Research ArticleCancer therapy

Loss of SPDEF and gain of TGFBI activity after androgen deprivation therapy promote EMT and bone metastasis of prostate cancer

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Science Signaling  15 Aug 2017:
Vol. 10, Issue 492, eaam6826
DOI: 10.1126/scisignal.aam6826
  • Fig. 1 TGFBI is associated with TGFβ signaling and EMT markers in prostate cancer.

    (A) Western blotting for TGFBI in AC3 cells in the presence (E+) or absence (E) of EpCAM. (B and C) Quantification of TGFBI, VIM, and SLUG mRNAs in AC3 cells after 24 hours treatment with TGFβ (B) or a TGFβ inhibitor (SB431542) (C) relative to vehicle (Veh)– or dimethyl sulfoxide (DMSO)–treated cells. (D and E) Quantification of TGFBI mRNA in a panel of prostate cancer cells after treatment with TGFβ (D) or SB431542 (E) as in (B) and (C). (F) Western blotting of lysates from cells after expression by transient transfection with TGFBI or empty vector (EV). (G) Western blotting of lysates from RasB1 cells transfected with control (Luc) or TGFBI-targeted shRNA and treated with vehicle (−) or TGFβ. Blots are representative of three independent experiments. Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 AR signaling negatively regulates TGFBI.

    (A) TGFBI mRNA expression in 22Rv1 or LNCaP cells treated with a control (NC) or AR-targeted siRNA (siAR) for 48 hours. (B) Western blotting of LNCaP cells after AR knockdown and TGFβ inhibition (SB431542) for 24 hours. (C) Western blotting of AR-negative cells stably transfected with either a control (EV) or AR-expressing (AR) vector. (D) Western blotting of RasB1 cells stably expressing AR (or empty vector) and treated with TGFβ for 24 hours. (E and F) Abundance of TGFβ at the mRNA (E) or protein (F) level in control or AR-overexpressing LNCaP cells and treated with DHT or vehicle (−) for 24 hours. (G and H) As in (E) and (F) in cells treated with AR inhibitor MDV3100 (MDV) or vehicle for 24 hours. (I) Western blotting of LNCaP cells transfected with AR-targeted siRNA (siAR) for 48 hours then DHT for 24 hours. (J) Western blotting of parental cultures of PC3 cells and those transiently transfected with AR then treated with DHT. (K) Mean TGFBI expression in TCGA prostate cancer data set grouped by relative AR activity based on a gene set [Nelson et al. (31) or Wang et al. (32); n = 207 per group]. Significance was determined by Student’s t test. Western blots are representative of three independent experiments. Data are means ± SEM. n = 3 biological replicates. **P < 0.01, ***P < 0.001.

  • Fig. 3 Reduction of TGFBI inhibits metastasis and growth of prostate cancer cells.

    (A) Migration assay in stable cell lines containing either a control (shLacZ) or a TGFBI-targeted shRNA (shTGFBI) vector, n = 3 biological replicates. Selected images are shown on the right. (B) Confirmation of the knockdown efficiency by shTGFBI in stable cell lines. (C to F) Survival analysis by a log-rank test (C) and bioluminescence imaging (BLI) analysis (D and E) and histology (F) of prostate tumor cell lesions in the bone and brain in mice 30 days after receiving intracardiac injection of RasB1 cells stably transfected with control (shLacZ) or TGFBI-targeted shRNA (shTGFBI). n = 6 mice per group. In (F): B, bone; T, tumor; BM, bone marrow. Scale bars, 100 μm. (G and H) Proliferation (G) and colony formation (H) by RasB1 cells stably transfected with shLacZ or shTGFBI. OD, optical density. (I to K) Growth (I), images (J), and weight (K) of tumor xenografts in mice 4 weeks after subcutaneous inoculation with RasB1 cells stably expressing shLacZ or shTGFBI. n = 6 mice per group. Data are means ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 SPDEF induction mediates suppression of TGFBI by AR signaling.

    (A) Western blotting for SPDEF and TGFBI in AR-negative (DU145, RasB1, and PC3) and AR-positive cells (22Rv1, LNCaP, and LNCaP-AR) in prostate cancer cell lines. (B and C) Abundance of SPDEF at the mRNA (B) and protein (C) levels in LNCaP and LNCaP-AR cells after a 24-hour treatment with DHT or MDV3100 relative to controls (vehicle or DMSO, respectively). (D to F) Effect of DHT, relative to that of vehicle, on the expression of SPDEF (D) or TGFBI (E) or protein abundance (F) in LNCaP-AR cells transiently transfected with vectors expressing control (shLuc) or SPDEF-targeted shRNA (shSPDEF). (G) Pearson correlation analysis between TGFBI and SPDEF expression in TCGA (n = 414) and Taylor (n = 111) prostate carcinoma data sets. Blots are representative of three independent experiments. Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 AR signaling inhibits TGFBI through SPDEF-dependent transcription.

    (A and B) Abundance of SPDEF and TGFBI mRNA (A) and protein (B) in the LNCaP-AR cells transfected with control (shLuc) or SPDEF-targeted shRNA (S1, shSPDEF-1; S2, shSPDEF-2). (C and D) Abundance of SPDEF and TGFBI mRNA (C) and protein (D) in PC3 and RasB1 cells transiently transfected with a control (EV) or SPDEF-expressing vector. (E) Top: Schematic of the predicted SREs in the promoter of human TGFBI. Bottom: Schematic of TGFBI expression reporter constructs showing the wild-type and mutant sequences of SRE1 and SRE5. (F and G) ChIP assays in LNCaP-AR cells treated with DHT (F) or MDV3100 (G). Antibody against GAPDH served as the control. Enrichment is given as a percentage of the total input and then normalized to immunoglobulin G (IgG). (H) Relative MFI of the TGFBI reporters (SRE1 and SRE5) in LNCaP-AR cells after treatment with DHT, MDV, and their combination (DHT + MDV). (I) Relative MFI of wild-type or mutant-SRE (M1, M5) TGFBI reporters in LNCaP-AR cells. (J and K) TGFBI reporter activity in response to DHT in LNCaP-AR cells (J) or transient SPDEF overexpression in RasB1 cells (K). Western blots are representative of three independent experiments. Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 TGFBI promotes malignant phenotypes in prostate cancer cells.

    (A and B) Effect of SPDEF knockdown in LNCaP-AR cells on proliferation (A) and migration (B) (shLuc, control; S1, shSPDEF-1; S2, shSPDEF-2). (C) Proliferation of RasB1 cells cotransfected with control vector (EV/con), SPDEF (SPDEF/con), and/or TGFBI (SPDEF/TGFBI) (D) Western blotting of samples from cells assessed in (C), representative of three independent experiments. (E and F) Migration (E) and invasion (F) abilities in RasB1 cells described in (C). Representative images are shown. (G to J) Effect of transient expression of TGFBI on proliferation in LNCaP-AR cell lines grown in CSS-containing medium (G and I) and treated with MDV3100 (H and J). Images are representative of n = 6 experiments. Data are means ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 7 Patients receiving ADT show reduced SPDEF and increased TGFBI and p-SMAD2.

    (A) Pearson correlation analysis of TGFBI and SPDEF mRNA in clinical tissue samples containing 15 cases collected from the Taipei Medical University Joint Human Biological Database. (B and C) Representative images of immunohistochemistry (IHC) staining of consecutive sections of a prostate cancer tissue array from US Biomax; n = 40. Scale bars, 100 μm (B). Association analysis of SPDEF and TGFBI positive staining status in patient samples with Gleason score by a χ2 test (C). (D to F) IHC staining for SPDEF, TGFBI, and phosphorylated SMAD2 (D) and analysis of nuclear SPDEF (E) and cytoplasmic TGFBI (F) in prostate cancer tissue sections from patients before and after ADT. n = 17 samples from Taipei Medical University–Wan Fang Hospital. Scale bars, 100 μm. Statistical analysis by a two-tailed Student’s t test. (G) Survival analysis of patients in a TCGA prostate cancer data set (n = 414) based on TGFBI and SPDEF expression. A log-rank test was used for the survival curve analysis. (H) Proposed model for how AR inhibits TGFβ signaling, EMT and metastatic progression of prostate cancer cells through repression of the TGFBI promoter by SPDEF binding, and how ADT may induce metastatic progression by inhibiting this tumor suppressive AR signaling axis.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/492/eaam6826/DC1

    Fig. S1. Induction of TGFBI is involved in the activated EMT, activated TGFβ signaling, and inactivated AR signaling signature in prostate cancer.

    Fig. S2. The effect of TGFBI in AR-positive prostate cancer cells.

    Fig. S3. Induction of TGFBI is associated with decreased androgen-activated SPDEF expression in prostate cancer patient samples.

    Fig. S4. Role of TGFBI in EMT after SPDEF knockdown in AR-inhibited prostate cancer cells.

    Table S1. Primer sequences of the promoter reporter constructs.

    Table S2. Real-time qPCR primer sequences.

    Table S3. Western blotting antibodies.

    Table S4. ChIP antibodies and primer sequences.

    Table S5. IHC staining antibodies.

  • Supplementary Materials for:

    Loss of SPDEF and gain of TGFBI activity after androgen deprivation therapy promote EMT and bone metastasis of prostate cancer

    Wei-Yu Chen, Yuan-Chin Tsai, Hsiu-Lien Yeh, Florent Suau, Kuo-Ching Jiang, Ai-Ning Shao, Jiaoti Huang, Yen-Nien Liu*

    *Corresponding author. Email: liuy{at}tmu.edu.tw

    This PDF file includes:

    • Fig. S1. Induction of TGFBI is involved in the activated EMT, activated TGFβ signaling, and inactivated AR signaling signature in prostate cancer.
    • Fig. S2. The effect of TGFBI in AR-positive prostate cancer cells.
    • Fig. S3. Induction of TGFBI is associated with decreased androgen-activated SPDEF expression in prostate cancer patient samples.
    • Fig. S4. Role of TGFBI in EMT after SPDEF knockdown in AR-inhibited prostate cancer cells.
    • Table S1. Primer sequences of the promoter reporter constructs.
    • Table S2. Real-time qPCR primer sequences.
    • Table S3. Western blotting antibodies.
    • Table S4. ChIP antibodies and primer sequences.
    • Table S5. IHC staining antibodies.

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    Citation: W.-Y. Chen, Y.-C. Tsai, H.-L. Yeh, F. Suau, K.-C. Jiang, A.-N. Shao, J. Huang, Y.-N. Liu, Loss of SPDEF and gain of TGFBI activity after androgen deprivation therapy promote EMT and bone metastasis of prostate cancer. Sci. Signal. 10, eaam6826 (2017).

    © 2017 American Association for the Advancement of Science

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