Within the tumor microenvironment, cancer cells receive a multitude of signals through direct cell-cell interactions with normal stromal cells, other cancer cells, and cells comprising the tumor vasculature via juxtacrine signaling pathways. Chemotherapeutic agents that are dependent on cell cycle progression for the induction of cell death, such as cisplatin, BCNU, and taxol, are known to be less effective in densely grown cultures with abundant cell-cell contacts, though this is widely attributed to growth arrest rather than pro-survival juxtacrine signaling. We observed an inhibition of cell death in densely-grown cells treated with tyrosine kinase inhibitors, a class of agents not known to be dependent on cell cycle progression for the induction of cell death. Therefore, this project aims to uncover the mechanisms by which the effects of tyrosine kinase inhibition are circumvented in dense cultures, a growth mode that is likely to be more reflective of tumors in situ. Because high-throughput screens for drug discovery typically use low density cultures not subject to juxtacrine survival signaling, this work may lead to the development of drugs with better in vivo efficacy. Plans and goals: 1. Define the molecular mechanisms of TKI resistance in high density cell cultures. Using primary glioma cell lines, we will profile gene expression, protein tyrosine phosphorylation, and oncoprotein abundance in cells grown at low and high density. Candidate pro-survival genes will then be validated by knocking down expression in the appropriate growth mode, then determining which RNAis restore TKI sensitivity in dense cultures. Bioinformatics analyses of the candidate genes will also be used to determine which pathways are essential for cell-cell contact-dependent TKI resistance. 2. Screen for drugs that cooperate with TKIs in high density cell cultures. Using the Approved Oncology Drugs set from the NCI Developmental Therapeutics Program, we will screen for drugs that restore TKI sensitivity in high density cell cultures. We will then determine the mechanism by which these new drugs impact TKI sensitivity by examining the effect of these drugs on the candidate genes and phospho-proteins revealed in Aim 1. 3. Validate the drug or pathway for use in TKI combination therapy in vivo. We predict that the pathways that confer TKI resistance in high density cultures may also contribute to poor performance of these drugs in vivo. To test this hypothesis, we will compare the survival of mice orthotopically implanted with human primary glioma cells treated with a single TKI to those concurrently treated with the TKI plus a drug revealed in Aim 2. We will also examine whether knocking down the genes or pathways induced by high density growth prolongs survival of the TKI-treated xenograft-implanted mice. Finally, we will perform hierarchical clustering of genes expressed in low and high density in vitro cultures to orthotopic implants and patient samples to determine whether high density cultures more closely recapitulate in vivo tumors and are thus more suited for high-throughput drug screens.
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