Lee B. Rivera

Lack of host SPARC enhances vascular function and tumor spread in an orthotopic murine model of pancreatic carcinoma

SUMMARY Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and rich in cysteine) as a key component of the stromal response, where it regulated tumor size, angiogenesis and extracellular matrix deposition. In the present study, we demonstrate that pancreatic tumors grown orthotopically in Sparc-null (Sparc−/−) mice are more metastatic than tumors grown in wild-type (Sparc+/+) littermates. Tumors grown in Sparc−/− mice display reduced deposition of fibrillar collagens I and III, basement membrane collagen IV and the collagen-associated proteoglycan decorin. In addition, microvessel density and pericyte recruitment are reduced in tumors grown in the absence of host SPARC. However, tumors from Sparc−/− mice display increased permeability and perfusion, and a subsequent decrease in hypoxia. Finally, we found that tumors grown in the absence of host SPARC exhibit an increase in alternatively activated macrophages. These results suggest that increased tumor burden in the absence of host SPARC is a consequence of reduced collagen deposition, a disrupted vascular basement membrane, enhanced vascular function and an immune-tolerant, pro-metastatic microenvironment.

The regulatory function of SPARC in vascular biology

SPARC is a matricellular protein, able to modulate cell/ECM interactions and influence cell responses to growth factors, and therefore is particularly attuned to contribute to physiological processes involving changes in ECM and cell mobilization. Indeed, the list of biological processes affected by SPARC includes wound healing, tumor progression, bone formation, fibrosis, and angiogenesis. The process of angiogenesis is complex and involves a number of cellular processes such as endothelial cell proliferation, migration, ECM degradation, and synthesis, as well as pericyte recruitment to stabilize nascent vessels. In this review, we will summarize current results that explore the function of SPARC in the regulation of angiogenic events with a particular emphasis on the modulation of growth factor activity by SPARC in the context of blood vessel formation. The primary function of SPARC in angiogenesis remains unclear, as SPARC activity in some circumstances promotes angiogenesis and in others is more consistent with an anti-angiogenic activity. Undoubtedly, the mercurial nature of SPARC belies a redundancy of functional proteins in angiogenesis as well as cell-type-specific activities that alter signal transduction events in response to unique cellular milieus. Nonetheless, the investigation of cellular mechanisms that define functional activities of SPARC continue to contribute novel and exciting paradigms to vascular biology.

Losartan Slows Pancreatic Tumor Progression and Extends Survival of SPARC-Null Mice by Abrogating Aberrant TGFβ Activation

Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFβ1 were increased significantly in tumors grown in SPARC-null mice. TGFβ1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFβ1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFβ1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFβ induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFβ1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFβ availability and activation.

TGF-β and αvβ6 Integrin Act in a Common Pathway to Suppress Pancreatic Cancer Progression

The TGF-β pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-β inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-β in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-β inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvβ6 integrin acted as a key upstream activator of TGF-β in evolving pancreatic cancers. In addition, TGF-β or αvβ6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-β signaling. Therefore, our findings indicate that αvβ6 and TGF-β act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.

SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity

SPARC prevents endoglin association with αV integrin, which blocks the activation of TGF-β signaling and promotes pericyte migration to nascent blood vessels.

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.

Neutralizing Murine TGFβR2 Promotes a Differentiated Tumor Cell Phenotype and Inhibits Pancreatic Cancer Metastasis

Elevated levels of TGFβ are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result, the TGFβ pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGFβ remains challenging because TGFβ has tumor suppressor functions in many epithelial malignancies, including pancreatic cancer. In fact, direct neutralization of TGFβ promotes tumor progression of genetic murine models of pancreatic cancer. Here, we report that neutralizing the activity of murine TGFβ receptor 2 using a monoclonal antibody (2G8) has potent antimetastatic activity in orthotopic human tumor xenografts, syngeneic tumors, and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density, and vascular function. These stromal-specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGFβ signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGFβ-dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors. Cancer Res; 74(18); 4996-5007. ©2014 AACR.

Rgs16 and Rgs8 in embryonic endocrine pancreas and mouse models of diabetes

SUMMARY Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic β-cells. Although β-cells can replicate in younger adults, the available diabetes therapies do not specifically target β-cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and β-cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates β-cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal-associated cells and islet β-cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated β-cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and β-cell expansion in embryos and metabolically stressed adults.

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