Clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant histological type of kidney cancer, for which there are no effective methods of treatment for metastatic disease. ccRCC is characterized by early loss of the von Hippel-Lindau tumor-suppressor gene (VHL) in a majority (60%-80%) of tumors. One major oncogenic effect of VHL loss is the induction of hypoxia-inducible factor (HIF) and HIF-regulated genes leading to angiogenesis, which supports tumor growth by providing nutrients from extracellular sources. In our previous work, we discovered that loss of VHL regulates pathways that secure nutrients from intracellular sources through autophagy: VHL, by inducing expression of miR-204, targets the autophagic regulator, LC3B, and inhibits oncogenic autophagy. By inhibiting HIF, VHL induces expression of LC3C, an LC3B ortholog, which has tumor suppressive activity. This proposal is focused on the TRPM3 channel (transient receptor potential M3 Ca2+-permeable, nonselective cation channel), which is the protein product of the host gene that encodes miR-204 in its intron 6. We discovered that TRPM3 plays a pro-oncogenic role in ccRCC. TRPM3 is overexpressed in ccRCC and regulates two autophagic pathways;it is necessary for LC3B/LC3A autophagy and inhibits LC3C-mediated autophagy. In contrast to miR-204, TRPM3 is repressed by VHL at the level of protein accumulation. Our hypothesis is that the effects of TRPM3 on autophagic programs play an essential role in its ability to promote RCC tumor growth, and that the effects of TRPM3 on autophagic programs are mediated through regulation of intracellular calcium.
In Aim 1, we will investigate the molecular mechanism by which VHL represses TRPM3, with the leading hypothesis that VHL-induced miR-204 targets and represses expression of its host gene product.
In Aim 2, by manipulating expression of different autophagic regulators affected by TRPM3, we will identify the autophagic pathways essential for the oncogenic activity of TRPM3.
In Aim 3 we will manipulate extracellular and intracellular Ca2+ and Ca2+-regulated kinases implicated in the regulation of autophagy and determine the effects on autophagy in cells that are VHL(-) or VHL(-) with TRPM3 knocked down.
In Aim 4 we will study the mechanisms of transcriptional induction of LC3C in cells with knockdown of TRPM3. With this proposal, we expect to further establish VHL as a major regulator of autophagy.

Public Health Relevance

Renal clear-cell carcinoma (RCC) is the most prevalent and malignant histological type of kidney cancer. Currently, there are no effective methods of treatment for RCC once it has spread from the kidney to other parts of the body. In this project, we will study newly identified pathways which contribute to tumor growth. We expect that this project will lead to much-needed new strategies for treating RCC. DESCRIPTION (provided by applicant): Clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant histological type of kidney cancer, for which there are no effective methods of treatment for metastatic disease. ccRCC is characterized by early loss of the von Hippel-Lindau tumor-suppressor gene (VHL) in a majority (60%-80%) of tumors. One major oncogenic effect of VHL loss is the induction of hypoxia-inducible factor (HIF) and HIF-regulated genes leading to angiogenesis, which supports tumor growth by providing nutrients from extracellular sources. In our previous work, we discovered that loss of VHL regulates pathways that secure nutrients from intracellular sources through autophagy: VHL, by inducing expression of miR-204, targets the autophagic regulator, LC3B, and inhibits oncogenic autophagy. By inhibiting HIF, VHL induces expression of LC3C, an LC3B ortholog, which has tumor suppressive activity. This proposal is focused on the TRPM3 channel (transient receptor potential M3 Ca2+-permeable, nonselective cation channel), which is the protein product of the host gene that encodes miR-204 in its intron 6. We discovered that TRPM3 plays a pro-oncogenic role in ccRCC. TRPM3 is overexpressed in ccRCC and regulates two autophagic pathways;it is necessary for LC3B/LC3A autophagy and inhibits LC3C-mediated autophagy. In contrast to miR-204, TRPM3 is repressed by VHL at the level of protein accumulation. Our hypothesis is that the effects of TRPM3 on autophagic programs play an essential role in its ability to promote RCC tumor growth, and that the effects of TRPM3 on autophagic programs are mediated through regulation of intracellular calcium. In Aim 1, we will investigate the molecular mechanism by which VHL represses TRPM3, with the leading hypothesis that VHL-induced miR-204 targets and represses expression of its host gene product. In Aim 2, by manipulating expression of different autophagic regulators affected by TRPM3, we will identify the autophagic pathways essential for the oncogenic activity of TRPM3. In Aim 3 we will manipulate extracellular and intracellular Ca2+ and Ca2+-regulated kinases implicated in the regulation of autophagy and determine the effects on autophagy in cells that are VHL(-) or VHL(-) with TRPM3 knocked down. In Aim 4 we will study the mechanisms of transcriptional induction of LC3C in cells with knockdown of TRPM3. With this proposal, we expect to further establish VHL as a major regulator of autophagy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA122346-07
Application #
8611902
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Salnikow, Konstantin
Project Start
2006-07-01
Project End
2018-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
7
Fiscal Year
2014
Total Cost
$283,353
Indirect Cost
$104,582
Name
University of Cincinnati
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Hall, Daniel P; Cost, Nicholas G; Hegde, Shailaja et al. (2014) TRPM3 and miR-204 establish a regulatory circuit that controls oncogenic autophagy in clear cell renal cell carcinoma. Cancer Cell 26:738-53
Czyzyk-Krzeska, M F; Zhang, X (2014) MiR-155 at the heart of oncogenic pathways. Oncogene 33:677-8
Bastola, Prabhat; Stratton, Yiwen; Kellner, Emily et al. (2013) Folliculin contributes to VHL tumor suppressing activity in renal cancer through regulation of autophagy. PLoS One 8:e70030
Mikhaylova, Olga; Stratton, Yiwen; Hall, Daniel et al. (2012) VHL-regulated MiR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal clear cell carcinoma. Cancer Cell 21:532-46
Yi, Ying; Mikhaylova, Olga; Mamedova, Aygun et al. (2010) von Hippel-Lindau-dependent patterns of RNA polymerase II hydroxylation in human renal clear cell carcinomas. Clin Cancer Res 16:5142-52
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