Kristina Y. Aguilera

Collagen Signaling Enhances Tumor Progression after Anti-VEGF Therapy in a Murine Model of Pancreatic Ductal Adenocarcinoma

There is growing evidence that antiangiogenic therapy stimulates cancer cell invasion and metastasis. However, the underlying molecular mechanisms responsible for these changes have not been fully defined. Here, we report that anti-VEGF therapy promotes local invasion and metastasis by inducing collagen signaling in cancer cells. We show that chronic VEGF inhibition in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) induces hypoxia, a less differentiated mesenchymal-like tumor cell phenotype, TGF-β expression, and collagen deposition and signaling. In addition, we show that collagen signaling is critical for protumorigenic activity of TGF-β in vitro. To further model the impact of collagen signaling in tumors, we evaluated PDA in mice lacking Sparc, a protein that reduces collagen binding to cell surface receptors. Importantly, we show that loss of Sparc increases collagen signaling and tumor progression. Together, these findings suggest that collagen actively promotes PDA spread and that enhanced disease progression associated with anti-VEGF therapy can arise from elevated extracellular matrix-mediated signaling.

Recruitment and retention: factors that affect pericyte migration

Pericytes are critical for vascular morphogenesis and contribute to several pathologies, including cancer development and progression. The mechanisms governing pericyte migration and differentiation are complex and have not been fully established. Current literature suggests that platelet-derived growth factor/platelet-derived growth factor receptor-β, sphingosine 1-phosphate/endothelial differentiation gene-1, angiopoietin-1/tyrosine kinase with immunoglobulin-like and EGF-like domains 2, angiopoietin-2/tyrosine kinase with immunoglobulin-like and EGF-like domains 2, transforming growth factor β/activin receptor-like kinase 1, transforming growth factor β/activin receptor-like kinase 5, Semaphorin-3A/Neuropilin, and matrix metalloproteinase activity regulate the recruitment of pericytes to nascent vessels. Interestingly, many of these pathways are directly affected by secreted protein acidic and rich in cysteine (SPARC). Here, we summarize the function of these factors in pericyte migration and discuss if and how SPARC might influence these activities and thus provide an additional layer of control for the recruitment of vascular support cells. Additionally, the consequences of targeted inhibition of pericytes in tumors and the current understanding of pericyte recruitment in pathological environments are discussed.

SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations

BackgroundTumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination.MethodsM and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer.ResultsComparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases.ConclusionsThe matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer.

Inhibition of discoidin domain receptor 1 reduces collagen-mediated tumorigenicity in pancreatic ductal adenocarcinoma

The extracellular matrix (ECM), a principal component of pancreatic ductal adenocarcinoma (PDA), is rich in fibrillar collagens that facilitate tumor cell survival and chemoresistance. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that specifically binds fibrillar collagens and has been implicated in promoting cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. We found that collagen-induced activation of DDR1 stimulated protumorigenic signaling through protein tyrosine kinase 2 (PYK2) and pseudopodium-enriched atypical kinase 1 (PEAK1) in pancreatic cancer cells. Pharmacologic inhibition of DDR1 with an ATP-competitive orally available small-molecule kinase inhibitor (7rh) abrogated collagen-induced DDR1 signaling in pancreatic tumor cells and consequently reduced colony formation and migration. Furthermore, the inhibition of DDR1 with 7rh showed striking efficacy in combination with chemotherapy in orthotopic xenografts and autochthonous pancreatic tumors where it significantly reduced DDR1 activation and downstream signaling, reduced primary tumor burden, and improved chemoresponse. These data demonstrate that targeting collagen signaling in conjunction with conventional cytotoxic chemotherapy has the potential to improve outcome for pancreatic cancer patients. Mol Cancer Ther; 16(11); 2473–85. ©2017 AACR.

Discoidin domain receptor 1 activity drives an aggressive phenotype in gastric carcinoma

BackgroundDiscoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that utilizes collagen as a ligand, is a key molecule in the progression of solid tumors as it regulates the interaction of cancer cells with the tumor stroma. However, the clinical relevance of DDR1 expression in gastric carcinoma is yet to be investigated. Here, we assessed the role of DDR1 in mediating the aggressive phenotype of gastric carcinoma and its potential as a therapeutic target.MethodsWe conducted DDR1 immunohistochemistry using a tissue microarray of 202 gastric carcinoma specimens. We examined the effect of collagen-induced activation of DDR1 on cell signaling, tumorigenesis, and cell migration in gastric cancer cell lines, and tumor growth in a xenograft animal model of gastric cancer.ResultsOur results showed that 50.5% of gastric cancer tissues are positive for DDR1 expression, and positive DDR1 expression was significantly correlated with a poor prognosis (P = 0.015). In a subgroup analysis, DDR1 expression was prognostically meaningful only in patients receiving adjuvant treatment (P = 0.013). We also demonstrated that collagen was able to activate DDR1 and increase the clonogenicity and migration of gastric cancer cells. We observed that a DDR1 inhibitor, 7rh benzamide, suppressed tumor growth in gastric cancer xenografts.ConclusionsOur findings suggest a key role for DDR1 signaling in mediating the aggressive phenotype of gastric carcinoma. Importantly, inhibition of DDR1 is an attractive strategy for gastric carcinoma therapy.

Abstract 626: BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models

The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers including lung and breast adenocarcinomas. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer (NK) cells and tumor-associated macrophages. Hence AXL resides uniquely at the nexus between tumor and microenvironmental anti-tumor immune suppression mechanisms. We report that BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in aggressive adenocarcinoma models with limited immunogenicity by targeting both tumor intrinsic and microenvironmental immune suppression. Immune therapy with anti-CTLA4/PD1 in the 4T1 model increased AXL and EMT-marker expression correlating with a lack of response. Combination with BGB324 resulted in durable primary tumor clearance versus anti-CTLA4/PD1 alone. In a separate study, BGB324 + anti-CTLA4 treatment resulted in significant long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of the 4T1 model was not detected in animals responding to the combination treatment. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. In the LL2 Lewis Lung model, BGB324 in combination with anti-PD1/PDL1 significantly prevented tumor growth compared to treatment with anti-PD1/PDL1. Tumors from mice treated with BGB324 in combination with immune checkpoint inhibitors displayed reduced EMT traits, altered cytokine expression, enhanced tumor infiltration of effector cells and decreased number of mMDSC. Also, BGB324 significantly reduced IL10 secretion by isolated human macrophages and enhanced human NK-cell mediated NSCLC tumor cell lysis. Collectively these results support a prominent role for AXL in resistance to immune therapy and support clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment. Citation Format: Katarzyna Wnuk-Lipinska, Kjersti Davidsen, Magnus Blo, Agnete Engelsen, Jing Kang, Linn Hodneland, Maria Lie, Sebastien Bougnaud, Kristina Aguilera, Lavina Ahmed, Agata Rybicka, Eline Milde Naevdal, Paulina Deyna, Anna Boniecka, Straume Oddbjorn, Salem Chouaib, Rolf Brekken, Gro Gausdal, James B. Lorens. BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models [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 626. doi:10.1158/1538-7445.AM2017-626

Abstract 182: SPARC as a regulator of collagen signaling in pancreatic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The extracellular matrix (ECM) is a principal component of pancreatic ductal adenocarcinoma (PDA), which is rich in fibrillar collagens, and provides structural support that facilitates tumor cell survival and chemoresistance. Collagen expression and deposition is a complex process that is orchestrated in part by the matricellular protein SPARC. SPARC expression in human patients with PDA correlates with improved chemoresponse; however, this mechanism is unclear. We propose that SPARC controls collagen binding to the cell surface and reduces collagen-mediated signaling via Discoidin domain receptors (DDR1, DDR2). DDR1/DDR2 are homologous receptor tyrosine kinases that bind specifically to fibrillar collagens and have been implicated regulating cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. Additionally, collagen has been shown to promote chemoresistance in pancreatic tumor cells. We hypothesize that SPARC promotes chemoresponse by reducing collagen-mediated DDR signaling. Structural studies identified that SPARC and DDRs share the same collagen binding site. We have demonstrated that SPARC inhibits collagen binding to DDR1/DDR2 via in vitro binding assays and cell based activity assays. To determine the functional relevance of SPARC expression and collagen-mediated DDR activation in PDA, Sparc-null (Sparc-/-) mice were crossed with KIC (LSL-KrasG12D; Cdkn2alox/lox; p48Cre/+) mice. Survival was reduced and tumors were more aggressive in Sparc-/- KIC along with an elevation in DDR-mediated signaling in the tumor microenvironment. Primary PDA cell lines isolated from Sparc+/+ and Sparc-/- KIC animals were used to probe collagen signaling cascades revealing that collagen activated Ddr1 and downstream intermediates including protein tyrosine kinase 2 (Pyk2) and pseudopodium-enriched atypical kinase 1 (Peak1) in Sparc-/- cell lines. Furthermore, recombinant SPARC or neutralizing antibodies to Ddr1 abrogated collagen induced Ddr1 signaling in vitro implicating Ddr1 as a putative therapeutic target in PDA. Pharmacologic inhibition of Ddr1 was evaluated with nilotinib, a BCR-Abl inhibitor that shows high activity against Ddr1, and 7rh, a novel inhibitor with high specificity for Ddr1. In vitro nilotinib and 7rh suppress collagen-induced Ddr1 signaling, cell migration, and colony formation. In vivo nilotinib reduced the growth of implanted pancreatic tumors in Sparc-/- animals with little effect on tumor growth in Sparc+/+ mice. Studies with 7rh in vivo demonstrate an inhibition of Ddr1 signaling in syngeneic pancreatic tumors and is well tolerated at doses up to 30 mg/kg given daily via oral gavage. Therapy studies combining standard chemotherapy (gemcitabine + Nab-paclitaxel) with 7rh are ongoing. Our results suggest that Ddr1 enhances tumor cell survival and tumor progression and that Sparc functions to reduce PDA progression by blunting collagen-induced DDR signaling. Citation Format: Kristina Y. Aguilera, Courtney D. Goldstein, Lee B. Rivera, Amy D. Bradshaw, Ke Ding, Rolf A. Brekken. SPARC as a regulator of collagen signaling in pancreatic cancer. [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 182. doi:10.1158/1538-7445.AM2014-182

Abstract P2-04-08: BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma

The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. Axl signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response. AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is required for tumor immune evasion in mammary adenocarcinoma models and EMT-mediated resistance to cytotoxic T cell and natural killer (NK)-cell mediated cell killing. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms in breast cancer. We evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in the aggressive mammary adenocarcinoma (4T1) syngeneic (Balb/C) mouse modelthat display limited immunogenicity. Immune therapy with anti-CTLA-4/anti-PD-1 increased AXL and EMT-marker expression in 4T1 tumors, and correlated with lack of response to immune therapy. Combination treatment with BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 4 doses) in Balb/C mice bearing established 4T1 tumors. The combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in durable primary tumor clearance in 23 % of treated mice versus 5.6% obtained with anti-CTLA-4/anti-PD-1 alone (p=0.0157). In a separate study, BGB324 + anti-CTLA-4 treated resulted in 22% long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of this model were concomitantly abrogated in the animals responding to the combination treatment. In addition, BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes (CTLs). Enhanced presence of CTLs was also detected in spleens from animals responding to treatment. BGB324 + anti-CTLA-4/anti-PD-1 treatment increased the number of NK cells, macrophages and polymorphonuclear neutrophils, but decreased the number of mMDSC. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. Together with recent results in other tumor types that support a prominent role for AXL in resistance to immune therapy and encouraging results from ongoing clinical trials with BGB324, support combining BGB324 with immune checkpoint inhibitors to improve treatment of breast cancer. Citation Format: Lorens JB, Lipinska KW, Davidsen K, Blo M, Hodneland L, Engelsen A, Kang J, Lie MK, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Naevdal EM, Deyna P, Boniecka A, Straume O, Chouaib S, Brekken RA, Gausdal G. BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-08.

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.