (HR) was 1.06 [95 % confidence interval (CI) 0.91–1.23; p = 0.23], indicating no significant benefit for the patients received cetuximab–gemcitabine regimen. Other regimens of cetuximab with gemcitabine-oxaliplatin or gemcitabineradiotherapy are ongoing for clinical trials. Erlotinib (Tarceva, OSI774) is an oral tyrosine kinase inhibitor, selectively suppresses EGFR tyrosine kinase. In 2007, a phase 3 clinical trial for erlotinib–gemcitabine was published.11 The RCT enrolling 569 patients demonstrated a significant synergistic effect of the combination, with an HR of 0.82 (95 % CI 0.69–0.99; p = 0.038). Although the erlotinib–gemcitabine arm showed a minimal 2-week increase in MST (6.24 months) compared with the gemcitabine-only arm (5.91 months), erlotinib is the first molecular-targeting agent with a statistical effect for pancreatic ductal adenocarcinoma. Erlotinib in combination

Molecular Pathogenesis and Therapy FIG. 3 EGFR signaling pathway. Targeted agents for pancreatic cancer appear in italics

with gemcitabine was received approval for use in patients with unresectable pancreatic adenocarcinoma. RAS/RAF/MEK SIGNAL TRANSDUCTION: THE INTRACELLULAR DOMINO DOWNSTREAM The activated EGFR tyrosine kinase stimulated mainly SH2-conatining proteins to transduce several intracellular signaling pathways (Fig. 3).8 An adaptor SH2/SH3 protein Grb2 stimulates SOS, leading to the activation of Ras that is farnesylated and then localized under the cellular membrane. Tipifarnib (R115777), a farnesyl transferase inhibitor, was applied for clinical trials of pancreatic ductal adenocarcinoma. According to the phase 3 RCT in 688 patients enrolled, 1-year survival rate was 27 % (MST 193 days) for the tipifarnib-gemcitabine combination therapy, compared to 24 % (MST 182 days) for the gemcitabine monotherapy, resulting in no statistical significance (p = 0.75).12 Activated form of Ras then stimulates Raf serine–threonine kinase. Sorafenib (Nexabar) is an oral multikinase inhibitor targeting Raf kinase as well as angiogenic receptors such as VEGFR and PDGFR. A phase 3 RCT in 104 patients revealed MST was 8 months for the sorafenib–gemcitabine therapy, compared to 9.2 months for the gemcitabine monotherapy (p = 0.231). The median progression-free time was 3.8 months for the combination therapy, compared to 5.7 months for the monotherapy (p = 0.902), showing apparently opposite outcome.13 It should be mentioned that Raf kinase inhibitor can induce dimerization of Raf proteins in the presence of oncogenic Ras, and substantially activation of Raf downstream signals.14 The complexities of Raf

function should be assessed to avoid adverse clinical effects in Ras-mutant cancers. Activated Raf kinase then phosphorylates MEK kinases, which finally activate Erk 1/2 of the MAPK family. Once activated, Erk 1/2 translocate to the nucleus where it acts as a regulator of gene expression for cell cycle progression, apoptosis resistance, and cellular motility. MEK kinase inhibitors, selumetinib (AZD6244) and trametinib (Mekinist, GSK1120212), were evaluated for pancreatic duct adenocarcinoma as gemcitabine combination therapy, but no clinical benefit has been reported in the clinical trials. PI3K/AKT SIGNAL TRANSDUCTION: THE PARADOX OF MOLECULAR TARGETS It should be clarified why EGFR is the therapeutic target of pancreatic duct adenocarcinoma, usually mutated in the downstream KRAS oncogene ([95 %). Indeed, the effect of cetuximab is clinically limited to the patients with colorectal cancer with wild-type KRAS.9 To solve the paradox, a series of important studies was reported using genetically engineered mice of pancreatic cancer.15,16 In pancreatic KRAS-mutant mice, EGFR knockout induced mutant KRAS-specific apoptosis and completely abrogated IPMN development. Furthermore, EGFR inhibitor delayed tumor initiation in KRAS-mutant mice with conditional TP53 knockout. These studies support a requirement for EGFR in KRAS-driven pancreatic tumorigenesis. Indeed, overexpression of EGFR is frequently detected in KRAS-mutant pancreatic ductal adenocarcinoma in clinical human samples and genetically engineered mice.16 Beyond Ras

S. Tanaka

pathways, other EGFR downstream signals should play a critical role in pancreatic carcinogenesis, such as PI3K, STAT, and Grb7 Fig. 3). We have cloned human Grb7 as a cell migration gene in cancer cells and reported a preclinical study of Grb7 peptide inhibitor targeting pancreatic cancer metastasis.17 PI3K consists of p85 adaptor and p110 kinase subunits.8 Followed by association with Gab1 after activation of EGFR, PI3K phosphorylates PIP2 to generate PIP3 transducing PDK, which in turn activates a serine/threonine kinase Akt. PIP3 is dephosphorylated by PTEN, a tumor suppressor that reverses this pathway. Once activated, Akt regulates multiple cellular target proteins including mTOR (mammalian target of rapamycin) and IKK. A serinethreonine kinase mTOR regulates the phosphorylation of p70 ribosomal protein S6 K and 4E-BP1/eukaryotic translation initiation factor 4E (eIF4E) pathway. Everolimus (Afinitor, RAD001), an inhibitor of mTOR developed as one of the rapamycin derivates, was applied for phase 2 clinical trials of pancreatic ductal adenocarcinoma. According to the phase 2 study in 33 patients with gemcitabine-refractory pancreatic cancer, MST was only 4.5 months and median progression-free time was 1.8 months; neither complete nor partial response was obtained.18 IKK, another downstream protein of Akt, provokes the subsequent activation of NF-jB transcription factor (Fig. 3). NF-jB promotes cell survival by activating transcription of target genes normally repressed by binding of the specific inhibitor IjB, which sequesters the NF-B p50/p65 heterodimer in the cytoplasm.8 Inhibition is reversed in response to several intracellular stimuli, resulting in targeted, ubiquitin/ proteasome-mediated degradation of IjB. Bortezomib (Velcade, PS-341), a potent and selective inhibitor of the proteasome, received approval for treatment of patients with progressive multiple myeloma. One of the actions of bortezomib is pleiotropic and includes inhibition of NF-jB activation by preventing IjB degradation. According to a phase 1 study of for advanced solid tumors including pancreatic cancer, the combination of bortezomib and irinotecan generally was tolerated well with manageable toxicities.19 A phase 2 trial for bortezomib and HDAC inhibitor panobinostat revealed a median progression-free time of only

2.1 months and severe adverse events, resulting in early termination of the clinical trial in patents with gemcitabinerefractory pancreatic cancer.20 Because abnormalities of intracellular signal transduction pathways should play essential roles in pancreatic cancer progression, further studies including other signals should be carried out. WNT AND HEDGEHOG SIGNAL TRANSDUCTIONS: CANCER STEMNESS IN THE NICHE Refractory of cancers might be caused by their heterogeneity in functional and molecular phenotypes. One of the essential models for generation of heterogeneity is dependent on a small subpopulation of stem-like cancer cells, socalled cancer stem cells (CSC).2 CSC have the ability to selfrenew and diversification to hierarchically organize the bulk with respect to tumor growth initiation and maintenance.8 CSC lying at the apex of the hierarchy are naturally resistant to chemotherapeutic agents, and function as a source to metastasizing and relapsing. As CSC of pancreatic ductal adenocarcinoma, the subpopulations of CD44?CD24?EpCAM?, c-MethighCD44?, CD133?CXCR4?, and ALDHhigh CD44?CD24? have been reported (Table 1).21–24 Using visualization of low proteasome addiction, we have identified human pancreatic stemlike cancer cells that were extremely resistant to gemcitabine and cisplatin, and these CSC demonstrated intrinsically specific activation of Wnt/bcatenin signaling (Fig. 4).25,26 Wnt, Hedgehog, and Notch signaling pathways are known to direct growth and patterning during embryonic development, as well as postembryonic regulation of stem cells in epithelia undergoing renewal.8 Signaling is initiated by the secreted Wnt proteins, which bind to a class of seven transmembrane receptors Frizzled (Fig. 5). We isolated human Frizzled-7 as one of the therapeutic targets in various cancer cells.27 Wnt binding to the Frizzled receptor leads to phosphorylation of Dishevelled which prevents the formation of APC-Axin-GSK3b complex. In the absence of Wnt signals, b-catenin phosphorylation by GSK3b complex allows recognition by b-TRCP, an E3 ubiquitin ligase with subsequent degradation of b-catenin by proteasome system. By contrast, in the presence of Wnt signals,

TABLE 1 Candidates of therapeutic targets in pancreatic cancer stem cells Cancer stem markers ?

?

?

CD44 CD24 ESA CD133?CXCR4? high

?

ALDH CD44 CD24 c-MethighCD44? Proteasomelow

?

Molecular targets

Study

Hedgehog

Cancer Res 200721

CXCR4

Cell Stem Cell 200723

– c-Met

J Natl Cancer Inst 201024 Gastroenterology 201122

Wnt/b-catenin

Gastroenterology 201226

Molecular Pathogenesis and Therapy

FIG. 4 Visualization of pancreatic CSC.26 a Asymmetric cell division of green fluorescent pancreatic CSC observed by time-lapse microscopy. b Immunocytochemical analysis of b-catenin in human

pancreatic CSC and non-CSC. CSChigh indicates nuclear accumulation of b-catenin was detected; CSClow, b-catenin was located to cell membrane but not translocated into nucleus

inhibition of GSK3b complex results in the cytoplasmic accumulation of unphosphorylated b-catenin, which translocates to the nucleus where it binds to CBP and engages the TCF family of transcription factors to activate genes like CCND1 and MYC. Nuclear translocation of bcatenin is recognized in 10–60 % of PanIN and pancreatic cancers. b-Catenin mutations at the GSK3b recognition site are detected in almost all cases of solid pseudopapillary tumor, an unusual form of pancreatic cystic tumors.28 Currently, several small molecule compounds, recombinant proteins and antibodies are under intense study for the development of agents targeting Wnt/b-catenin pathway (Fig. 5). LGK974 is a unique inhibitor of porcupine acyltransferase that be involved in lipid modification of Wnt proteins in the ER membrane to secrete Wnt protein.29 A phase 1 clinical study of Wnt secretion inhibitor LGK974 has been commenced for patients with pancreatic and breast cancers (NCT01351103). OMP-54F28, a recombinant fusion protein of the extracellular domain of the human Frizzled receptor and a human IgG1 Fc fragment, functions as a decoy-type inhibitor of Wnt signals. Patient enrollment is ongoing for a phase 1 clinical study of OMP-54F28 to treat advanced pancreatic cancer (NCT02050178). Vantictumab (OMP-18R5), a human monoclonal antibody targeting

Frizzled protein, is examined in a phase 1 study for advanced pancreatic cancer (NCT02005315).30 In Japan, PRI-724 has been developed as an inhibitor of b-catenin/CBP complex formation. A phase 1 clinical study of PRI-724 is ongoing in patients with advanced pancreatic cancer (NCT01764477). Not only Wnt but also Hedgehog pathways play an essential role in regulating stem cell fate.8 Hedgehog signal transduction, a critical regulator of self-renewal potential of stem cells, is up-regulated in CD44?CD24?EpCAM? pancreatic CSC.21 Overexpression of Sonic Hedgehog (Shh) is sufficient to initiate PanIN-like precursor lesions in the transgenic mice. Secreted Shh binds to the receptor Patched to activate Smoothened (SMO), inducing nuclear translocation of Gli transcriptional activator that targets genes including cyclin D, cyclin E, Myc, Gli1, and Patched (Fig. 6). As specific inhibitors of Hedgehog signaling, SMO antagonists such as vismodegib (GDC-0449), saridegib (IPI-926), and sonidegib (LDE225) have been examined in clinical trials to treat patients with advanced pancreatic cancer. The experimental model of mouse pancreatic cancer revealed that Shh inhibition targets mainly stroma cells through a paracrine pathway rather than pancreatic cancer cells per se through an autocrine pathway. In a preclinical

S. Tanaka FIG. 5 Wnt signaling pathway. Targeted agents for pancreatic cancer appear in italics

FIG. 6 Hedgehog signaling pathway. Targeted agents for pancreatic cancer appear in italics

study, Hedgehog inhibitor saridegib depleted tumor-associated stromal tissue, and saridegib–gemcitabine combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to suppression of primary and metastatic tumors.31 Within pancreatic cancer tissues, overexpression of Shh is recognized in pancreatic cancer cells, but the Shh target gene Gli1 is expressed dominantly in the stroma cells.32 A paracrine mechanism of Shh-mediated pancreatic carcinogenesis should clarify the essential role of microenvironmental stroma in generation of cancer stem niche. Because cross-talk between CSC and the stromal niche provides a physical barrier to treatment of pancreatic cancer, stromal deletion is a promising target of other clinical trials.33 A phase 1 study of CD40 agonist CP-870,893 showed regression of stromal tissues, resulting in suppression of primary and

metastatic tumors.34 PEGPH20 (pegylated recombinant human hyaluronidase) targeting stromal desmoplasia is examined in a phase 1 study to treat advanced pancreatic cancer in combination with gemcitabine (NCT01839487). Nanoparticle albumin-bound paclitaxel (nab-paclitaxel; Abraxane), a unique anticancer drug, can bind to SPARC protein expressed in stromal tissue and disrupt the desmoplasia associated with pancreatic cancer.35 A phase 3 RCT enrolling 861 patients with metastatic pancreatic cancer was conducted to study the combination of nab-paclitaxel and gemcitabine compared to gemcitabine monotherapy.36 The median overall survival was 8.5 versus 6.7 months (HR 0.72, 95 % CI 0.62–0.84; p = 0.001), the median progression-free survival was 5.5 versus 3.7 months (HR 0.69, 95 % CI 0.58–0.82; p = 0.001), and 1-year survival was 35 versus 22 %, all in favor of the combination of nab-paclitaxel and gemcitabine. Nab-paclitaxel

Molecular Pathogenesis and Therapy

is currently approved by U.S. Food and Drug Administration in the treatment of advanced pancreatic cancer patients, and it will accelerate the development of niche-targeting therapy. Interestingly, more recent studies demonstrated that targeting the stroma resulted in undifferentiated, aggressive pancreatic cancer that responds to Treg checkpoint blockade or antiangiogenic therapy, uncovering a protective role by tumor stoma.37,38 Given the complexity and plasticity of the stroma and associated immunocytes, further investigations are required to more clearly define detrimental and beneficial aspects of niche biology in pancreatic cancer stroma. PANCREATIC NEUROENDOCRINE TUMOR Pancreatic neuroendocrine tumor (PNET), comprising 1–3 % of all pancreatic neoplasms, is the second common

FIG. 7 Comparison of the mutation rates of driver gene candidates in PNET and pancreatic ductal adenocarcinoma. Blue indicates PNET; red, pancreatic ductal adenocarcinoma (PDA). Data based on exome sequencing reported by Jiao et al.41

FIG. 8 Molecular roles of PNET tumor suppressors in chromatin regulation. Top Menin protein encoded by MEN1 gene complexes with MLL histone methyltransferase. Bottom ATRX is an ATP-dependent SWI/SNFlike chromatin remodeler that complexes with DAXX, direct histone chaperone for H3.3 enriched in heterochromatin at telomere

primary malignancy of the pancreas.39 Generally slow progression of the PNET may understate their malignant potentials, but in fact, the majority of patients (*70 %) present with metastatic lesion or lesions, resulting in an overall 5-year survival rate of *60 % and a 10-year survival rate of only 45 % even after surgical resection of the PNET. Conventional systemic treatment options for PNET include somatostatin analogs or cytotoxic chemotherapy. Because PNET are frequently diagnosed as hypervascular tumors, the inhibition of angiogenic signaling is a promising therapeutic approach in PNET. Vascular endothelial growth factor (VEGF) is one of the potent factors stimulating the growth and migration of the endothelial cells.8 Among several inhibitors of VEGF receptor kinases, sunitinib (Sutent, SU11248) was approved for use in advanced PNET in accordance with significantly improved survival in a phase 3 RCT study.40 According to the recent data of whole exome sequencing, mutations in genes in the PI3K/mTOR pathway were detected in 15 % of sporadic PNET: PIK3CA, PTEN, and TSC2 (Fig. 7).41 Indeed, the mTOR inhibitor everolimus was received approval for use in patients with advanced PNET (Fig. 3).42 TP53 mutations were detected in only 3 % of PNET but in [90 % of neuroendocrine carcinoma.40,43 The most frequently mutated gene in PNET was MEN1 (44 %), the gene responsible for the hereditary syndrome as multiple endocrine neoplasia type 1 (MEN-1). Additionally, recurrent somatic mutations in mutually exclusive genes ATRX and DAXX were newly identified in 43 % of PNET.41 It is of note that these frequently mutated genes specify proteins implicated in epigenetic regulation of chromatin modification. Menin protein encoded by MEN1 is one of the essential components of a histone methyltransferase complex (Fig. 7). ATRX is an ATP-dependent SWI/SNF-like chromatin remodeler that complexes with DAXX. Because the recent evidence points toward DAXX as the direct histone

S. Tanaka

chaperone for H3.3 enriched in heterochromatin at telomere (Fig. 8), inactivating mutations of ATRX or DAXX may promote tumorigenesis through altered gene expression, telomere dysfunction, and genomic instability.44,45 Chromatin regulators should be worthy of further investigation as the next key to solve the mechanism of such complex diseases. Application of the synthetic lethal strategy on these abnormalities must be actively engaged in developing a novel targeted therapy of aggressive pancreatic tumors.46

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12.

13. ACKNOWLEDGMENT I sincerely appreciate the opportunity to write this review article, by the recommendation of the Japanese Society of Gastroenterological Surgery (JSGS) and the Society of Surgical Oncology. I thank all of my colleagues and collaborators at Tokyo Medical and Dental University, Osaka University, Kyushu University, Tokushima University, and Brown Medical School. This work was supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Research (A), Project of Development of Innovative Research on Cancer Therapeutics from Ministry of Education, Culture, Sports, Science and Technology of Japan, and Health and Labour Sciences Research Grant from Ministry of Health Labour and Welfare of Japan. I was a recipient of JSGS Award Science for the year 2013. CONFLICT OF INTEREST interest.

14.

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17.

The author declares no conflict of

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