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. 2018 Nov 22;8(1):17259.
doi: 10.1038/s41598-018-35332-4.

Analysis of regulator of G-protein signalling 2 (RGS2) expression and function during prostate cancer progression

Anna Linder  1 , Malin Hagberg Thulin  2 , Jan-Erik Damber  3 , Karin Welén  3
Affiliations

Analysis of regulator of G-protein signalling 2 (RGS2) expression and function during prostate cancer progression

Anna Linder et al. Sci Rep. .

Abstract

Prostate cancer (PC) represents the second highest cancer-related mortality among men and the call for biomarkers for early discrimination between aggressive and indolent forms is essential. Downregulation of Regulator of G-protein signaling 2 (RGS2) has been shown in PC, however the underlying mechanism has not been described. Aberrant RGS2 expression has also been reported for other carcinomas in association to both positive and negative prognosis. In this study, we assessed RGS2 expression during PC progression in terms of regulation and impact on tumour phenotype and evaluated its prognostic value. Our experimental data suggest that the RGS2 downregulation seen in early PC is caused by hypoxia. In line with the common indolent phenotype of a primary PC, knockdown of RGS2 induced epithelial features and impaired metastatic properties. However, increased STAT3, TWIST1 and decreased E-cadherin expression suggest priming for EMT. Additionally, improved tumour cell survival and increased BCL-2 expression linked decreased RGS2 levels to fundamental tumour advantages. In contrast, high RGS2 levels in advanced PC were correlated to poor patient survival and a positive metastatic status. This study describes novel roles for RGS2 during PC progression and suggests a prognostic potential discriminating between indolent and metastatic forms of PC.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
RGS2 protein expression in patient specimens. RGS2 Immunohistochemistry (IHC) staining of TURP specimens. (a) Representative images of RGS2 expression in BPH and hormone-naïve PC specimens in stage T1b, showing the expression pattern of epithelial and stromal cells. Bar = 100 µm. (b) Quantitative comparisons of RGS2 expression of epithelial cells of BPH (n = 36) and PC (n = 28) (p < 0.00001, by Mann-Whitney test). (c) Intra sectional RGS2 difference in RGS2 expression between PC areas and adjacent normal areas (n = 8) (p = 0.12, Wilcoxon W paired test). (d) Quantitative comparison of RGS2 staining in BPH (n = 36) and PC associated stroma (n = 27) (p = 0.007, Mann-Whitney test).
Figure 2
Figure 2
RGS2 expression is inhibited by hypoxia. (a) Overview of RGS2 expression in LNCaP descending mouse tumours representative of RGS2 expression in correlation to blood vessels, proliferation and hypoxia (Bar = 100 µm). (b) Representative western blot from cell lysates obtained from cells cultured in a hypoxic atmosphere (H) (1% O2) for 72 hours and corresponding normoxic controls (N) (21% O2). HIF1A controls for hypoxic response and β-actin included for protein loading. (c) Representative WB from cell lysates obtained after 72 hours of CoCl2 treatment (T) and respective control (C). The blots were cropped. LNCaP and LNCaP-19 was run on the same gel for both experiments (Hypoxia and CoCl2) and PC-3 and WPMY were run on the same gel. All gels were run under the same conditions. The original pictures of the full-length western blots can be found in Supplementary Fig. S3.
Figure 3
Figure 3
High RGS2 expression is correlated with metastasis and poor prognosis. (a) Assessment of RGS2 staining in relation to metastasis in patients with advanced PC. Metastasis positive (n = 27) and negative (n = 11). (b) Representative images of RGS2 staining of matched specimens of M0 and M1. Bar represents 100 µm. Kaplan-Meier curves and log rank of (c) cancer specific survival and high respective low RGS2 expression (p = 0.041, by Log-rank test) and (d) cancer specific survival and M-stage (nM1 = 24, nM0 = 12, nMx = 7, p = 0.018, by Log-rank test).
Figure 4
Figure 4
Increased epithelial features in response to RGS2 downregulation. (a) Quantitative comparison of migrating properties of the sh-clones, presented as percentage of area of gap closure compared to the starting point. (n = 4; 24 h p = 0.042, 48 h p = 0.017, by two-tailed Student’s t test). (b) Representative images of scratch assay at analysed time points. (c) Magnification of the scratch for visualization of migration pattern. (d) Phase imaging of the Sh-clones for demonstration of morphology and growth pattern. (e) Quantification of clone formation (n = 4, p = 0.0051, by two-tailed Student’s t test). (f) Images of colony formation ability of low seeded clones stained with crystal violet.
Figure 5
Figure 5
Impact of decreased RGS2 expression on tumour survival and cell growth. (a) Total cell growth of the RGS2 knockdown clone (ShRGS2) and corresponding non-target control clone (ShNT) evaluated with trypan blue dye positive cell count. (b) Flow cytometric analysis of cell cycle profile of PI-stained ShRGS2 and ShNT cells cultured under standard conditions. (n = 3, SubG1 p = 0.019, G0/G1 p = 0.070 student’s t test) (c) qRT-PCR evaluation of RGS2 and BCL2 mRNA expression in vitro (n = 3; RGS2 p < 0.00001, BCL-2 p = 0.040, Student’s t test). (d) RGS2 and BCL-2 protein expression in vitro analysed with western blot. The blots were cropped. The original pictures for the evaluation of the clones can be found in Supplementary Fig. S42. (e) RGS2 and BCL-2 gene expression in subcutaneous tumours after implantation of ShRGS2 (n = 4) and ShNT (n = 4) evaluated with qRT-PCR (n = 4; RGS2 p = <0.00001, p = 0.0168, student’s t test). (f) Relative tumour growth in vivo (n = 4) and ShNT (n = 4) in BALB/c nude mice. (g) Tumour weight at experiment endpoint. (h) Comparison of Ki67 positive cells (p = 0.0277, student’s t test). (i) Comparison of total cell count per hot spots (p = 0.028, student’s t test). (j) Assessment of Ki67 negative cells (p = 0.045, student’s t test).
Figure 6
Figure 6
Affected Pathways and EMT initiation in response to RGS2 depletion. (a) Evaluation of STAT3 and AKT proteins in response to RGS2 knockdown using WB. (b) Overview of evaluated EMT markers from a PC gene expression panel (this data represent a single replicate). (c) qRT-PCR quantification of mRNA expression of EMT associated TWIST1 and E-cadherin of TWIST1 and CDH1 (n = 3; TWIST1 p = 0.021, CDH1 p = 0.034, student’s t test). (d) Representative western blots for corresponding protein expression. The blots were cropped and gels were run at the same conditions. The original pictures for the evaluation of the clones can be found in Supplementary Fig. S4). (e) Immunocytochemistry (ICC) analysis of localization of E-cadherin expression (red) and nuclei counterstained with DAPI (blue).
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