ERRα Maintains Mitochondrial Oxidative Metabolism and Constitutes an Actionable Target in PGC1α-Elevated Melanomas

Abstract

The uncontrolled growth of tumors provides metabolic dependencies that can be harnessed for therapeutic benefit. Although tumor cells exhibit these increased metabolic demands due to their rapid proliferation, these metabolic processes are general to all cells, and furthermore, targeted therapeutic intervention can provoke compensatory adaptation that alters tumors' characteristics. As an example, a subset of melanomas depends on the transcriptional coactivator PGC1α function to sustain their mitochondrial energy-dependent survival. However, selective outgrowth of resistant PGC1α-independent tumor cells becomes endowed with an augmented metastatic phenotype. To find PGC1α-dependent components that would not affect metastasis in melanomas, an unbiased proteomic analyses was performed and uncovered the orphan nuclear receptor ERRα, which supports PGC1α's control of mitochondrial energetic metabolism, but does not affect the antioxidant nor antimetastatic regulatory roles. Specifically, genetic or pharmacologic inhibition of ERRα reduces the inherent bioenergetic capacity and decreases melanoma cell growth, but without altering the invasive characteristics. Thus, within this particularly aggressive subset of melanomas, which is characterized by heighted expression of PGC1α, ERRα specifically mediates prosurvival functions and represents a tangible therapeutic target.Implications: ERRα, a druggable protein, mediates the bioenergetic effects in melanomas defined by high PGC1α expression, suggesting a rational means for therapeutic targeting of this particularly aggressive melanoma subtype. Mol Cancer Res; 15(10); 1366-75. ©2017 AACR.

Figure 1. ERRα is associated with PGC1α in melanoma cells

A. A list of the most abundant proteins co-immunoprecipitated with Flag/HA-tagged PGC1α in A375P melanoma cells as identified by mass spectrometry. B. Endogenous PGC1α is interacting with ERRα in PGC1α-positive melanoma cell lines A375P and G361. Immunoprecipition of PGC1α was followed by immunoblotting using the indicated antibodies. C. Pearson-based correlation between PGC1α and ERRα expression levels across metastatic melanoma samples within TCGA (N=366). D. Based on 2-sample, 2-sided t-test statistics, the 20 percentile highest and lowest PGC1a expression levels associate with highest and lowest ERRα expression levels, respectively. E. Based on Mantel-Cox log rank test, the top 20 percentile mutual expression rank (MER) of PGC1α:ERRα segregates poorer overall survival relative to the metastatic melanoma cohort average (p < 0.022).

Figure 2. ERRα is required to support mitochondrial oxidative metabolism

A. Immunoblotting of melanoma cells upon ERRα depletion by CRISPR. Small guide control vector: sgCtrl; small guide RNA for ERRα: sgERRα. B. Expression of mitochondrial related genes at the mRNA level in melanoma cells upon ERRα depletion. C. Immunoblotting of mitochondrial related genes in ERRα-depleted melanoma cells. D. Mitochondrial activity of melanoma cells with ERRα depletion presented as oxygen consumption rate (OCR) measured by seahorse flux assay. E. Intracellular ATP levels in cells with ERRα depletion. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by t-test (B) or one-way ANOVA (D, E).

Figure 3. Depletion of ERRα compromises the growth of human melanomas

A. Growth curve of various PGC1α -positive melanoma cell lines with ERRα depletion. B. End-point tumor weight of PGC1α-positive melanoma cells with ERRα depletion after inoculated into nude mice. Data were presented as mean with SD. C. Immunoblotting of PGC1α-negative melanoma cells upon ERRα depletion by CRISPR. D. Growth curve of PGC1α -negative melanoma cell lines with ERRα depletion. E. End-point tumor weight of PGC1α-melanoma cell line A375 with ERRα depletion in nude mice. *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) as determined by t-test if not otherwise indicated.

Figure 4. Inhibition of ERRα activity phenocopies ERRα depletion in melanoma cells

A. Immunoblotting of melanoma cells upon treatment with ERRα antagonist Cpd29. B. Expression of oxidative genes at the mRNA level in melanoma cells upon ERRα antagonist Cpd29. C. Mitochondrial activity of melanoma cells treated with ERRα antagonist Cpd29 as measured by seahorse flux assay. D. Intracellular ATP levels in cells treated with Cpd29. E. Growth curve of various melanoma cell lines treated with Cpd29. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by t-test (B, E) or one-way ANOVA (C, D).

Figure 5. Inhibition of ERRα activity suppresses melanoma growth in vivo

A. Growth curve of melanomas in nude mice upon treatment with 30 mg/kg ERRα antagonist Cpd29 (G361: n=10 for vehicle, n=7 for Cpd29; MeWo: n=12 for vehicle, n=13 for Cpd29). Data were presented as mean with SD. B. End-point tumor weight of melanoma xenografts upon Cpd29 treatment. Data were presented as mean with SD. C. A schematic model depicting the functional roles of ERRα in the mediating of PGC1α effects in melanoma cells. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by t-test if not otherwise indicated.