Quite technical but idea is that by targeting genes which are located next door to her2 in cases of herceptin resistance once can still decrease cell proliferation and induce some apoptosis(cell death). Note that this worked in both ER+ and ER- her2+ cell lines and that one of the two genes tested had a role in cholesterol metabolism/trafficking (I am sure RB would spot this)
GRB7 is one of the genes utilized in the mathematical model which established the ONCODX test.
Research Article
RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes
Jessica Kao, Jonathan R. Pollack *
Department of Pathology, Stanford University, Stanford, California
email: Jonathan R. Pollack (pollack1@stanford.edu)
*Correspondence to Jonathan R. Pollack, Department of Pathology, Stanford University School of Medicine, 269 Campus Drive, CCSR-3245A, Stanford, CA 94305-5176, USA
Abstract
DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes driving amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20-30% of tumors exhibit amplification within chromosome band 17q12, containing the ERBB2 oncogene. Analysis of array-based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon core) at 17q12 includes two other genes, GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell-cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell-cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified with ERBB2 at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes.
Using RNAi methods to knock down the expression of amplified genes, we have shown here that in some cell contexts genes coamplified together with ERBB2 at 17q12 contribute to cancer-relevant phenotypes. Specifically, we have found that knockdown of GRB7 and STARD3 expression leads to reduced cell proliferation, which appears to result at least in part from decreased cell-cycle progression. Therefore, we infer that overexpression of these genes due to amplification contributes to the elevated proliferation rates that characterize SKBR3 and BT474 cells. Indeed, preliminary experiments using other breast cancer cell lines with 17q12 amplification (e.g., ZR75-30) demonstrate the same to hold true (data not shown). Although previously speculated (Stein et al.,[1994]; Akiyama et al.,[1997]; Moog-Lutz et al.,[1997]
, this is to our knowledge the first experimental support for a functional contribution of coamplified genes within the 17q12 amplicon.
GRB7 is an SH2-domain containing adapter protein that has been shown to interact with EGFR and ERBB2 (Han et al.,[2001]). Amplification and overexpression of GRB7 may enhance signaling through EGFR-family receptor pathways in some contexts. STARD3 functions in cholesterol trafficking (Strauss et al.,[2003]), and has been shown to stimulate steroidogenesis (Watari et al.,[1997]) where breast tumor growth is often responsive to steroids (Flototto et al.,[2001]). While plausible then, the mechanisms through which GRB7 and STARD3 overexpression promote cell proliferation remain to be elucidated. The differential effect of GRB7 and STARD3 knockdown observed in BT474 compared with that in SKBR3 cells (where the effect is more reproducibly observed in suboptimal growth conditions using low serum) also remains to be investigated, but might be attributable to differences in the residual levels of expressed protein, or to differences in the genetic/epigenetic alterations of the cell lines.
Our findings indicate that combined targeting of ERBB2/GRB7 or ERBB2/STARD3 provides little additive effect on reducing proliferation levels beyond targeting ERBB2 alone, at least in the context of BT474 cells. This finding underscores the prominent role of the ERBB2 oncogene within this amplicon, and from a practical standpoint suggests that therapies directed against GRB7 and STARD3 would likely provide little if any additional benefit over targeted ERBB2 therapies alone for patients with tumors carrying 17q12 amplification. Nonetheless, there may be utility for such therapies in ERBB2 therapy (e.g., trastuzamab)-resistant cases.
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