b-Arrestins are classic attenuators of G-protein-coupled receptor signaling. However, they have multiple roles in cellular physiology, including carcinogenesis. This work shows for the first time that b-arrestins have prognostic significance for predicting metastasis and response to chemotherapy in bladder cancer. b-Arrestin-1 (ARRB1) and b-arrestin-2 (ARRB2) mRNA levels were measured by quantitative RT-PCR in two clinical specimen cohorts (n ¼ 63 and 43). The role of ARRBs in regulating a stem cell-like phenotype and response to chemotherapy treatments was investigated. The consequence of forced expression of ARRBs on tumor growth and response to Gemcitabine in vivo were investigated using bladder tumor xenografts in nude mice. ARRB1 levels were significantly elevated and ARRB2 levels downregulated in cancer tissues compared with normal tissues. In multivariate analysis only ARRB2 was an independent predictor of metastasis, diseasespecific-mortality, and failure to Gemcitabine þ Cisplatin (GþC) chemotherapy; $80% sensitivity and specificity to predict clinical outcome. ARRBs were found to regulate stem cell characteristics in bladder cancer cells. Depletion of ARRB2 resulted in increased cancer stem cell markers but ARRB2 overexpression reduced expression of stem cell markers (CD44, ALDH2, and BMI-1), and increased sensitivity toward Gemcitabine. Overexpression of ARRB2 resulted in reduced tumor growth and increased response to Gemcitabine in tumor xenografts. CRISPR-Cas9-mediated gene-knockout of ARRB1 resulted in the reversal of this aggressive phenotype. ARRBs regulate cancer stem cell-like properties in bladder cancer and are potential prognostic indicators for tumor progression and chemotherapy response.
The atypical C-X-C chemokine receptor 7 (CXCR7) has been implicated in supporting aggressive cancer phenotypes in several cancers including prostate cancer. However, the mechanisms driving overexpression of this receptor in cancer are poorly understood. This study investigates the role of androgen receptor (AR) in regulating CXCR7. Androgen deprivation or AR inhibition significantly increased CXCR7 expression in androgen-responsive prostate cancer cell lines, which was accompanied by enhanced epidermal growth factor receptor (EGFR)-mediated mitogenic signaling, promoting tumor cell survival through an androgen-independent signaling program. Using multiple approaches we demonstrate that AR directly binds to the CXCR7 promoter, suppressing transcription. Clustered regularly interspaced short palindromic repeats (CRISPR) directed Cas9 nuclease-mediated gene editing of CXCR7 revealed that prostate cancer cells depend on CXCR7 for proliferation, survival and clonogenic potential. Loss of CXCR7 expression by CRISPR-Cas9 gene editing resulted in a halt of cell proliferation, severely impaired EGFR signaling and the onset of cellular senescence. Characterization of a mutated CXCR7-expressing LNCaP cell clone showed altered intracellular signaling and reduced spheroid formation potential. Our results demonstrate that CXCR7 is a potential target for adjuvant therapy in combination with androgen deprivation therapy (ADT) to prevent androgen-independent tumor cell survival.
BackgroundThe clinical manifestation of benign prostatic hyperplasia (BPH) is causally linked to the inflammatory microenvironment and proliferation of epithelial and stromal cells in the prostate transitional zone. The CXC‐chemokine interleukin‐8 (IL‐8) contributes to inflammation. We evaluated the expression of inflammatory cytokines in clinical specimens, primary cultures, and prostatic lineage cell lines. We investigated whether IL‐8 via its receptor system (IL‐8 axis) promotes BPH.MethodsThe messenger RNA and protein expression of chemokines, including components of the IL‐8 axis, were measured in normal prostate (NP; n = 7) and BPH (n = 21), urine (n = 24) specimens, primary cultures, prostatic lineage epithelial cell lines (NHPrE1, BHPrE1, BPH‐1), and normal prostate cells (RWPE‐1). The functional role of the IL‐8 axis in prostate epithelial cell growth was evaluated by CRISPR/Cas9 gene editing. The effect of a combination with two natural compounds, oleanolic acid (OA) and ursolic acid (UA), was evaluated on the expression of the IL‐8 axis and epithelial cell growth.ResultsAmong the 19 inflammatory chemokines and chemokine receptors we analyzed, levels of IL‐8 and its receptors (CXCR1, CXCR2), as well as, of CXCR7, a receptor for CXCL12, were 5‐ to 25‐fold elevated in BPH tissues when compared to NP tissues (P ≤ .001). Urinary IL‐8 levels were threefold to sixfold elevated in BPH patients, but not in asymptomatic males and females with lower urinary tract symptoms (P ≤ .004). The expression of the IL‐8 axis components was confined to the prostate luminal epithelial cells in both normal and BPH tissues. However, these components were elevated in BPH‐1 and primary explant cultures as compared to RWPE‐1, NHPrE1, and BHPrE1 cells. Knockout of CXCR7 reduced IL‐8, and CXCR1 expression by 4‐ to 10‐fold and caused greater than or equal to 50% growth inhibition in BPH‐1 cells. Low‐dose OA + UA combination synergistically inhibited the growth of BPH‐1 and BPH primary cultures. In the combination, the drug reduction indices for UA and OA were 16.4 and 7852, respectively, demonstrating that the combination was effective in inhibiting BPH‐1 growth at significantly reduced doses of UA or OA alone.ConclusionThe IL‐8 axis is a promotor of BPH pathogenesis. Low‐dose OA + UA combination inhibits BPH cell growth by inducing autophagy and reducing IL‐8 axis expression in BPH‐epithelial cells.
<p>Supplementary Table S1: Specimen and patient characteristics; Supplementary Table S2: Determination of the association between adjuvant treatment failure and demographic/clinical parameters or marker levels; Supplementary Table S3: qPCR primers and ARRB1-specific CRISPR guide RNA sequences; Supplementary Table S4: Source of various antibodies used for immunofluorescence and Western blotting; Supplementary Figure S1: Kaplan Meier plot for stratification of patients into higher risk and lower risk for Disease-Specific Mortality (DSM) based on the ARRB2 arrestin level; Supplementary Figure S2: Expression of cytokeratins and Bmi-1 following modulation of ARRB2 levels in 5637 cells; Supplementary Figure S3: Expression of SOX2 following modulation of ARRB2 levels in bladder cancer cell lines; Supplementary Figure S4: Response of ARRB2 overexpressing transfectants to Cisplatin; Supplementary Figure S5: Expression ABC transporters in ARRB2-modulated bladder cancer cells; Supplementary Figure S6: Effect of ARRB2 overexpression on Cancer Stem Cell markers in vivo; Supplementary Figure S7: ARRB1 depletion affects clonogenic growth, spheroid formation and expression of CSC markers</p>
<div>Abstract<p>β-Arrestins are classic attenuators of G-protein–coupled receptor signaling. However, they have multiple roles in cellular physiology, including carcinogenesis. This work shows for the first time that β-arrestins have prognostic significance for predicting metastasis and response to chemotherapy in bladder cancer. β-Arrestin-1 (ARRB1) and β-arrestin-2 (ARRB2) mRNA levels were measured by quantitative RT-PCR in two clinical specimen cohorts (<i>n</i> = 63 and 43). The role of ARRBs in regulating a stem cell-like phenotype and response to chemotherapy treatments was investigated. The consequence of forced expression of ARRBs on tumor growth and response to Gemcitabine <i>in vivo</i> were investigated using bladder tumor xenografts in nude mice. ARRB1 levels were significantly elevated and ARRB2 levels downregulated in cancer tissues compared with normal tissues. In multivariate analysis only ARRB2 was an independent predictor of metastasis, disease-specific-mortality, and failure to Gemcitabine + Cisplatin (G+C) chemotherapy; ∼80% sensitivity and specificity to predict clinical outcome. ARRBs were found to regulate stem cell characteristics in bladder cancer cells. Depletion of ARRB2 resulted in increased cancer stem cell markers but ARRB2 overexpression reduced expression of stem cell markers (CD44, ALDH2, and BMI-1), and increased sensitivity toward Gemcitabine. Overexpression of ARRB2 resulted in reduced tumor growth and increased response to Gemcitabine in tumor xenografts. CRISPR-Cas9–mediated gene-knockout of ARRB1 resulted in the reversal of this aggressive phenotype. ARRBs regulate cancer stem cell-like properties in bladder cancer and are potential prognostic indicators for tumor progression and chemotherapy response.</p></div>
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