Abstract 509: Hypoxia and HIF silencing mediated dysregulation of total choline, CD44 expression, and metastatic burden in MDA-MB-231 human breast cancers

Abstract

Abstract Hypoxic tumors frequently exhibit an aggressive phenotype due to dysregulated gene expression and metabolic changes. Hypoxia results in the stabilization of hypoxia inducible factors (HIF-1/2) that transcriptionally activate genes involved in invasion, metastasis, metabolism, and in the adaptation of cancer cells to their microenvironment. In breast cancer, stem-like breast cancer cells that survive, repopulate and metastasize to distant locations, have elevated expression of CD44. In a previous study, we observed elevated expression of CD44 in hypoxic tumor regions, and identified HIF-1α as a regulator of CD44 expression in breast cancer cells under hypoxic conditions [1]. Hypoxia has also been implicated in increasing the activity of choline kinase (Chk)-alpha, the enzyme responsible for elevated phosphocholine (PC) and total choline (tCho) consistently observed in cancers [2]. In previous studies, lentiviral transduction of MDA-MB-231 breast cancer cells (231 cells) with shRNA against Chk-alpha and the in vivo delivery of the Chk-shRNA virus into tumor bearing mice resulted in decreased CD44 message and expression together with effective silencing of Chk message and expression [3]. Here, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI), we have established the importance of HIF in reducing total choline and metastatic tumor burden, and have identified a role for CD44 in establishing lung metastasis. HIF silencing in MDA-MB-231 cells significantly delayed tumor growth in mice. Both, the in vitro 1H and 31P MR spectra and in vivo 1H MRS images of tumors derived from engineered cells showed decreased tCho levels and distribution. This decrease of tCho was statistically significant in tumors derived from double silenced cells. Western blot analysis of tumors detected a decrease in Chk expression in double silenced (HIF-1 and 2) tumors. Silencing HIF-1α, -2α or both resulted in a significant reduction of metastatic lung burden in mice. Additionally, HIF-2α silencing was more effective at reducing lung colonization than HIF-1α, while silencing both was the most effective. Although metastatic burden decreased in HIF-1α silenced cells, the percentage of cells with high CD44 expression in the metastatic foci was comparable to that in the wild type or empty vector foci. These data identify the importance of targeting HIF and CD44 to prevent lung colonization and disrupt the metastatic cascade. This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for valuable technical assistance.