Caroline H. Diep

Synergistic Effect between Erlotinib and MEK Inhibitors in KRAS Wild-Type Human Pancreatic Cancer Cells

Purpose: The combination of erlotinib and gemcitabine has shown a small but statistically significant survival advantage when compared with gemcitabine alone in patients with advanced pancreatic cancer. However, the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study, we sought to identify gene targets that, when inhibited, would enhance the activity of epidermal growth factor receptor (EGFR)-targeted therapies in pancreatic cancer cells. Experimental Design: A high-throughput RNA interference (RNAi) screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays. Results: Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits, mitogen-activated protein kinase (MAPK) 1, was selected for further mechanistic studies. Combination treatments of erlotinib and two MAP kinase kinase (MEK) inhibitors, RDEA119 and AZD6244, showed significant synergistic effect for both combinations (RDEA119–erlotinib and AZD6244–erlotinib) compared with the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wild-type cells but not in KRAS mutant cells. Conclusions: Overall, our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer. Clin Cancer Res; 17(9); 2744–56. ©2011 AACR.

Down-regulation of Yes Associated Protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells.

Background The Hippo pathway regulates organ size by inhibiting cell proliferation and promoting cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector of the Hippo pathway that promotes cell growth as a transcription co-activator. In human cancer, the YAP1 gene was reported as amplified and over-expressed in several tumor types. Methods Immunohistochemical staining of YAP1 protein was used to assess the expression of YAP1 in pancreatic tumor tissues. siRNA oligonucleotides were used to knockdown the expression of YAP1 and their effects on pancreatic cancer cells were investigated using cell proliferation, apoptosis, and anchorage-independent growth assays. The Wilcoxon signed-rank, Pearson correlation coefficient, Kendalls Tau, Spearmans Rho, and an independent two-sample t (two-tailed) test were used to determine the statistical significance of the data. Results Immunohistochemistry studies in pancreatic tumor tissues revealed YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the tumor cells, whereas the adjacent normal epithelial showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell density. YAP1 total protein expression increased in the nuclear fractions in BxPC-3 and PANC-1, while it declined in HPDE6 as cell density increased. Additionally, treatment of pancreatic cancer cell lines, BxPC-3 and PANC-1, with YAP1-targeting siRNA oligonucleotides significantly reduced their proliferation in vitro. Furthermore, treatment with YAP1 siRNA oligonucleotides diminished the anchorage-independent growth on soft agar of pancreatic cancer cells, suggesting a role of YAP1 in pancreatic cancer tumorigenesis. Conclusions YAP1 is overexpressed in pancreatic cancer tissues and potentially plays an important role in the clonogenicity and growth of pancreatic cancer cells.

Abstract POSTER-BIOL-1311: Mechanisms of differential activities of progesterone receptor (PR) isoforms in ovarian cancer models

Progesterone is proliferative in the breast, but inhibitory in the reproductive tract and ovaries. Elevated circulating progesterone levels observed during pregnancy or through the use of progestin-containing oral contraceptives are associated with decreased lifetime risk of ovarian cancer (OC). In contrast, low progesterone or the functional loss of PR are associated with increased risk. The detailed mechanisms of progesterone’s protective role in OC are not well understood. The actions of progesterone are mediated through two nuclear receptors, the full-length PR-B and the N-terminally truncated PRA. While PR-A and PR-B share the same amino acid sequence downstream of the extreme N-terminus (B-upstream segment or BUS) that is missing in PR-A, they are functionally distinct transcriptional regulators. While PR-isoform specificity has been extensively studied in the breast (PR-B is required for mammary gland development and proliferative in breast cancer models), little is understood about the specific actions of PR isoforms in the ovary. It has been suggested that PR-A is primarily an inhibitory isoform that acts to suppress transcriptional actions of ER-alpha and PR-B. Further complexity is conferred by compartmentalized PR. For example, in the reproductive track, progestins may act primarily through stromal PR-A as potent antagonists of estrogen-induced epithelial hyperplasia. More recently, we have demonstrated that PRB is an OC tumor suppressor that exhibits anti-proliferative properties by inducing cellular senescence. In OC cells, in the presence of progestins, PR-B induces cellular senescence through a mechanism that involves FOXO1-dependent induction of p21 expression. In contrast to PR-B, progestin stimulation of PR-A failed to upregulate p21 and FOXO1 mRNA and thus did not induce a cellular senescence phenotype. However, we detected either PR-B or PR-A and FOXO1 within the same PRE-containing region of the p21 upstream promoter, suggesting that PR-A may actively suppress the expression of this gene, while PR-B forms an active transcriptional complex. PR isoforms differentially associate with protein kinases that in turn alter their transcriptional activity at selected target genes. We speculated that PR isoforms directly associated with FOXO1 in transcriptional complexes alter the phosphorylation state of FOXO1 to dictate the degree of isoform-specific PR target gene expression, thereby determining cell fate. Future studies will define how the PR isoforms differentially cooperate with FOXO1 as either regulated (i.e. phosphorylated) activating or repressive transcription complexes on target genes that control cell fate. Overall, identifying the mechanisms governing PRA versus PR-B specific gene regulation may provide a means to therapeutically induce the protective actions of progesterone in hormone-driven cancers towards specific cell fates (e.g. senescence relative to proliferation). (Studies supported by NIH R01 CA159712, T32 CA009138, CTSA UL1TR000114, MN Ovarian Cancer Alliance, and Doctoral Dissertation Fellowship.) Citation Format: Caroline H. Diep, Carol A. Lange. Mechanisms of differential activities of progesterone receptor (PR) isoforms in ovarian cancer models [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1311.

Abstract 393: The chemokine receptor, CXCR2/IL8RB, contributes to the survival of pancreatic adenocarcinoma, and may play a role in stroma-tumor communication

The failure of most chemotherapeutic agents to treat pancreatic cancer could be related to poor delivery of drug due to the physical barrier of dense fibrosis surrounding the tumor. Targeting and attenuating the fibrotic stroma may result in better access of drug to tumor. Our current studies show that CXCR2/IL8RB is over-expressed in the surrounding stroma of pancreatic ductal adenocarcinoma tumors. CXCR2/IL8RB and its ligands are well known to be involved in fibrosis, and recently published papers suggest a role for CXCR2 in tumor proliferation, as well. Therefore, CXCR2 is a good target for pancreatic cancer therapy, because it has the potential to target both the stroma surrounding the tumor, as well as the tumor itself. When comparing matched samples of patient tumor and adjacent normal tissue, the mean IHC score of stroma surrounding normal pancreatic ducts is 0.61 (95% CI 0.38-0.84), while the mean IHC score of stroma surrounding ductal adenocarcinoma is 1.6 (95% CI: 1.3-1.9; Mann-Whitney test p This work was supported by NIH P01 Grant CA109552. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 393. doi:10.1158/1538-7445.AM2011-393

Abstract 1613: BTK as a drug target for cancers that harbor inactivating mutations in the DPC4 gene

The Deleted in Pancreatic Cancer locus 4 (DPC4) is a tumor suppressor gene that is often inactivated in pancreatic and colorectal cancers. In pancreatic adenocarcinomas, the frequency of inactivation of DPC4 is approximately 55%, while in colorectal carcinomas has been reported to be 10% to 35%. The loss of DPC4 has been shown to be associated with the progression and malignancy of pancreatic cancer along with decreased patient survival. We employed a small interfering (siRNA) library based screening strategy to identify potential synthetic lethal partners of the DPC4 gene. We used a kinase focused siRNA library that consisted of two siRNA oligonucleotides for each of the 624 protein kinase genes. A DPC4 isogenic pair, BxPC3-Vector and BxPC3-DPC4 (Wang et al. 2006) were treated by the siRNA oligonucleotides in parallel and the effects of the siRNA oligonucleotides on the growth of the cell lines were then compared. siRNA oligonucleotides that selectively inhibited the cell growth of the BxPC3-Vector cell line were selected as potential positive hits. These genes, once validated, represented potential drug targets that are very specific to cancer cells harboring mutations in the DPC4 gene. One of the top-ranked hits we identified from this screening is the Bruton agammaglobulinemia tyrosine kinase (BTK). With BTK siRNA treatment, BxPC3-Vector cells, which are DPC4 null, showed ∼30% more cell growth inhibition than the BxPC3-DPC4 cells, which are DPC4 wildtype. This selectivity of BTK siRNA against DPC4 null cells was further confirmed in a confirmation screen using two pancreatic cancer cell lines, BxPC3 and PANC-1, which are DPC4 null and DPC4 wildtype, respectively. BTK siRNA also showed significant selectivity against DPC4 deficiency in another pair of DPC4 isogenic colon cancer cell lines, HCT-116 and HCT-116-DPC4-knockout. To validate the BTK siRNA selectivity findings, we evaluated the BTK inhibitor PCI-32765 in our cancer cell line models and observed the same selectively against DPC4 null pancreatic and colon cancer cell lines. These results indicate that BTK is a potential molecular target for pancreas, colon, and other cancers harboring inactivating mutations in the DPC4 gene. (The work was supported by a NCI grant P01CA109552.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1613.

Abstract 1338: Chemokine (CXC motif) Ligand 5 protein expression in pancreatic tumors: Exploring chemostatic gradients in the tumor microenvironment

Due to lack of early presenting symptoms, pancreatic cancer is often diagnosed late when the tumor is highly invasive with aggressive clinical course and conventional therapies are limited in their effectiveness. Identifying molecular targets which contribute to tumor aggressiveness and metastasis could lead to development of better therapies to treat pancreas cancer patients. To identify potential molecular targets, we applied a specialized bioinformatics Breakpoint/Gene Aberration algorithm to analyze aCGH data and microarray expression data from 16 low-passage pancreas xenograft samples. Genes which were located near a putative breakpoint and whose expression was altered in a majority of the xenografts samples were considered putative targets. We report here preliminary data concerning one target, Chemokine (CXC motif) Ligand 5 (CXCL5) and its receptor CXCR2/IL8Rb. CXCL5 is a member of the ELR+ Chemokine family, a family of small, secreted ligands which are generally involved in cellular responses to injury and inflammation, but are also implicated in cancer progression and angiogenesis. CXCL5 is up regulated in 12 out of 16 xenograft samples analyzed (75%), with an average fold increase in expression of 41 (range 18 to 59). CXCL5 over-expression was further confirmed by IHC analysis of a custom made primary pancreas tumor microarray. Median IHC score (range 0-3) for CXCL5 in adenocarcinoma epithelial cells is 1.6, versus a median of 0.8 in the matched adjacent normal samples (N=34; p This work was supported by NIH P01 Grant CA109552. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1338.

Abstract B170: Downregulation of Yes‐associated protein 1 (YAP1) expression reduces cell proliferation and clonogenicity of pancreatic cancer cells

Recent genetic studies in Drosophila have shown the Hippo pathway to be a potential tumor suppressor pathway that could play vital role(s) in mammalian cancer development. The Hippo pathway regulates organ size by inhibiting cell proliferation and promotes cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector that promotes cell growth as a transcription co‐activator. In human cancer, YAP1 has been shown to be a candidate oncogene as the chromosomal region containing YAP1 (11q22) has been reported as amplified and over‐expressed in several tumor types, such as colon, liver, and lung (Overholtzer et al. 2006 PNAS, 103: 12405–12410). To determine the role of YAP1 in the tumorigenesis and progression of pancreatic cancer, here we examine YAP1 protein expression in pancreatic tumors and investigate the phenotypic effects of YAP1 down‐regulation in cultured pancreatic cancer cells. Initially, we evaluated YAP1 expression in pancreatic tumors through immunohistochemistry (IHC) using tissue microarrays (TMA) to determine relative protein abundance and localization at the tissue and cellular level. YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the cells, whereas the companion normal pancreas tissue showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell densities. YAP1 total protein expression and Ser127 phosphorylation increased concurrently with cell density in whole cell lysates and cytosolic fractions, but does not in nuclear fractions. Additionally, transfection of YAP1‐targeting siRNA oligonucleotides within pancreatic cancer cell lines, BxPC‐3 and PANC‐1, significantly decreased cell viability and reduced cell proliferation in vitro. An additional observation was the diminished anchorage‐independent growth on soft agar of BxPC‐3 and PANC‐1 cells upon transfection with YAP1 siRNA oligonucleotides. The number of colonies formed were approximately 5% in BxPC‐3 (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B170.

Abstract A30: Integrated analysis of genomic data and annotations in cancer research

Many cancer researchers are currently generating heterogeneous genomic data sets containing mRNA expression, array based comparative genomic hybridization (aCGH), and other information from multiple patients. In the near future, data sets will likely include proteomics, methylomics, and high throughput sequencing data. Analytical tools that integrate these different data types to prioritize genes for further study have lagged, however. With this in mind, we have implemented a standalone software application that integrates CGH and mRNA expression profiling data. This application implements several different methods to generate ranked lists of genes and genomic regions based on breakpoint frequency, aberration frequency, and the impact of copy number changes on mRNA expression. The software is Java GUI based, with abstracted object models that can be extended as new genomic data types and annotations become available. We used this application to analyze aCGH datasets generated from 22 primary pancreatic tumor xenografts and mRNA expression datasets from 16 of the 22 samples. Genes which were located near a putative breakpoint and whose expression levels were high in a majority of the xenografts samples were considered putative targets. Preliminary IHC analysis of pancreas primary tumor tissue microarrays confirms one of these putative targets, Chemokine (CXC motif) Ligand 5 (CXCL5). By allowing cancer researchers to correlate genomic aberrations with expression data, we believe that this application will generate more meaningful targets for further biomarker identification and/or drug target discovery. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A30.