Hidayatullah G. Munshi - US grants

DESCRIPTION (provided by applicant): The invasive behavior of oral squamous cell carcinoma (OSCC) requires coordinated cellular events including degradation of the extracellular matrix (ECM) and the acquisition of motility. Altered expression of cadherins, transmembrane proteins that promote cell-cell contacts, has been associated with progression of OSCC. In many tumors, loss of E-cadherin or aberrant expression of N-cadherin has been shown to correlate with increased invasive behavior. Production of ECM degrading proteases, for example matrix metalloproteinases (MMPs), is also an early event in the malignant progression. In OSCC, a correlation between enhanced expression of MMP-2 (gelatinase A), MMP-9 (gelatinase B), membrane-type 1 MMP (MT1-MMP) and tumor progression has been described. Our data demonstrate that E-cadherin can regulate the expression of MMP-2 and MMP-9. Moreover, we have shown that disruption of cell-cell adhesions can induce MMP-dependent cellular invasion. Furthermore, our preliminary data demonstrate the involvement of phosphatidylinositol 3-kinase (PI3-kinase) in the E-cadherin-mediated regulation of MMPs. Based on these results, it is the working hypothesis of this proposal that a functional link between cell-cell adhesion and proteolysis regulates OSCC invasive behavior. Specifically, we propose that cadherin engagement modulates MMP expression and consequently controls cell motility and invasion. To test this hypothesis, we will assess the role of E- and N-cadherin in the regulation of MMP expression. Immunohistochemical and biochemical analysis of tumor tissues will be employed to evaluate the expression patterns of E- and N-cadherins, and MMPs. The biochemical pathways that are involved in the regulation of MMPs by E- and N-cadherin will then be evaluated. The long-term goal of the proposed research is to provide a more detailed understanding of the functional link between cell-cell adhesion and proteolysis and the contribution of this interplay to regulation of metastasis.

[unreadable] DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related death in the U. S. The median survival time after diagnosis is less than 6 months, while 5-year disease-free survival is less than 5%. Poor outcome is attributed to the relatively advanced stage of disease at time of diagnosis, with approximately 80% of patients presenting with locally aggressive or metastatic disease. PDAC is associated with an intense fibrotic reaction around the tumor known as desmoplastic reaction. This reaction is composed of interstitial extracellular matrix (ECM), predominantly type I collagen, together with proliferating fibroblastic cells. However, the functional interactions among the pancreatic ductal cells, the interstitial ECM and the stromal fibroblasts are poorly understood. According to the NCI Pancreatic Cancer Progress Review Group 'a better understanding is needed of the basic mechanisms involved in the development of the stroma, its interaction with the pancreatic cancer cells and its role in the pathogenesis of pancreatic cancer'. We have demonstrated that extracellular matrix deposited by pancreatic fibroblasts promotes TGF-?1 expression by PDAC cells followed by increased MT1-MMP expression [Ottaviano AJ et al, Cancer Research 2006]. Our preliminary data also indicate that MT1-MMP increases cyclin D1 expression and enhances p38 MAPK signaling to promote growth in collagen-rich microenvironment. Moreover, our preliminary data suggest that expression of MT1- MMP in the pancreas using a transgenic mouse model increases fibrosis. Together these data support our central hypothesis that a feed-forward amplification loop involving cross-talk between type I collagen, TGF-?1 and MT1-MMP contributes to the aggressive phenotype of PDAC: the desmoplastic reaction promotes TGF-?1 expression and signaling to increase MT1-MMP expression, but this in turn contributes to expanded fibrosis and a further increase in TGF-?1 signaling and MT1-MMP expression, thus enhancing PDAC invasion and metastasis. Experiments proposed in Aim 1 have been designed to understand the first half of the amplification loop (fibrosis ? TGF-?1 ? MT1-MMP) by elucidating the mechanism by which the ECM, in particular type I collagen, increases TGF-?1 and MT1-MMP expression by PDAC cells. In Aim 2 we will examine the second half of the amplification loop (MT1- MMP ? fibrosis and growth in 3D collagen) by elucidating the mechanism by which MT1-MMP promotes fibrosis and facilitates pancreatic tumor growth in a collagen-rich microenvironment using organotypic, orthotopic and transgenic models of pancreatic cancer. Our rationale for these proposed studies is that once the mechanism of cross-talk between the fibrotic reaction and PDAC cells is fully determined, this information may ultimately lead to new treatment strategies that reduce the morbidity and mortality of pancreatic cancer. PUBLIC HEALTH RELEVANCE: The relatively high mortality resulting from pancreatic ductal adenocarcinoma (PDAC) is largely due to the fact that approximately 80% of patients present with locally invasive or metastatic disease at the time of diagnosis. PDAC is frequently associated with an intense area of collagen-rich fibrosis surrounding the tumor known as the desmoplastic reaction. While the desmoplastic reaction is likely an important precursor to the development of PDAC local invasion, and perhaps metastases as well, the precise mechanisms that contribute to these processes are currently not well understood. The objective of this application is to delineate the molecular mechanisms by which desmoplastic reaction contributes to PDAC progression, and once the mechanism of cross-talk between the fibrotic reaction and PDAC cells is fully determined, we predict that this will lead to new treatment strategies that reduce the morbidity and mortality of pancreatic cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The bromodomain (BRD) and extra terminal domain (BET) family of proteins, which function as 'readers' of histone acetylation marks, mediate growth of pancreatic ductal adenocarcinoma (PDAC) cells in 3D collagen. Additionally, PDAC cells in 3D collagen demonstrate chemoresistance through increased expression of high mobility group A2 (HMGA2), an architectural protein that regulates chromatin structure. The long-term goal is to help develop novel mechanism-based targeted therapies for the treatment of PDAC. The objective in this application is to determine how BET proteins mediate chemoresistance and contribute to fibrosis in vivo. The central hypothesis is that BET protein inhibition will decrease PDAC tumor growth and increase chemosensitivity by decreasing the cancer stem cell population and HMGA2 protein function, respectively. A second hypothesis is that BET inhibition will lead to an attenuation of fibrosis in PDAC tumors. These hypotheses are based on strong preliminary data demonstrating that BET inhibitors decrease growth of PDAC and stellate cells in 3D collagen. In addition, treatment of PDAC cells with BET inhibitors decreases cancer stem cell population and represses HMGA2. The rationale is that a determination of the role and underlying mechanism of BET proteins in PDAC progression in vivo is likely to contribute substantively to a conceptual framework whereby new clinically effective targeted therapies can ultimately be developed. Three specific aims are proposed: 1) Determine the role of BET proteins in PDAC progression in vivo; 2) Evaluate the ability of BET protein inhibition to increase chemotherapy sensitivity; and 3) Evaluate the ability of BET protein inhibition to attenuate fibrosis. Under the first aim, the effect of BET inhibitors on PDAC progression will be determined in mouse models. Further, the extent to which BET inhibitors decrease PDAC stem cell population in vivo will be evaluated. Also, the ability of gold nanoparticles (Au-NPs) coupled with BRD4 siRNA to inhibit tumor growth will be determined. For the second aim, the ability of BET inhibitors to increase chemotherapy efficacy will be evaluated in 3D collagen and in mouse models. Additionally, the role of BET proteins in DNA damage response and the contribution of HMGA2 to BET protein regulation of chemoresistance will be assessed. In the third aim, the mechanism by which BET inhibitors regulate stellate cell activation and collagen production will be determined. The effectiveness of BET inhibitors and Au-NPs functionalized with BRD4 siRNA to attenuate fibrosis in mouse models will also be evaluated. The research proposed is innovative because it utilizes complex models of pancreatic cancer, including in vitro organotypic cultures and in vivo orthotopic and transgenic mouse models to determine the role of BET proteins in PDAC progression. An additional innovation is the use of Au-NPs functionalized with siRNAs to downregulate BRD4 expression in the model systems. This proposed research is significant because it is expected to provide strong scientific justification for the continued development and future clinical trials of BET inhibitors in PDAC.

DESCRIPTION (provided by applicant): The bromodomain (BRD) and extra terminal domain (BET) family of proteins, which function as 'readers' of histone acetylation marks, mediate growth of pancreatic ductal adenocarcinoma (PDAC) cells in 3D collagen. Additionally, PDAC cells in 3D collagen demonstrate chemoresistance through increased expression of high mobility group A2 (HMGA2), an architectural protein that regulates chromatin structure. The long-term goal is to help develop novel mechanism-based targeted therapies for the treatment of PDAC. The objective in this application is to determine how BET proteins mediate chemoresistance and contribute to fibrosis in vivo. The central hypothesis is that BET protein inhibition will decrease PDAC tumor growth and increase chemosensitivity by decreasing the cancer stem cell population and HMGA2 protein function, respectively. A second hypothesis is that BET inhibition will lead to an attenuation of fibrosis in PDAC tumors. These hypotheses are based on strong preliminary data demonstrating that BET inhibitors decrease growth of PDAC and stellate cells in 3D collagen. In addition, treatment of PDAC cells with BET inhibitors decreases cancer stem cell population and represses HMGA2. The rationale is that a determination of the role and underlying mechanism of BET proteins in PDAC progression in vivo is likely to contribute substantively to a conceptual framework whereby new clinically effective targeted therapies can ultimately be developed. Three specific aims are proposed: 1) Determine the role of BET proteins in PDAC progression in vivo; 2) Evaluate the ability of BET protein inhibition to increase chemotherapy sensitivity; and 3) Evaluate the ability of BET protein inhibition to attenuate fibrosis. Under the first aim, the effect of BET inhibitors on PDAC progression will be determined in mouse models. Further, the extent to which BET inhibitors decrease PDAC stem cell population in vivo will be evaluated. Also, the ability of gold nanoparticles (Au-NPs) coupled with BRD4 siRNA to inhibit tumor growth will be determined. For the second aim, the ability of BET inhibitors to increase chemotherapy efficacy will be evaluated in 3D collagen and in mouse models. Additionally, the role of BET proteins in DNA damage response and the contribution of HMGA2 to BET protein regulation of chemoresistance will be assessed. In the third aim, the mechanism by which BET inhibitors regulate stellate cell activation and collagen production will be determined. The effectiveness of BET inhibitors and Au-NPs functionalized with BRD4 siRNA to attenuate fibrosis in mouse models will also be evaluated. The research proposed is innovative because it utilizes complex models of pancreatic cancer, including in vitro organotypic cultures and in vivo orthotopic and transgenic mouse models to determine the role of BET proteins in PDAC progression. An additional innovation is the use of Au-NPs functionalized with siRNAs to downregulate BRD4 expression in the model systems. This proposed research is significant because it is expected to provide strong scientific justification for the continued development and future clinical trials of BET inhibitors in PDAC.

Recent evidence indicates that invasion of pancreatic ductal adenocarcinoma (PDAC) cells in 3D collagen depends on G?13, a member of the G12 family of heterotrimeric G proteins, and can be reversed by the collagen-binding protein discoidin domain receptor 1 (DDR1). The long-term goal is to contribute toward the development of novel mechanism-based targeted therapies for the treatment of PDAC. The main objective in this application is to determine how G?13 contributes to PDAC progression in vivo. The central hypothesis is that G?13 enhances PDAC progression by disrupting DDR1-mediated cell-cell adhesion and by activating YAP1 signaling. A second hypothesis is that G?13 enhances inflammation that is present in PDAC tumors. These hypotheses are based on extensive preliminary data demonstrating that G?13 knockdown decreases invasion in 3D collagen, decreases YAP1 signaling, and enhances E-cadherin-mediated cell-cell adhesion. In addition, loss of the polarity protein Par3, which can function downstream of DDR1, enhances YAP1 signaling and promotes invasion of PDAC cells in 3D collagen. Moreover, G?13 regulates HMGA2, which can mediate chemoresistance, and also regulates in PDAC cells stem cell factor (SCF), which can mediate mast cell migration. The rationale for the proposed research is that a determination of the effect and underlying mechanism of G?13 in PDAC progression in vivo is likely to provide strong justification for the continued development of G?13 and its downstream effectors as targets for novel anti-PDAC therapy. Three specific aims are proposed: 1) Determine the role of G?13 in PDAC progression in vivo; 2) Determine the role of G?13 in mediating PDAC inflammation in vivo; and 3) Determine the mechanism by which DDR1 counteracts G?13 in PDAC cells in vivo. Under the first aim, the effects of knocking out G?13 on limiting tumor progression and increasing response to chemotherapy will be evaluated in mouse models and in human PDAC organoids. The role of YAP1 in G?13-mediated PDAC progression and the mechanism by which G?13 mediates chemo- resistance will be evaluated. For the second aim, the mechanism by which G?13 in PDAC cells enhances SCF expression and mast cell migration will be characterized. In addition, the effects of modulating G?13 in vivo on other inflammatory cells will also be determined. In the third aim, the role of Par3 in mediating tumor progression in mouse models and in human PDAC organoids will be evaluated. The extent to which Par3 functions downstream of DDR1 to attenuate the effects of G?13 on PDAC progression will also be determined. The research proposed is innovative because it utilizes complex models, including 3D acinar cultures, human PDAC organoids, and transgenic and orthotopic mouse models, to delineate the role of G?13 and Par3 in PDAC progression. This proposed research is significant because it will provide a mechanistic determination of the role of G?13 in mediating tumor progression and chemoresistance, and also PDAC inflammation, subsequently creating new opportunities for the development of innovative therapies to treat PDAC patients.

ABSTRACT ? TUMOR ENVIRONMENT AND METASTASIS The Tumor Environment and Metastasis (TEAM) Program of the Robert H. Lurie Comprehensive Cancer Center is a multidisciplinary basic science program that evolved from the Tumor Invasion, Metastasis and Angiogenesis (TIMA) Program to reflect recent conceptual advances in our understanding of tumor cell plasticity, heterogeneity and immunology. TEAM retains its strengths in areas of cell adhesion, migration and the role of extracellular matrix in cancer, while adding a new aim in the area of tumor immunology and inflammation. The objective of the TEAM Program is to elucidate how interactions between tumor cells, immune cells and components of the host stromal microenvironment mediate tumor development and progression. Toward achieving this objective, TEAM Program members are addressing the following three specific aims: (1) determine how cells interact with each other and their matrix, and define how adhesion-mediated signaling events affect tumor cell plasticity, invasion and metastasis; (2) elucidate the role of the extracellular matrix and the lympho-vascular system in tumor progression and therapeutic resistance; and (3) understand the role of innate and adaptive immune systems in cancer initiation and progression, and develop strategies to effectively activate the immune system against cancer. The TEAM Program has 35 core members from 14 different departments and 3 schools at Northwestern University. Hidayatullah Munshi, MD, with expertise in cell-matrix interactions and matrix-driven drug resistance, was appointed as the Leader of the TEAM Program to replace Dr. Kathleen Green who became Associate Director for Basic Sciences in conjunction with reorganization of the Basic Science division. Carole LaBonne, PhD, who is interested in understanding how neural crest-derived factors promote tumor progression and metastasis, remains the co-Leader of the TEAM Program. Program members are highly interactive intra- and inter- programmatically, collaborating on joint basic and translational research initiatives. During the last budget year, the TEAM Program received funding of $11,541,419 (direct) in cancer-relevant peer-reviewed grant support, with $2,275,130 (direct) from the NCI and $9,266,289 (direct) from other peer-reviewed sources. Over the course of the current funding period, program members have published 411 cancer-relevant scientific articles. Of these, 24% were in high impact journals, 16% were intra-programmatic, 34% were inter-programmatic, and 73% were inter-institutional collaborations. To successfully achieve the aims of the TEAM Program, future plans of the TEAM Program include strategic recruitment of faculty, particularly in the areas of tumor plasticity and immunology, and working with clinical partners to accelerate clinical translation of basic science discoveries by TEAM members.

There is an urgent need to evaluate immunotherapy combination regimens in pre-clinical models that faithfully represent the complex biology of human pancreatic ductal adenocarcinoma (PDAC) tumors. Since ex vivo human PDAC tumor slice cultures maintain the tumor morphology, stromal architecture, and immune cell composition of in vivo PDAC tumors, slice cultures have potential utility in the rapid screening of immunotherapy combination regimens for PDAC. The main objective in this application is to use human PDAC tumor slice cultures to identify combination regimens that enhance the response to immune checkpoint inhibitors in PDAC. The central hypothesis is that human PDAC tumor slice cultures will allow for rapid evaluation and mechanistic characterization of novel immunotherapy combination regimens against PDAC. Two specific aims are proposed: 1) Screen combination regimens in human pancreatic tumor slice cultures; and 2) Characterize the mechanisms by which the effective combination therapies promote immune responses in pancreatic tumors. Under the first aim, novel combination regimens with epigenetic and immune checkpoint inhibitors will be tested in human PDAC tumor slice cultures using small molecule inhibitors and antibodies that are currently in early phase human studies. The ability of these regimens to enhance cytolytic CD8+ T cell infiltration and function will be evaluated to identify the regimens most effective at cancer cell killing. In the second aim, the mechanisms by which the effective combination regimens successfully overcome immune, biochemical, and physical barriers to CD8+ T cell infiltration and function will be characterized. The extent to which the effective combination regimens decrease immunosuppressive cells, cytokines, and chemokines will be evaluated. The extent to which the effective combination regimens enhance the spatial interaction between CD8+ T cells and cancer cells to promote successful cancer cell killing will also be evaluated. There are several innovative elements in this proposal, including the combination of checkpoint inhibitors with small molecule inhibitors that have not been previously evaluated together, and the use of ex vivo slice cultures of human PDAC tumors to rapidly evaluate efficacy of immunotherapy regimens. This proposed research is significant because it will have important clinical- translational implications and should result in the development of mechanism-based novel combination therapies for PDAC.