Cellular fat metabolism and breast cancer
We have used shRNA-based functional genomics to study genes required for HER2/neu -positive breast cancer survival. Overexpression of the ERBB2 (HER2/neu) oncogene occurs in nearly 30% of breast cancers and is a prognostic indicator for aggressive disease and reduced survival. The humanized anti-ERBB2 monoclonal antibody trastuzumab (Herceptin) was developed as a targeted breast cancer therapy. Although it is one of our best weapons against advanced breast cancer at present, its success has been modest. Clinical trials of Herceptin as a single agent therapy have provided overall response rates ranging from 11 to 26% for patients with metastatic HER2/neu -positive breast cancer. Since a relatively large proportion of patients do not benefit from Herceptin, it has long been thought that other factors must influence therapy response in HER2/neu -positive tumors.
Using our shRNA library, we have identified genes related to fat synthesis and storage in these breast cancer cells as being required for their survival. Our results indicate that NR1D1 is among the most critical genes for HER2/neu -positive breast cancer cell survival. Importantly, NR1D1 is co-overexpressed with ERBB2 since it maps very closely to the ERBB2 locus and is usually found on the same 17q12-q21 amplicons which are responsible for ERBB2 overexpression. NR1D1 encodes an orphan nuclear receptor with roles in adipogenesis and circadian rhythm control. It has been proposed to represent a link between the circadian clock and cellular metabolism. We have shown that NR1D1 works in concert with the adipogenic transcription factor PPARgamma and the PPARgamma binding protein in regulating breast cancer cell metabolism. Our results demonstrate that the cells of this aggressive form of breast cancer are genetically preprogrammed to depend on fatty acid synthesis and storage for the energy production that is necessary for survival.
High-throughput gene analysis of breast cancer
We have also used an shRNA library to test each of the 90 tyrosine kinases in the human genome for effects on proliferation of breast cancer cells. Tyrosine kinases represent the largest group of structurally dominant oncogenes in the human genome. Nevertheless, we identified several that were required for the survival of breast cancer cells that were previously unknown to play roles in cancer. Among the most interesting was Bruton's tyrosine kinase (BTK) which has only been studied in the maturation of B-cells. Our work indicates that a novel isoform of this kinase is expressed at low levels in breast cancer cells. Down-regulation causes apoptosis.
Redox regulation of cell cycle progression
In one functional genomics study, we have used a retroviral human cDNA library to identify genes that are involved in the regulation of calcium signaling in the G1 phase of the mammalian cell cycle. Although this type of regulation has been appreciated for decades, the mechanisms at work are poorly understood. Although overexpression of the well studied ras and jun oncogenes was found to drive cells through an early calcium antagonist-induced cell cycle block, it was an amino acid transporter, the human cystine/ glutamate exchange facilitator, that had the largest impact. This transporter, xCT, carries out the rate limiting step of glutathione synthesis in many cell types and is frequently overexpressed in tumors and other rapidly proliferating cells. We found that overexpression of this gene produces an intracellular reducing environment that bypasses the requirement for calcium signaling in the early cell cycle. Cells overexpressing xCT have increased levels and activity of the AP-1 transcription factor in G1 that accounts for their ability to proliferate in the presence of calcium channel antagonists.