2008 — 2012 |
Coller, Hilary A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Micrornas in Cellular Quiescence
DESCRIPTION (provided by applicant): Many cells in the human body are quiescent;that is, they have temporarily stopped dividing but retain the capacity to divide when conditions are suitable, for instance, when the organism must grow or a damaged tissue must be repaired. Although quiescence is a common state for many somatic cells, including stem cells, we know remarkably little about the regulation of cellular quiescence, the changes that cells undergo upon becoming quiescent, and what quiescence looks like in the body. We demonstrated that quiescence is an active and evolving state characterized by extensive changes in gene expression patterns. We hypothesized that microRNAs are involved in regulating the large number of gene expression changes observed with quiescence. We identified specific microRNAs up- and down-regulated with quiescence. miR-31 is downregulated with quiescence and is upregulated in colon and pancreatic cancer, especially late stage tumors. We have shown that overexpression of miR-31 results in a faster and more robust cell cycle entry from quiescence. We propose here to identify miR-31 targets and define the mechanisms by which it affects quiescence. We also propose to define the sequences within the miR-31 promoter responsible for its downregulation with quiescence. Only a small number of molecules can hasten cell cycle entry from quiescence, including myc, E2F and cyclin E. We anticipate that elucidating the mechanisms by which miR-31 promotes proliferation will elucidate an important new signaling pathway. PUBLIC HEALTH RELEVANCE: Many common diseases likely result from failures of quiescence;that is, an inability of cells to temporarily stop dividing. These include developmental abnormalities, tumors, fibrosis, and ulcers. The ability to reenter the cell cycle after quiescence is a critical attribute of stem cells, and its decline likely contributes to aging. We propose here to characterize the role of one specific signaling pathway central to quiescence, and expect that the mechanisms identified will provide important insights into a wide range of medical conditions.
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1 |
2009 — 2012 |
Coller, Hilary A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Combined Computational and Experimental Approach to Defining Mechanisms of Micr
DESCRIPTION (provided by applicant): microRNAs (miRNAs) are central regulators of a wide array of biological processes including cancer, development, neurodegenerative and metabolic diseases and viral infection. Yet, the details of their molecular mechanisms remain controversial. In the course of our studies on cellular quiescence in human cells, we discovered that the let-7 miRNA likely regulates not only genes with recognition sequences in their 3'UTRs but also genes with recognition sequences in their coding regions. We further discovered evidence for mechanistic differences between the targeting of 3'UTR versus coding region recognition sites. Our results in combination with published data also raise the possibility of target-specificity in miRNA mechanisms in the context of quiescence. We propose here a combined computational and experimental approach to test our hypothesis that miRNAs have distinct mechanisms of action depending upon the interaction between the miRNA and the target. We expect that the results will be valuable for predicting physiologically relevant targets of miRNAs and for designing maximally effective miRNA therapeutics. In our first aim, we will use a combination of comparative genomics and reporter assays to determine whether there are multiple distinct mechanisms by which miRNAs target different portions of transcripts. In Specific aims #2 - #4, we will test our hypothesis that there are transcript-specific mechanisms that govern whether miRNAs regulate targets via transcript degradation or translation. We will analyze cells transfected with one of several miRNAs by performing microarray analysis on total transcripts and polysome-associated transcripts. These data will allow us to identify characteristics of miRNA-target interactions that result in transcript degradation and those that affect translation initiation. In specific aim #3, we will extend these experiments to test our hypothesis that there is a target-specific switch in miRNA activity from repression to activation as cells become quiescent. Finally, in specific aim #4, we will extend our analysis to endogenous miRNAs by monitoring the impact of anti-miRs, miRNA inhibitors, on total and polysome-associated transcripts. Our expectation is that deciphering the mechanisms of miRNA function, including the basis for specificity for miRNA-target interactions, will provide clarity to the field of miRNA research. The findings, which will be made available through our website, will also advance many related fields as researchers studying a wide array of biological processes and disease states are better able to associate miRNAs with their targets and functions. PUBLIC HEALTH RELEVANCE: While miRNAs are clearly central players in a wide array of biological processes including cancer, development, neurodegenerative and metabolic diseases and viral infection, linking miRNAs with the specific molecules that they regulate has been a challenge. We propose here a combined computational and experimental approach to elucidate the mechanisms of miRNA-target interactions for many different miRNA- target pairings. We anticipate that these findings will be of great value to researchers in a wide range of fields working to identify miRNAs targets, and those designing miRNAs as therapeutics.
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1 |
2018 — 2021 |
Coller, Hilary A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Stromal Autophagy in Cutaneous Melanoma @ University of California Los Angeles
Project Summary Melanoma is a highly aggressive cancer with a rising incidence and good therapeutic options exist for only a subset of patients. Recent studies have shown that treatment with an autophagy inhibitor can improve outcome for melanoma patients. Recognizing the importance of interactions between cancer cells and the host, we chose to investigate the role of autophagy in the stroma. We discovered a new connection between the metabolic/autophagic state of the host, and the immune response to a tumor. We found that the host fibroblast-rich stroma adjacent to a melanoma have higher levels of autophagy marker LC3 than cells distant from the tumor. Using co-culture models, we discovered that cancer cells induce autophagy in co-cultured fibroblasts in a TGF-?-dependent manner. Orthotopic melanoma allografts showed dramatic growth reduction in genetically-engineered mice in which autophagy protein Atg7 was inactivated in the entire animal, or only in fibroblast specific protein 1 (FSP1)- expressing cells, which includes fibroblasts and immune cells. Tumors grown in Atg7-deficient hosts had lower levels of proliferation and higher levels of apotosis than tumors hosted in control mice. We conclude that Atg7 expression in the host plays an important role in supporting tumor progression. To understand the mechanism through which host Atg7 inactivation affects tumor growth, we performed RNA-Seq analysis of tumors harbored in Atg7-inactivated and control mice and discovered a gene expression signature of inflammation. Analysis of cytokines in the plasma and T cells of Atg7-deficient mice revealed higher levels of Th1 type cytokines and no change in Th2 type cytokines. Memory CD4+ and CD8+ T cells from Atg7-deficient mice transitioned from naïve to effector states. Tumor infiltrating lymphocytes in Atg7-deficient mice contained higher levels of CD8+ T cells than in control mice. Treatment with antibodies that deplete T cells resulted in larger tumors in Atg7-deficient mice when tested in the whole body conditional knockout model or in mice with FSP1-specific autophagy inactivation. We have therefore discovered a new mechanism regulating the immune response to tumors. We will investigate the role for additional cell types in anti-tumor immunity in Atg7-deficient mice, and the role of Atg7 in anti-tumor and anti-antigen effector responses. We will test whether small molecule autophagy inhibitors also induce anti-tumor immunity and whether autophagy inactivation sensitizes mice to immune checkpoint inhibitors. We will also test whether the levels of host autophagy in specific cell types correlates with the extent of immune cell infiltration into melanomas in patient samples. We anticipate our findings will suggest new approaches for activating a patient's immune response to fight cancer.
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