Amanda Kirane

Apricoxib, a Novel Inhibitor of COX-2, Markedly Improves Standard Therapy Response in Molecularly Defined Models of Pancreatic Cancer

Purpose: COX-2 is expressed highly in pancreatic cancer and implicated in tumor progression. COX-2 inhibition can reduce tumor growth and augment therapy. The precise function of COX-2 in tumors remains poorly understood, but it is implicated in tumor angiogenesis, evasion of apoptosis, and induction of epithelial-to-mesenchymal transition (EMT). Current therapeutic regimens for pancreatic cancer are minimally effective, highlighting the need for novel treatment strategies. Here, we report that apricoxib, a novel COX-2 inhibitor in phase II clinical trials, significantly enhances the efficacy of gemcitabine/erlotinib in preclinical models of pancreatic cancer. Experimental Design: Human pancreatic cell lines were evaluated in vitro and in vivo for response to apricoxib ± standard-of-care therapy (gemcitabine + erlotinib). Tumor tissue underwent posttreatment analysis for cell proliferation, viability, and EMT phenotype. Vascular parameters were also determined. Results: COX-2 inhibition reduced the IC50 of gemcitabine ± erlotinib in six pancreatic cancer cell lines tested in vitro. Furthermore, apricoxib increased the antitumor efficacy of standard combination therapy in several orthotopic xenograft models. In vivo apricoxib combination therapy was only effective at reducing tumor growth and metastasis in tumors with elevated COX-2 activity. In each model examined, treatment with apricoxib resulted in vascular normalization without a decrease in microvessel density and promotion of an epithelial phenotype by tumor cells regardless of basal COX-2 expression. Conclusions: Apricoxib robustly reverses EMT and augments standard therapy without reducing microvessel density and warrants further clinical evaluation in patients with pancreatic cancer. Clin Cancer Res; 18(18); 5031–42. ©2012 AACR.

Smac mimetic-derived augmentation of chemotherapeutic response in experimental pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is highly resistant to conventional chemotherapy, in part due to the overexpression of inhibitors of apoptosis proteins (IAPs). Smac is an endogenous IAP-antagonist, which renders synthetic Smac mimetics attractive anticancer agents. We evaluated the benefits of combining a Smac mimetic, JP1201 (JP), with conventional chemotherapy agents used for PDAC management.MethodsCell viability assays and protein expression analysis were performed using WST-1 reagent and Western blotting, respectively. Apoptosis was detected by annexin V/propidium iodide staining. In vivo tumor growth and survival studies were performed in murine PDAC xenografts.ResultsJP and gemcitabine (Gem) inhibited PDAC cell proliferation with additive effects in combination. The percentage of early apoptotic cells in controls, JP, Gem and JP + Gem was 17%, 26%, 26% and 38%, respectively. JP-induced apoptosis was accompanied by PARP-1 cleavage. Similar additive anti-proliferative effects were seen for combinations of JP with doxorubicin (Dox) and docetaxel (DT). The JP + Gem combination caused a 30% decrease in tumor size in vivo compared to controls. Median animal survival was improved significantly in mice treated with JP + Gem (38 d) compared to controls (22 d), JP (28 d) or Gem (32 d) (p = 0.01). Animal survival was also improved with JP + DT treatment (32 d) compared to controls (16 d), JP (21 d) or DT alone (27 d).ConclusionsThese results warrant further exploration of strategies that promote chemotherapy-induced apoptosis of tumors and highlight the potential of Smac mimetics in clinical PDAC therapy.

Epithelial–mesenchymal transition increases tumor sensitivity to COX-2 inhibition by apricoxib

Although cyclooxygenase-2 (COX-2) inhibitors, such as the late stage development drug apricoxib, exhibit antitumor activity, their mechanisms of action have not been fully defined. In this study, we characterized the mechanisms of action of apricoxib in HT29 colorectal carcinoma. Apricoxib was weakly cytotoxic toward naive HT29 cells in vitro but inhibited tumor growth markedly in vivo. Pharmacokinetic analyses revealed that in vivo drug levels peaked at 2-4 µM and remained sufficient to completely inhibit prostaglandin E(2) production, but failed to reach concentrations cytotoxic for HT29 cells in monolayer culture. Despite this, apricoxib significantly inhibited tumor cell proliferation and induced apoptosis without affecting blood vessel density, although it did promote vascular normalization. Strikingly, apricoxib treatment induced a dose-dependent reversal of epithelial-mesenchymal transition (EMT), as shown by robust upregulation of E-cadherin and the virtual disappearance of vimentin and ZEB1 protein expression. In vitro, either anchorage-independent growth conditions or forced EMT sensitized HT29 and non-small cell lung cancer cells to apricoxib by 50-fold, suggesting that the occurrence of EMT may actually increase the dependence of colon and lung carcinoma cells on COX-2. Taken together, these data suggest that acquisition of mesenchymal characteristics sensitizes carcinoma cells to apricoxib resulting in significant single-agent antitumor activity.

The importance of surgical margins in retroperitoneal sarcoma.

Surgery is the “gold‐standard” treatment for retroperitoneal sarcomas, but local recurrence is common, and can cause disease‐related death. Complete gross resection is associated with improved survival, but debate exists as to whether resection of adjacent organs to improve margins or prescription of neoadjuvant radiation leads to better outcomes. This review summarizes data addressing prognostic value of margin, extent of surgery necessary to optimize treatment of retroperitoneal sarcomas, and role of histology in optimizing therapy. J. Surg. Oncol. 2016;113:270–276.

Abstract B78: Warfarin blocks Gas6-mediated Axl activation required for pancreatic tumor plasticity and metastasis

Warfarin, a vitamin K antagonist anti-coagulant in clinical use for over 50 years, is reported to exert anti-cancer effects. We hypothesized that the molecular mechanism underlying the observed anti-tumor effects of warfarin is unrelated to generalized anti-coagulation, but rather due to inhibition of the Axl receptor tyrosine kinase. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here we document that inhibiting Gas6-mediated Axl activation with low dose warfarin blocks pancreatic cancer progression and spread. Warfarin and other Axl-targeting agents inhibit tumor progression and block spontaneous metastasis in multiple murine models of pancreatic cancer. Warfarin inhibited Axl-dependent tumor cell migration, invasiveness and proliferation while increasing apoptosis and sensitivity to chemotherapy. We demonstrate that Axl signaling is necessary for pancreatic tumor cell epithelial plasticity which is potently reversed by warfarin or selective Axl inhibition in vitro and in vivo. We anticipate Axl is a critical driver of pancreatic cancer progression and its inhibition with low dose warfarin or Axl-selective targeting agents may significantly improve outcome in patients. Citation Format: Amanda Kirane, Kathleen W. Ludwig, Gry Haaland, Tone Sandal, Renata Ranaweera, Jason E. Toombs, Laura A. Sullivan, Miao Wang, Noah Sorrelle, Sean P. Dineen, Michael T. Dellinger, James B. Lorens, Rolf A. Brekken. Warfarin blocks Gas6-mediated Axl activation required for pancreatic tumor plasticity and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B78.

Intraduodenal sarcoma recurrence of retroperitoneal origin: an unusual cause for a duodenal obstruction

Soft tissue sarcomas are uncommon tumors, and intraduodenal soft tissue sarcoma manifestation is even more rare. Only three cases of intraduodenal sarcomas have been reported in the literature thus far. Here, we report a case of an intraduodenal recurrence of a retroperitoneal sarcoma causing bowel obstruction. This unusual recurrence pattern likely relates to the patient’s previous resection and radiation treatment, and highlights the benefits, limitations and follow-up strategies after multimodality treatment.

Abstract 3819: COX-2 inhibition with apricoxib drives mesenchymal to epithelial transition and synergizes with anti-VEGF therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Resistance to standard therapy remains a challenge in treatment of pancreatic ductal adenocarcinoma (PDAC) resulting in need for novel therapeutic strategies. Anti-VEGF therapy with r84 delays PDAC progression; however, chronic hypoxia ultimately results in transition to a mesenchymal phenotype and rapid progression at a later timepoint. COX-2 inhibition with apricoxib, a novel antagonist in Phase II trials, reverses EMT in PDAC cells. We investigate the relationship between COX-2 and VEGF production in PDAC cell lines to evaluate the efficacy of a combination strategy in preclinical models of PDAC. In vitro, changes in VEGF production by PDAC cells following apricoxib were assessed by ELISA at baseline and following forced induction of EMT. The effect of r84, apricoxib, or combination on tumor growth and metastatic incidence was determined in SCID mice with established orthotopic pancreatic xenografts. Improvement of survival and reduction in tumor burden was assessed in a murine genetic model of PDAC using p48-Cre; KrasG12D;Cdkn2alox/lox mice, tissue was collected at 4 weeks of therapy with control antibody, r84 or apricoxib for analysis. PDAC cell lines grown in the presence of TGF-β and collagen demonstrated increased Zeb1 and decreased ECAD expression by Western blot. Treatment with clinically-achievable doses of apricoxib reversed this change in phenotype. High expression of COX-2 correlated with high levels of VEGF in conditioned media in human PDAC cell lines. VEGF production was initially sensitive to COX-2 inhibition, with complete depletion of VEGF in the first 6 hours, but VEGF levels returned to baseline by 16 hours and continued to rise throughout the first 72 hours post apricoxib. Upon repeat dosing at 24 hours, VEGF production had become independent of COX-2 and no changes were observed. Similar results were seen following induction of EMT, however, baseline VEGF production was dramatically increased, and while there was an initial decrease after apricoxib, production remained more than 2-fold higher than in cells under normal culture conditions. In vivo, apricoxib and r84 had minimal effect on primary tumor growth as single agents; however each agent resulted in a reduction of metastatic incidence. Combination therapy reduced primary tumor size and virtually eliminated metastases. Overall survival was significantly improved with apricoxib as a single agent. Apricoxib treated animals also demonstrated a delay in progression of disease and decreased collagen deposition in the microenvironment compared to r84, which increased collagen deposition.We conclude that COX-2 inhibition by apricoxib delays progression of PDAC and results in mesenchymal to epithelial transition. Combination with anti-VEGF therapy results in potent antitumor and antimetastatic effects in our models and warrants further evaluation as a strategy to augment chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3819. doi:1538-7445.AM2012-3819

Abstract A24: Strategies to combat hypoxia-driven PDAC invasion after antiangiogenic therapy.

Evidence that antiangiogenic therapy can enhance local invasion of primary tumors and metastasis is mounting. However, the molecular mediators responsible for these events are unclear. The goal of this study was to assess the efficacy of different therapies on hypoxia-induced changes in the tumor microenvironment after targeting VEGF in murine models of pancreatic ductal adenocarcinoma (PDAC). We report that VEGF-blockade controlled primary PDAC growth similar to chemotherapy. Unlike chemotherapy, VEGF-blockade induced tumor hypoxia, resulted in a mesenchymal primary tumor phenotype, and did not control liver dissemination. Further, VEGF-blockade enhanced collagen deposition and collagen signaling in primary PDAC. In vitro, collagen promoted PDAC epithelial to mesenchymal transition (EMT) and protected PDAC cells from TGFβ1-induced apoptosis. The matricellular protein SPARC orchestrates collagen deposition and reduces collagen-cell interaction. We show that SPARC inhibits collagen binding to discoidin domain receptors (DDRs) and that collagen itself can drive EMT in the absence of SPARC in vitro. To model the effect of enhanced collagen signaling in the absence of other alterations in vivo we assessed tumor growth in SPARC- deficient mice. Tumor growth was accelerated and more aggressive in the absence of SPARC in implant and genetic models of PDAC. Additionally, inhibition of collagen signaling reduced pancreatic tumor growth selectively in SPARC-deficient animals. These findings implicate that collagen signaling through DDRs participates in PDAC progression. To perturb tumor progression upstream of collagen signaling we evaluated HIF inhibition with metronomic doxorubicin and Cox-2 inhibition with apricoxib. These strategies blunted tumor growth and hypoxia induced EMT. Together, our findings suggest that multiple control points are plausible to combat hypoxia driven tumor progression and enhance the efficacy of anti-angiogenic therapy. This work was supported by NIH RO1 CA118240 and the Effie Marie Cain Scholarship in Angiogenesis Research. Citation Format: Lee B. Rivera, Juliet G. Carbon, Kristina Aguilera, Jason E. Toombs, Amanda Kirane, Michael T. Dellinger, Gaurab Chakrabarti, Jordan Braunfeld, Rolf A. Brekken. Strategies to combat hypoxia-driven PDAC invasion after antiangiogenic therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A24.

Abstract 1599: Inhibition of COX-2 with apricoxib enhances the efficacy of standard therapy in preclinical models of human pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cyclo-oxygenase-2 (COX-2) is expressed at a high level in pancreatic cancer and is implicated in pancreatic tumor progression. Inhibition of COX-2 can decrease tumor growth and augment cytotoxicity induced by either gemcitabine or EGFR inhibition. COX-2 while poorly understood has been implicated in tumor angiogenesis, tumor cell apoptosis and epithelial to mesenchymal transition (EMT). We investigated apricoxib, a novel COX-2 inhibitor, to further define the function of COX-2 in pancreatic cancer. Baseline expression and functional response of EGFR and COX-2 was determined in seven human pancreatic cancer cell lines. The level of phosphorylated EGFR (p-EGFR) and prostaglandin E2 (PGE2) production was determined by ELISA. Cytotoxicity was determined for gemcitabine, erlotinib, and apricoxib independently and in combination by MTS assay. The effect of standard therapy alone and in combination with apricoxib on AsPC-1 and Colo357 tumor growth in vivo was determined in SCID mice bearing orthotopic pancreatic xenografts. Microenvironmental parameters including apoptosis, proliferation, vessel density, and EMT were analyzed by IHC and VEGF levels were determined by ELISA. All cell lines expressed EGFR and COX-2; however expression was not predictive of p-EGFR level, PGE2 production or response to drug. AsPC-1 cells had negligible baseline COX-2 activity in vitro, whereas Colo357 cells expressed relatively high levels COX-2 and PGE2. Apricoxib decreased cell growth and COX-2 activity in all cell lines and addition of apricoxib improved response to chemotherapy in vitro. In vivo, addition of apricoxib to standard therapy significantly reduced tumor growth and, strikingly, almost eradicated metastases in mice bearing Colo357 but not AsPC-1 xenografts. COX-2 expression decreased in a dose-dependent fashion in apricoxib-treated animals. Plasma VEGF levels were unaffected by apricoxib in ASPC-1 -bearing animals, but were suppressed to undetectable levels in Colo357-bearing animals. Microvessel density was unaltered between groups but tumors from animals treated with combination therapy displayed increased pericyte coverage of vessels. Apoptosis was increased by addition of apricoxib, while markers of proliferation were decreased. In addition, combination of standard therapy with apricoxib reduced EMT, evidenced by elevation of epithelial markers and reduction of Zeb1 and vimentin, markers of a mesenchymal phenotype. We conclude that apricoxib significantly enhances the activity of gemcitabine and erlotinib, specifically in tumors with elevated COX-2 activity. Inhibition of COX-2 by apricoxib results in increased apoptosis, decreased proliferation, vascular normalization and a decrease in the mesenchymal phenotype in COX-2-dependent models. We believe clinical evaluation of apricoxib is warranted in pancreatic cancer patients. A Phase 2 study is ongoing. 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 1599. doi:10.1158/1538-7445.AM2011-1599