Kinase suppressor of ras 1 (KSR1) regulates PGC1α and estrogen-related receptor α to promote oncogenic Ras-dependent anchorage-independent growth

Abstract

Kinase suppressor of ras 1 (KSR1) is a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase (ERK) cascade that enhances oncogenic Ras signaling. Here we show KSR1-dependent, but ERK-independent, regulation of metabolic capacity is mediated through the expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and estrogen-related receptor α (ERRα). This KSR1-regulated pathway is essential for the transformation of cells by oncogenic Ras. In mouse embryo fibroblasts (MEFs) expressing H-Ras(V12), ectopic PGC1α was sufficient to rescue ERRα expression, metabolic capacity, and anchorage-independent growth in the absence of KSR1. The ability of PGC1α to promote anchorage-independent growth required interaction with ERRα, and treatment with an inhibitor of ERRα impeded anchorage-independent growth. In contrast to PGC1α, the expression of constitutively active ERRα (CA-ERRα) was sufficient to enhance metabolic capacity but not anchorage-independent growth in the absence of KSR1. These data reveal KSR1-dependent control of PGC1α- and ERRα-dependent pathways that are necessary and sufficient for signaling by oncogenic H-Ras(V12) to regulate metabolism and anchorage-independent growth, providing novel targets for therapeutic intervention.

Fig. 1.

KSR1 enhances the expression of metabolic regulators PGC1α and ERRα only in the presence of H-Ras^V12. KSR1^−/− MEFs were infected with recombinant retroviruses encapsulating bicistronic vectors carrying genes for KSR1 and GFP, GFP alone (control), H-Ras^V12 and puromycin resistance, or puromycin resistance alone (control). (A) Whole-cell extracts were immunoblotted with the indicated antibodies to confirm the expression of KSR1 and H-Ras^V12 in cells prior to testing. (B) KSR1^−/− MEFs ± H-Ras^V12 ± KSR1 were assessed for glucose uptake by the incorporation of [³H]-2-deoxyglucose. (C) KSR1^−/− MEFs ± H-Ras^V12 ± KSR1 were lysed and separated into cytoplasmic and nuclear extracts and immunoblotted with the indicated antibodies. (D) KSR1^−/− MEFs expressing both H-Ras^V12 and KSR1 were treated with 20 μM U0126 or PD0325901 for 24 h and lysed and separated into cytoplasmic and nuclear extracts and immunoblotted with the indicated antibodies. (E) Whole-cell extracts from KSR1^−/− MEFs expressing H-Ras^V12 and either KSR1 and GFP or GFP alone (control) were probed on Western blots with the indicated antibodies. (F and G) The rates of oxygen consumption (F) and extracellular acidification (G) were assessed in KSR1^−/− MEFs expressing H-Ras^V12 ± KSR1. Data shown are the result of triplicate measurements for ECAR and OCR ± standard deviation taken at baseline and after increasing doses of FCCP. n.s., not significant (P > 0.05); *, P < 0.05; ****, P < 0.0001 by two-tailed Student's t test.

Fig. 2.

PGC1α stimulates anchorage-independent growth and metabolism in KSR1^−/− MEFs expressing H-Ras^V12. KSR1^−/− MEFs with stable expression of H-Ras^V12 were infected with recombinant retroviruses encapsulating bicistronic retroviral vectors carrying genes for PGC1α constructs and GFP or GFP alone (control). (A) Nuclear extracts were immunoblotted with the indicated antibodies to confirm the expression of PGC1α constructs and to determine ERRα expression. (B and C) Cells were assessed for anchorage-independent growth by plating 5,000 cells/dish in 0.4% agar and counting colonies over 100 μm after 14 days. Representative photomicrographs of colonies (B) and colony count (C) are shown. (D) Whole-cell extracts from KSR1^−/− MEFs expressing H-Ras^V12 and either PGC1α constructs and GFP or GFP alone (control) were probed on Western blots with the indicated antibodies. (E and F) The rates of oxygen consumption (E) and extracellular acidification (F) were assessed in KSR1^−/− MEFs expressing H-Ras^V12 and either PGC1α constructs and GFP or GFP alone (control). Data shown are the result of triplicate measurements for ECAR and OCR ± standard deviation taken at baseline and after increasing doses of FCCP. n.s., not significant (P > 0.05); ****, P < 0.0001 by two-tailed Student's t test.

Fig. 3.

Constitutively active ERRα (CA-ERRα) stimulates metabolism but not anchorage-independent growth in KSR1^−/− MEFs expressing H-Ras^V12. KSR1^−/− MEFs with stable expression of H-Ras^V12 were infected with recombinant retroviruses encapsulating bicistronic retroviral vectors carrying genes for CA-ERRα and GFP or VP16 and GFP (control). (A) Nuclear extracts were immunoblotted with the indicated antibodies to confirm the expression of CA-ERRα and to determine endogenous PGC1α and ERRα expression. (B and C) Cells were assessed for anchorage-independent growth by plating 5,000 cells/dish in 0.4% agar and counting colonies over 100 μm after 14 days. Representative photomicrographs of colonies (B) and colony count (C). (D) Whole-cell extracts from KSR1^−/− MEFs expressing H-Ras^V12 and either CA-ERRα and GFP or VP16 and GFP (control) were probed on Western blots with the indicated antibodies. (E and F) The rates of oxygen consumption (E) and extracellular acidification (F) were assessed in KSR1^−/− MEFs expressing H-Ras^V12 and either CA-ERRα and GFP or VP16 and GFP (control). Data shown are the result of triplicate measurements for ECAR and OCR ± standard deviation taken at baseline and after increasing doses of FCCP. n.s., not significant (P > 0.05 by two-tailed Student's t test); ****, P < 0.0001 by 2-way analysis of variance (ANOVA).

Fig. 4.

ERRα inverse agonist XCT790 inhibits anchorage-independent growth in KSR1^−/− MEFs expressing H-Ras^V12 and KSR1. KSR1^−/− MEFs with stable expression of H-Ras^V12 and KSR1 were treated with 10 μM XCT790 or DMSO (control) for the indicated times and then assayed. (A) Nuclear extracts were immunoblotted with the indicated antibodies to determine the expression of ERRα 24 h after treatment. (B) Cells were assessed for anchorage-independent growth by plating 5,000 cells/dish in 0.4% agar and counting colonies over 100 μm after 14 days. Cells were pretreated with drug for 24 h before plating and drug was included in both top and bottom layers of agar. (C) 50,000 cells were plated in the presence of XCT790 or DMSO, and cell numbers were determined at the indicated times. (D and E) The rates of oxygen consumption (D) and extracellular acidification (E) were assessed in KSR1^−/− MEFs expressing H-Ras^V12 and KSR1 treated with either XCT790 or DMSO for 24 h. Data shown are the result of triplicate measurements for ECAR and OCR ± standard deviation taken at baseline and after increasing doses of FCCP. n.s., not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by two-tailed Student's t test.