The pro-cell death Bcl-2 family member, BNIP3 is mutated in breast tumors and this mutation increases resistance to hypoxia-induced cell death in breast cancer cell lines

4078 BNIP3 (Bcl-2/adenovirus E1B 19 kDa Interacting Protein) is a pro-cell death member of the Bcl-2 family of proteins. It has been shown that BNIP3 expression is increased in hypoxic regions of breast tumors. This increased expression is paradoxical since BNIP3 induces cell death, yet these tumor cells remain viable. One possible explanation is the presence of mutations in the BNIP3 gene leading to the inactivation of the BNIP3 protein. We have previously found that BNIP3 is over-expressed in brain glioblastoma multiforme (GBM) tumors and is mutated in 17% of these tumors inactivating its cell death function. Therefore, we are investigating whether mutations in the BNIP3 gene are present in breast tumors and whether these mutations block hypoxia-induced cell death. To determine whether BNIP3 is mutated in breast tumors, RNA is isolated from frozen high grade breast tumor sections from the Manitoba Breast Tumor Bank. Reverse-transcriptase PCR is performed and the resultant cDNA sequenced. Of the 17 tumors sequenced to date, four mutations have been found, including two nonsense mutations (single nucleotide insertions) and two missense mutations. These mutations were confirmed using a single nucleotide primer extension assay. Immuno-fluorescent staining for BNIP3 expression in breast tumors revealed BNIP3 expression in tumors where the BNIP3 gene is mutated. We also determined that the ovarian cancer cell line SkOv3 has a mutation in BNIP3 resulting in a truncated protein. To determine if the mutations detected in breast tumors affect BNIP3 protein function, the breast cancer cell line MCF-7 and the human embryonic cell line HEK293 were transfected with expression constructs containing wild type or mutant BNIP3 cDNA. The expression of mutant BNIP3 renders these cells more resistant to hypoxia-induced cell death compared to cells expressing empty vector or wild type BNIP3. Since BNIP3 induces cell death by localizing with the mitochondria, we evaluated the ability of mutant BNIP3 to localize to the mitochondria. We demonstrated that mutant BNIP3 localizes to the mitochondria. Ongoing studies will demonstrate whether breast cancer cells expressing mutant BNIP3 injected into nude mice form tumors faster than cells expressing empty vector. Overall, our results indicate that BNIP3 is mutated in breast tumors and these mutations render breast cancer cells resistant to hypoxia-induced cell death.

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