Julie L. Wilson

DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes in vivo tumorigenicity. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.

Quantitative Analysis of Three-dimensional Conformal Radiotherapy Techniques for Posterior Fossa Treatment in Children

Numerous beam directions using 3-D conformal techniques can be employed in treating tumors in the posterior fossa, each with characteristic normal tissue exposure along the entrance and exit trajectory. A representative variety of beam configurations were modeled in a modern computer planning system initially with the entire posterior fossa as the target. These beams were quantitatively scored using criteria based on integral doses for both low dose and high dose effects encompassing a variety of critical normal structures, thus identifying strengths and weaknesses of each beam. By blocking portions of a particular beam accounting for unfavorable scores, a map of “zones” within the posterior fossa ideally treated by a certain beam or beams could be constructed. No universally ideal photon beam arrangement for the entire posterior fossa target could be identified. However, using single beam analysis, the strengths and weaknesses of particular strategies could be quantified. For example, vertex beams treating the cerebellar hemispheres allow the greatest sparing of cochlea and hypothalamus but at the cost of increased low to moderate dose to the supratentorial brain. Using the constructed maps identifying “zones” appropriately treated by a given beam or beams, three-dimensional conformal treatment plans with favorable dose-volume statistics can be designed based on previously defined normal tissue tolerance considerations. It is shown how this approach can be individualized based on specific patient characteristics (e.g., age). We conclude that radiotherapy directed to the posterior fossa can be optimized based on systematic assessment of individual beam contributions to normal tissues. This technique allows fast selection of treatment beams based on known normal tissue anatomical and tolerance information. Further studies will be required regarding long term effects of various radiation doses on specific volumes of normal tissue in order to individualize beam selection. When treating children, knowledgeable consideration of these beam characteristics can help avoid late effects.

Pancreatic cancer ascites xenograft–an expeditious model mirroring advanced therapeutic resistant disease

Pancreatic ductal adenocarcinoma has limited treatment options. There is an urgent need for developing appropriate pre-clinical models recapitulating metastatic disease, the most common clinical scenario at presentation. Ascites accumulation occurs in up to 20–30% of patients with pancreatic cancer; this milieu represents a highly cellular research resource of metastatic peritoneal spread. In this study, we utilized pancreatic ascites/pleural effusion cancer cells to establish patient derived xenografts. Ascites/pleural effusion-patient derived xenografts were established from twelve independent cases. Xenografts were serially passed in nude mice and tissue bio-specimen banking has been established. Histopathology of emergent tumors demonstrates poorly to moderately differentiated, glandular and mucin producing tumors, mirroring morphology of primary pancreatic cancer tumors. Whole genome sequencing of six patient derived xenografts samples demonstrates common mutations and structural variations similar to those reported in primary pancreatic cancer. Xenograft tumors were dissociated to single-cells and in-vitro drug sensitivity screen assays demonstrated chemo-resistance, correlating with patient clinical scenarios, thus serving as a platform for clinically relevant translational research. Therefore, establishment of this novel ascites/pleural effusion patient derived xenograft model, with extensive histopathology and genomic characterization, opens an opportunity for the study of advanced aggressive pancreatic cancer. Characterization of metastatic disease and mechanisms of resistance to therapeutics may lead to the development of novel drug combinations.

Meta-analysis of 1,200 transcriptomic profiles identifies a prognostic model for pancreatic ductal adenocarcinoma

Background With a dismal 8% median 5-year overall survival (OS), pancreatic ductal adenocarcinoma (PDAC) is highly lethal. Only 10-20% of patients are eligible for surgery, and over 50% of these will die within a year of surgery. Identify molecular predictors of early death would enable the selection of PDAC patients at high risk. Methods We developed the Pancreatic Cancer Overall Survival Predictor (PCOSP), a prognostic model built from a unique set of 89 PDAC tumors where gene expression was profiled using both microarray and sequencing platforms. We used a meta-analysis framework based on the binary gene pair method to create gene expression barcodes robust to biases arising from heterogeneous profiling platforms and batch effects. Leveraging the largest compendium of PDAC transcriptomic datasets to date, we show that PCOSP is a robust single-sample predictor of early death (≤1 yr) after surgery in a subset of 823 samples with available transcriptomics and survival data. Results The PCOSP model was strongly and significantly prognostic with a meta-estimate of the area under the receiver operating curve (AUROC) of 0.70 (P=1.9e-18) and hazard ratio (HR) of 1.95(1.6-2.3) (P=2.6e-16) for binary and survival predictions, respectively. The prognostic value of PCOSP was independent of clinicopathological parameters and molecular subtypes. Over-representation analysis of the PCOSP 2619 gene-pairs (1070 unique genes) unveiled pathways associated with Hedgehog signalling, epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) signalling. Conclusion PCOSP could improve treatment decision by identifying patients who will not benefit from standard surgery/chemotherapy and may benefit from alternate approaches. Abbreviations AUROC Area under the receiver operating curve GO Gene annotation OS Overall survival PCOSP Pancreatic cancer overall survival predictor PDAC Pancreatic ductal adenocarcinoma TSP Top scoring pairs.

Abstract 4180: TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is projected to become the 2nd leading cause of cancer deaths by 2030. PDACs are associated with a high frequency of KRAS mutations (95%) and overexpression of many pro-angiogenic cytokines and growth factors. Using a genetically engineered mouse model (GEMM) that we established in which oncogenic Kras is combined with loss of RB (KRC), we have determined that PDACs arising in these mice (mPDACs) harbor many endothelial cells (ECs) and sinusoidal-like vessels that have blood flow, as determined by intravital confocal microscopy. Array analysis of pancreatic cancer cells (PCCs) derived from KRC tumors revealed gene expression profiles that correlate with active TGF-β signaling pathways, as determined by gene set enrichment analysis (GSEA), and include increased expression of multiple pro-angiogenic genes. In silico analysis indicated that many of these pro-angiogenic genes were TGF-β targets, and inhibition of the type I TGF-β receptor (TβRI) with SB505124 confirmed that TGF-βs drive pro-angiogenic gene expression in KRC PCCs. Moreover, TGF-β increased the levels of pro-angiogenic cytokines in conditioned media (CM) prepared from KRC PCCs, which when added to ECs, activated canonical TGF-β signaling pathways and stimulated EC proliferation and migration. Although SB505124 blocked TGF-β pathway activation in ECs, it failed to suppress CM-enhanced EC proliferation and migration. By contrast, SB505124 attenuated tumor angiogenesis, growth and metastasis in a syngeneic orthotopic mouse model using KRC PCCs. Taken together, these results suggest that TGF-β promotes angiogenesis in an indirect manner, by up-regulating pro-angiogenic factors in PCCs that act on ECs in a paracrine manner. Therefore, targeting TGF-β in PDAC could be a beneficial anti-angiogenic strategy. Citation Format: Jesse Gore, Kelly E. Craven, Julie L. Wilson, Murray Korc. TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2015-4180

Abstract 969: TGF-beta cross-talks with the EGF receptor family to promote proliferation of pancreatic cancer cells with dysfunctional RB

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, associated with a high frequency of KRAS mutations (95%) and loss of negative growth constraints, due, in part, to frequent CDKN2A (85%), TP53 (75%) and SMAD4 (55%) mutations. PDACs also overexpress all three TGF-β isoforms, and high levels of TGF-βs correlates with decreased survival. Recently, we showed that in human PDAC and in murine PDAC (mPDAC) arising in biologically aggressive, oncogenic Kras-driven genetically engineered mouse models (GEMMs), proliferating pancreatic cancer cells (PCCs) often exhibit abundant phosphorylated Smad2 and RB indicating that in these PCCs, canonical TGF-β signaling pathways are active, whereas RB is inactive. Using PCCs derived from a GEMM that expresses oncogenic Kras, but lacks RB (KRC cells), we showed that TGF-β1 activates canonical and non-canonical (MEK, Src, PI3K) pathways, and enhances growth in 3-dimensional (3D) culture. This mitogenic effect is abrogated by type I TGF-β receptor (TβRI) kinase inhibition with SB505124, combined inhibition by MEK/Src or MEK/PI3K, or re-expression of wild-type, but not mutant RB. Thus, loss of RB function enables TGF-β to directly enhance PCC growth. Given that the EGF receptor (EGFR) family can activate these non-canonical pathways and that TGF-β cross-talks with EGFR, we investigated the role of EGFR family members in mediating the mitogenic effects of TGF-β on RB-deficient PCCs. In 3D culture, TGF-β1-enhanced proliferation was inhibited by targeting EGFR with erlotinib and completely suppressed by EGFR/HER2 inhibition with lapatinib. Moreover, lentiviral-mediated silencing of HER2 markedly attenuated TGF-β1-stimulated KRC growth, and this inhibitory effect was potentiated by erlotinib. Thus, TGF-β19s mitogenic effects are mediated, in part, through transactivation of EGFR/HER2, raising the possibility that concomitant use of TβRI inhibitors and lapatinib to inhibit EGFR/HER2 and may represent a novel therapeutic approach in PDAC. Citation Format: Jesse Gore, Samantha L. Deitz, Julie L. Wilson, Murray Korc. TGF-beta cross-talks with the EGF receptor family to promote proliferation of pancreatic cancer cells with dysfunctional RB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 969. doi:10.1158/1538-7445.AM2014-969