Janusz Franco-Barraza

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

BackgroundAlterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions.We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer.MethodsWe generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses.ResultsWe observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated.ConclusionCancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.

A Chemomechanical Model of Matrix and Nuclear Rigidity Regulation of Focal Adhesion Size

In this work, a chemomechanical model describing the growth dynamics of cell-matrix adhesion structures (i.e., focal adhesions (FAs)) is developed. We show that there are three regimes for FA evolution depending on their size. Specifically, nascent adhesions with initial lengths below a critical value that are yet to engage in actin fibers will dissolve, whereas bigger ones will grow into mature FAs with a steady state size. In adhesions where growth surpasses the steady state size, disassembly will occur until their sizes are reduced to the equilibrium state. This finding arises from the fact that polymerization of adhesion proteins is force-dependent. Under actomyosin contraction, individual integrin bonds within small FAs (i.e., nascent adhesions or focal complexes) must transmit higher loads while the phenomenon of stress concentration occurs at the edge of large adhesion patches. As such, an effective stiffness of the FA-extracellular matrix complex that is either too small or too large will be relatively low, resulting in a limited actomyosin pulling force developed at the edge that is insufficient to prevent disassembly. Furthermore, it is found that a stiffer extracellular matrix and/or nucleus, as well as a stronger chemomechanical feedback, will induce larger adhesions along with a higher level of contraction force. Interestingly, switching the extracellular side from an elastic half-space, corresponding to some widely used in vitro gel substrates, to a one-dimensional fiber (as in the case of cells anchoring to a fibrous scaffold in vivo) does not qualitative change these conclusions. Our model predictions are in good agreement with a variety of experimental observations obtained in this study as well as those reported in the literature. Furthermore, this new model, to our knowledge, provides a framework with which to understand how both intracellular and extracellular perturbations lead to changes in adhesion structure number and size.

Structural and Mechanistic Insights into the Recruitment of Talin by RIAM in Integrin Signaling

Plasma membrane (PM)-bound GTPase Rap1 recruits the Rap1-interacting-adaptor-molecule (RIAM), which in turn recruits talin to bind and activate integrins. However, it is unclear how RIAM recruits talin and why its close homolog lamellipodin does not. Here, we report that, although RIAM possesses two talin-binding sites (TBS1 and TBS2), only TBS1 is capable of recruiting cytoplasmic talin to the PM, and the R8 domain is the strongest binding site in talin. Crystal structure of an R7R8:TBS1 complex reveals an unexpected kink in the TBS1 helix that is not shared in the homologous region of lamellipodin. This kinked helix conformation is required for the colocalization of RIAM and talin at the PM and proper activation of integrin. Our findings provide the structural and mechanistic insight into talin recruitment by RIAM that underlies integrin activation and explain the differential functions of the otherwise highly homologous RIAM and lamellipodin in integrin signaling.

Matrix-regulated integrin αvβ5 maintains α5β1-dependent desmoplastic traits prognostic of neoplastic recurrence

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5β1-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvβ5-integrin redistribution of pFAK-independent active α5β1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5β1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool. DOI: http://dx.doi.org/10.7554/eLife.20600.001

Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

The mechanisms by which the extreme desmoplasia observed in pancreatic tumors develops remain unknown and its role in pancreatic cancer progression is unsettled. Chemokines have a key role in the recruitment of a wide variety of cell types in health and disease. Transcript and protein profile analyses of human and murine cell lines and human tissue specimens revealed a consistent elevation in the receptors CCR10 and CXCR6, as well as their respective ligands CCL28 and CXCL16. Elevated ligand expression was restricted to tumor cells, whereas receptors were in both epithelial and stromal cells. Consistent with its regulation by inflammatory cytokines, CCL28 and CCR10, but not CXCL16 or CXCR6, were upregulated in human pancreatitis tissues. Cytokine stimulation of pancreatic cancer cells increased CCL28 secretion in epithelial tumor cells but not an immortalized activated human pancreatic stellate cell line (HPSC). Stellate cells exhibited dose- and receptor-dependent chemotaxis in response to CCL28. This functional response was not linked to changes in activation status as CCL28 had little impact on alpha smooth muscle actin levels or extracellular matrix deposition or alignment. Co-culture assays revealed CCL28-dependent chemotaxis of HPSC toward cancer but not normal pancreatic epithelial cells, consistent with stromal cells being a functional target for the epithelial-derived chemokine. These data together implicate the chemokine CCL28 in the inflammation-mediated recruitment of cancer-associated stellate cells into the pancreatic cancer parenchyma.

NetrinG1/NGL-1 Axis promotes pancreatic tumorigenesis through cancer associated fibroblast derived nutritional supply and immunosuppression

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+ cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+ CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stunted in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. Significance PDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1 in vivo by limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.

Lyophilized açaí pulp (Euterpe oleracea Mart) attenuates colitis-associated colon carcinogenesis while its main anthocyanin has the potential to affect the motility of colon cancer cells

This study evaluated the possible protective effects of lyophilized açaí pulp (AP) in a colitis-associated carcinogenesis (CAC) rat model and the modifying effect of cyanidin 3-rutinoside (C3R) on the motility of RKO colon adenocarcinoma cells, using the wound healing assay. Male Wistar rats were induced to develop CAC using 1,2-dimethylhydrazine (DMH) and 2,4,6-trinitrobenzene acid (TNBS). Animals were randomly assigned to different groups that received basal diet or basal diet supplemented with 5.0% or 7.5% lyophilized AP. The findings indicate: 1) C3R (25 μM) has the potential to reduce RKO cell motility in vitro; 2) ingestion of lyophilized AP reduces the total number of aberrant crypt foci (ACF), ACF multiplicity, tumor cell proliferation and incidence of tumors with high grade dysplasia; 3) AP increases the gene expression of negative regulators of cell proliferation such as Dlc1 and Akt3, as well as inflammation (Ppara). Thus, lyophilized AP could exert a potential antitumor activity.

Abstract 3379: Metabolic influences of pancreatic tumor microenvironment on pancreatic cancer cell's metabolism

Pancreatic cancer, the most lethal of solid tumors, is associated with a five-year survival rate and high mortality. The lethality of this tumor stems from lack of early symptoms, inability for detection of cancerous pancreatic lesions, and a diagnosis window that is accompanied by tumor resistance and metastasis. The bulk of the tumor mass, the fibrotic stroma, has been deemed an active player in the initiation and progression of pancreatic ductal adinocarcinoma (PDAC). A number of studies have elucidated the interactions between stromal cells and pancreatic cancer cells (PCCs), and the extracellular matrix (ECM) and PCCs. However, the role of the tumor microenvironment on the metabolic machinery of PCCs remains an active field of investigation. In this study, we investigate the effect of pancreatic stellate cells (PSCs), cancer-associated fibroblasts (CAFs), with or without ECM components, in a two-dimensional or three-dimensional setting on the glycolytic and mitochondrial pathways of patient-derived PCCs. The modulation of the expression levels of metabolic enzymes by the tumor microenvironment was also investigated. The metabolic reprogramming induced in PCCs by normal and reactive pancreatic stroma was investigated by isotopomer flux analysis. Our results reveal that pancreatic reactive stroma differentially upregulates glutamine and arginine metabolism in PCCs. The insights obtained from our work will lead to the development of targeted therapies for stroma and pancreatic cancer cells. Citation Format: Joelle Baddour, Lifeng Yang, Juan C. Marini, Janusz Franco-Barraza, Edna Cukierman, Chaoxin Hu, Anirban Maitra, Deepak Nagrath. Metabolic influences of pancreatic tumor microenvironment on pancreatic cancer cell9s metabolism. [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 3379. doi:10.1158/1538-7445.AM2014-3379

Abstract 1115: A discrete tuning of α5β1 integrin activity sustains the tumor-ECM induced fibroblastic activation in pancreatic cancer stroma

Pancreatic adenocarcinoma (PDAC) presents one of the highest mortality rates amongst all neoplasias. The desmoplastic microenvironment characteristic of PDAC plays a pivotal role in this aggressive cancer progression. Using an in vivo-mimetic 3D human stroma system, we have shown that quiescent fibroblasts become activated, undergoing a desmoplastic (i.e., myofibroblastic) phenotypic switch in response to tumor-associated (TA) but not to normal fibroblast-derived extracellular matrix (ECM). Nevertheless, the molecular mechanisms responsible for this process remain unclear. Using syngeneic human fibroblasts harvested from patient-matched normal and tumor samples, we produced a human 3D pancreatic stromal system. The system was used to investigate the role of integrins during the TA-ECM-induced phenotypic switch, focusing on two integrins associated with myofibroblastic differentiation, αvβ5 and α5β1. We evaluated the role of well-known integrin signaling effectors, i.e., non-receptor tyrosine kinases FAK and Src family kinases (SFK), in the observed TA-ECM-induced phenotypic switch. Approaches included the use of specific inhibitors or activators of integrins and SFK or FAK chemical or genetic inhibitors. Our data strongly suggest a αvβ5/α5β1-integrin cross-talk necessary to maintain the TA-ECM-induced phenotypic switch. This fine-tuned integrin cross-talk participates in the TA-ECM-induced overexpression and stress fiber localization of desmoplastic stroma marker, α-smooth muscle actin (α-SMA). Our findings correlate with clustering of α5β1-integrin at TA-ECM-altered 3D adhesion structures, together with differences in α5β1 activation. Interestingly, we uncovered a mechanism whereby αvβ5-integrin activity, in a SFK/FAK-dependent manner, inhibits a FAK-independent α5β1-integrin activity, preventing it from inhibiting the TA-ECM-induced phenotypic switch. Finally, using patient samples, we verified our in vitro generated hypothesis, while results suggested that αvβ5-integrin inhibition may constitute a valid inhibitory PDAC-associated stroma clinical treatment/approach. Likewise, this work also concluded that the TA-ECM-regulated mechanism studied may comprise a clinically relevant occurrence, suggestive of a noteworthy value in assessing stromal activity in PDAC patients. Funding provided by The Commonwealth of Pennsylvania, The Bucks County Chapter Board of Associates and NCI/NIH grants CA113451 and CA06927 Citation Format: Janusz Franco-Barraza, Tiffany Luong, Neelima Shah, Raj Madhani, Katherine Alpaugh, John Hoffman, Edna Cukierman. A discrete tuning of α5β1 integrin activity sustains the tumor-ECM induced fibroblastic activation in pancreatic cancer stroma. [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 1115. doi:10.1158/1538-7445.AM2014-1115

Abstract 137: Tumor-associated ECM induces renal cell carcinoma tumorigenic responses via α6β1-integrin regulation of ITPKA

It is estimated that over 65,150 patients were diagnosed with renal cell carcinoma (RCC) with 13,680 deaths in 2013. Surgery and targeted therapies have limited impact on survival in patients with advanced metastatic RCC. Tumor-stroma plays a pivotal role in RCC tumorigenesis including metastasis. One of the striking features of RCC is a fibro vascular stromal reaction with an altered tumor-associated extracellular matrix (TA-ECM) that directly intercalates with the tumoral epithelia playing a functional role in RCC progression. We previously suggested that stromal activation characterized by the expression of specific tumor-associated stromal proteins and the TA-ECM not only constitutes an independent worse prognostic marker, but also comprises a predictive risk indicator of recurrent RCC. Our work is based on the fact that stromal-tumor interactions are most readily studied in in vivo-mimetic 3D models. Among the most promising systems are in vitro 3D stromal models developed by our group in which tumor cells are plated within TA-ECMs derived from fibroblasts harvested from surgical tissue samples comprised of patient-matched normal and tumor-associated fibroblasts. We have demonstrated the physiological relevance of this system in vivo and found that mesenchymal RCC stromal cells and matrices are modified during epithelial tumor progression. Here we show that ECMs produced by normal fibroblasts are restrictive, while syngeneic tumor-associated ECMs induce increased RCC tumorigenic responses including tumor growth, resistance to apoptosis and promotion of invasion. An unbiased gene expression array was conducted using RNA obtained from RCC cells cultured within the assorted ECMs. Classic RCC signaling pathways were evident, and in addition proteins previously unsuspected to play important roles in stromal regulation of RCC progression were identified. Amid these, we observed significant increases in the levels of inositol 1,4,5-trisphosphate 3-kinase-A (IP3KA; also known as ITPKA). Our data suggests that ITPKA9s activity is necessary for the above-stated TA-ECM induced tumorigenic responses. The study describes how, in response to tumor-associated but not normal ECM, α6β1-integrin activity regulates ITPKA via increased cytosolic Ca2+ stabilizing constitutive Raf1 and Erk1/2 activities to promote tumorigenic responses. Moreover, in vitro experiments were confirmed using the original patient samples while clinical relevance was established in a well-annotated RCC tissue microarray cohort. We propose that activated stromal RCC triggers a novel α6β1/ITPKA/Erk1/2 pro-tumorigenic signaling pathway. In consequence, patients presenting activated stroma, together with high tumoral α6β1-integrin and/or ITPKA expression, are predicted to present unfavorable outcomes. This work is expected to facilitate future clinical testing of the new targets to aid development of novel RCC therapeutics. Citation Format: Vivekanand Gupta, Janusz Franco-Barraza, Neelima Shah, Essel Dulaimi, Yan Zhou, Karthik Devarajan, Kathy Q. Cai, Katherine R. Alpaugh, Robert G. Uzzo, Edna Cukierman. Tumor-associated ECM induces renal cell carcinoma tumorigenic responses via α6β1-integrin regulation of ITPKA. [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 137. doi:10.1158/1538-7445.AM2014-137