The CCL2-CCR2 Axis Contributes to Migration of Cabazitaxel-resistant Prostate Cancer Cells.

BACKGROUND/AIM: Developing resistance to cabazitaxel is a major challenge in patients with docetaxel- and castration-resistant prostate cancer (CRPC) since it is frequently administered as a last resort. We have previously reported that CCL2 induces resistance to the antiproliferative effect of cabazitaxel in DU145-TxR/CxR prostate cancer cell lines. However, how CCL2 induces resistance to the antimigration effect of cabazitaxel remains unclear. MATERIALS AND METHODS: We established a cabazitaxel-resistant cell line, DU145-TxR/CxR, from a previously established paclitaxel-resistant cell line, DU145-TxR, which was confirmed to show docetaxel resistance. We performed migration assay and analyzed the expression of epithelial-mesenchymal transition markers using DU145-TxR/CxR with or without CCL2 silencing with small interfering RNA (siRNA) transfection. RESULTS: Cabazitaxel inhibited the migration of DU145 cells through the inactivation of STAT3. A CCR2 (a specific receptor of CCL2) antagonist suppressed the migration of DU145-TxR and DU145-TxR/CxR cells under cabazitaxel treatment. Western blotting revealed that the CCR2 antagonist inhibited STAT3 phosphorylation in DU145-TxR and DU145-TxR/CxR cells under cabazitaxel treatment. CCL2 silencing with siRNA in DU145-TxR and DU145-TxR/CxR cells decreased migration through STAT3 and p38 inactivation. Furthermore, CCL2 activated AKT, and CCR2 antagonist inhibited AKT phosphorylation in DU145-TxR and DU145-TxR/CxR cells with recovery of sensitivity to cabazitaxel under cabazitaxel treatment. CONCLUSION: The CCL2-CCR2 axis is a key contributor to resistance to the antimigration effect of cabazitaxel in prostate cancer cells. CCL2-CCR2 axis inhibition may be a potential therapeutic target against chemoresistant CRPC in combination with cabazitaxel.

CCL2 induces resistance to the antiproliferative effect of cabazitaxel in prostate cancer cells.

Understanding the mechanism of chemoresistance and disease progression in patients with prostate cancer is important for developing novel treatment strategies. In particular, developing resistance to cabazitaxel is a major challenge in patients with docetaxel-resistant and castration-resistant prostate cancer (CRPC) because cabazitaxel is often administered as a last resort. However, the mechanism by which cabazitaxel resistance develops is still unclear. C-C motif chemokine ligands (CCL) were shown to contribute to the castration resistance of prostate cancer cells via an autocrine mechanism. Therefore, we focused on CCL as key factors of chemoresistance in prostate cancer cells. We previously established a cabazitaxel-resistant cell line, DU145-TxR/CxR, from a previously established paclitaxel-resistant cell line, DU145-TxR. cDNA microarray analysis revealed that the expression of CCL2 was upregulated in both DU145-TxR and DU145-TxR/CxR cells compared with DU145 cells. The secreted CCL2 protein level in DU145-TxR and DU145-TxR/CxR cells was also higher than in parental DU145 cells. The stimulation of DU145 cells with CCL2 increased the proliferation rate under treatments with cabazitaxel, and a CCR2 (a specific receptor of CCL2) antagonist suppressed the proliferation of DU145-TxR and DU145-TxR/CxR cells under treatments of cabazitaxel. The CCL2-CCR2 axis decreased apoptosis through the inhibition of caspase-3 and poly(ADP-ribose) polymerase (PARP). CCL2 is apparently a key contributor to cabazitaxel resistance in prostate cancer cells. Inhibition of the CCL2-CCR2 axis may be a potential therapeutic strategy against chemoresistant CRPC in combination with cabazitaxel.

Coffee diterpenes kahweol acetate and cafestol synergistically inhibit the proliferation and migration of prostate cancer cells.

BACKGROUND: Coffee inhibits the progression of prostate cancer; however, the direct mechanism through which coffee acts on prostate cancer cells remains unclear. This study aimed to identify the key compounds of coffee that possess anti-cancer effects and to investigate their mechanisms of action. METHODS: The anti-proliferation and anti-migration effects of six potentially active types of coffee compounds, including kahweol acetate, cafestol, caffeine, caffeic acid, chlorogenic acid, and trigonelline hydrochloride, were evaluated using LNCaP, LNCaP-SF, PC-3, and DU145 human prostate cancer cells. The synergistic effects of these compounds were also investigated. Apoptosis-related and epithelial-mesenchymal transition-related proteins, androgen receptor in whole cell and in nucleus, and chemokines were assessed. A xenograft study of SCID mice was performed to examine the in vivo effect of coffee compounds. RESULTS: Among the evaluated compounds, only kahweol acetate and cafestol inhibited the proliferation and migration of prostate cancer cells in a dose-dependent manner. The combination treatment involving kahweol acetate and cafestol synergistically inhibited proliferation and migration (combination index <1) with the induction of apoptosis, the inhibition of epithelial-mesenchymal transition, and decrease in androgen receptor, resulting in the reduction of nuclear androgen receptor in androgen receptor-positive cells. Moreover, kahweol acetate and cafestol downregulated CCR2 and CCR5 without an increase in their ligands, CCL2 and CCL5. The xenograft study showed that oral administration of kahweol acetate and cafestol significantly inhibited tumor growth. CONCLUSION: Kahweol acetate and cafestol synergistically inhibit the progression of prostate cancer. These coffee compounds may be novel therapeutic candidates for prostate cancer.

Effectiveness and Safety of Pegfilgrastim in BEP Treatment for Patients with Germ Cell Tumor.

BACKGROUND: The effectiveness and safety of pegfilgrastim during bleomycin, etoposide and cisplatin (BEP) chemotherapy have not yet been investigated. PATIENTS AND METHODS: Patients with germ cell tumors (GCTs) who received pegfilgrastim during BEP at the Kanazawa University Hospital between January 2014 and December 2016 were retrospectively analyzed. The frequency of adverse events and effectiveness in inhibiting neutropenia were compared between cycles using pegfilgrastim and those using filgrastim. RESULTS: Pegfilgrastim and filgrastim were administered in 13 and 22 cycles, respectively. The absolute neutrophil count at the nadir was significantly lower in patients receiving pegfilgrastim than in those receiving filgrastim (p=0.003). The duration of grade 2-4 neutropenia in cycles using filgrastim was significantly longer than that in those pegfilgrastim (p=0.01). No significant differences in the incidence of febrile neutropenia and serious adverse events were observed. CONCLUSION: Pegfilgrastim can be safely and effectively administrated during BEP for patients with GCT.

Establishment and characterization of two cabazitaxel-resistant prostate cancer cell lines.

Once castration-resistant prostate cancer (CRPC) become resistant for cabazitaxel treatment, the patients are obliged to best supportive care. Therefore, the elucidation of the mechanism of the cabazitaxel-resistance and the conquest are important themes to improve the prognosis of the patients. Then we tried to establish cabazitaxel-resistant CRPC cell lines and characterized them. We established two cabazitaxel-resistant cell lines, PC-3-TxR/CxR and DU145-TxR/CxR from PC-3-TxR and DU145-TxR cell lines previously we established. PC-3-TxR/CxR and DU145-TxR/CxR cells became resistant for cabazitaxel by 11.8-fold and 4.4-fold, respectively. The TxR/CxR cells showed cabazitaxel-resistant using SCID mice in vivo. Although expression of multi-drug resistance gene 1 (MDR1) was up-regulated in DU145-TxR compared with DU145 cells, it was not up-regulated in DU145-TxR/CxR cells any more. In contrast, expression of MDR1 gene was up-regulated in PC-3-TxR compared with PC-3 cells and it was further up-regulated in PC-3-TxR/CxR compared with PC-3-TxR cells. Comparison of cDNA microarray between PC-3-TxR and PC-3-TxR/CxR cells or between DU145-TxR and DU145-TxR/CxR cells revealed that many genes were up-regulated or down-regulated. Finally, knockdown of MDR1 recovered the sensitivity to cabazitaxel not only in PC-3-TxR/CxR cells but also DU145-TxR/CxR cells. Together, regulation of MDR1 gene is important for conquest of the cabazitaxel-resistance.

Crosstalk Between Androgen-sensitive and Androgen-insensitive Prostate Cancer Cells.

AIM: To investigate how androgen-sensitive LNCaP cells crosstalk with androgen-insensitive DU145 or PC-3 cells. MATERIALS AND METHODS: The numbers of LNCaP cells were counted when co-cultured with DU145 or PC-3 cells and vise versa. Androgen receptor (AR) activity in LNCaP cells was examined by luciferase reporter assay after transfection with a luciferase reporter driven by PSA promoter in the presence of DU145 or PC-3 cells. Concentration of androgens in the medium was measured by liquid chromatography-mass spectrometry (LC-MS/MS). The ability of migration and invasion of PC-3 and DU145 cells was investigated using a 2-layer chamber, in the presence of LNCaP cells. RESULTS: Co-culture of LNCaP cells with DU145 cells resulted in the conversion of dehydroepiandrosterone (DHEA) to dihydrotestosterone (DHT), which stimulated cell proliferation and PSA promoter activity in LNCaP cells. The increased cell proliferation rate and AR activity, induced in LNCaP cells after DHT treatment, was further enhanced by co-culture with DU145 cells. LNCaP cells also stimulated the proliferation of DU145 and PC-3 cells, via secreting soluble factors. Finally, LNCaP cells promoted migration and invasion of PC-3 cells, in a co-culture system; however inhibited migration and invasion of DU145 cells. CONCLUSION: Crosstalk between androgen-sensitive PCa cells and androgen-insensitive PCa cells might develop the progression of PCa.

Significance of Perioperative Chemotherapy in Squamous Cell Carcinoma of the Upper and Lower Urinary Tract.

BACKGROUND/AIM: To evaluate the clinicopathological characteristics and prognosis of squamous cell carcinoma (SCC) of the urinary tract (UT) urothelium, and investigate the utility of perioperative chemotherapy. PATIENTS AND METHODS: Thirty-three patients diagnosed with SCC in renal pelvis, ureter and bladder at the Kanazawa University between 2007 and 2017 were included in this retrospective study. Cancer-specific survival (CSS) rates according to cancer stage and neoadjuvant chemotherapy (NAC) were determined. RESULTS: Among the entire cohort, two-year CSS rates were 100%, 75%, 47% and 0% in stages I, II, III and IV, respectively, with a significant correlation between CSS and cancer stage (p=0.0063). Sixteen patients underwent radical surgery, and seven patients received NAC; however, there were no significant differences in CSS rates among patients stratified by NAC (p=0.7043). CONCLUSION: Worse cancer stage for SCC of UT urothelium was associated with worse prognosis. Given the poor efficacy of NAC in these patients, surgical treatment should be preferred.

Tumor necrosis factor-α induces prostate cancer cell migration in lymphatic metastasis through CCR7 upregulation.

Understanding the mechanism of lymph node metastasis, a poor prognostic sign for prostate cancer, and the further dissemination of the disease is important to develop novel treatment strategies. Recent studies have reported that C-C chemokine receptor 7 (CCR7), whose ligand is CCL21, is abundantly expressed in lymph node metastasis and promotes cancer progression. Tumor necrosis factor-α (TNF-α) is chronically produced at low levels within the tumor microenvironment. The aim of this study was to determine whether TNF-α promotes prostate cancer dissemination from metastatic lymph nodes through activation of the CCL21/CCR7 axis. First, human prostate cancer cells were determined to express both TNF-α and CCR7. Second, low concentrations of TNF-α were confirmed to induce CCR7 in prostate cancer cells through phosphorylation of ERK. Finally, CCL21 was found to promote the migration of prostate cancer cells through phosphorylation of the protein kinase p38. Our results suggest that TNF-α leads to the induction of CCR7 expression and that the CCL21/CCR7 axis might increase the metastatic potential of prostate cancer cells in lymph node metastasis.

C-C motif ligand 5 promotes migration of prostate cancer cells in the prostate cancer bone metastasis microenvironment.

Chemokines and their receptors have key roles in cancer progression. The present study investigated chemokine activity in the prostate cancer bone metastasis microenvironment. Growth and migration of human prostate cancer cells were assayed in cocultures with bone stromal cells. The migration of LNCaP cells significantly increased when co-cultured with bone stromal cells isolated from prostate cancer bone metastases. Cytokine array analysis of conditioned medium from bone stromal cell cultures identified CCL5 as a concentration-dependent promoter of LNCaP cell migration. The migration of LNCaP cells was suppressed when C-C motif ligand 5 (CCL5) neutralizing antibody was added to cocultures with bone stromal cells. Knockdown of androgen receptor with small interfering RNA increased the migration of LNCaP cells compared with control cells, and CCL5 did not promote the migration of androgen receptor knockdown LNCaP. Elevated CCL5 secretion in bone stromal cells from metastatic lesions induced prostate cancer cell migration by a mechanism consistent with CCL5 activity upstream of androgen receptor signaling.

Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22-CCR4 axis.

Previous studies have found that tumor-associated macrophages (TAMs) promote cancer progression. We previously reported that TAMs promote prostate cancer metastasis via activation of the CCL2-CCR2 axis. The CCR4 (receptor of CCL17 and CCL22) expression level in breast cancer was reported to be associated with lung metastasis. The aim of this study was to elucidate the role of CCR2 and CCR4 in prostate cancer progression. CCR2 and CCR4 were expressed in human prostate cancer cell lines and prostate cancer tissues. In vitro co-culture of prostate cancer cells and macrophages resulted in increased CCL2 and CCR2 levels in prostate cancer cells. The addition of CCL2 induced CCL22 and CCR4 production in prostate cancer cells. The migration and invasion of prostate cancer cells via enhanced phosphorylation of Akt were promoted by CCL17 and CCL22. CCR4 may be a potential candidate for molecular-targeted therapy.

Metastasectomy Improves Survival in Patients with Metastatic Urothelial Carcinoma.

Metastatic urothelial carcinoma is one of the most fatal urological malignancies. Cisplatin-based systemic chemotherapy is the standard treatment for metastatic urothelial carcinoma, and there is little evidence to support metastasectomy. The aims of the study were to evaluate the efficacy of metastasectomy and to investigate the prognoses of the patients. The study included 436 patients with urothelial carcinoma who were treated at our hospital. Of these, we included and retrospectively analyzed 29 patients who received curative treatment for the primary tumor and had been treated for metastases. Seven of these patients underwent metastasectomy. In a multivariate analysis, a serum C-reactive protein level before treatment for metastasis of <1 mg/dl and metastasectomy were independent significant predictors of both better progression-free survival and better overall survival. Metastasectomy may be considered a potential treatment for patients with metastases from urothelial carcinoma.

Serum chemokine (CC motif) ligand 2 level as a diagnostic, predictive, and prognostic biomarker for prostate cancer.

Prostate-specific antigen (PSA) is regarded as the most sensitive biomarker for prostate cancer. Although androgen/androgen receptor (AR) signaling promotes prostate cancer progression, suppression of AR signaling induces chemokine (CC motif) ligand 2 (CCL2), which enables prostate cancer cells to gain metastatic potential. AR-controlled PSA alone may be an unreliable biomarker for patients receiving androgen deprivation therapy. Therefore, we investigated the validity of CCL2 as a complementary biomarker to PSA for prostate cancer. Our in vitro approach of enriching for prostate cancer cells with higher migration potential showed that CCL2 activated cellular migration. Importantly, we found that CCL2 levels were significantly different between men (n = 379) with and without prostate cancer. Patients with CCL2 ≥ 320 pg/mL had worse overall survival and prostate cancer -specific survival than those with CCL2 < 320 pg/mL. A novel risk classification was developed according to the risk factors CCL2 ≥ 320 pg/mL and PSA ≥ 100 ng/mL, and scores of 2, 1, and 0 were defined as poor, intermediate, and good risk, respectively, and clearly distinguished patient outcomes. CCL2 may serve as a novel biomarker for prostate cancer. The novel risk classification based on combining CCL2 and PSA is more reliable than using either alone.