Abstract 5312: Identification of novel HIF-1-dependent target genes in a murine model of breast cancer

Abstract

Abstract Introduction: Hypoxia is a hallmark of most solid tumors. Under hypoxic stress tumor cells adapt by regulating survival, metabolism and angiogenesis. The heterodimeric Hypoxia-Inducible Factor (HIF)-1 transcription factor is a master regulator of this response, and is comprised of HIF-1α, the oxygen-regulated subunit, and aryl hydrocarbon nuclear receptor translocator (ARNT), which is constitutively expressed. HIF-1α protein is over-expressed in ∼30% of primary breast tumors and ∼70% of metastases. Over-expression of HIF-1α is independently correlated with poor prognosis and decreased survival in breast cancer patients. In agreement with these observations, conditional deletion of Hif1a represses tumor initiation and lung metastasis in the MMTV-polyoma virus middle T (MMTV-PyMT) model of breast cancer. Methods: Primary mammary tumor epithelial cells (MTECs) were established from late stage carcinomas originating in PyMT+; Hif1a floxed mice maintained on an inbred background (FVB/Nj). MTECs were exposed ex vivo to Adenovirus-β-gal or -Cre to create wild type (WT) and knockout (KO) cells, respectively; cells were then re-introduced into the mammary fat pad of FVB/Nj recipients. HIF-1α deletion reduced primary tumor growth by ∼60% and lung macrometastases by ∼90%. To identify HIF-dependent target genes, microarray profiling was performed. Several genes were differentially expressed between both WT and KO cells cultured at normoxia (21% O2) or hypoxia (0.5% O2) and end-stage WT and KO tumors. Of particular interest, creatine kinase brain isoform (Ckb) mRNA levels were reduced >100-fold in KO cells and >2-fold in KO end-stage tumors; down-regulation at the protein level was confirmed by western blotting. Two independent shRNA constructs (shc59 and shc61) were generated to stably knockdown Ckb in WT cells to assay for effects on cell proliferation and metastasis. Results: In 2-D culture, no significant difference in cell growth was observed in either knockdown line versus the WT cells. In contrast, when one million cells were introduced into the circulation by tail vein injection, 100% of mice injected with WT cells developed macrometastases, whereas no macromets were derived from shc61 cells and only 20% of mice injected with shc59 cells developed macromets. Four putative hypoxic response elements (HREs) have been identified in the Ckb promoter; therefore, ChIP experiments are in progress to determine if Ckb is a direct HIF-1 target. Additional experiments will test for a role of Ckb in regulating primary tumor growth, cell migration or invasion, and, if cyclocreatine, a CKB inhibitor, is effective in reducing primary tumor growth and/or metastasis. Conclusions: Further characterization of the function of Ckb, and other genes downstream of HIF-1α that drive metastasis, may identify pathways amenable to therapeutic intervention to treat patients with metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5312. doi:1538-7445.AM2012-5312