GRB7 is amplified and over-expressed in breast cancer with chromosome 17q11-12 amplification. GRB7 can interact with multiple signaling molecules and is implicated in breast cancer biology. GRB7 can facilitate HER-2 mediated signaling and tumor formation. Studies, including that of ours, have shown GRB7 protein overexpression, rather than that of HER-2, is an adverse prognostic factor in breast cancer. Moreover, GRB7 protein is the only adverse prognostic marker, from among 374 studied, in triple negative breast cancer. We hypothesize that GRB7 directed signaling is functionally important in breast cancer with chromosome 17q11-12 amplification. Targeting GRB7 signaling therefore represents an important and novel therapeutic strategy for HER-2 positive and triple negative breast cancers that are otherwise clinically aggressive. The objectives are: (1) to investigate the dependency of breast cancer cells on GRB7 signaling by determining if inhibition of GRB7 signaling functions leads to suppression of breast cancer cell growth; (2) to develop isogenic pairs of breast cancer cell lines with shRNA mediated GRB7 knockdown and perform Induced Essentiality screening with small molecule inhibitor libraries; (3) to perform screening with a siRNA library that covers the whole actionable kinome with cell spot microarrays and a novel reverse transfection technology to identify and validate targets whose knock down displays GRB7-dependent effects on proliferation, apoptosis, and/or migration/invasion; and (4) to investigate the functional involvement of candidate cell cycle kinase(s) in GRB7 signaling and their roles as therapeutic targets. Our approaches are the following: GRB7 knock down has been achieved by transient siRNA transfection in multiple breast cancer cell lines with chromosome 17q11-12 amplification. GRB7 protein depletion decreases cell proliferation in cell lines irrespective of their sensitivityto currently available HER-2 targeted therapies. Stable GRB7 knock down has been achieved by shRNA expression in breast cancer cell lines and its effect on cell growth in culture and in orthotopic mouse xenograft models will be assessed. Stable GRB7 knock down creates a 'sensitized' cellular context to screen for therapeutic targets and molecules whose signaling functions depend on GRB7 expression. We will screen for signaling molecules whose inhibition is sensitized by GRB7 over-expression- i.e. induced essentiality. In addition, signaling molecules whose inhibition displays differential sensitivity according to their GRB7 status will unravel the functional complexities of GRB7 signaling in breast cancer. The novel cell spot microarray coupled with siRNA reverse transfection will be used to screen actionable targets of the whole human kinome. We are able to expand this cell based functional genetic screen to include more siRNA constructs, allow combinatorial siRNA analyses, and obtain multi-parametric phenotypic readouts and comparative analysis of multiple cell types simultaneously. We will also perform screening of small molecule inhibitor libraries that comprise of compounds that are well characterized and undergoing early clinical development. With screening of small molecule inhibitor and siRNA libraries made up by actionable targets, we are looking for candidates whose inhibition produces distinct phenotypes that depend on the GRB7 expression status. Screen positives will be validated with independent functional assays and in additional cell lines and animal models. Since screen positives are molecules with actionable features or compounds already undergoing early phase clinical development, they can be readily moved forwards in drug development expeditiously. Thorough investigation of these GRB7 dependent signaling molecules and therapeutic targets will produce new therapies and reveal why GRB7 expression predicts adverse prognosis. High throughout screening of actionable siRNA and small molecule inhibitor libraries represents a novel paradigm to elucidate the function of an adaptor molecule and, more importantly, to effectively produce clinically useful, non-toxic therapeutics specifically directed against an oncogenic signaling molecule that is otherwise difficult to target.
Breast cancer is a leading cause of cancer for American women and a major issue of women's health, which is a priority research area of specific interest to BLR&D. Currently 15% of American military are women and the number is rising. 20-25% of breast cancers carry amplification of a segment of chromosome 17 that contains the GRB7 gene. GRB7 amplification and over-expression contribute to the aggressive behaviors of breast cancer. Study of GRB7 signaling will reveal why breast cancer with GRB7 over-expression is a more aggressive disease and lead to development of novel targeted therapy. GRB7 amplification and over- expression has been described in 20-25% of gastro-esophageal (GE) junction cancer. Though of lower incidence, GE junction cancer is a much more lethal disease than colon or prostate cancer and its incidence is rapidly climbing. Our work in understanding GRB7 signaling and development of novel therapy targeting GRB7 therefore will benefit both aggressive forms of cancer. Our work is important for the VA mission.
