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. 2011;6(8):e23057.
doi: 10.1371/journal.pone.0023057. Epub 2011 Aug 3.

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Jonas Sjölund  1 , Anna-Karin Boström, David Lindgren, Sugata Manna, Aristidis Moustakas, Börje Ljungberg, Martin Johansson, Erik Fredlund, Håkan Axelson
Affiliations

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Jonas Sjölund et al. PLoS One. 2011.

Abstract

Background: Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/principal findings: We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student's t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion: An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Notch inhibition in CCRCC cells attenuates TGF-β signaling.
(A) Western blot analysis of icNotch1 in 786-O and SKRC-10 cells treated for 24 h with DAPT (+) or vehicle control (−). Cell extracts were separated by SDS-PAGE and after transfer the membrane was probed with an anti-cleaved Notch1 antibody. The blot was reprobed with an anti-ACTIN antibody to ensure equal loading. (B) GSEA of a TGF-β upregulated gene set classified as upregulated by TGF-β1 treatment of skin fibroblasts at 1–4 h in the SKRC-10 microarray data. Isolated and purified RNA from SKRC-10 cells, treated with DAPT or vehicle control in 1% FCS supplemented media for 24 h, was used in oligomer microarray experiments. Genes in the SKRC-10 data list were ranked for downregulation upon DAPT treatment by measure of rank product analysis FDR. Gene sets with a q-value below 0.25 were considered significantly enriched. Genes upregulated by TGF-β1 from the gene set were significantly enriched in the list ranked by downregulated by DAPT (NES = 1.75, q = 0.09). (C) Gene expression analysis of indicated genes reported to be activated in response to TGF-β in DAPT or vehicle control (c) treated SKRC-10 cells. Cells were treated as indicated in (B). Data represents mean log2 ratios+95% confidence intervals of three separate experiments. (D) Gene expression analysis of indicated genes reported to be repressed in response to TGF-β in DAPT or vehicle control (c) treated SKRC-10 cells. Cells were treated as indicated in (B). Data represents mean log2 ratios+95% confidence intervals of three separate experiments. (E) Notch inhibition in CCRCC cells decreases the mRNA expression of the Notch primary target HES1 and the TGF-β target genes SERPINE1 and SKIL. Q-PCR analyses of HES1, SKIL and SERPINE1 mRNA levels in 786-O or SKRC-10 cells treated for 24 h with DAPT or vehicle control (c). mRNA levels were normalized to SDHA, YWHAZ and UBC expression and data represents mean+95% confidence intervals of three separate experiments. ***, ** and * indicates statistical significant changes (two-sided Student's t-test, p<0.001, p<0.01 and p<0.05 respectively).
Figure 2
Figure 2. High TGF-β signaling pathway activity is associated with decreased patient survival.
(A) Kaplan-Meier curves comparing disease-specific survival among 176 CCRCC patients divided into quartiles based on the TGF-β activity score. High TGF-β pathway activity score (76–100%) was significantly (log-rank, p = 0.006) associated with a worse disease-specific survival. (B) Immunohistochemical analyses using phospho-specific SMAD2 (pSMAD2) antibody of control (c) or SB431542 treated 786-O cells. Sections were counterstained with hematoxylin-eosin. Original magnification, ×40. (C) pSMAD2 is expressed in vivo. Immunohistochemistry of pSMAD2 in nine CCRCC tumors, that all showed positive nuclear staining. Sections were counterstained with hematoxylin-eosin. Original magnification, ×40.
Figure 3
Figure 3. The TGF-β signaling pathway is functional in CCRCC cells.
(A) Immunoblotting using TGFBR1 and TGFBR2 antibodies of cell lysates from 786-O and SKRC-10 cells. (B and C) pSMAD2 and SMAD-1/2/3 protein levels in cell lysates from 786-O (B) and SKRC-10 (C) cells stimulated with vehicle control (−) or 0.25 ng/ml TGF-β1 (+) for the indicated time points. (D) Western blot analysis using pSMAD2 antibody and SMAD2 antibody, of cell lysates from 786-O and SKRC-10 cells treated with vehicle (−) or SB431542 (+) for 4 h. (E) ELISA measurement of TGF-β1 levels in serum free media from 786-O and SKRC-10 cells grown for 48 h. Data represents mean+95% confidence intervals of three separate experiments. (F and G) 786-O (F) and SKRC-10 (G) cells were transfected with the SMAD regulated (CAGA)12-Luc reporter construct, treated with vehicle control (c), increasing concentrations of TGF-β1 or 0.25 ng/ml TGF-β1 and SB431542 for 24 h and analyzed for relative luciferase activity. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (H) 786-O or SKRC-10 cells were transfected with the (CAGA)12-Luc reporter construct, treated with vehicle control (c) or SB431542 for 24 h and analyzed for relative luciferase activity. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (I) Q-PCR analyses of JUNB and SERPINE1 mRNA levels in SKRC-10 cells treated with vehicle control (c), SB431542 and/or 0.25 ng/ml TGF-β1 for 4 h. mRNA levels were normalized to SDHA, YWHAZ and UBC expression and data represents mean+95% confidence intervals of three separate experiments. ***, ** and * indicates statistical significant changes (two-sided Student's t-test, p<0.001, p<0.01 and p<0.05 respectively).
Figure 4
Figure 4. Notch inhibition alters endogenous and TGF-β1 induced activation of the TGF-β signaling pathway.
(A) Immunoblotting using pSMAD2 antibody and SMAD2 antibody, of cell lysates from 786-O cells stimulated with vehicle control (−) or DAPT (+) for the indicated time points. (B) Immunoblotting using Notch1 antibody and pSMAD2 antibody of cell lysates from 786-O and SKRC-10 cells transfected with control siRNA (c-si) or siRNA against Notch1 (siN-1). Cells were harvested after 24 h of transfection. (C) Western blot analysis, using pSMAD2 antibody and SMAD-1/2/3 antibody of cell lysates from 786-O and SKRC-10 cells stimulated with 0.25 ng/ml TGF-β1 and vehicle (−) or 0.25 ng/ml TGF-β1 and DAPT (+) for 4 h. (D) 786-O and SKRC-10 cells transfected with the (CAGA)12-Luc reporter construct and treated with vehicle control (c) or DAPT for 24 h and followed by measurment of relative luciferase activity. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (E) Relative luciferase activity in extracts from 786-O cells transfected with the (CAGA)12-Luc reporter construct and vector control (Vector C) or icNotch1 expression vector followed by treatment with DAPT or vechicle control (c) for 24 h. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated and vector control transfected cells. (F and G) 786-O (F) and SKRC-10 (G) cells were transfected with the (CAGA)12-Luc reporter construct, treated with vehicle control (c), 0.25 ng/ml TGF-β1 and vehicle control or 0.25 ng/ml TGF-β1 and DAPT for 24 h and analyzed for relative luciferase activity. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (H) Q-PCR analyses of JUNB and SERPINE1 mRNA levels in SKRC-10 cells treated with vehicle control (c), 0.25 ng/ml TGF-β1 and vehicle (c) or 0.25 ng/ml TGF-β1 and DAPT for 4 h. mRNA levels were normalized to SDHA, YWHAZ and UBC expression and data represents mean+95% confidence intervals of three seperate experiments. ***, ** and * indicates statistical significant changes (two-sided Student's t-test, p<0.001, p<0.01 and p<0.05 respectively).
Figure 5
Figure 5. Notch inhibition attenuates CCRCC cell migration and invasion.
(A) [3H]thymidine incorporation of 786-O and SKRC-10 cells grown for indicated hours (h) in the presence of 1.0 ng/ml TGF-β1 or treated with vehicle control (c). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (B) Cell migration assessed by Boyden chamber assays of 786-O and SKRC-10 cells treated with vehicle control (c) or DAPT. The cells were allowed to migrate towards the lower compartment for 4 h (786-O) or 5 h (SKRC-10). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (C) Cell migration as determined by Boyden chamber assays of 786-O and SKRC-10 cells transfected with non-specific control (c-si) or Notch1 (siN-1) specific siRNAs. After 24 h of transfection, cells were counted and seeded into the Boyden chamber. The cells were allowed to migrate towards the lower compartment for 4 h (786-O) or 5 h (SKRC-10). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (D) Cell migration as determined by Boyden chamber assays of SKRC-10 cells treated with vehicle control (c), 2 µM SB431542, 10 µM DAPT and control. The cells were allowed to migrate towards the lower compartment for 5 h. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (E) Cell migration analyses of SKRC-10 cells treated with vehicle control (c) or treated with 0.25 ng/ml TGF-β1 and vehicle (c) or 0.25 ng/ml TGF-β1 and DAPT. The cells were allowed to migrate towards the lower compartment for 5 h. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (F) Cell invasion as determined by Matrigel coated Boyden chamber assays of 786-O and SKRC-10 cells treated with vehicle control (c) or DAPT. The cells were allowed to invade and migrate towards the lower compartment containing 10% FCS for 16 h (786-O) or 21 h (SKRC-10). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. ***, ** and * indicates statistical significant changes (two-sided Student's t-test, p<0.001, p<0.01 and p<0.05 respectively). (G) TGF-β pathway activity correlates to metastatic spread of primary CCRCCs. CCRCCs from patients with either metastatic disease at diagnosis or that later developed metastasis (Metastatic, n = 13) showed a significantly elevated TGF-β signaling activity based on the 145-gene TGF-β signature as compared to tumors from patients with a localized disease and with no documented metastases during follow-up (Localized, n = 9) (two-sided Student's t-test, p = 0.044).
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