Prostate cancer stem-like cells/cancer-initiating cells have an autocrine system of hepatocyte growth factor

Abstract

Prostate cancer cells include a small population of cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) that have roles in initiation and progression of the cancer. Recently, we isolated prostate CSCs/CICs as aldehyde dehydrogenase 1-highh (ALDH1(high) ) cells using the ALDEFLUOR assay; however, the molecular mechanisms of prostate CSCs/CICs are still elusive. Prostate CSCs/CICs were isolated as ALDH1(high) cells using the ALDEFLUOR assay, and the gene expression profiles were analyzed using a cDNA microarray and RT-PCR. We found that prostate CSCs/CICs expressed higher levels of growth factors including hepatocyte growth factor (HGF). Hepatocyte growth factor protein expression was confirmed by enzyme linked immunosorbent assay and Western blotting. On the other hand, c-MET HGF receptor was expressed in both CSCs/CICs and non-CSCs/CICs at similar levels. Hepatocyte growth factor and the supernatant of myofibloblasts derived from the prostate augmented prostasphere formation in vitro, and prostasphere formation was inhibited by an anti-HGF antibody. Furthermore, c-MET gene knockdown by siRNA inhibited the prostasphere-forming ability in vitro and tumor-initiating ability in vivo. Taken together, the results indicate that HGF secreted by prostate CSCs/CICs and prostate myofibroblasts has a role in the maintenance of prostate CSCs/CICs in an autocrine and paracrine fashion.

Figure 1

Expression of growth factors and receptors in prostate cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs). (A) Reverse transcription‐polymerase chain reaction (RT‐PCR) of HGF,IGF1 and MET in ALDH1^high cells and ALDH1^low cells. HGF,IGF1 and MET mRNA expression was evaluated by RT‐PCR. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as a positive control. (B) Western blot analysis of ALDH1, hepatocyte growth factor (HGF) and c‐MET. Western blot analysis using 5 × 10⁴ ALDH1^high cells and ALDH1^low cells was performed. Anti‐β‐Actin was used as an internal control. (C) ALDH1 and HGF expression in primary prostate CSCs/CICs. Prostate adenocarcinoma tissue was stained by anti‐SOX2 and anti‐ALDH1 (left panel) and anti‐SOX2 and anti‐HGF (right panel). Nucleus brown staining was judged as positive staining for SOX2, and cytoplasm red staining was judged as positive staining for ALDH1 and HGF. Arrows indicate SOX2 and ALDH1 double positive cells and SOX2 and HGF double positive cells. Magnification, ×400. (D) Hepatocyte growth factor secretion by ALDH1^high cells and ALDH1^low cells. Isolated ALDH1^high cells and ALDH1^low cells were incubated in a 96‐well plate for 2 days. Then HGF was evaluated by enzyme linked immunosorbent assay (ELISA). Control represents only culture medium. Asterisks represents statistically significant difference (P < 0.05, t‐test).

Figure 2

Growth factors enhance prostasphere formation in vitro. (A) Prostasphere formation was increased by hepatocyte growth factor (HGF). Prostasphere formation was performed in serum‐free Dulbecco's modified eagle medium (DMEM)/F12 media. Growth factors were added to the prostasphere culture. Representative pictures of prostaspheres are shown. Magnification, ×100. (B) Representative pictures of prostaspheres formed by ALDH1^high cells and ALDH1^low cells. ALDH1^high cells and ALDH1^low cells were cultured with or without HGF and IGF1. Representative pictures are shown. Magnification ×100. (C) Prostasphere formation of ALDH1^high cells and ALDH1^low cells with or without growth factors (HGF and IGF1). ALDH1^high cells and ALDH1^low cells were cultured in serum‐free media with or without HGF and IGF1. The numbers of prostaspheres were evaluated at day 3. Asterisks represents statistically significant difference (P < 0.05, t‐test).

Figure 3

Prostasphere formation was inhibited by anti‐hepatocyte growth factor (HGF) antibody. ALDH1^high cells were cultured in Dulbecco's modified eagle medium (DMEM)/F12 medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Anti‐HGF antibody and control antibody were added to the culture. Representative pictures are shown (A). Magnification, ×100. The numbers of prostaspheres were evaluated at day 3 (B). Asterisks represents statistically significant difference (P < 0.05, t‐test).

Figure 4

Prostasphere formation was increased by supernatant of prostate myofibroblasts. ALDH1^high cells were cultured in Dulbecco's modified eagle medium (DMEM)/F12 medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Fifty percent (v/v) of myofibroblast culture supernatant was added to culture. Anti‐HGF antibody and control antibody were added to the culture. Representative pictures are shown (A). Magnification, ×100. The numbers of prostaspheres were evaluated at day 3 (B). Asterisks represents statistically significant difference (P < 0.05, t‐test).

Figure 5

c‐MET has a role in the maintenance of prostate cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs). (A) Reverse transcription‐polymerase chain reaction (RT‐PCR) of MET knockdown cells. MET‐specific siRNA and control siRNA were transfected into 22Rv1 cells. Two days after transfection, total RNA was purified and analyzed by RT‐PCR. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as an internal control. (B) ALDEFLUOR assay of MET knockdown cells. MET siRNA3‐ and control siRNA‐transfected 22Rv1 cells were analyzed by ALDEFLUOR assay. Percentages indicate rates of ALDH1^high cells. (C) MET gene knockdown inhibited cell growth in vitro. MET siRNA3‐ and control siRNA‐transfected 22Rv1 cells were seeded into a six‐well plate, and cell numbers were counted at 24, 48 and 72 h. An asterisk represents a statistically significant difference (P < 0.05, t‐test). (D) MET gene knockdown inhibited prostasphere formation in vitro. MET siRNA3‐ and control siRNA‐transfected 22Rv1 cells were seeded into an Ultra‐Low Attachment plate and cultured for 3 days. (E) MET gene knockdown inhibited tumor initiation in vivo. MET siRNA3‐ and control siRNA‐transfected 22Rv1 cells were transplanted into male NOD/SCID mice subcutaneously. Tumor growth was measured every week. Tumor growth curve and a representative picture are shown. An asterisk represents a statistically significant difference (P < 0.05, t‐test). (F) ALDH1 expression in MET knockdown tumor. ALDH1 expression was investigated by immunohistochemical staining. Representative pictures are shown. Magnification, ×100.

Figure 6

Model of the maintenance of prostate cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs)through hepatocyte growth factor (HGF)/c‐MET signaling.