The purpose of this proposal is to study a novel uncharacterized GPCR. The hypothesis to be tested is that this orphan GPCR specifically demarcates activated epithelial stem cells and multipotent progenitors and critically regulates the unique proliferative characteristics of these cell populations. Preliminary analysis of the expression of a lacZ reporter knocked into the locus of the GPCR shows highly specific localization to well-characterized epithelial stem cell niches during embryonic and pubertal mammary and hair development.
Aim 1 is designed to establish the physiological role of the GPCR. Characterization of the reporter expression pattern throughout the mammary cycle will be completed. Cells that express the reporter will be isolated by FACS and their stem/progenitor cell properties will be tested functionally. The regenerative capacity of reporter-positive cells will be tested in transplantation studies carried out at limiting dilution and their potency tested by analysis of the outgrowth phenotypes. Their relationship to other putative mammary stem/progenitor cell populations will be determined by FACS and immunohistochemical marker analysis.
In aim 2 we will study the in vivo functional effects of loss of the GPCR signaling capability. The phenotypic effects of homozygous deletion of the GPCR cytoplasmic domain will be studied on mammary development and on murine mammary cancer models that arise from stem/progenitor cell expansion. The impact on stem/progenitor content, proliferative activity and tumor-promotion will be determined. These experiments are warranted by the interaction of GPCR with the tumor suppressor protein Discs large (Dlg), which regulates cell polarity, asymmetric division, proliferation and migration and by preliminary data indicating retardation in ductal outgrowth. ?
This novel orphan G-protein coupled receptor provides a highly specific marker of epithelial stem cells as they become activated to proliferate. As such it may have considerable clinical relevance as a prognostic marker of tumors from many different epithelial sources that arise from deregulated stem/progenitor cell proliferation. As a cell surface protein, reagents against this G-protein coupled receptor will provide powerful tools to identify, monitor and eliminate cancer-initiating cells that manifest stem cell-like behaviors such as the ability to undergo asymmetrical division. One quarter of all drugs in clinical trial target G-protein coupled receptors. Thus the development of antibodies and small molecule inhibitors against this protein could yield important translational initiatives to target breast cancer stem cells. ? ? ?
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