CCL2 as a potential therapeutic target for clear cell renal cell carcinoma

We previously reported that the pVHL-atypical PKC-JunB pathway contributed to promotion of cell invasiveness and angiogenesis in clear cell renal cell carcinoma (ccRCC), and we detected chemokine (C-C motif) ligand-2 (CCL2) as one of downstream effectors of JunB. CCL2 plays a critical role in tumorigenesis in other types of cancer, but its role in ccRCC remains unclear. In this study, we investigated the roles and therapeutic potential of CCL2 in ccRCC. Immunohistochemical analysis of CCL2 expression for ccRCC specimens showed that upregulation of CCL2 expression correlated with clinical stage, overall survival, and macrophage infiltration. For functional analysis of CCL2 in ccRCC cells, we generated subclones of WT8 cells that overexpressed CCL2 and subclones 786-O cells in which CCL2 expression was knocked down. Although CCL2 expression did not affect cell proliferation in vitro, CCL2 overexpression enhanced and CCL2 knockdown suppressed tumor growth, angiogenesis, and macrophage infiltration in vivo. We then depleted macrophages from tumor xenografts by administration of clodronate liposomes to confirm the role of macrophages in ccRCC. Depletion of macrophages suppressed tumor growth and angiogenesis. To examine the effect of inhibiting CCL2 activity in ccRCC, we administered CCL2 neutralizing antibody to primary RCC xenografts established from patient surgical specimens. Inhibition of CCL2 activity resulted in significant suppression of tumor growth, angiogenesis, and macrophage infiltration. These results suggest that CCL2 is involved in angiogenesis and macrophage infiltration in ccRCC, and that CCL2 could be a potential therapeutic target for ccRCC

Functional and genomic characterization of patient‐derived xenograft model to study the adaptation to mTORC1 inhibitor in clear cell renal cell carcinoma

Resistance to the mechanistic target of rapamycin (mTOR) inhibitors, which are a standard treatment for advanced clear cell renal cell carcinoma (ccRCC), eventually develops in most cases. In this study, we established a patient-derived xenograft (PDX) model which acquired resistance to the mTOR inhibitor temsirolimus, and explored the underlying mechanisms of resistance acquisition. Temsirolimus was administered to PDX model mice, and one cohort of PDX models acquired resistance after repeated passages. PDX tumors were genetically analyzed by whole-exome sequencing and detected several genetic alterations specific to resistant tumors. Among them, mutations in ANKRD12 and DNMT1 were already identified in the early passage of a resistant PDX model, and we focused on a DNMT1 mutation as a potential candidate for developing the resistant phenotype. While DNMT1 expression in temsirolimus-resistant tumors was comparable with the control tumors, DNMT enzyme activity was decreased in resistant tumors compared with controls. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated heterozygous knockdown of DNMT1 in the temsirolimus-sensitive ccRCC (786-O) cell line was shown to result in a temsirolimus-resistant phenotype in vitro and in vivo. Integrated gene profiles using methylation and microarray analyses of PDX tumors suggested a global shift for the hypomethylation status including promotor regions, and showed the upregulation of several molecules that regulate the mTOR pathway in temsirolimus-resistant tumors. Present study showed the feasibility of PDX model to explore the mechanisms of mTOR resistance acquisition and suggested that genetic alterations, including that of DNMT1, which alter the methylation status in cancer cells, are one of the potential mechanisms of developing resistance to temsirolimus

Amino-terminal enhancer of split gene AES encodes a tumor and metastasis suppressor of prostate cancer

A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration-resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR-defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium-specific inactivation of Aes in Ptenflox/flox mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways

Decreased expression of lysophosphatidylcholine (16:0/OH) in high resolution imaging mass spectrometry independently predicts biochemical recurrence after surgical treatment for prostate cancer.

Article first published online: 1 SEP 2015[Background]Human prostate cancers are highly heterogeneous, indicating a need for various novel biomarkers to predict their prognosis. Lipid metabolism affects numerous cellular processes, including cell growth, proliferation, differentiation, and motility. Direct profiling of lipids in tissue using high-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS) may provide molecular details that supplement tissue morphology. [Methods]Prostate tissue samples were obtained from 31 patients, with localized prostate cancer who underwent radical prostatectomy. The samples were assessed by HR-MALDI-IMS in positive mode, with the molecules identified by tandem mass spectrometry (MS/MS). The effect of identified molecules on prostate specific antigen recurrence free survival after radical prostatectomy was determined by Cox regression analysis and by the Kaplan–Meier method. [Results]Thirteen molecules were found to be highly expressed in prostate tissue, with five being significantly lower in cancer tissue than in benign epithelium. MS/MS showed that these molecules were [lysophosphatidylcholine (LPC)(16:0/OH)+H]+, [LPC(16:0/OH)+Na]+, [LPC(16:0/OH)+K]+, [LPC(16:0/OH)+matrix+H]+, and [sphingomyelin (SM)(d18:1/16:0)+H]+. Reduced expression of LPC(16:0/OH) in cancer tissue was an independent predictor of biochemical recurrence after radical prostatectomy. [Conclusions]HR-MALDI-IMS showed that the expression of LPC(16:0/OH) and SM(d18:1/16:0) was lower in prostate cancer than in benign prostate epithelium. These differences in expression of phospholipids may predict prostate cancer aggressiveness, and provide new insights into lipid metabolism in prostate cancer

Experimental Evidence of Persistent Androgen-Receptor-Dependency in Castration-Resistant Prostate Cancer

Abstract: In the majority of castration-resistant prostate cancer (CRPC), prostate-specific antigen (PSA), product of a gene that is almost exclusively regulated by the androgen receptor (AR), still acts as a serum marker reflecting disease burden, indicating that AR signaling is activated even under castrate level of serum androgen. Accumulated evidence shows that transcriptional ability of AR is activated both in ligand-dependent and-independent manners in CRPC cells. Some androgen-independent sublines derived from originally androgen-dependent LNCaP prostate cancer cells overexpress the AR and PSA, for which silencing the AR gene suppresses cellular proliferation. The overexpression of the AR confers androgen-independent growth ability on androgen-dependent prostate cancer cells. Some patient-derived prostate cancer xenograft lines also acquire castration-resistant growth ability secreting PSA. More recent publications have shown that the AR activated in CRPC cells regulates distinct gene sets from that in androgen-dependent status. This concept provides very important insights in the development of novel anti-prostate cancer drugs such as new generation anti-androgens and CYP17 inhibitors

Up-regulation of miR-582-5p regulates cellular proliferation of prostate cancer cells under androgen-deprived conditions.

[BACKGROUND]MicroRNAs are noncoding small RNA that negatively regulate target gene expression by binding to the 3′-UTR of mRNA. Previous studies have shown that several microRNAs play a pivotal role in prostate cancer by acting as oncogenes or tumor suppressors. This study was aimed at identifying microRNAs that contribute to the progression to castration resistant prostate cancer. [METHODS]MicroRNAs expression profiles of a xenograft model and cell lines were examined by microarray analysis and real-time PCR. Functional analysis of miR-582-5p in cellular proliferation was examined by cell counting. Furthermore, in order to investigate a candidate target of miR-582-5p, microarray analysis and analysis in silico were utilized. [RESULTS]MiR-582-5p was identified to be up-regulated at the castration resistant stage of a xenograft model, KUCaP2 and in castration resistant cell line, AILNCaP#1. Overexpression of miR-582-5p increased the number and the percentage of S phase of LNCaP cells under androgen deprived condition. Moreover, suppression of miR-582-5p decreased the number and the percentage of S phase of AILNCaP#1 cells. Furthermore, we identified that miR-582-5p down-regulates EFNB2 expression, which is down-regulated at the castration resistant stage of a xenograft model, KUCaP2 and in castration resistant cell line, AILNCaP#1. [CONCLUSIONS]Our results suggest that up-regulation of miR-582-5p contributes to an increase in the proliferation of prostate cancer cells under androgen deprived conditions

A pilot study of highly hypofractionated intensity-modulated radiation therapy over 3 weeks for localized prostate cancer

The purpose of this pilot study was to evaluate the feasibility of highly hypofractionated intensity-modulated radiation therapy (IMRT) in 15 fractions over 3 weeks for treating localized prostate cancer based on prostate position-based image-guided radiation therapy. Twenty-five patients with National Comprehensive Cancer Network (NCCN) very low- to unfavorable intermediate-risk prostate cancer were enrolled in this study from April 2014 to September 2015 to receive highly hypofractionated IMRT (without intraprostatic fiducial markers) delivering 54 Gy in 15 fractions over 3 weeks. Patients with intermediate-risk disease underwent neoadjuvant androgen suppression for 4–8 months. Twenty-four patients were treated with highly hypofractionated IMRT, and one was treated with conventionally fractionated IMRT because the dose constraint of the small bowel seemed difficult to achieve during the simulation. Seventeen percent had very low- or low-risk, 42% had favorable intermediate-risk, and 42% had unfavorable intermediate-risk disease according to NCCN guidelines. The median follow-up period was 31 months (range, 24–42 months). No Grade ≥3 acute toxicity was observed, and the incidence rates of Grade 2 acute genitourinary and gastrointestinal toxicities were 21% and 4%, respectively. No Grade ≥2 late toxicity was observed. Biochemical relapse was observed in one patient at 15 months, and the biochemical relapse-free survival rate was 95.8% at 2 years. A prostate-specific antigen bounce of ≥0.4 ng/ml was observed in 11 patients (46%). The highly hypofractionated IMRT regimen is feasible in patients with localized prostate cancer and is more convenient than conventionally fractionated schedules for patients and health-care providers

Outcomes of curative nephrectomy against renal cell carcinoma based on a central pathological review of 914 specimens from the era of cytokine treatment.

[Background]The purpose of this study was to determine the state of modern practice with regard to renal cell carcinoma (RCC) outcomes and to assess the effects on survival of such clinical and pathological factors such as histological subtype (HS) and nuclear grade by conducting a central pathological review based on the current World Health Organization classification and the staging system of the American Joint Committee on Cancer/Union for International Cancer Control. [Methods]We collected glass slides and clinical data sets for 914 cases of RCC treated with curative nephrectomy from 1995 to 2000. Overall (OS), cancer-specific (CSS), and relapse-free (RFS) survival were compared for HS and nuclear grades determined by a central pathology review board comprising 5 board-certified pathologists, pathological staging, and a variety of clinical factors. [Results]The 5 and 7-year CSS in this study were 96 and 93 %, respectively, values superior to those reported in Western countries. Concordance between the original and reviewed HS and nuclear grades were 90.9 and 21.1 %, respectively. HS correlated with OS (P = 0.043) but was not an independent prognostic factor in the multivariate analysis (P = 0.820). Tumor size, Fuhrman grade, and infiltration type were common independent prognostic factors for OS, CSS, and RFS. [Conclusions]This study revealed RCC outcomes in the era of cytokine treatment for metastasis. Central pathological review is an essential component of a multicenter study with long-term follow-up. Tumor size, Fuhrman grade, and infiltration type had much greater effects than HS on survival after curative nephrectomy

Prognosis of Japanese patients with previously untreated metastatic renal cell carcinoma in the era of molecular-targeted therapy

A multicenter cooperative study was conducted to clarify the prognosis of Japanese patients with metastatic renal cell carcinoma in the era of molecular-targeted therapy and the clinical usefulness of the Japanese metastatic renal cancer (JMRC) prognostic classification. Of 389 consecutive patients for whom treatment was started between 2008 and 2010 at 23 hospitals in Japan, 357 patients who received vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGFR-TKI) or cytokine as initial systemic therapy were the subject of the present study. Patients were classified into three prognostic groups according to the JMRC prognostic classification. The endpoints were progression-free survival (PFS) and overall survival (OS) after the start of the initial treatment. The median PFS and OS for the entire cohort of 357 patients were 9.1 and 27.2months, respectively. VEGFR-TKI were selected for patients with multiple organ metastases, those with liver metastasis, and those with bone metastasis. The median PFS and OS were 11.0 and 23.2months and 5.4 and 38.2months in the VEGFR-TKI group and the cytokines group, respectively. The JMRC prognostic classification was useful as a prognostic model for PFS and OS (c-indexes: 0.613 and 0.630 in patients who initially received VEGFR-TKI and 0.647 and 0.642 in patients who received cytokines, respectively). The present study showed for the first time the prognosis of Japanese patients with metastatic renal cell carcinoma in the era of molecular-targeted therapy. The JMRC prognostic classification may be clinically useful as a prognostic model