Elucidating the molecular mechanisms that regulate stem cell numbers in vivo

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DESCRIPTION (provided by applicant): The molecular mechanisms controlling stem cell self-renewal versus differentiation hold great potential for advances in cancer biology, aging and regenerative medicine. Increasing the pool of stem cells provides a condition for oncogenesis; tumors have "cancer stem cells" that self-renew and establish metastases. A critical regulator of stem cell numbers in mammals is the JAK/STAT pathway. Inappropriate activation of mammalian JAKs and STATs cause cancer and inhibiting their activity blocks the growth of primary human cancer cells. These data suggests that developing drugs which specifically inhibit JAKs or STATs, could offer novel treatments for cancer patients. Despite these compelling observations, the mechanisms utilized by this pathway to regulate stem cell numbers in mammals have not yet been elucidated. Mammals have multiple jak and stat genes, making the analysis of their in vivo function difficult. Drosophila offers an ideal system to address this issue, as its control of stem cell numbers is conserved in several tissues, including the testis and eye. Drosophila has only one jak and one stat (called stat92E) and offers facile in vivo analysis. This application concerns the dissection of molecular mechanisms used by the JAK/STAT pathway to regulate stem cell number in Drosophila. Three distinct processes must occur in a coordinated manner for stem cell populations to be maintained: increase in cellular mass, mitosis and self-renewal. Since Stat92E is a transcription factor, discrete Stat92E target genes should mediate its effects on these processes. Three such genes with human homologs, cyclin E (cycE), dmyc and chinmo, appear to reside directly downstream of Stat92E. CycE is the rate-limiting regulator of the cell cycle and its expression is increased by Stat92E in a cell- autonomous manner. dMyc is the sole c-Myc family member in Drosophila and is a critical regulator of cellular growth. Stat92E increases protein synthesis and may act through dMyc to do so. chinmo encodes a novel nuclear protein and is regulated by Stat92E in a cell-autonomous manner. Like Stat92E, Chinmo is required within testis stem cells for their self-renewal. The identification of Stat92E-regulated targets with links to mitosis, cellular growth and self-renewal is a major advance in our understanding of the growth-regulatory properties of the JAK/STAT pathway. Aim 1 (Characterize the effects of JAK/STAT signaling on proliferation) employs molecular methods and FACS analysis to measure the effects of JAK/STAT signaling on proliferation rates, cell cycle phasing and expression of critical cell cycle factors. Aim 2 (Determine if Stat92E regulates cellular growth through dMyc) uses genetic approaches and FACS analysis to measure the effects of Stat92E on cellular growth and to determine if it regulates this process through dmyc. Aim 3 (Determine if Stat92E regulates self-renewal in through chinmo) uses genetic and molecular approaches to determine if Stat92E regulates self-renewal in testis stem cells through chinmo alone or through additional target genes. PUBLIC HEALTH RELEVANCE Due to evolution, JAK and STAT genes exist in very similar forms in other organisms, such as the fruit fly Drosophila. The fruit fly is an excellent genetic organism that has been used for many years in research laboratories and with great success to identify and characterize genes critical for basic biological processes. Our study is designed to eludicate how the JAK/STAT pathway regulates stem cell numbers in Drosophila.