Epigenetic alterations in tumor microenvironment

Abstract

The tumor microenvironment plays an important role in the initiation and progression of tumors. We hypothesized that since stromal cells do not generally carry oncogenic mutations, they are reprogrammed via epigenetic alterations. To evaluate this hypothesis we utilized 3 models of premalignant and malignant conditions. At first, we analyzed genomewide alterations in DNA methylation in carcinogenesis associated fibroblasts (CAFs) from pancreatic adenocarcinomas. We also generated CAFs from mesenchymal stem cells (MSCs) after exposure to pancreatic cancer cell conditioned medium and analyzed their methylome at higher resolution using the HELP-Tagging assay. Both primary CAFs and in vitro reprogrammed MSCs were characterized with global demethylation and integrative analysis revealed a common epigenetic signature of 427 differentially methylated regions. Overexpression and demethylation of CXCR4 was seen in CAFs and inhibition of this receptor led to decreased tumor cell invasion implicating it as a potential therapeutic target for interrupting stromal-tumor cell interactions in pancreatic cancer. Next, we evaluated primary marrow stromal cells from patients with Myelodysplastic Syndrome (MDS) and compared them with healthy controls. We observed that MDS marrow stroma was epigenetically distinct with significant hypermethylation of various genetic pathways involved in cell signaling and metabolism. Stromal cells from MDS patients treated with the DNMT inhibitor, 5-Azacytidine (5-Aza), did not contain widespread hypermethylation demonstrating that these drugs also affect the microenvironment. Finally, co-culture of healthy CD34+ hematopoietic cells with MDS stromal cells led to dysplastic colony growth that was abrogated after pretreatment of the stroma with 5-Aza. In our third project we determined that microRNA-21 is overexpressed in MDS hematopoietic cells and binds to 3'UTR of SMAD7 gene thus leading to its downregulation. TGF-beta is an important mediator of stromal-tumor interactions and decreased expression of negative regulator, SMAD7, leads to overactivation of the TGF-beta receptor kinase, thus leading to dysplastic maturation of hematopoietic stem cells. Inhibition of miR-21 led to significant increases in hematocrit in a mouse model of bone marrow failure, thus implicating this approach as a potential therapeutic strategy in MDS. Overall, these studies demonstrate widespread and context-specific epigenetic changes in the tumor microenvironment.