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Transcription Profiling of Protein Kinase C Activation in a Panel of Breast Cancer Cells Identifies a Strong Genetic Signature Consistent with Invasion and Metastasis
Background. Metastasis is the most important determinant of survival for breast cancer patients yet one of few clinical parameters that dictate treatment options. It is firmly established that Protein Kinase C (PKC) activity correlates strongly with breast tumor progression, inversely correlates with ER status, is predictive of tamoxifen failure, and thus a strong therapeutic candidate. Using DNA microarrays, we investigated the transcriptional response of PKC activation in 9 breast cancer cell lines with variable invasiveness. A strong expression signature consistent with increased invasive potential was identified, and several candidates warrant prognostic evaluation in patient samples. Interestingly, the receptor for activated C Kinase (RACK) which exhibits tumor suppressive properties was strongly downregulated in invasive lines. Lastly, a proportion of our candidate markers are also predictors of metastasis in published tumor expression profiles. Objective/Hypothesis. To validate whether invasive signature genes predict development of metastases in tumors, and elucidate the role of individual PKC isozymes and associated signaling pathway interactions in the development of aggressive phenotypes. We hypothesize that increasing our mechanistic understanding of the transcriptional consequences of deregulated PKC isoforms and manifestation of these signatures in tumors will significantly enhance our prognostic accuracy. Specific Aims. 1. Confirm that expression signatures for invasive potential functionally correlate with invasive potential in all cell lines. 2. Validate highly-weighted biomarkers consistent with increased in vitro invasive potential in a panel of primary tumors with known metastatic outcome. 3. Mechanistically investigate the role of individual PKC isoforms and RACKs in phorbol-ester induced invasiveness in MCF7 cells. Study Design. Chemotaxis and invasion assays of all cell lines will be conducted to confirm literature reports of increased invasive potential mediated by PKC. A tumor tissue microarray will be constructed by our core facility from primary breast tumors with known metastatic outcome for imunohistochemical analysis of candidate biomarkers. In vitro analyses using PKC-specific inhibitors and siRNAs will elucidate the mechanistic role of individual PKC isoforms on the development of aggressive phenotypes. Potential Outcomes. Our rationalized molecular analysis of PKC-mediated metastasis mechanisms will facilitate identification of new clinical diagnostic, prognostic biomarkers and reduce unnecessary and costly patient therapy.
Decisions regarding therapeutic management of newly diagnosed breast cancers remains a clinical judgment based on classical pathology, and surgical staging. Clinical parameters considered include tumor size, lymph node involvement and whether tumor cells have spread to other organs of the body, skin, or lymph nodes above the collarbone. When breast cancer is first diagnosed and found to be confined to the breast itself, with no spread to lymph nodes, the likelihood of long term survival is good. However once tumors have spread from the primary site there is a significant increase in likelihood that secondary tumors will arise at distant sites throughout the body (known as metastasis) and a dramatic drop in patient cure rate. Our current understanding of why breast tumors spread and become progressively more aggressive over time is relatively poor. In addition, there are very few clinical diagnostic tests available to examine a patient's tumor at the molecular level to make a rationalized determination of, for example, what type of therapy may be most efficacious for their individual disease, or indeed predict whether a patient’s disease is destined to progress. This unsatisfactory situation has lead researchers to study molecules within breast tumor cells that display differences in identifiable features such as tumor invasion into surrounding tissues. Recent advances in sophisticated robotics and completion of the human genome project has given rise to a fantastic and revolutionary new technology wherein we can analyse in parallel, the total molecular complexity of a tumor and begin to unravel why cell growth is likely to be deregulated in one tumor versus another. This study involves the use of this technology, and by using laboratory models of breast cancer, a number of novel candidate molecules have been identified that appear to only associate with those tumors that display aggressive behaviour. The goal of this research is to evaluate the potential of a selection of these candidate molecules for more accurate diagnosis of a patient's disease stage, and prediction of disease progression. Our study design involves an investigation of the presence of these molecules or 'markers' in tumors that eventually 'spread' verses those that did not, to validate them as a potentially useful clinical predictive test. Lastly, investigation of why the identified molecules are deregulated in the first place will be examined using laboratory models of disease, in the hope we may eventually prevent tumor invasion and metastasis with novel targeted therapeutic agents.