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TGFβ-induced phosphorylation of Par6 promotes migration and invasion in prostate cancer cells.

Mu Y, Zang G, Engström U, Busch C, Landström M - Br. J. Cancer (2015)

Bottom Line: Transforming growth factor β (TGFβ)-induced phosphorylation of Par6 on the conserved serine 345 is implicated in epithelial-to-mesenchymal transition (EMT) in breast cancer.Subcellular localisation of p-Par6(345) in migrating TGFβ-treated PC-3U cells was analysed by confocal imaging.TGFβ induced Par6 phosphorylation on Ser345 and its recruitment to the leading edge of the membrane ruffle in migrating PC-3U cells, where it colocalised with aPKCζ.

View Article: PubMed Central - PubMed

Affiliation: Pathology Section, Department of Medical Biosciences, Umeå University, Umeå SE-901 85, Sweden.

ABSTRACT

Background: The Par complex - comprising partition-defective 6 (Par6), Par3, and atypical protein kinase C (aPKC) - is crucial for cell polarisation, the loss of which contributes to cancer progression. Transforming growth factor β (TGFβ)-induced phosphorylation of Par6 on the conserved serine 345 is implicated in epithelial-to-mesenchymal transition (EMT) in breast cancer. Here we investigated the importance of phosphorylated Par6 in prostate cancer.

Methods: We generated a p-Par6(345)-specific antibody and verified its specificity in vitro. Endogenous p-Par6(345) was analysed by immunoblotting in normal human prostate RWPE1 and prostate cancer (PC-3U) cells. Subcellular localisation of p-Par6(345) in migrating TGFβ-treated PC-3U cells was analysed by confocal imaging. Invasion assays of TGFβ-treated PC-3U cells were performed. p-Par6 expression was immunohistochemically analysed in prostate cancer tissues.

Results: TGFβ induced Par6 phosphorylation on Ser345 and its recruitment to the leading edge of the membrane ruffle in migrating PC-3U cells, where it colocalised with aPKCζ. The p-Par6-aPKCζ complex is important for cell migration and invasion, as interference with this complex prevented prostate cancer cell invasion. High levels of activated Par6 correlated with aggressive prostate cancer.

Conclusions: Increased p-Par6Ser(345) levels in aggressive prostate cancer tissues and cells suggest that it could be a useful novel biomarker for predicting prostate cancer progression.

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Related in: MedlinePlus

p-Par6Ser345 expression in prostate cells and tissues and its clinical correlation. p-Par6Ser345 expression was evaluated by immunohistochemical staining in tissue microarray cores (TMA) of human prostate cancer tissues. (A) Representative images of p-Par6Ser345 staining in normal prostate tissue, moderately differentiated prostatic adenocarcinoma with a Gleason score (GS) of 3+3 (GS<7), prostatic adenocarcinoma with pseudoglandular morphology and GS of 4+3 (GS=7), and poorly differentiated prostatic adenocarcinoma with GS of 4+4 (GS>7). (B) p-Par6Ser345 staining was quantified using ImageJ. Mean±s.e.m. of estimated grey-scale values obtained from the following groups: normal tissue (n=50), GS<7 (n=21), GS=7 (n=26), and GS>7 (n=30). *P<0.05 for between-group differences, Student's t-tests. (C) The prostate cancer cell line PC-3U and the noncancerous prostate epithelial cell line RWPE1 were treated with TGFβ as indicated, lysed, and immunoblotted as indicated. (D) Bar graphs showing relative levels of p-Par6Ser345 present the mean±s.d. from three independent experiments. *P<0.05, Student's t-test. (E) PC-3U and RPWE1 cells were treated with TGFβ followed by fixation in 4% paraformaldehyde and immunofluorescence staining for E-cadherin and N-cadherin. Nuclei were stained with DAPI. Scale bar=20 μm.
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fig5: p-Par6Ser345 expression in prostate cells and tissues and its clinical correlation. p-Par6Ser345 expression was evaluated by immunohistochemical staining in tissue microarray cores (TMA) of human prostate cancer tissues. (A) Representative images of p-Par6Ser345 staining in normal prostate tissue, moderately differentiated prostatic adenocarcinoma with a Gleason score (GS) of 3+3 (GS<7), prostatic adenocarcinoma with pseudoglandular morphology and GS of 4+3 (GS=7), and poorly differentiated prostatic adenocarcinoma with GS of 4+4 (GS>7). (B) p-Par6Ser345 staining was quantified using ImageJ. Mean±s.e.m. of estimated grey-scale values obtained from the following groups: normal tissue (n=50), GS<7 (n=21), GS=7 (n=26), and GS>7 (n=30). *P<0.05 for between-group differences, Student's t-tests. (C) The prostate cancer cell line PC-3U and the noncancerous prostate epithelial cell line RWPE1 were treated with TGFβ as indicated, lysed, and immunoblotted as indicated. (D) Bar graphs showing relative levels of p-Par6Ser345 present the mean±s.d. from three independent experiments. *P<0.05, Student's t-test. (E) PC-3U and RPWE1 cells were treated with TGFβ followed by fixation in 4% paraformaldehyde and immunofluorescence staining for E-cadherin and N-cadherin. Nuclei were stained with DAPI. Scale bar=20 μm.

Mentions: To confirm the potential role of p-Par6 as a predictive biomarker, we analysed p-Par6 expression by using immunohistochemical staining in TMA sections from 127 prostate cancer samples, including 50 samples of adjacent normal prostatic tissue, 21 samples with GS<7, 26 samples with GS=7, and 30 samples with GS⩾7. Figure 5A shows representative images of p-Par6 expression. In normal prostate tissue, low levels of p-Par6 were detected in the glandular epithelium and strictly localised in the apical membrane towards the lumen and the basolateral border (Figure 5A). In contrast, samples of intermediate-grade (GS<7) and high-grade prostate cancer (GS⩾7) showed increased p-Par6 expression throughout the malignant epithelium. Interestingly, the p-Par6 level corresponded with the volume of malignant epithelium. Furthermore, p-Par6 expression was dramatically increased in poorly differentiated carcinoma where cancer cells proliferated and invaded into glandular lumens and the surrounding mesenchymal tissue (GS⩾7) (Figure 5A). Figure 5B presents the average grey-scale values for intensity of immunohistochemical staining for p-Par6, showing a significant greater p-Par6 expression in tumour samples compared with normal tissue samples (P<0.05).


TGFβ-induced phosphorylation of Par6 promotes migration and invasion in prostate cancer cells.

Mu Y, Zang G, Engström U, Busch C, Landström M - Br. J. Cancer (2015)

p-Par6Ser345 expression in prostate cells and tissues and its clinical correlation. p-Par6Ser345 expression was evaluated by immunohistochemical staining in tissue microarray cores (TMA) of human prostate cancer tissues. (A) Representative images of p-Par6Ser345 staining in normal prostate tissue, moderately differentiated prostatic adenocarcinoma with a Gleason score (GS) of 3+3 (GS<7), prostatic adenocarcinoma with pseudoglandular morphology and GS of 4+3 (GS=7), and poorly differentiated prostatic adenocarcinoma with GS of 4+4 (GS>7). (B) p-Par6Ser345 staining was quantified using ImageJ. Mean±s.e.m. of estimated grey-scale values obtained from the following groups: normal tissue (n=50), GS<7 (n=21), GS=7 (n=26), and GS>7 (n=30). *P<0.05 for between-group differences, Student's t-tests. (C) The prostate cancer cell line PC-3U and the noncancerous prostate epithelial cell line RWPE1 were treated with TGFβ as indicated, lysed, and immunoblotted as indicated. (D) Bar graphs showing relative levels of p-Par6Ser345 present the mean±s.d. from three independent experiments. *P<0.05, Student's t-test. (E) PC-3U and RPWE1 cells were treated with TGFβ followed by fixation in 4% paraformaldehyde and immunofluorescence staining for E-cadherin and N-cadherin. Nuclei were stained with DAPI. Scale bar=20 μm.
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fig5: p-Par6Ser345 expression in prostate cells and tissues and its clinical correlation. p-Par6Ser345 expression was evaluated by immunohistochemical staining in tissue microarray cores (TMA) of human prostate cancer tissues. (A) Representative images of p-Par6Ser345 staining in normal prostate tissue, moderately differentiated prostatic adenocarcinoma with a Gleason score (GS) of 3+3 (GS<7), prostatic adenocarcinoma with pseudoglandular morphology and GS of 4+3 (GS=7), and poorly differentiated prostatic adenocarcinoma with GS of 4+4 (GS>7). (B) p-Par6Ser345 staining was quantified using ImageJ. Mean±s.e.m. of estimated grey-scale values obtained from the following groups: normal tissue (n=50), GS<7 (n=21), GS=7 (n=26), and GS>7 (n=30). *P<0.05 for between-group differences, Student's t-tests. (C) The prostate cancer cell line PC-3U and the noncancerous prostate epithelial cell line RWPE1 were treated with TGFβ as indicated, lysed, and immunoblotted as indicated. (D) Bar graphs showing relative levels of p-Par6Ser345 present the mean±s.d. from three independent experiments. *P<0.05, Student's t-test. (E) PC-3U and RPWE1 cells were treated with TGFβ followed by fixation in 4% paraformaldehyde and immunofluorescence staining for E-cadherin and N-cadherin. Nuclei were stained with DAPI. Scale bar=20 μm.
Mentions: To confirm the potential role of p-Par6 as a predictive biomarker, we analysed p-Par6 expression by using immunohistochemical staining in TMA sections from 127 prostate cancer samples, including 50 samples of adjacent normal prostatic tissue, 21 samples with GS<7, 26 samples with GS=7, and 30 samples with GS⩾7. Figure 5A shows representative images of p-Par6 expression. In normal prostate tissue, low levels of p-Par6 were detected in the glandular epithelium and strictly localised in the apical membrane towards the lumen and the basolateral border (Figure 5A). In contrast, samples of intermediate-grade (GS<7) and high-grade prostate cancer (GS⩾7) showed increased p-Par6 expression throughout the malignant epithelium. Interestingly, the p-Par6 level corresponded with the volume of malignant epithelium. Furthermore, p-Par6 expression was dramatically increased in poorly differentiated carcinoma where cancer cells proliferated and invaded into glandular lumens and the surrounding mesenchymal tissue (GS⩾7) (Figure 5A). Figure 5B presents the average grey-scale values for intensity of immunohistochemical staining for p-Par6, showing a significant greater p-Par6 expression in tumour samples compared with normal tissue samples (P<0.05).

Bottom Line: Transforming growth factor β (TGFβ)-induced phosphorylation of Par6 on the conserved serine 345 is implicated in epithelial-to-mesenchymal transition (EMT) in breast cancer.Subcellular localisation of p-Par6(345) in migrating TGFβ-treated PC-3U cells was analysed by confocal imaging.TGFβ induced Par6 phosphorylation on Ser345 and its recruitment to the leading edge of the membrane ruffle in migrating PC-3U cells, where it colocalised with aPKCζ.

View Article: PubMed Central - PubMed

Affiliation: Pathology Section, Department of Medical Biosciences, Umeå University, Umeå SE-901 85, Sweden.

ABSTRACT

Background: The Par complex - comprising partition-defective 6 (Par6), Par3, and atypical protein kinase C (aPKC) - is crucial for cell polarisation, the loss of which contributes to cancer progression. Transforming growth factor β (TGFβ)-induced phosphorylation of Par6 on the conserved serine 345 is implicated in epithelial-to-mesenchymal transition (EMT) in breast cancer. Here we investigated the importance of phosphorylated Par6 in prostate cancer.

Methods: We generated a p-Par6(345)-specific antibody and verified its specificity in vitro. Endogenous p-Par6(345) was analysed by immunoblotting in normal human prostate RWPE1 and prostate cancer (PC-3U) cells. Subcellular localisation of p-Par6(345) in migrating TGFβ-treated PC-3U cells was analysed by confocal imaging. Invasion assays of TGFβ-treated PC-3U cells were performed. p-Par6 expression was immunohistochemically analysed in prostate cancer tissues.

Results: TGFβ induced Par6 phosphorylation on Ser345 and its recruitment to the leading edge of the membrane ruffle in migrating PC-3U cells, where it colocalised with aPKCζ. The p-Par6-aPKCζ complex is important for cell migration and invasion, as interference with this complex prevented prostate cancer cell invasion. High levels of activated Par6 correlated with aggressive prostate cancer.

Conclusions: Increased p-Par6Ser(345) levels in aggressive prostate cancer tissues and cells suggest that it could be a useful novel biomarker for predicting prostate cancer progression.

Show MeSH
Related in: MedlinePlus