RUNX2 overexpression and PTEN haploinsufficiency cooperate to promote CXCR7 expression and cellular trafficking, AKT hyperactivation and prostate tumorigenesis

Abstract

Rationale: The overall success rate of prostate cancer (PCa) diagnosis and therapy has been improved over the years. However, genomic and phenotypic heterogeneity remains a major challenge for effective detection and treatment of PCa. Efforts to better classify PCa into functional subtypes and elucidate the molecular mechanisms underlying prostate tumorigenesis and therapy resistance are warranted for further improvement of PCa outcomes. Methods: We generated Cre⁺;Runx2-cTg;Pten^p/+ (Runx2-Pten double mutant) mice by crossbreeding Cre⁺;Runx2-cTg males with Pten conditional (Pten^p/p) females. By using Hematoxylin and Eosin (H&E) staining, SMA and Masson's Trichrome staining, we investigated the effect of PTEN haploinsufficiency in combination with Runx2 overexpression on prostate tumorigenesis. Moreover, we employed immunohistochemistry (IHC) to stain Ki67 for cell proliferation, cleaved caspase 3 for apoptosis and AKT phosphorylation for signaling pathway in prostate tissues. Chromatin immunoprecipitation coupled quantitative PCR (ChIP-qPCR), reverse transcription coupled quantitative PCR (RT-qPCR), western blot (WB) analyses and immunofluorescence (IF) were conducted to determine the underlying mechanism by which RUNX2 regulates CXCR7 and AKT phosphorylation in PCa cells. Results: We demonstrated that mice with prostate-specific Pten heterozygous deletion and Runx2 overexpression developed high-grade prostatic intraepithelial neoplasia (HGPIN) and cancerous lesions at age younger than one year, with concomitant high level expression of Akt phosphorylation and the chemokine receptor Cxcr7 in malignant glands. RUNX2 overexpression induced CXCR7 transcription and membrane location and AKT phosphorylation in PTEN-deficient human PCa cell lines. Increased expression of RUNX2 also promoted growth of PCa cells and this effect was largely mediated by CXCR7. CXCR7 expression also positively correlated with AKT phosphorylation in PCa patient specimens. Conclusions: Our results reveal a previously unidentified cooperative role of RUNX2 overexpression and PTEN haploinsufficiency in prostate tumorigenesis, suggesting that the defined RUNX2-CXCR7-AKT axis can be a viable target for effective treatment of PCa.

Keywords: CXCR7; PTEN; RUNX2; prostate cancer; tumorigenesis.

Figure 1

Generation of Runx2 conditional transgenic (Runx2-cTg) mice. (A) Western blot showing Runx2 levels in “wild-type” (Cre^-;Pten^p/+) or Pten heterozygous (Cre⁺;Pten^p/+) murine prostate tissues. Erk2 was used as a loading control. (B) Schematic of HA-Runx2 conditional transgenic construct pLSL-HA-Runx2. (C) Western blot for HA-tagged Runx2 in LNCaP cells transfected with CMV-Runx2 (positive control) or the pLSL-HA-Runx2 plasmid in combination with or without CMV-Cre. (D) Agarose gel shows the genotyping results of two independent transgenic founder Runx2-cTg mice. (E), Western blot showing Runx2 levels in “wild-type” (Cre^-;Runx2-cTg) and Runx2 transgenic (Cre⁺;Runx2-cTg) murine prostate tissues.

Figure 2

Runx2 overexpression and Pten heterozygous deletion cooperate to induce HGPIN and cancerous lesions in mice. (A) Quantitative data for 4-month-old mice from each genotype (n = 10/group) with LGPIN, HGPIN and/or cancerous lesions (HGPIN/Ca) or no lesion (nonmalignant). (B) H&E staining of DLP and VP from mice with indicated genotypes (n = 10 mice/genotype) at 8 months of age. The high magnification image is corresponding to the framed area in low magnification image in each genotype. Scale bars are indicated in the images. (C) Quantitative analysis of LGPIN, HGPIN and/or cancerous lesion (HGPIN/Ca) or no lesion (nonmalignant) in 8-month-old mice with indicated genotypes (n = 10 mice/genotype). (D) H&E, IHC for smooth muscle actin (SMA) and Masson's Trichrome staining (pointed by red asterisks respectively) in the prostate of mice with five different genotypes (n = 5 mice/genotype) at age of 8 months. Scale bars are indicated in the images.

Figure 3

Runx2 regulates proliferation of neoplastic prostatic cells in vitro and in mice. (A) MTS assay measuring proliferation of PC-3 cells transfected with control or RUNX2-specific siRNAs at different time points. Inset, western blot showing the effectiveness of RUNX2 knockdown. ERK2 was used as a loading control. (B) IHC of proliferation marker Ki-67 in prostate tissues collected from mice with the indicated genotypes at age of 8 months. Arrows indicate Ki-67 positive cells. (C) Quantification of Ki-67 positive cells in each indicated mouse genotype. Data are shown as mean ± SD (n = 6 mice/genotype). * P < 0.01, NS, no significance. (D) IHC analysis of cleaved Caspase 3 expression in the prostate of mice with the indicated genotypes at age of 8 months (n = 6 mice/genotype). Cleaved Caspase 3 IHC in PCa tissues from castrated prostate-specific Pten homozygous deletion mice was included as positive control. Photos are representatives of results from 6 mice in each group (n = 6).

Figure 4

Akt is hyperphosphorylated in neoplastic lesions in Runx2-Pten mice. (A) Quantitative data of Akt S473 phosphorylation (p-Akt-S473) IHC in the prostate of 4-month-old mice with the indicated genotypes (n = 10 mice/genotype). (B) Representative images of IHC for p-Akt-S473 in DLP and VP of 8 month-old mice with the indicated genotypes (n = 10 mice/genotype). The high magnification image (400X) is corresponding to the framed area in low magnification image (100X) in each genotype. Scale bars are indicated in the images. Scale bars are shown in the images. (C) Quantitative data of p-Akt-S473 IHC in the prostate of 8-month-old mice with the indicated genotypes (n = 10 mice/genotype).

Figure 5

Co-regulation of the CXCR7 gene by RUNX2 and PTEN in PCa. (A) UCSC genome browser screenshots showing signal profiles of RUNX2 ChIP-seq (reported previously 32) in the ACKR3 (CXCR7) gene locus in C4-2B cells. H3K4me1 and H3K4me3 ChIP-seq data were acquired from LNCaP cells as reported previously . P, promoter, NC, negative control region, E, enhancer. (B, C) PC-3 cells were transfected with empty vector (EV) or HA-Runx2 for 24 h for western blot (B) and ChIP-qPCR analysis of RUNX2 binding at the CXCR7 gene promotor and enhancers (C). ERK2 was used as a loading control. All data are shown as mean values ± SD (n = 3). * P < 0.05 comparing RUNX2 binding in EV transfected cells. (D-G) LAPC-4 cells were transfected with EV and HA-Runx2 expression vector in combination with control or PTEN-specific siRNAs (siPTEN) for 48 h followed by ChIP-qPCR analysis of RUNX2 binding at the CXCR7 gene promoter and enhancers (D), RT-qPCR assessment of CXCR7 mRNA expression (E), and western blot analysis of effectiveness of murine Runx2 transfection and PTEN knockdown on expression of p-AKT-473 (F) and CXCR7 protein level in LAPC-4 cells. Quantified data (G) was resulted from the normalization by ERK2 western blot intensity.

Figure 6

CXCR7 expression correlates with AKT phosphorylation in PCa in Runx2-Pten mice and patient PCa specimens and CXCR7 is required for Runx2-induced PCa cell growth. (A) H&E and IHC for Cxcr7 and S473 phosphorylated Akt (p-Akt-S473) in the prostate tissues of mice with five different genotypes at age of 8 months. Scale bars are indicated in the images. Photos are representatives of results from 3 mice in each group (n = 3). (B-D) IHC for CXCR7 and p-AKT-S473 in PCa specimens in a cohort of 55 patients. Representative CXCR7 and p-AKT-S473 IHC images from two cases of PCa are shown in (B). Heatmap is utilized to summarize IHC of CXCR7 and p-AKT-S473 protein expression in all cases of PCa analyzed (C). Spearman correlation analysis exhibits a positive correlation between CXCR7 and p-AKT-S473 expression in this cohort of patients and the correlation is statistically significant (D). (E-G) PC-3 cells were infected with lentivirus for empty vector or HA-Runx2 in combination with control shRNA (shC) and CXCR7-specific shRNAs (shCXCR7). At 48 h after infection, cells were harvested for western blot analysis of indicated proteins (E) and colony formation assays with representatives of colonies shown in (F) and quantification data shown in (G). Data represent mean values ± SD (n = 3). * P < 0.05; **, P < 0.001.

Figure 7

RUNX2 overexpression facilitates CXCR7 endocytic recycling. (A-B), Co-localization of immunostaining of CXCR7 and two endocytic recycling markers TFRC (A) and RAB11 (B) in LNCaP cells. (C) LNCaP cells were transiently transfected with HA-Runx2 and treated with vehicle (DMSO) and the lysosome inhibitor concanamycin A (ConA) for 16 h. The cells were fixed and immunostained for CXCR7. (D) Overexpression of Runx2 enhanced the distribution of CXCR7 on the plasma membrane as shown by IFC. (E-F) PC-3 cells were treated with vehicle (0.1% BSA) or CXCL12 (200 ng/ml). After 48 h treatment, cells were harvested for western blot analysis of indicated proteins (E) and colony formation assays with representatives of colonies and quantification data shown in (F). *, p85 form of S6 kinase. Data represent mean values ± SD (n = 3). P value was indicated in the image. (G) A hypothetical model wherein RUNX2 overexpression in combination of PTEN lose (PTEN^+/- or PTEN^-/-) elicits a feed forward signaling loop that leads to increased expression and cellular trafficking of CXCR7, hyperphosphorylation of AKT, and prostate tumorigenesis and progression.