Anna R. Lay

HOXB7 promotes invasion and predicts survival in pancreatic adenocarcinoma

The homeobox gene HOXB7 is overexpressed across a range of cancers and promotes tumorigenesis through varying effects on proliferation, survival, invasion, and angiogenesis. Although published microarray data suggest HOXB7 is overexpressed in pancreatic ductal adenocarcinoma (PDAC), its function in pancreatic cancer has not been studied.

WNT7B mediates autocrine Wnt/β-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma.

Developmental and cancer models show Wnt/β-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/β-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/β-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression-based assays revealed wide heterogeneity in Wnt/β-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally generated, pancreas-specific gene expression signature of Wnt/β-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early-stage PDAC tumors (N=41). In this cohort, higher Wnt/β-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease-specific survival (median survival time 20.3 versus 43.9 months, log-rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/β-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small-molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/β-catenin activity. Gene-knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B-mediated cell autonomous Wnt/β-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/β-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/β-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.

The CREB binding protein inhibitor ICG-001 suppresses pancreatic cancer growth

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer due in part to a lack of highly robust cytotoxic or molecular-based therapies. Recent studies investigating ligand-mediated Wnt/β-catenin signaling have highlighted its importance in pancreatic cancer initiation and progression, as well as its potential as a therapeutic target in PDAC. The small-molecule ICG-001 binds cAMP-responsive element binding (CREB)-binding protein (CBP) to disrupt its interaction with β-catenin and inhibit CBP function as a coactivator of Wnt/β-catenin–mediated transcription. Given its ability to inhibit Wnt/β-catenin–mediated transcription in vitro and in vivo, as well as its efficacy in preclinical models of colorectal cancer and other Wnt-driven diseases, we examined ICG-001 and its potential role as a therapeutic in PDAC. ICG-001 alone significantly inhibited anchorage-dependent and -independent growth of multiple PDAC lines, and augmented in vitro growth inhibition when used in combination with gemcitabine. ICG-001 had only variable modest effects on PDAC apoptosis and instead mediated PDAC growth inhibition primarily through robust induction of G1 cell-cycle arrest. These effects, however, seemed decoupled from its inhibition of Wnt/β-catenin–mediated transcription. DNA microarrays performed on PDAC cells in the context of ICG-001 treatment revealed ICG-001 altered the expression of several genes with well-established roles in DNA replication and cell-cycle progression, including direct actions on SKP2 and CDKN1A. ICG-001 also significantly prolonged survival in an in vivo orthotopic xenograft model of PDAC, indicating ICG-001 or derived compounds that disrupt CBP activity are potentially useful small-molecule therapeutics for pancreatic cancer. Mol Cancer Ther; 13(10); 2303–14. ©2014 AACR.

Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFβ-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that ‘High-RPS8 and Low-PRELP’ was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46–4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated.

Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy in need of more effective treatment approaches. One potential therapeutic target is Wnt/β-catenin signaling, which plays important roles in PDAC tumor initiation and progression. Among Wnt inhibitors with suitable in vivo biologic activity is vitamin D, which is known to antagonize Wnt/β-catenin signaling in colorectal cancer and have antitumor activity in PDAC. For this study, the relationship between vitamin D signaling, Wnt/β-catenin activity, and tumor cell growth in PDAC was investigated through the use of calcipotriol, a potent non-hypercalcemic vitamin D analogue. PDAC tumor cell growth inhibition by calcipotriol was positively correlated with vitamin D receptor expression and Wnt/β-catenin activity. Furthermore, vitamin D and Wnt signaling activity were found to be reciprocally linked through feedback regulation. Calcipotriol inhibited autocrine Wnt/β-catenin signaling in PDAC cell lines in parallel with decreased protein levels of the low-density lipoprotein receptor-related protein 6 (LRP6), a requisite coreceptor for ligand-dependent canonical Wnt signaling. Decrease in LRP6 protein seen with calcipotriol was mediated through a novel mechanism involving transcriptional upregulation of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1). Finally, changes in LRP6 or LDLRAP1 expression directly altered Wnt reporter activity, supporting their roles as regulators of ligand-dependent Wnt/β-catenin signaling. Implications: This study provides a novel biochemical target through which vitamin D signaling exerts inhibitory effects on Wnt/β-catenin signaling, as well as potential biomarkers for predicting and following tumor response to vitamin D–based therapy. Mol Cancer Res; 13(11); 1509–19. ©2015 AACR.

Plectin-1 as a biomarker of malignant progression in intraductal papillary mucinous neoplasms a multicenter study

Objective This study aimed to evaluate Plectin-1 expression as a biomarker of malignant risk for intraductal papillary mucinous neoplasms (IPMNs). Methods Plectin-1 immunohistochemistry (IHC) was performed retrospectively on surgical (n = 71) and cytological (n = 33) specimens from Mayo Clinic Jacksonville and UCLA Medical Center, including IPMNs with low-grade dysplasia, high-grade dysplasia (HGD), or an associated invasive adenocarcinoma. Results Plectin-1 expression was increased in invasive adenocarcinoma compared with adjacent in situ IPMN (P = 0.005), as well as the in situ HGD component of IPMNs with invasive cancer compared with HGD of IPMNs without invasive cancer (P = 0.02). Plectin IHC discriminated IPMNs with invasive adenocarcinoma from noninvasive IPMN (area under the curve [AUC] of 0.79, 75% sensitivity, and 85% specificity) but was insufficient for discriminating HGD IPMN from low-grade dysplasia IPMNs in surgical resections (AUC of 0.67, 56% sensitivity, and 64% specificity) or fine-needle aspiration specimens (AUC of 0.45). Conclusions Although Plectin-1 IHC has insufficient accuracy to be used as a definitive biomarker for malignant risk in the evaluation of IPMN biopsy or cytological specimens, increased Plectin-1 expression observed in both invasive cancer and in situ HGD of malignant IPMNs suggests that it might be successfully leveraged as a cyst fluid biomarker or molecular imaging target.

Thioredoxin system-mediated regulation of mutant Kras associated pancreatic neoplasia and cancer

Peroxiredoxin-1 (Prdx1), a member of the thioredoxin (Txn) system, is overexpressed and correlates with poor prognosis in pancreatic cancer patients and can suppress Kras signaling through redox-mediated inhibition of ERK and AKT in lung and breast cancer. Its redox function is maintained by Txn and sulfiredoxin (Srxn), and its tumor promoting functions are activated by post-translational modification. We studied the role of the Txn system in pancreatic neoplasia and cancer by determining how it regulates the phosphorylation of Kras effectors and by determining its association with patient survival. We found that elevated Prdx1 nuclear localization significantly correlated with better patient survival. Our data also demonstrate that the expression of the Txn system is dysregulated, with elevated Prdx1 expression and significantly decreased Txn and Srxn expression in pancreatic lesions of targeted mutant Kras mouse models. This correlated with distinct differences in the interconversion of Prdx1 oligomers that affect its ability to regulate ERK and AKT phosphorylation. Our data also suggest that Prdx1 post-translational modification and oligomerization suppress Prdx1 mediated redox regulation of ERK phosphorylation. We observed distinct differences in Txn expression and in the ability of pTyr-Prdx1 to bind to pERK in a PanIN model of pancreatic neoplasia as compared to an IPMN model, indicating a distinct difference in the function of post-translationally modified Prdx1 in cells with less Txn expression. Modified Txn system function and post-translational regulation may therefore play a significant role in pancreatic tumorigenesis by altering Kras effector phosphorylation and inhibiting the tumor suppressive redox functions of Prdx1.

Abstract 4011: Wnt/β-catenin transcriptional activation promotes tumorigenesis and predicts survival in pancreatic cancer.

Mutations in key regulators of Wnt/β-catenin signaling lead to its aberrant hyperactivation and promote oncogenesis in several cancer types. While Wnt/β-catenin signaling is variably increased in pancreatic adenocarcinoma (PDAC), there is a near absence of activating mutations in its key regulatory genes in PDAC tumors. As the precise role of Wnt/β-catenin signaling in pancreatic tumorigenesis is unclear, we sought to clarify the mechanisms by which it is altered and determine its specific consequences on gene transcription and phenotype in PDAC. Using a luciferase-based Wnt/β-catenin reporter platform to determine relative levels of Wnt/β-catenin transcriptional activation, we found pathway activation was highly variable across 22 PDAC cell lines, including distinct subsets with either low or much high levels of baseline reporter activity. Supervised analysis of microarray data was performed on these defined subsets to generate a pancreatic-specific gene expression signature able to discriminate levels of Wnt/β-catenin transcriptional activation in PDAC. This analysis identified a set of 208 genes (median FDR 3.4%) representing a robust candidate list of mediators or downstream transcriptional targets. Several genes were validated as bona fide downstream transcriptional targets of Wnt/β-catenin in PDAC cell lines by observing expected changes in their expression following genetic and pharmacologic manipulations that activate or inhibit Wnt signaling. WNT7B, FZD5 and TCF7L2 were among genes commonly overexpressed in Wnt high lines, representing potential positive mediators of Wnt/β-catenin signaling in PDAC. Likewise, NLK and CSNK1E were among genes overexpressed in Wnt low lines, representing potentially important negative regulators of the pathway in PDAC. When primary patient tumors were dichotomized based on a pancreas-specific Wnt/β-catenin transcriptional signature, PDAC tumors with higher Wnt/β-catenin transcriptional activation had worse disease-specific survival (median survival time 20.3 versus 43.9 months, log rank P=0.03), suggesting a possible direct link between Wnt/β-catenin transcriptional activation and aggressive tumor behavior. Furthermore, genetic and pharmacologic inhibition of Wnt/β-catenin signaling in PDAC cell lines significantly reduced non-adherent growth and tumorsphere formation in vitro. Inhibition of Wnt/β-catenin signaling also increased survival and reduce metastatic burden in an in vivo orthotopic xenograft model using PDAC cell lines. This study has defined a pancreatic cancer-specific Wnt/β-catenin transcriptional signature able to predict more aggressive clinical behavior and with potential utility for stratifying patients most likely to benefit from Wnt-targeted therapy. Citation Format: Michael Arensman, Anna R. Lay, Rima M. Kulikauskas, Andy J. Chien, David W. Dawson. Wnt/β-catenin transcriptional activation promotes tumorigenesis and predicts survival in pancreatic cancer. [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 4011. doi:10.1158/1538-7445.AM2013-4011

Abstract 366: Disruption of TCF7L1 mediated transcriptional repression promotes pancreatic tumorigenesis

While phenotypic studies demonstrate Wnt/β-catenin signaling is critical for the initiation and progression of pancreatic adenocarcinoma (PDA), the downstream transcriptional effectors tied to its effects are largely unknown. Previous work from our group has identified differential patterns of LEF/TCF expression linked to variations in Wnt activation and function in PDA, including reduced TCF7L1 (aka TCF3) message and protein levels in pancreatic tumors with elevated Wnt pathway activity. Consistent with this observation, we found TCF7L1 protein levels rapidly diminished in PDA lines after Wnt pathway activation mediated by either Wnt3A ligand or GSK3β inhibitor. RNAi-mediated knockdown of TCF7L1 in PDA lines phenocopied most of the pro-tumorigenic effects seen with Wnt3A ligand treatment. Because TCF7L1 commonly represses transcription and is rapidly downregulated in PDA upon Wnt activation, we explored its transcriptional derepression as a potentially important mechanism through which Wnt signaling promotes pancreatic tumorigenesis. Candidate genes linked to TCF7L1-mediated transcriptional repression in PDA were identified by RNA-sequencing of MiaPaCa-2 and PANC1 lines following RNAi-mediated knockdown of TCF7L1. A total of 196 genes upregulated at least 1.5-fold in both cell lines were identified, representing potential direct targets of TCF7L1-mediated transcriptional repression. Gene ontology analysis among these 196 upregulated genes revealed enrichment of categories including small GTPase-mediated signal transduction, RAS protein signal transduction, regulation of axon extension, and regulation of synaptic plasticity. Among top upregulated genes was prostaglandin E synthase (PTGES), an enzyme catalyzing the isomerization of prostaglandin H2 to prostaglandin E2 (PGE2) as the final step in PGE2 synthesis from arachidonic acid. Notably, PGE2 stimulates pancreatic cancer proliferation, invasion, angiogenesis and metastasis. Validation experiments in TCF7L1-expressing PDA lines confirmed PTGES message and protein levels rapidly increased in response to TCF7L1 depletion and were associated with a corresponding increase in PGE2 production as measured by ELISA. PGE2 increased Wnt reporter activity in PDA cell lines, a potentially important feedforward mechanism whereby Wnt signaling may be reinforced upon its activation. In conclusion, TCF7L1 represses target genes with known roles in PDA, while reduction in TCF7L1 through Wnt pathway activation or alternative means is apparently tied to a program of transcriptional derepression linked to the promotion of pancreatic tumorigenesis. Citation Format: Kathleen M. Kershaw, Bridgette T. Ho, Missael I. Vasquez, Anna R. Lay, David W. Dawson. Disruption of TCF7L1 mediated transcriptional repression promotes pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2017-366

Abstract 4603: TCF7L1 constrains Wnt/β-catenin signaling and tumor growth in pancreatic cancer

Wnt/β-catenin signaling plays important roles in pancreatic adenocarcinoma (PDA) tumor initiation and progression through its promotion of various pro-tumorigenic phenotypes. While RNF43 mutations increase Wnt signaling activity and confer Wnt growth-dependency in a subset of PDA, other key driver mutations in the Wnt pathway are only rarely encountered in PDA. To gain additional insights into alternative mechanisms contributing to increased Wnt pathway activity in PDA, we examined the expression and activity of LEF/TCF family members, the key transcriptional binding partners for β-catenin in canonical Wnt signaling. Luciferase-based β-catenin-activated reporter (BAR) assays demonstrated variable Wnt activity across a large panel of PDA cell lines, which was used to dichotomize cell lines into groups with high or lower β-catenin/TCF-dependent transcription. Supervised analysis of PDA cell line microarray data and parallel western blot analyses identified distinct patterns of TCF/LEF expression, including increased TCF7 and TCF7L2 (aka TCF4) expression in high BAR lines and increased TCF7L1 (aka TCF3) expression in low BAR lines. RNAi-mediated knockdown of individual or combinations of TCF/LEFs in PDA lines revealed TCF7L1 and TCF7L2 both function to repress β-catenin-activated reporter activity. TCF7L1 protein was also found to be targeted for rapid downregulation following Wnt pathway activation by treatment with either Wnt3A ligand or GSK3β inhibitor. RNAi knockdown of TCF7L1 in PDA lines increased anchorage-independent growth and tumor cell migration in vitro and increased in vivo PDA growth in an orthotopic xenograft tumor model. In summary, differential patterns of TCF/LEF expression are correlated with and functionally responsible for differences in Wnt pathway activation and function in PDA. Furthermore, these data implicate abrogation of TCF7L1 growth suppressive phenotypes via β-catenin-mediated downregulation of TCF7L1 as a potentially important mechanism through which Wnt signaling promotes pancreatic tumorigenesis. Citation Format: Kathleen M. Kershaw, Michael D. Arensman, Bridgette Ho, Anna R. Lay, Nicole A. Dawson, David W. Dawson. TCF7L1 constrains Wnt/β-catenin signaling and tumor growth in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4603.