Abstract P1-05-15: Identification of a novel Hypoxia Inducible Factor-1 regulated gene involved in breast cancer growth and metastasis

Introduction: Hypoxia is a hallmark of most solid tumors. In response to 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. HIF-1alpha protein is over-expressed in ∼30% of primary breast tumors and ∼70% of metastases, which independently correlates with poor prognosis and decreased survival in patients. Moreover, conditional deletion of Hif1a in the mammary epithelium of the MMTV-polyoma middle T (MMTV-PyMT) transgenic model of breast cancer significantly delays mammary tumor initiation and lung metastasis burden. Methods: Our lab has established primary mammary tumor epithelial cells (MTECs) from late stage carcinomas originating in PyMT+; Hif1α floxed mice (FVB/Nj strain, F11). These MTECs were exposed to either Adenovirus-eta-βgal or -Cre to create wild-type (WT) and knock-out (KO) cells, respectively. PyMT MTECs are transplantable into syngeneic FVB/Nj female recipients. Deletion of HIF-1 activity reduced primary tumor growth by ∼60% and the formation of lung macrometastases originating from mammary fat pad tumors by >90%. Microarray profiling was conducted to identify genes differentially expressed between WT and KO cells cultured at normoxia (21% O2) or hypoxia (0.5% O2) as well as between end-stage WT and KO tumors derived from the parental cell lines. Several genes were down-regulated in both gene sets in response to deletion of Hif1a. One mRNA of particular interest, creatine kinase brain isoform (Ckb) was down-regulated in KO cells >100 fold and >2 fold in end-stage tumors. Pools of stable Ckb knockdown (KD) PyMT cells were generated via lentiviral transduction of two independent shRNA constructs (pLKO.1 shc59 or shc61, each producing >75% knockdown) to determine the effects of Ckb knockdown on cell proliferation, migration and metastasis. Results: No differences in cell proliferation were observed between WT and Ckb KD cells when cultured in standard culture conditions. In contrast, when cells were introduced to the mammary fat pad, initiation of palpable tumors was delayed by >30 days for each shRNA KD line, suggesting that CKB function may be modulated by the tumor microenvironment. A significant decrease in invasion through Matrigel was also observed for each shRNA Ckb cell line. Likewise, when cells were introduced into circulation via tail vein injection, 20% of mice injected with either shc59 or shc61 cells developed lung metastases whereas 100% of mice injected with WT cells developed metastases. Bioinformatic analyses indicate that multiple putative HIF-1 binding sites (HREs) are present in both the mouse and human CKB gene promoters; chromatin immunoprecipiation (ChIP) experiments are in progress to validate that CKB is a direct HIF-1 target gene. Conclusions: HIF-1alpha strongly promotes metastasis, the major cause of mortality in breast cancer patients. Further characterization of genes downstream of HIF-1alpha, such as CKB, that play a key role in driving invasion and tumor initiation may identify new pathways amenable to therapeutic intervention for patients with metastatic breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-15.