Epigenetic regulation of the tumor microenvironment in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) contains a highly desmoplastic stroma which not only promotes aggressive local growth of the tumor but plays a key role in the failure of traditional chemotherapeutic approaches. Despite being a major component of the tumor stroma, the extracellular matrix (ECM) of PDAC has been poorly characterized. The ECM is a meshwork of cross-linked proteins that function, at least in part, as a reservoir for growth factors, cytokines and ECM-remodeling enzymes. Collectively, the proteins that make up the ECM have been termed the matrisome. The production and remodeling of the ECM, largely mediated by cancer-associated fibroblasts (CAFs), promote tumor growth and migration of cancers cells. Work by the host laboratory characterizing epigenetic regulators in pancreatic cancer has revealed an important role for the BET family of chromatin adaptors in the growth of PDAC cells and CAFs in both xenograft and genetically engineered mouse models of PDAC. Gene expression profiling of PDAC xenograft tumors revealed that BET proteins regulate the expression of a large portion of the genes that comprise the matrisome, which will be the focus of this grant proposal.Experiments in the first aim will employ an unbiased proteomic approach to define the PDAC matrisome, as well as transcriptomic analysis to determine the extent by which BET proteins regulate the expression of matrisome components. Experiments proposed in the second aim will interrogate whether pharmacological inhibition of BET proteins results in a reorganization of the ECM that can be exploited to prevent metastatic dissemination of the primary tumor and investigate if structural differences in patient samples correlate with patient outcome. The experiments proposed in this application will examine the regulation of the PDAC matrisome by BET proteins and determine whether this regulation can be therapeutically targeted to alter the biology of the tumor. This will ultimately contribute to the understanding of pancreatic cancer as a whole.

In this research project, we characterized the non-cellular content of the PDAC stroma for the first time. For this we utilized three different patient derived xenografts that were orthotopically implanted into pancreata of immunodeficient mice. This model allowed for separation of the mouse derived stroma from the human derived cancer. Importantly, we found that matrisome gene signatures found in these PDX models were very similar to signatures found in patient tumors, that we had laser-micro dissected. Suprisingly, stromal cells were not the only major producing matrix cells, but also cancer cells contributed substantially (40%) to the make-up of the PDAC matrisome. In fact, most of the genes were expressed by both compartments (stroma and cancer), and not as previously described majorly by the stromal cells. Furthermore, we deciphered the epigenetic role of BET bromodomain proteins for the expression of the PDAC matrisome. BET proteins were able to abrogate a stroma-cancer cross-talk induced effect on matrisome expression in a co-culture model. We also found that this effect is likely driven by contactdependent communication. Finally, the alteration of tumor-stroma crosstalk led to significant growth reduction in an orthotopic tumor model, in which the tumor cells themselves were insensitive to BETi in culture, suggesting the importance of modulating the cancer-stroma crosstalk in PDAC tumors. We were also able to calculate a matrisome signature classifier to predict long-versus short term survival in an independent PDAC cohort with 532 worldwide resected PDAC patients, again strengthening the importance of the PDAC matrisome to the biology of tumors. In summary, we have newly characterized a new compartment of PDAC tumors, and defined one pharmacological target to alter a large percentage of extracellular matrix genes in order to slow tumor growth. These data will potentially lead to a new understanding of these deadly tumors and offers multiple new targets for anticancer treatment. This project has led to multiple talks and awards for the DFG scholar. It also offers a great basis to continue research as a surgeon-scientist.