RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes

ArticleinGenes Chromosomes and Cancer 45(8):761-9 · August 2006with6 Reads
DOI: 10.1002/gcc.20339 · Source: PubMed
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.
    • "A minimal amplified region around ERRB2 oncogene has been defined in several studies [19, 20], and some of the genes have been found to play a role in tumorigenesis [14, 21, 22]. These genes include STARD3 and GRB7, whose products promote the proliferation of HER2-positive breast cancer cell lines [18]. Thus, GRB7 over-expression has been identified as an independent prognostic factor [23], and the co-amplification of ERBB2 with the non-core gene topoisomerase II (TOP2A) predicts sensitivity to anthracycline therapy [21, 24, 25]. "
    [Show abstract] [Hide abstract] ABSTRACT: Around, 30-40% of HER2-positive breast cancers do not show substantial clinical benefit from the targeted therapy and, thus, the mechanisms underlying resistance remain partially unknown. Interestingly, ERBB2 is frequently co-amplified and co-expressed with neighbour genes that may play a relevant role in this cancer subtype. Here, using an in silico analysis of data from 2,096 breast tumours, we reveal a significant correlation between Gasdermin B (GSDMB) gene (located 175 kilo bases distal from ERBB2) expression and the pathological and clinical parameters of poor prognosis in HER2-positive breast cancer. Next, the analysis of three independent cohorts (totalizing 286 tumours) showed that approximately 65% of the HER2-positive cases have GSDMB gene amplification and protein over-expression. Moreover, GSDMB expression was also linked to poor therapeutic responses in terms of lower relapse free survival and pathologic complete response as well as positive lymph node status and the development of distant metastasis under neoadjuvant and adjuvant treatment settings, respectively. Importantly, GSDMB expression promotes survival to trastuzumab in different HER2-positive breast carcinoma cells, and is associated with trastuzumab resistance phenotype in vivo in Patient Derived Xenografts. In summary, our data identifies the ERBB2 co-amplified and co-expressed gene GSDMB as a critical determinant of poor prognosis and therapeutic response in HER2-positive breast cancer.
    Full-text · Article · Jul 2016
    • "Amplification of the HER2 gene without overexpression of the HER2 protein is clinically important, because it prevents patients with breast cancer from benefitting from targeted HER2 therapy [10]. The GRB7 gene may amplify and cause GRB7 protein overex- pression [11]. The GRB7 gene plays an important role in the growth and migration of cancer. "
    [Show abstract] [Hide abstract] ABSTRACT: The HER2 gene is responsible for the formation of the HER2 receptor on the surface of epithelial cells. Increased numbers of this receptor are associated with a worse prognosis in cancer. Increased numbers of copies of the HER2 gene occur in about 20-30% of breast cancer patients, so determining HER2 receptor levels is important in the current diagnosis and treatment of breast cancer. One diagnostic technique is the immunohistochemical (IHC) method, which permits indirect measurement of overexpression of HER2 receptors, based on subjective determination of the intensity of the color reaction. Another technique is the use of fluorescent in situ hybridization (FISH), which permits the exact number of copies of the HER2 gene to be specified. Based on the results of FISH tests, patients can be qualified for treatment with antibodies that partially block HER2 receptors. This treatment causes inhibition of tumor growth signals. Determining the HER2 status in breast cancer with the FISH method allows the further progress of the disease to be predicted, the right treatment to be chosen and the response to the treatment to be foreseen. Because of the widespread use of the FISH and IHC methods, comparing the advantages and disadvantages of these two methods seems to be relevant.
    Full-text · Article · Jan 2015
    • "Grb7 is an adaptor protein that acts as a pivotal signal mediator, connecting RTK signalling to a variety of downstream signal transduction pathways involved in cancer progression. It is now recognised as an independent prognostic marker for tumour cell progression and a potential new target for the development of novel anticancer therapies192021. Several previous studies have explored the role of Grb7 in the progression of cancer cells and determined that Grb7 is important in both HER2?ve cells as well as TNBC. "
    [Show abstract] [Hide abstract] ABSTRACT: Grb7 is an adapter protein, aberrantly co-overexpressed with HER2 and identified as an independent prognostic marker in breast cancer. It has been established that Grb7 exacerbates the cellular growth and migratory behaviour of HER2+ve breast cancer cells. Less is known about Grb7's role in the context of HER2-ve cells. Here we directly compare the effect of stable Grb7 knockdown in oestrogen sensitive (T47D), HER2+ve (SKBR3) and triple-negative (MDA-MB-468 and MDA-MB-231) breast cancer cell lines on anchorage dependent and independent cell growth, wound healing and chemotaxis. All cell lines showed reduced ability to migrate upon Grb7 knockdown, despite their greatly varied endogenous levels of Grb7. Decreased cell proliferation was not observed in any of the cell lines upon Grb7 knockdown; however, decreased ability to form colonies was observed for all but the oestrogen sensitive cell line, depending upon the stringency of the growth conditions. The data reveal that Grb7 plays an important role in breast cancer progression, beyond the context of HER2+ve cell types.
    Full-text · Article · Jan 2014
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