Background & Aims Activin, a member of the transforming growth factor-β (TGFB) family, might be involved in pancreatic tumorigenesis, like other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis. Methods We disrupted Acvr1b specifically in pancreata of mice (Acvr1bflox/flox;Pdx1-Cre mice) and crossed them with LSL-KRASG12D mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1bflox/flox;LSL-KRASG12D;Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16flox/flox;LSL-KrasG12D;Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse transcriptase PCR, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry. Results Loss of ACVR1B from pancreata of mice increased proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes, compared with control mice. Disruption of Acvr1b in LSL-KRASG12D; Pdx1-Cre mice accelerated growth of pancreatic IPMNs, compared with LSL-KRASG12D;Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells and with expansion of IPMN lesions in Acvr1bflox/flox;LSL-KRASG12D;Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1bflox/flox;LSL-KrasG12D;Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades. Conclusion Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway might therefor be a therapeutic target for pancreatic cancer.

Objectives Mucinous cystic neoplasms (MCNs) are rare, potentially curable, mucin-producing neoplasms of the pancreas. We have previously reported PIK3CA (phosphoinositide-3-kinase catalytic subunit, p110α) mutations in intraductal papillary mucinous neoplasms, another mucin-producing neoplasm of the pancreas. In this study, we analyzed the presence of PIK3CA and AKT1/PKB (V-akt murine thymoma viral oncogene homolog 1) hot-spot mutations in MCN specimens. Methods Using the genomic DNA sequencing of tumor tissues isolated by laser capture microdissection, we evaluated 15 well-characterized MCNs for the E542K, E545K(exon 9), and H1047R (exon 20) hot-spotmutations in the PIK3CA gene and the E17K mutation in the AKT1 gene. Results A hot-spotmutation (E545K) of the PIK3CA gene was detected in 1 of the 15 MCNs and further confirmed by a mutant-enriched method. Interestingly, this mutation was found to be present only in the high-grade but not in low-grade dysplastic epithelium obtained from this neoplasm and coexisted with a KRASG12D mutation. No mutations were identified in the AKT1 gene. Conclusions Our data, when combined with previous reports on intraductal papillary mucinous neoplasms, indicate that oncogenic activation of the PI3K pathway involving PIK3CA gene mutations can contribute to the progression of mucin-producing neoplasms but not pancreatic intraepithelial neoplasia. PIK3CA status could be useful for understanding their progression to malignancy.

PanINs and IPMNs are the two most common precursor lesions that can progress to invasive pancreatic ductal adenocarcinoma (PDA). DCLK1 has been identified as a biomarker of progenitor cells in PDA progressed from PanINs. To explore the potential role of DCLK1-expressing cells in the genesis of IPMNs, we compared the incidence of DCLK1-positive cells in pancreatic tissue samples from genetically-engineered mouse models (GEMMs) for IPMNs, PanINs, and acinar to ductal metaplasia by immunohistochemistry and immunofluorescence. Mouse lineage tracing experiments in the IPMN GEMM showed that DCLK1 cells originated from a cell lineage distinct from PDX1 progenitors. The DCLK1 cells shared the features of tuft cells but were devoid of IPMN tumor biomarkers. The DCLK1 cells were detected in the earliest proliferative acinar clusters prior to the formation of metaplastic ductal cells, and were enriched in the "IPMN niches". In summary, DCLK1 labels a unique pancreatic cellular lineage in the IPMN GEMM. The clustering of DCLK1 cells is an early event in Kras-induced pancreatic tumorigenesis and may contribute to IPMN initiation.

The association of TGF-β pathway with human tumorigenesis has been rigorously demonstrated. However, little is known about activin signaling, part of the TGF-β family, in pancreatic tumorigenesis. We have previously reported sporadic mutations of the Activin receptor type 1B (ACVR1B) in human pancreatic ductal adenocarcinoma (PDAC). To investigate the significance of ACVR1B in pancreatic tumorigenesis, Acvr1bflox/flox; Pdx1-Cre mice were generated and examined. Chronic pancreatitis-like histological changes such as inflammatory cell infiltration, acinar to ductal metaplasia, and fibrosis were observed in Acvr1b mutant alone mice older than 8 months of age. In combination with mutant KrasG12D in the pancreas, Acvr1b deletion accelerated the development of pancreatic intraductal papillary mucious neoplasms (IPMN), but not the pancreatic intraepithelial neoplasias (PanINs). The IPMN progressed to invasive and metastatic cancer was observed in this model. The expressions of Cox-2 and phospho-PDK1 are dramatically increased in preneoplastic and neoplastic lesions, but activated Notch4 expression is exclusively observed in the IPMN lesions by immunohistochemistry, suggesting that Notch4 signaling may play a role in the formation or maintenance of the mucin-rich ductal phenotype in this model. Interestingly, the progression of IPMN to invasive cancer appears to require additional p16 loss in this double mutant mouse model. In human IPMN, loss of p16 expression is associated with increasing grade. Our data provide the first evidence that Acvr1b acts as a tumor-suppressor in vivo and that activin signaling plays a predominant role in the development of pancreatic IPMNs. Significance: PDAC can arise from PanINs or IPMN. It has been reported previously that disruption of TGF-β signaling promotes the progression of PanIN to PDAC in the TGFβRII knockout background. In contrast, here we present that activin signaling deficiency favors the development of IPMNs and enhances their progression to PDAC. This phenotype overlaps with those observed in the Smad4 knockout background, suggesting that the involvement of activin signaling in the IPMN to PDAC sequence is Smad4-dependent. Together these data demonstrate that TGF-β superfamily ligands play critical roles in regulating the IPMN-derived versus PanIN-derived carcinogenesis. Citation Format: Wanglong Qiu, Sophia Tang, Sohyae Lee, Andrew T. Turk, Anthony Sireci, Anne Qiu, Ralph H. Hruban, Helen E. Remotti, Gloria H. Su. Inactivation of activin signaling pathway accelerates the development of pancreatic intraductal papillary mucinous neoplasms in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2735. doi:10.1158/1538-7445.AM2013-2735