KIBRA promotes prostate cancer cell proliferation and motility

Abstract

KIBRA is a regulator of the Hippo-yes-associated protein (YAP) pathway, which plays a critical role in tumorigenesis. In the present study, we show that KIBRA is a positive regulator in prostate cancer cell proliferation and motility. We found that KIBRA is transcriptionally upregulated in androgen-insensitive LNCaPC4-2 and LNCaP-C81 cells compared to parental androgen-sensitive LNCaP cells. Ectopic expression of KIBRA enhances cell proliferation, migration and invasion in both immortalized and cancerous prostate epithelial cells. Accordingly, knockdown of KIBRA reduces migration, invasion and anchorage-independent growth in LNCaP-C4-2/C81 cells. Moreover, KIBRA expression is induced by androgen signaling and KIBRA is partially required for androgen receptor signaling activation in prostate cancer cells. In line with these findings, we further show that KIBRA is overexpressed in human prostate tumors. Our studies uncover unexpected results and identify KIBRA as a tumor promoter in prostate cancer.

Keywords: AR signaling; KIBRA; motility; proliferation; prostate cancer.

Fig.1. KIBRA is transcriptionally upregulated in androgen-insensitive prostate cancer cells

(A, B) RWPE-1, LNCaP, LNCaP-C4-2, LNCaP-C33, and LNCaP-C81 cells lines were cultured as described in ‘Material and methods’. The total cell lysates were probed with the indicated antibodies. SE: short exposure; LE: long exposure. (C) Quantitative RT-PCR of WWC1/2/3 in LNCaP and its sublines. (D) LNCaP and LNCaP-C4-2 cells were treated with cycloheximide (CHX, 50 µg/ml) at the indicated time points and the total cell lysates were analyzed with the indicated antibodies. The relative intensity was shown from the average of three blots (Image J). (E) LNCaP-C4-2 cell lines expressing control shRNA or shRNA against YAP were utilized to determine the indicated protein levels by Western blotting. Data were obtained from three (n=3) independent experiments (A–E) and expressed as mean ± s.e.m (C). *: p<0.05; **: p<0.01; ***: p<0.001 (t-test).

Fig.2. KIBRA promotes proliferation, migration and invasion in RWPE-1 cells

(A) Establishment of RWPE-1 cells expressing vector (control) or KIBRA. WWC2 protein and WWC3 mRNA levels were determined in these cells. (B) The proliferation curve of the cell lines established in (A). (C, D) Cell migration effect was determined with the cell lines in (A). Representative photos for migrating cells are shown in (D). (E, F) Cell invasion effect was determined with the cell lines in (A). Representative photos for invading cells are shown in (F). (G) Anchorage-independent growth (colony assay in soft agar) was determined with the cell lines established in (A). No colony was formed in RWPE-1-vector and –KIBRA cells. (H) Total cell lysates from cell lines established in (A) were probed with the indicated antibodies. Data were obtained from three (n=3) independent experiments (A–H) and expressed as mean ± s.e.m (B, C). *: p<0.05; **: p<0.01; ***: p<0.001 (t-test).

Fig.3. KIBRA promotes proliferation, migration and invasion in LNCaP cells

(A) Establishment of LNCaP cells expressing vector (control) or KIBRA. WWC2 protein and WWC3 mRNA levels were determined in these cells. (B) The proliferation curve of the cell lines established in (A). (C, D) Cell invasion effect was determined with the cell lines established in (A). Representative photos for invading cells are shown in (D). (E, F) Cell migration effect was determined with the cell lines established in (A). Representative photos for invading cells are shown in (F). (G, H) Anchorage-independent growth (colony assay in soft agar) was determined with the cell lines established in (A). (I) Representative photos of LNCaP-vector or LNCaP-KIBRA cells that have been cultured in normal medium (FBS) or androgen deprivation medium (CSS) for 4 days. FBS: fetal bovine serum; CSS: charcoal striped serum. Data were obtained from three (n=3) independent experiments (A–I) and expressed as mean ± s.e.m (B, C, E, G). *: p<0.05; **: p<0.01; ***: p<0.001 (t-test).

Fig.4. KIBRA knockdown impairs motility and anchorage-independent growth in prostate cancer cells

(A) Establishment of LNCaP-C4-2/C81 cells expressing control shRNA (shControl) or shRNA against KIBRA (shKIBRA). (B) The proliferation curves (determined by MTT assays) of the cell lines established in (A). (C, D) Cell migration effect was determined with the cell lines established in (A). Representative photos for migrating cells are shown in (C). (E) Cell invasion effect was determined with the cell lines established in (A). (F) Colony formation assays in LNCaP-C4-2 cells established in (A). Data were obtained from three (n=3) independent experiments (A–F) and expressed as mean ± s.e.m (B, D-F). *: p<0.05; **: p<0.01; ***: p<0.001 (t-test).

Fig. 5. KIBRA is induced by R1881 and is required for AR signaling activation

(A) Total cell lysates from various stable cell lines were probed with the indicated antibodies. (B) LNCaP cells were treated with R1881 as indicated. Total protein lysates were subjected to Western blot analysis. (C) LNCaP-C4-2 and LNCaP-C81 cells were treated with R1881 (1 nM) for 24 h and total protein lysates were subjected to Western blot analysis with the indicated antibodies. (D, E) Quantitative RT-PCR in LNCaP or LNCaP-C81 cells treated or not treated with R1881 (1 nM) for 24 h. (F) LNCaP-C81 cells were treated with cycloheximide (CHX, 50 µg/ml) at the indicated time points and the total cell lysates were analyzed with the indicated antibodies. The relative intensity was shown from the average of three blots (Image J). (G) Quantitative RT-PCR for PSA in LNCaP-C4-2 cells with control or KIBRA knockdown. (H) Total cell lysates from various stable cell lines were probed with the indicated antibodies. Data were obtained from three (n=3) independent experiments (A–H) and expressed as mean ± s.e.m (D–G). *: p<0.05; ***: p<0.001 (t-test).

Fig. 6. KIBRA is overexpressed in prostate cancer

(A–D) KIBRA mRNA is increased in clinical samples. Data were mined from published studies through biogps.org (A) and oncomine.org (B–D). The original references are: [47] (A), [48] (B), [49] (C), and [50] (D). The box (B–D) extends from the 25th to 75th percentiles. The line is plotted at the median and the whiskers go to the smallest and the largest value for each group (B–D). (E) Total protein lysates from prostate tumors and normal prostate tissue were subjected to Western blot analysis with the indicated antibodies.