Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis - PubMed (original) (raw)

. 2015 Mar 2;125(3):1174-88.

doi: 10.1172/JCI76029. Epub 2015 Feb 9.

Nagireddy Putluri, Weiwen Long, Bin Zhang, Jianghua Wang, Akash K Kaushik, James M Arnold, Salil K Bhowmik, Erin Stashi, Christine A Brennan, Kimal Rajapakshe, Cristian Coarfa, Nicholas Mitsiades, Michael M Ittmann, Arul M Chinnaiyan, Arun Sreekumar, Bert W O'Malley

Coactivator SRC-2-dependent metabolic reprogramming mediates prostate cancer survival and metastasis

Subhamoy Dasgupta et al. J Clin Invest. 2015.

Abstract

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2-driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer.

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Figures

Figure 9

Figure 9. SRC-2 promotes prostate cancer metastasis.

(A) PC-3 cells stably expressing shNT and sh19 (n = 5) were orthotopically implanted into the left ventral prostate lobe of SCID mice and imaged 8 weeks after surgery using a UVP Biospectrum imager. Arrows indicate the primary tumors, and arrowheads show the location of metastatic spreading. (B and C) Each primary tumor was measured using slide calipers, and relative tumor volumes (B) and metastases (C) were plotted for each mouse (horizontal line represents the mean). (D and E) Targeted MS analysis of xenograft tumor extracts from PC-3 shNT and sh19 cells depicted in A showing the relative levels of glutamate and oleic acids. (F–H) Fragments per kilobase of exon per million fragments mapped (FPKM) values of SRC2, FASN, and SCD from a cohort of benign adjacent (n = 16), organ-confined prostate cancer (n = 68) and metastatic prostate cancer (n = 48). (I and J) Log2-transformed data depicting the levels of glutamate and oleic acid from benign adjacent prostate (n = 16), clinically localized prostate cancer (n = 12, PCA), and metastatic prostate cancer (n = 14) tissues (46). This cohort is a subset of the RNA-seq cohort shown in F–H. *P < 0.05 and **P < 0.001 by Student’s t test. For metabolomic analyses, we calculated a permutation-based P value (10,000 permutations of sample labels) to define the significance of metabolites in different categories (45).The data were plotted as a box plot in R language and show the 5% and 95% quantiles (whiskers), 25% and 75% quartiles (box), and the median (horizontal line). *q < 0.05 and **q < 0.001 by 2-sided Student’s t test, with an FDR-corrected P value of less than 0.05 considered significant.

Figure 8

Figure 8. SRC-2 is essential for prostate cancer cell survival.

(A) Representative images depicting the growth of the stable C4-2 cells shNT, sh18, and sh19 in soft agar assay 2 weeks after plating. (B) Quantification of the total number of stable C4-2 cell colonies that survived after 2 weeks. *P <0.05 by 1-way ANOVA. (C) H&E-stained sections of mouse lungs from experimental lung metastasis assay. Nude mice were injected via the tail vein with PC-3 cells stably expressing shNT, sh18, and sh19 (n = 7), and growth and survival of the cells in mouse lungs were analyzed after 5 weeks. T, tumor. Scale bar: 100 μm. (D) Quantification of Ki67-stained cells (antibody epitope reacts with human Ki67 protein) in mouse lung sections from shNT-, sh18-, and sh19-injected animals. *P < 0.05 and **P < 0.001 by 2-tailed Student’s t test. Refer also to Supplemental Figure 11C. (E) Western blot analysis showing expression levels of SRC-2, FASN, SCD, and actin in the stable C4-2 cells shNT and sh19, and reexpression of SRC-2 in sh19 cells infected with SRC-2 adenovirus. Actin was normalized to the protein loading control. The full, uncut gels are shown in the Supplemental Material. (F) Clonogenic survival assay in the stable PC-3 cells shNT, sh18, and sh19 expressing GFP adenovirus, SRC-2 adenovirus, or FASN adenovirus to rescue the defective survival phenotype in SRC-2–depleted cells. (G) Relative growth of C4-2 and PC-3 cells stably expressing shNT that were either untreated or treated with DMSO or BPTES (1 μM) for 4 days. sh19 cells were used to monitor the effect of SRC-2 knockdown (n = 6/group). Data represent the mean ± SEM. *P < 0.05 compared with DMSO and **P < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test.

Figure 7

Figure 7. SRC-2 defines the metabolic and energetic program of human prostate cancer cells.

(A) Real-time measurement of basal and maximal OCRs in the stable C4-2 cells shNT, sh18, and sh19 (n = 3/group). *P < 0.05 and **P < 0.001 versus shNT by 2-way ANOVA with Tukey’s multiple comparisons test. (B and C) Basal and maximal OCRs in the stable C4-2 cells shNT, sh18, and sh19 were measured in DMSO control (B) or FCCP-treated (C) cells cultured in the presence of high glucose (11 mM)/low glutamine (0.2 mM) or low glucose (5 mM)/high glutamine (2 mM) concentrations (n = 3/group). Box and whisker plots indicate the minimum to maximum values (whiskers), 25% and 75% quartiles (boxes), and the median (horizontal lines). **P < 0.001 by 2-tailed Student’s t test. (D) Intracellular ATP levels in the stable C4-2 cells shNT, sh18, and sh19 cultured in complete media (n = 3/group). Box and whisker plot shows the minimum to maximum values (whiskers), 25% and 75% quartiles (boxes), and the median (horizontal lines). *P < 0.05 and **P < 0.001 by 1-way ANOVA. (E) Clonogenic survival assay showing the number of C4-2 cells stably expressing shNT, sh18, and sh19 clones that survived a 2-week period of nutritional stress. (F) Total number of colonies observed in the clonogenic survival assay shown in E. Data represent the mean ± SEM. *P < 0.05 and **P < 0.001 by Student’s t test.

Figure 6

Figure 6. Glutamine stimulation enhances the transcriptional activity of SRC-2.

(A) PC-3 cells expressing either GFP adenovirus or SRC-2 adenovirus were transfected with an FASN-luciferase construct and stimulated with high glucose (11 mM)/low glutamine (0.2 mM) or high glutamine (2 mM)/low glucose (5 mM) concentrations, followed by treatment with DMSO or torin (250 nM). A luciferase assay was then performed to measure FASN promoter activity, and data were normalized to total protein (n = 4). (B and C) ChIP of SRC-2 from C4-2 cells showing the differential recruitment of SRC-2 on the FASN and SCD promoters upon glutamine stimulation (2 mM) in the presence or absence of torin (250 nM). The amplicons tested are indicated in the figure. IgG antibody was used as a control, and data are presented as the percentage of input chromatin (n = 3). (D) Immunoprecipitation of HA–SRC-2 followed by Western immunoblotting to detect the phosphorylation status of SRC-2 using phosphorylated serine/threonine antibody. Input lysates were obtained from C4-2 cells expressing GFP adenovirus or SRC-2 adenovirus and subsequently stimulated with increasing concentrations of glutamine (0.2 mM and 2 mM), followed by treatment with DMSO or the mTORC1 inhibitor torin (250 nM). Cell-permeable octyl-α-ketoglutarate was used to rescue the effects of low glutamine levels on SRC-2 phosphorylation. Actin was used to normalize the loading input. The full, uncut gels are shown in the Supplemental Material. (E) Schematic depicting the proposed glutamine/mTORC1 signaling pathway, with SRC-2 as the key downstream mediator regulating transcriptional functions that coactivate SREBP-1. Data represent the mean ± SEM. *P < 0.05 and **P < 0.001 by Student’s t test with Holm-Sidak multiple comparisons test.

Figure 5

Figure 5. Glutamine stimulation induces mTORC1-dependent phosphorylation of SRC-2.

(A) Western blot analysis of FASN, SCD, SRC-2, and actin in the stable C4-2 cells shNT, sh18, and sh19 grown with or without glutamine (2 mM). (B) Immunoprecipitation of HA–SRC-2 followed by Western immunoblotting to detect the phosphorylation status of SRC-2 using phosphorylated serine/threonine (p-Ser/Thr) antibody. Input lysates were obtained from C4-2 cells expressing GFP adenovirus or SRC-2 adenovirus and subsequently stimulated with increasing concentrations of glutamine (0.2 mM, 2 mM, and 4 mM), followed by treatment with DMSO or the mTORC1 inhibitor torin (250 nM). Actin was used to normalize the input loading control. Semiquantitative levels of phosphorylated SRC-2 were analyzed by densitometry, and relative values (compared with those in lane 2) normalized to actin are indicated numerically under each lane. (C) Western blot analysis showing the effect of 3 different siRNAs on mTOR expression. (D) C4-2 cells expressing SRC-2 adenovirus were transfected with nontargeting siRNA or 3 different mTOR siRNAs, followed by treatment with or without glutamine (2 mM). HA–SRC-2 was immunoprecipitated followed by Western immunoblotting to detect the phosphorylation status of SRC-2. GFP adenovirus was used as a negative control. (E) C4-2 cells expressing HA-tagged WT SRC-2 or the phosphorylation-deficient mutants S499A, S699A, and S493A were stimulated with or without glutamine (2 mM). HA was used to immunoprecipitate WT SRC-2 or mutants, followed by Western immunoblotting to detect the phosphorylation status of SRC-2. Actin was used to normalize the input loading control (HA–SRC-2). Actin was used as a control to normalize the protein loading control, and GFP adenovirus was used as a control virus. The full, uncut gels are shown in the Supplemental Material.

Figure 4

Figure 4. SRC-2 represses transcription of the zinc transporter ZIP1 to stimulate ACO activity.

(A–C) Enzymatic activity of ACO, IDH, and CS in the stable C4-2 cells shNT, sh18, and sh19 (n = 5). (D) qRT-PCR analysis of SRC2 and ZIP1 (SLC39A1) gene expression in the stable C4-2 cells shNT and sh18 and in C4-2 shNT cells expressing SRC-2 adenovirus (n = 4/group). *P < 0.05 by 2-way ANOVA with Tukey’s multiple comparisons test. (E) ChIP of SRC-2 from C4-2 cells showing the recruitment of SRC-2 on the ZIP1 proximal promoter (–175 to 70 bp) compared with the –5,000 bp upstream region from the start site. (F) Luciferase reporter assay in HeLa cells transiently transfected with empty pGL3 vector (Empty luciferase) and a pGL3-ZIP1-luciferase construct (–246 to +82 bp) in the presence of vector alone (ZIP1 luciferase) or of the SRC-2 construct (ZIP1-luc + SRC-2) (n = 6). (G) Mass isotopomer distribution of stearate labeling from [5-13C]glutamine in C4-2 cells ectopically expressing human ZIP1 (Adv ZIP1) or control virus (Adv GFP) (n = 3). The mass spectrometric method used in this study failed to detect higher fatty acid isotopomers. Data represent the mean ± SEM. *P < 0.05 and **P < 0.001 by Student’s t test (A, E, and F), with Holm-Sidak multiple comparisons test in G.

Figure 3

Figure 3. SRC-2 coactivates SREBP1 to promote lipogenesis.

(A) Luciferase reporter assay in HeLa cells transiently transfected with a FASN-luciferase construct (–220 to +25 bp) in the presence of vector alone, SREBP-1, SRC-2, or a combination of both SRC-2 and SREBP-1 (n = 6). (B) Luciferase reporter assay in HeLa cells transiently transfected with an SCD-luciferase construct (–1,280 to +174 bp) in the presence of vector alone, SREBP-1, SRC-2, or a combination of both SRC-2 and SREBP-1 (n = 6). (C and D) Western blot analysis followed by luciferase reporter assay in PC-3 cells transfected with an FASN-luciferase construct in the presence of control siRNA (siNT), SRC-2-siRNA (siSRC-2), SREBP-1 siRNA (siSREBP-1), or a combination of both SRC-2 and SREBP-1. Semiquantitative levels of each band were analyzed by densitometry using UVP Vision Works LS software, and the relative values (compared with untreated) normalized to actin are indicated numerically under each lane. The full, uncut gels are shown in the Supplemental Material. (E) ChIP of SRC-2 and SREBP-1 from the stable C4-2 cells shNT and sh18 showing the recruitment of these 2 proteins on the FASN promoter. The amplicons tested were either from a proximal promoter region (–140 to –40 bp) or an unconserved upstream region (–2,000 bp) from the transcriptional start site. IgG antibody was used as a control, and data are presented as a percentage of input chromatin (n = 4). Schematic shows the SRE, GC box, E box, and TATA elements on the FASN promoter. Data represent the mean ± SEM. *P < 0.05 by 2-way ANOVA with Tukey’s multiple comparisons test (E) and **P < 0.001 by Student’s t test (A, B, and D).

Figure 2

Figure 2. SRC-2 promotes lipogenesis by reductive glutamine metabolism.

(A) Mass isotopomer distribution of citrate extracted from the stable C4-2 cells shNT (nontargeting) and SRC-2 shRNA clones sh18 and sh19 cultured in the presence of 2 mM L-[U-13C]glutamine and 11 mM unlabeled glucose for 24 hours (n = 6). *P < 0.05 and **P < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test. (B and C) Citrate and α-ketoglutarate (α-KG) labeling from [1-13C]glutamine ([1-13C]gln) in the stable C4-2 cells shNT and sh19 (n = 3). (D) Mass isotopomer distribution of stearate labeling from [5-13C]glutamine ([5-13C]gln) in the stable C4-2 cells shNT and sh19 (n = 3). The mass spectrometric method used in this study failed to detect higher fatty acid isotopomers. *P < 0.05 by Student’s t test with Holm-Sidak multiple comparisons test. (E) qRT-PCR analysis of FASN, SCD, and SRC2 gene expression in the stable C4-2 cells shNT, sh18, and sh19 (n = 4). Relative mRNA expression was normalized to actin (housekeeping gene). (F) qRT-PCR analysis of FASN, SCD, and SRC2 gene expression in C4-2 cells expressing GFP (Adv GFP) and SRC-2 (Adv SRC-2) adenovirus (n = 3). Relative mRNA expression was normalized to actin (housekeeping gene). (G) Western blot analysis of FASN, SCD, and SRC-2 in the stable C4-2 cells shNT, sh18, and sh19 and in C4-2 cells expressing GFP adenovirus or SRC-2 adenovirus (n = 3). Actin was used to normalize protein loading. The full, uncut gels are shown in the Supplemental Material. Data are represented as the mean ± SEM. *P < 0.05 and **P < 0.001 by Student’s t test (B–F).

Figure 1

Figure 1. SRC-2 promotes lipogenesis in prostate tumor cells primarily from glutamine sources.

(A) Western blot showing expression of SRC-2 and actin in C4-2 cells stably expressing shNT and 2 different clones of SRC-2 shRNA (sh18 and sh19). Actin was used to normalize protein loading. The full, uncut gels are shown in the Supplemental Material. (B) Oil Red O staining of the stable C4-2 cells shNT, sh18, and sh19 showing the neutral lipid content of the cells. Cells were counterstained with the nuclear marker DAPI, and merged images are shown in the right panels. Scale bar: 10 μm. (C) Quantitative analysis of Oil Red O staining by measuring the absorbance (OD at 490 nm) of extracted dye (n = 4). (D–G) Targeted MS–based metabolomic analyses demonstrating the relative content of palmitic, stearic, palmitoleic, and oleic acids in C4-2 cells treated with control siRNA (siGFP) or 2 different SRC-2 siRNAs (siSRC-2 #1 and siSRC-2 #2) (n = 4/group). Data are represented as the mean ± SEM. **P < 0.001 by Student’s t test versus shNT or siGFP.

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