An epigenetic reprogramming strategy to re-sensitize radioresistant prostate cancer cells

Radiotherapy is a mainstay of curative prostate cancer treatment, but risks of recurrence after treatment remain significant in locally advanced disease. Given that tumor relapse can be attributed to a population of cancer stem cells (CSC) that survives radiotherapy, analysis of this cell population might illuminate tactics to personalize treatment. However, this direction remains challenging given the plastic nature of prostate cancers following treatment. We show here that irradiating prostate cancer cells stimulates a durable upregulation of stem cell markers that epigenetically reprogram these cells. In both tumorigenic and radioresistant cell populations, a phenotypic switch occurred during a course of radiotherapy that was associated with stable genetic and epigenetic changes. Specifically, we found that irradiation triggered histone H3 methylation at the promoter of the CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), stimulating its gene transcription. Inhibiting this methylation event triggered apoptosis, promoted radiosensitization, and hindered tumorigenicity of radioresistant prostate cancer cells. Overall, our results suggest that epigenetic therapies may restore the cytotoxic effects of irradiation in radioresistant CSC populations

CXCR4 Expression in Prostate Cancer Progenitor Cells

Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44+/CD133+ prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy

Aldehyde Dehydrogenase Is Regulated by beta-Catenin/TCF and Promotes Radioresistance in Prostate Cancer Progenitor Cells

Radiotherapy is a curative treatment option in prostate cancer. Nevertheless, patients with high-risk prostate cancer are prone to relapse. Identification of the predictive biomarkers and molecular mechanisms of radioresistance bears promise to improve cancer therapies. In this study, we show that aldehyde dehydrogenase (ALDH) activity is indicative of radioresistant prostate progenitor cells with an enhanced DNA repair capacity and activation of epithelial-mesenchymal transition (EMT). Gene expression profiling of prostate cancer cells, their radioresistant derivatives, ALDH(+) and ALDH(-) cell populations revealed the mechanisms, which link tumor progenitors to radioresistance, including activation of the WNT/beta-catenin signaling pathway. We found that expression of the ALDH1A1 gene is regulated by the WNT signaling pathway and co-occurs with expression of beta-catenin in prostate tumor specimens. Inhibition of the WNT pathway led to a decrease in ALDH(+) tumor progenitor population and to radiosensitization of cancer cells. Taken together, our results indicate that ALDH(+) cells contribute to tumor radioresistance and their molecular targeting may enhance the effectiveness of radiotherapy. Cancer Res; 75(7); 1482-94. (c)2015 AACR

CXCR4⁺ populations in DU145 and PC3 cell lines are multipotent.

<p>FACS purified CXCR4⁺ and CXCR4⁻ DU145 and PC3 cells were cultured under differentiation conditions in the presence of 10% FBS.CXCR4⁺ cells differentiate toCK5⁺ basal epithelial cells (10.7%), CK5⁺/CK18⁺ intermediate cells (32.2%), and CK18⁺ luminal epithelial cells (57.1%). In contrast, CXCR4⁻ cells differentiate to CK18⁺cells (91.9% of total cell population) and CK5⁺/CK18⁺ cells (8% of total cell population) but not basal epithelial cells. Scale bars indicate 15 µm.</p

Overexpression of CXCR4 inprostate cancer progenitors.

<p>(A) Cells grown under sphere forming conditions showed an increased expression level of CXCR4 as analyzed by RT-PCR and Western blot analysis. For sphere formation, single cells were plated at 500 cells/mL in 10-cm dishes with an ultralow attachment surface and grown in serum-free epithelial basal medium for 7 days. (B) Flow cytometry analysis showed significant enrichment of the CXCR4⁺ population within CD44⁺/CD133⁺ cells compared to the total cell population for DU145 and PC3 cells (p<0.001). The cells were triple stained and analyzed with a BD LSR II flow cytometer. (C) Representative fluorescent images of CD133 and CD44 co-immunostaining showing that CXCR4⁺ DU145 and PC3 cells have a higher proportion of CD44⁺/CD133⁺ cells compared to CXCR4⁻ cells. Cells expressing high of low levels of CXCR4 were FACS-purified, plated in 384 well black clear bottom plates at a density of 100 cells/well in serum-free epithelial basal medium. After 18 hours, the cells were fixed with 3.7<b>%</b> formaldehyde in PBS and stained with anti-CD133 and anti-CD44 antibodies. Cells in at least five randomly selected fields of view were counted for each condition. Arrows show the triple positive cells. Scale bars indicate 15 µm. *- p value<0.05. (D) Immunostaining of paraffin-embedded sections of xenograft tumors formed by FACS purified CD44⁺/CD133⁺ and CD44⁻/CD133⁻ cells showed more than 13% CXCR4⁺ cells in tumors derived from CD44⁺/CD133⁺ cells compared to 2.2% CXCR4⁺ cells in xenograft tumors derived from CD44⁻/CD133⁻ cells. A total of 10³ FACS-sorted DU145 CD133⁺/CD44⁺ or CD133⁻/CD44⁻ cells embedded in BD matrigel were injected s.c. into NOD/SCID mice. Tumors were allowed to grow for 42 days until the tumors produced by DU145 CD133⁺/CD44⁺ reached a size of 400 mm³ and the tumors produced by DU145 CD133⁻/CD44⁻ reached a size of 125 mm³. Cells in at least five randomly selected fields of view were counted for each condition. Scale bars indicate 30 µm. (E) CXCR4 immunostaining on paraffin-embedded sections of xenograft tumors made by the cells grown under sphere forming and monolayer conditions showed more than 6% CXCR4⁺ cells in sphere-derived tumors as compared to 1.4% of CXCR4⁺ cells in monolayer-derived xenograft tumors. **- p value<0.01.Scale bars indicate 30 µm.</p

Targeting the tumor initiating and differentiated populations within DU145 and PC3 carcinoma cell lines by CXCR4 antagonist and conventional therapy.

<p>(A) Treatment with CXCR4 antagonist AMD3100 decreases the CD133⁺/CD44⁺ population but did not significantly affect the CD133⁻/CD44⁻ population within prostate cancer cells. The use of cytotoxic drugs alone results in a decrease in proliferating tumor cells, but leads to an overall increase in the relative population of tumor initiating cells. Combinatorial treatment of PC3 cells with AMD3100 and the conventional chemotherapeutic drug 5-fluouracil decreases both undifferentiated and differentiated cells populations. PC3 cells were grown in serum-free, EBM medium with supplements and treated with the indicated concentrations of AMD3100 and 5-fluouracil. On the 5^th day the cells were subjected to flow cytometry analysis; *-p value<0.05. (B) Combinatorial therapy <i>in vivo</i> demonstrates significant inhibition of tumor growth compared to single drug treatment. The mice were treated with Taxotere (20 mg/kg, 1q.w., p.o) as described (19). Alzet pumps were used to deliver AMD3100 at a constant rate of 0.25 µg/kg/hour. The pumps loaded with AMD3100 or saline were implanted subcutaneously. The mice were observed for 8 weeks for appearance and development of tumors. (C) CD133 and CD44 immunostaining on frozen sections of xenograft tumors treated with combinatorial or mono-therapy from (B) revealed selective inhibition of the CD133⁺/CD44⁺ population by the CXCR4 antagonist AMD3100; *-p value<0.05. (D) Western blot analysis showed a significant decrease in FOXO3A phosphorylation in the tumors treated with AMD3100.</p

CXCR4 regulates tumor progenitor cell adhesion.

<p>(A) DU145 CD44⁺/CD133⁺/CXCR4⁺ cells have significantly higher CXCL12-dependent adhesion to fibronectin than CD44⁺/CD133⁺/CXCR4⁻ or CD44⁻/CD133⁻/CXCR4⁻ cells populations. Cells were FACS sorted and plated on fibronectin coated plates and allowed to adhere for 1 h. The cells were counted in three wells per condition using a phase-contrast microscope.*-p value<0.05. (B) The expression level of α2, α5, and β3 integrins subunits is strongly upregulated in CD133⁺/CD44⁺ DU45 compared to CD133⁻/CD44⁻ DU45 cells. (C) Inactivation of the PI3K pathway with NVP-BEZ235 significantly decreases CXCR4-dependent adhesion of CD133⁺/CD44⁺ DU45 cells to fibronectin. The adhesion of CD133⁺/CD44⁺ DU45 cells could be restored in the presence of CXCL12. The adhesion of CD133⁻/CD44⁻ DU45 did not respond to the CXCR4 and PI3K signaling modulation, *-p value<0.05.</p

CXCR4 neutralization leads to attenuation of the CD44⁺/CD133⁺ prostate progenitor population.

<p>(A) DU145 cells were plated in 96-well low-attachment plates at 100 cells per well (5 replicates) and the spheres were grown in serum-free, EBM medium with supplements. The antibodies were replenished daily. Cells were imaged with an Acumen eX3 microplate cytometer and spheres were detected using image analysis software. The sphere size was measured by GFP intensity. The spheres were discriminated from cell debris using a Gaussian filter. The spheres included in the analysis are outlined in red and indicated by arrows. Representative data from one of two independent experiments is shown; *- p value<0.05. (B) Flow cytometry analysis revealed attenuation of CD44⁺/CD133⁺ population in DU145 cells treated with 10 µg/ml neutralizing anti-CXCR4 (mouse monoclonal IgG, clone 44716, R&D Systems) or 10 µg/ml control antibody (mouse IgG isotype control, Lifespan Bioscience Inc.) for 5 days. The cell were grown in medium supplemented with 2% FBS. Culture medium was refreshed every second day; *- p value<0.05. (C) Western blot analysis of DU145 cells treated with 10 µg/ml neutralizing anti-CXCR4 antibody for 5 days demonstrated downregulation of the PI3K/AKT pathway activation compared to the cells treated with 10 µg/ml control antibody. The cell were grown in medium supplemented with 2% FBS. Culture medium was refreshed every second day. (D) Preincubation of prostate cancer cells with neutralizing anti-CXCR4 antibody significantly delays tumor growth. 5×10⁵ DU145 cells pretreated with neutralizing anti-CXCR4 or control antibody for 5 days were embedded in BD matrigel and injected s.c. into NOD/SCID mice.*- p value<0.01.</p