Epigenetic silencing of SDF1: Roles in cancer metastasis

Chemokines are chemotactic cytokines that function through specific interactions with their receptors on target cells. Stromal cell derived factor-1 ( SDF1) also referred to systematically as CXC chemokine ligand 12 ( CXCL12) is a primordial chemokine, highly conserved throughout evolution, with homologues in organisms as primitive as Drosophila. The importance of this signaling axis is emphasized in the mammalian system by the embryonic lethality of mice containing a systemic deletion for either SDF1 or its cognate receptor, CXCR4. Prior to this work, CXCR4 expression was well established on numerous human epithelial cell types and their derived carcinomas. Further, endocrine SDF1 signaling through CXCR4 was known to influence the metastasis of epithelial derived carcinoma cells. However, data addressing the autocrine expression of SDF1 in cancer cells and the role it played in CXCR4 mediated metastasis were lacking.;Data from our lab had indicated that constitutive SDF1 expression by normal intestinal epithelium was absent in in vitro colorectal carcinoma cell lines. Here we define a mechanism of SDF1 silencing in cancer and show evidence to support the hypothesis that lack of SDF1 expression in carcinoma cells promotes their metastasis. We demonstrate that DNA hypermethylation of the human SDF1 promoter is strictly confined to cancerous tissues both of colonic and mammary epithelia. Further, pharmacological and genetic inhibition of DNA methyltransferase enzymes decreased promoter methylation and restored expression of SDF1 in carcinoma cell lines. Using in vivo bioluminescent imaging we show that re-establishment of SDF1 expression in colorectal and mammary carcinoma cells inhibited in vivo liver and lung metastasis, respectively. Moreover, SDF1 re-expression inhibited orthotopic primary tumor establishment of colorectal carcinoma cells upon intestinal engraftment. As a potential mechanism for these observed phenotypes we defined an SDF1-dependent increase in apoptotic cell death of colorectal carcinoma cells upon detachment from the extracellular matrix, a process termed anoikis. Anoikis of colorectal carcinoma cells re-expressing SDF1 is characteristic of normal intestinal epithelial cell death involving deceased maintenance of extracellular matrix, breakdown of focal adhesions, caspase activation and DNA degradation. Further, the MAP kinase ERK1/2 was defined as a key signaling molecule in both normal intestinal epithelial cell and SDF1-induced anoikis of colorectal carcinoma cells. These data suggest that through increased resistance to anoikis, SDF1 silencing in colorectal carcinoma participates in the exit from normal intestinal epithelial cell turnover, aiding both in tumor formation as well as metastasis.;Decreased metastasis was not restricted to colonic carcinoma as this phenotype was also observed in breast cancer cells upon SDF1 re-expression. Highlighting the heterogeneous nature of epithelial-derived tumors decreased metastasis of mammary carcinoma cells was associated with increased proliferation with no affect on apoptosis. Consistent with this increased proliferation, in vivo tumor growth of mammary carcinoma cells engrafted onto the mammary fat-pad was increased upon SDF1 re-expression. These seemingly contrasting results of one molecule both increasing tumor growth, but decreasing metastasis were explained by in vitro calcium flux and chemotaxis assays showing a decrease in responsiveness to ectopic SDF1 upon autocrine re-expression of that molecule. Together, our data in mammary carcinoma suggest that primary tumor cells in which SDF1 has been silenced are at selective advantage for metastasis due an increased ability to sense ectopic ligand.;The data in this dissertation significantly increase our understanding of the functional consequences of aberrant epigenetic modifications in the progression of carcinoma to malignancy. Previous to this work, increased CXCR4 expression on metastatic carcinoma cells was accepted as the driving force for chemokine-mediated metastasis of solid tumors. We have expanded this paradigm by documenting that the silencing of autocrine SDF1 establishes a migratory, CXCR4-restricted phenotype that fosters pathological utilization of this homeostatic signaling axis by cancer cells. Further, these data present evidence for both diagnostic and therapeutic disease interventions through the respective detection of SDF1 promoter hypermethylation, and the re-establishment of SDF1 in tumors via DNA methylation inhibitors or targeted gene replacement.