Masatoshi Eto

Macrophage infiltration and its prognostic relevance in clear cell renal cell carcinoma

Most malignant tumors evidence infiltration of many macrophages. In this study, we investigated an anti‐inflammatory macrophage phenotype (M2) in clear cell renal cell carcinoma (RCC) using CD163 and CD204 as markers. Immunostaining showed a correlation between the number of CD163+ cells and age, sex, nuclear grade, and TNM classification. High infiltration of CD163+ cells was significantly associated with poor clinical prognosis in univariate analysis but not in multivariate analysis. We also carried out in vitro studies to examine cell–cell interactions between macrophages and cancer cells. Culture supernatants from RCC cell lines induced polarization of macrophages toward the M2 phenotype. Coculture of macrophages with cancer cells significantly induced activation of signal transducers and activators of transcription‐3 (Stat3) in the cancer cells. Direct coculture of RCC cells with macrophages led to stronger activation of Stat3 in the cancer cells than did indirect coculture using Transwell chamber dishes. Because RCC cells expressed membrane‐type macrophage colony‐stimulating factor (mM‐CSF) on the cell surface, we suggested that this mM‐CSF plays an important role in direct cell–cell interactions. Stat3 activation in cancer cells that was induced by coculture with macrophages was suppressed by downregulation of the M‐CSF receptor (M‐CSFR) in macrophages and by an inhibitor of M‐CSFR. In conclusion, investigation of CD163+ tumor‐associated macrophages would be useful for assessment of the clinical prognosis of patients with ccRCC. Cell–cell interactions mediated by mM‐CSF and M‐CSFR binding could contribute to cancer cell activation. (Cancer Sci 2011; 102: 1424–1431)

Castration resistance of prostate cancer cells caused by castration-induced oxidative stress through Twist1 and androgen receptor overexpression

There are few successful therapies for castration-resistant prostate cancer (CRPC). Recently, CRPC has been thought to result from augmented androgen/androgen receptor (AR) signaling pathway, for most of which AR overexpression has been observed. In this study, Twist1, a member of basic helix-loop-helix transcription factors as well as AR was upregulated in response to hydrogen peroxide, and the response to which was abolished by an addition of N-acetyl-L-cysteine and Twist1 knockdown. In addition, castration-resistant LNCaP derivatives and hydrogen peroxide-resistant LNCaP derivatives exhibited a similar phenotype to each other. Then, both castration and AR knockdown increased intracellular reactive oxygen species level. Moreover, Twist1 was shown to regulate AR expression through binding to E-boxes in AR promoter region. Silencing of Twist1 suppressed cell growth of AR-expressing LNCaP cells as well as castration-resistant LNCaP derivatives by inducing cell-cycle arrest at G1 phase and cellular apoptosis. These findings indicated that castration-induced oxidative stress may promote AR overexpression through Twist1 overexpression, which could result in a gain of castration resistance. Modulation of castration-induced oxidative stress or Twist1/AR signaling might be a useful strategy for developing a novel therapeutics in prostate cancer, even in CRPC, which remains dependent on AR signaling by overexpressing AR.

Tip60 promotes prostate cancer cell proliferation by translocation of androgen receptor into the nucleus.

There are currently few effective therapies for castration‐resistant prostate cancer (CRPCa). CRPC which is resistant to castration is thought to result from increased activation of the androgen/androgen receptor (AR) signaling pathway, which may be augmented by AR coactivators.

IL-17 production by γδ T cells is important for the antitumor effect of Mycobacterium bovis bacillus Calmette-Guérin treatment against bladder cancer.

Intravesical inoculation of Mycobacterium bovis bacillus Calmette‐Guérin (BCG) has been used for the treatment of bladder cancer. Recent studies implied the requirement of neutrophil infiltration for the antitumor effect. In this study, we found that IL‐17 was produced in the bladder after BCG treatment, preceding the infiltration of neutrophils. Neutrophils in the bladder after BCG treatment were reduced in IL‐17‐deficient mice, in which BCG‐induced antitumor effect against intravesically inoculated bladder cancer was abolished. Notably, the level of IL‐17 production and the number of neutrophils in BCG‐treated bladder was reduced in γδ T‐cell‐deficient mice but not in CD4‐depleted mice. Survival of bladder cancer‐inoculated γδ T‐cell‐deficient mice was not improved by BCG treatment. These results suggest that IL‐17‐producing γδ T cells play a key role in the BCG‐induced recruitment of neutrophils to the bladder, which is essential for the antitumor activity against bladder cancer.

Phase III Trial of Everolimus in Metastatic Renal Cell Carcinoma: Subgroup Analysis of Japanese Patients from RECORD-1

Objective To assess the efficacy and safety of everolimus in Japanese patients with metastatic renal cell carcinoma. Methods A subgroup analysis of the pivotal Phase III, randomized, double-blind, placebo-controlled trial of everolimus 10 mg/day in patients with disease progression after treatment with sorafenib, sunitinib or both assessed outcomes in Japanese participants. Results were compared with those for the overall study population. Results The final trial analysis included 24 Japanese patients (everolimus, n= 15; placebo, n = 9). Median progression-free survival in the Japanese subpopulation was 5.75 months (95% confidence interval, 4.90 months to not reached) with everolimus and 3.61 months (95% confidence interval, 1.91–9.03 months) with placebo (hazard ratio, 0.19; 95% confidence interval, 0.05–0.83). Median overall survival was not reached with everolimus and was 14.9 months (95% confidence interval, 11.0–16.8 months) with placebo (hazard ratio, 0.30; 95% confidence interval, 0.07–1.27). Overall, efficacy and safety were similar when comparing the Japanese and overall populations. In the Japanese subpopulation, the most common adverse events with everolimus were stomatitis, infections and rash. Four Japanese subjects (27%) developed Grade 1 (n = 2) or 2 (n = 2) pneumonitis (all reversible and allowing for continuation of therapy, after interruption, steroids and dose reduction for both Grade 2 cases), with a lower pneumonitis incidence of 14% in the overall population (albeit associated with a Grade 3 incidence of 4%). Conclusions These findings suggest that the demonstrated benefits of everolimus in the overall trial population are similar in Japanese patients with metastatic renal cell carcinoma.

Enhancement of Human Cancer Cell Motility and Invasiveness by Anaphylatoxin C5a via Aberrantly Expressed C5a Receptor (CD88)

Purpose: The anaphylatoxin C5a is a chemoattractant that induces leukocyte migration via C5a receptor (C5aR). There is emerging evidence that C5a is generated in the cancer microenvironment. We therefore sought C5aR expression and a direct influence of the C5a–C5aR axis on cancer cells. Experimental Design: C5aR expression was investigated in human cancer tissues and cell lines. Effects of C5a stimulation on cancer cells were studied by cytoskeletal rearrangement, time-lapse analysis, Matrigel chamber assay, and invasion in nude mouse in a comparison of C5aR-expressing cancer cells with control cells. Results: C5aR was aberrantly expressed in various human cancers. Several cancer cell lines also expressed C5aR. C5a triggered cytoskeletal rearrangement and enhanced cell motility three-fold and invasiveness 13-fold of C5aR-expressing cancer cells. Such enhancement by C5a was not observed in control cells. Cancer cell invasion was still enhanced in the absence of C5a concentration gradient and even after the removal of C5a stimulation, suggesting that random cell locomotion plays an important role in C5a-triggered cancer cell invasion. C5a increased the release of matrix metalloproteinases (MMP) from cancer cells by two- to 11-fold, and inhibition of MMP activity abolished the C5a-enhancing effect on cancer cell invasion. Compared with control cells, C5aR-expressing cells spread 1.8-fold more broadly at implanted nude mouse skin sites only when stimulated with C5a. Conclusions: These results illustrate a novel activity of the C5a–C5aR axis that promotes cancer cell invasion through motility activation and MMP release. Targeting this signaling pathway may provide a useful therapeutic option for cancer treatment. Clin Cancer Res; 19(8); 2004–13. ©2013 AACR.

Randomised phase III study of neoadjuvant chemotherapy with methotrexate, doxorubicin, vinblastine and cisplatin followed by radical cystectomy compared with radical cystectomy alone for muscle-invasive bladder cancer: Japan Clinical Oncology Group Study JCOG0209

BACKGROUND This study aimed to determine the clinical benefit of neoadjuvant methotrexate, doxorubicin, vinblastine, and cisplatin (MVAC) in patients with muscle-invasive bladder cancer (MIBC) treated with radical cystectomy. PATIENTS AND METHODS Patients with MIBC (T2-4aN0M0) were randomised to receive two cycles of neoadjuvant MVAC followed by radical cystectomy (NAC arm) or radical cystectomy alone (RC arm). The primary end point was overall survival (OS). Secondary end points were progression-free survival, surgery-related complications, adverse events during chemotherapy, proportion with no residual tumour in the cystectomy specimens, and quality of life. To detect an improvement in 5-year OS from 45% in the RC arm to 57% in the NAC arm with 80% power, 176 events were required per arm. RESULTS Patients (N = 130) were randomly assigned to the RC arm (N = 66) and the NAC arm (N = 64). The patient registration was terminated before reaching the initially planned number of patients because of slow accrual. At the second interim analysis just after the early stoppage of patient accrual, the Data and Safety Monitoring Committee recommended early publication of the results because the trial did not have enough power to draw a confirmatory conclusion. OS of the NAC arm was better than that of the RC arm, although the difference was not statistically significant [hazard ratio 0.65, multiplicity adjusted 99.99% confidence interval 0.19-2.18, one-sided P = 0.07]. In the NAC arm and the RC arm, 34% and 9% of the patients had pT0, respectively (P < 0.01). In subgroup analyses, OS in almost all subgroups was in favour of NAC. CONCLUSIONS This trial showed a significantly increased pT0 proportion and favourable OS of patients who received neoadjuvant MVAC. NAC with MVAC can still be considered promising as a standard treatment. UMIN CLINICAL TRIALS REGISTRY IDENTIFIER C000000093.

Inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1) modulates the acquisition of cisplatin resistance in bladder cancer cell lines.

To investigate the molecules that regulate the acquisition of cis-diamminedichloroplatinum (II) (cisplatin) resistance, we performed cDNA microarrays using two pairs of parental and cisplatin-resistant bladder cancer cell lines. We found a markedly reduced expression of inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1), endoplasmic reticulum membrane protein, in cisplatin-resistant cells. The suppression of IP3R1 expression using small interfering RNA in parental cells prevented apoptosis and resulted in decreased sensitivity to cisplatin. Contrarily, overexpression of IP3R1 in resistant cells induced apoptosis and increased sensitivity to cisplatin. These results suggest that cisplatin-induced downregulation of IP3R1 expression was closely associated with the acquisition of cisplatin resistance in bladder cancer cells.

Oral administration of xanthan gum enhances antitumor activity through Toll-like receptor 4

PURPOSE Xanthan gum (XG) is a complex exopolysaccharide produced by the plant-pathogenic bacterium Xanthomonas campestris pv. and is widely used as a thickener or viscosifier. We examined in this study the antitumor effects of XG. EXPERIMENTAL DESIGN Cytokine production by XG-stimulated murine macrophage cell lines, J772 and RAW264.7, and peritoneal adherent cells from wild type C57BL/6 mice, TLR2 or MyD88-deficient mice, C3H/HeN, and TLR4-mutant C3H/HeJ mice were examined. In order to examine in vivo antitumor effects of XG, mice were inoculated subcutaneously with tumor cells and administered orally with XG once every 5days from 1day before the tumor inoculation. Tumor growth, mouse survival, NK activity, and tumor-specific cytotoxicity were examined. RESULTS In vitro culture with XG induced interleukin-12 and tumor necrosis factor-alpha production from macrophages. XG stimulated macrophages in a MyD88-dependent manner and was mainly recognized by Toll-like receptor 4 (TLR4). Oral administration of XG significantly retarded tumor growth and prolonged survival of the mice inoculated subcutaneously with B16K(b) melanoma cells. NK activity as well as tumor-specific cytotoxicity of CD8 T cells was augmented in the XG-treated mice. The in vivo antitumor effects of XG were also dependent on TLR-4, as C3H/HeJ mice, which lack TLR4 signaling, exhibited no effect of XG on the growth of syngeneic bladder tumor, MBT-2. CONCLUSION These results suggest beneficial effects of oral administration of XG on immune-surveillance against neoplasms.

Importance of C16 ceramide accumulation during apoptosis in prostate cancer cells

Aim:  Adenocarcinoma of the prostate is one of the most frequently diagnosed non‐cutaneous cancers and the second leading cause of cancer‐related deaths among men in the United States. To fully understand the role of ceramide during apoptosis induced by androgen ablation, we modified the levels of intracellular ceramide by pharmacological agents as well as through serum deprivation in androgen‐dependent and independent cell lines.