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Regulation Of Her2/Neu And Her3 Membrane Expression In Breast Cancer Therapy
Investigator Initiated Research
Cancer stem cells appear to be the underlying source of many refractory malignancies including breast cancer. Effective anti-cancer therapies will originate from an understanding of how signaling pathways regulate cancer stem cell proliferation. The signaling pathway comprised of Smoothened (Smo), the Smo inhibitor Patched (Ptc), the Ptc binding protein/ligand Hedgehog (Hh), G protein-coupled receptors kinases (GRKs), and beta-arrestin is critical to normal embryonic patterning and organ development. In normal non-replicating adult tissues Hedgehog/Smo signaling remains relatively quiescent unless activated by exogenous stimuli, for instance in the inflammatory response during tissue repair. However, in cancerous tissue three compelling lines of evidence indicate a significant role for Hh/Smo signaling. First, in isolated human breast cancer stem cells Hedgehog signaling is inappropriately up regulated in a dramatic fashion. Second, patients with Gorlin syndrome harbor germline mutations in the Ptc gene, and they tend to develop multiple neoplasms (basal cell carcinoma and medulloblastoma) due to inappropriate Smo activation. Third, cyclopamine, a Smo antagonist, dramatically represses the in vivo growth of tumor Xenografts.
In recognizing a need to develop novel therapies targeting the underlying molecular basis of many refractory malignancies, we have instituted in our laboratory a systematic program for discovering novel compounds with therapeutic potential against stem cell driven cancers. In particular, we have already identified some promising compounds that block Hh signaling based on findings from our basic research program [Chen, W. et al. Science. 306:2257-60]. We have identified by primary and secondary screening 32 potent small molecule inhibitors of Hh signaling that inhibit the Smo receptor and we anticipate more hit compounds will be available as we continue expanding our screening capacity. However, the success of anti-HER2 antibodies for breast cancer treatment indicates alternative approaches to small molecules for targeting Hh signaling can be equally as effective and moreover complements our current strategy. We therefore also propose using techniques analogous to those applied to HER2 antibody development to generate anti-Smo antibodies for antagonizing Hh/Smo activity.
We hypothesize based upon the role of Hh signaling in embryonic development, tissue regeneration and repair, and cancer stem cell renewal that inhibition of Hedgehog/Smoothened signaling by using small molecules and/or antibodies will prevent breast cancer cell (stem) growth. Our objective is to develop and first apply both small molecules and antibodies to breast cancer cells and ultimately to clones of breast cancer stem cells in order to inhibit or prevent their growth sufficiently well to either eradicate tumors entirely or make them susceptible to the application of combination therapies. Accordingly we propose three specific aims:
1. To optimize and synthesize new anti-Hedgehog/Smoothened compounds available in our laboratory using structure-activity relationships (SAR) analysis as a guide. We will use a variety of techniques including competition binding and Gli-luciferase reporter assays to confirm their pharmacological properties with cyclopamine as a baseline standard.
2. To develop and assess anti-Smo antibody inhibitors of cancer cell (stem) growth in vitro and in vivo. This will allow us to develop novel combination therapies to block tumor growth.
3. To identify the small molecule and antibody binding sites on Smoothened targeted by the most efficacious of the anti-Smo inhibitors of cancer cell (stem) growth. Potent compounds and antibodies showing efficacy against Smo promoted cell growth will be used to map functional motifs of Smoothened by employing site-directed mutagenesis and peptide spot array assays that produce peptide fingerprint images.
These studies will provide the biological rationale and preclinical information to help guide the clinical development of two different classes of Hh/Smo inhibitors. We anticipate advancing these inhibitors to the clinic within a 3-5 year timeline.
Hedgehog (Hh) signaling is a process that normal cells use to maintain growth. However, when Hh signaling becomes abnormally regulated it can sustain the growth of cancer cells in the breast and in metastases. Therefore blocking Hh signaling in breast cancer cells can provide a road to improve its medical therapy in its early, late, or recurrent stage. In our work studying the basic biology of Hedgehog signaling, we have discovered new Hedgehog signaling inhibitors. They provide a new avenue for developing cancer therapies for cells that need this pathway to sustain their viability. We have instituted in our laboratory a systematic program for discovering these novel compounds as well as developing antibodies with therapeutic potential against Hedgehog driven breast cancers by blocking Smoothened, a key receptor protein in mediating Hedgehog signaling. We characterize these newly discovered inhibitors in cellular assays and compare their effectiveness against the plant compound cyclopamine which is a natural Hh signaling inhibitor. We additionally will utilize breast cancer cell lines and appropriate cancer models to assess the therapeutic efficacy and toxicity of the candidate compounds. These studies will provide needed new drugs for treating breast cancer. We believe that our compounds and antibodies will form the foundations for new medical therapies that will change breast cancer from a life threatening disease into a curable illness.