Conserved coamplification event in HER2+ breast cancer increases metastasis

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PROJECT SUMMARY Breast cancer is the most common cancer diagnosed among women and is estimated to be the leading cause of death for women worldwide. Approximately one fifth of breast cancer patients’ tumors overexpress the human epidermal growth factor receptor 2 (HER2) protein; this is the result of an amplification event on the long arm of human chromosome 17, leading to high copy number gains of the HER2/Neu gene (ERBB2). Metastatic HER2+ breast cancer is more aggressive than other subtypes of breast cancer, responds poorly to general chemotherapy, and is invariably fatal. Lack of understanding of the molecular mechanisms that underpin the metastatic cascade leads to limited therapy options and poor clinical outcomes. To study the mechanisms of metastasis in HER2+ breast cancer, we utilize a transgenic mouse model which overexpresses Neu, the rat isoform of HER2, under control of the mouse mammary tumor virus (MMTV) promoter, henceforth known as MMTV-Neu. Interestingly, our recent paper describing whole genome sequencing (WGS) of MMTV-Neu primary tumors revealed a coamplification event centered on mouse chromosome 11qD, near Erbb2, in a majority of samples. Comparing these results to human genomic data through The Cancer Genome Atlas (TCGA) reveals an analogous amplification event on the highly syntenic human chromosome 17q21.33 near, but separate from, the ERBB2 amplicon. This secondary amplicon was found to be present in 25% of HER2+ patients, 9% of all breast cancers, and contains a number of candidate genes that putatively mediate metastasis. Preliminary data addresses the potential for these candidate genes in mediating metastasis, but the molecular mechanisms for how this occurs remains unknown. The central hypothesis of this proposal is that upregulation of three genes in this amplicon, collagen type 1 alpha 1 chain (Col1A1), chondroadherin (CHAD), and prohibitin (PHB), increases metastasis through alterations to the tumor microenvironment and oncogenic signaling pathways. This hypothesis will be evaluated using three independent aims that will collectively identify key events and dependencies in the HER2+ metastatic cascade. Aim 1 identifies the stage of metastasis affected by the 17q21.33 amplicon by knockout/knockdown and overexpression of the three candidate genes in HER2+/Neu overexpressing cell lines injected into syngeneic mice. Aim 2 utilizes transcriptomic differences between tumors, immunohistochemistry (IHC), and co-immunoprecipitation (Co-IP) to determine signaling network differences between tumors +/- for candidate genes and spatial expression of genes within the tumor. Aim 3 determines what genes in the amplicon are necessary or sufficient to increase metastasis by overexpressing candidate genes alone or in tandem and measuring effects on migration and metastasis. The long-term objectives and broad goals of this hypothesis driven proposal are to provide an increased understanding of why some breast cancers become metastatic, ultimately to improve patient clinical outcomes.