Should Patients with Renal Cell Carcinoma and Pathological Nodal Invasion Be Classified As Having Stage IV Disease?

BACKGROUND: Lymph node invasion is associated with poor outcome in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: Patients with RCC within a single center from 2001 to 2018 were retrospectively obtained from the Chang Gung Research Database. Patient gender, physical status, Charlson Comorbidity Index, tumor side, histology, age at diagnosis, and body mass index (BMI) were compared. The overall survival (OS) and cancer-specific survival (CSS) of each group were estimated using the Kaplan-Meier method. Log-rank tests were used to compare between the subgroups. RESULTS AND CONCLUSIONS: A total of 335 patients were enrolled, of whom 76 had pT3N0M0, 29 had pT1-3N1M0, 104 had T1-4N0M1, and 126 had T1-4N1M1 disease. Significant OS difference was noted between pT3N0M0 and pT1-3N1M0 groups with 12.08 years [95% confidence interval (CI), 8.33-15.84] versus 2.58 years (95% CI, 1.32-3.85), respectively (P < 0.005). No significant difference was observed in OS between pT1-3N1M0 and T1-4N0M1 groups with 2.58 years (95% CI, 1.32-3.85) versus 2.50 years (95% CI, 1.85-3.15, P = 0.72). The OS of N1M1 group was worse than that of N0M1 group with 1.00 year (95% CI, 0.74-1.26) versus 2.50 years (95% CI, 1.85-3.15, P < 0.05). Similar results were also observed in CSS. In summary, we claim that RCC with lymph node (LN) invasion should be reclassified as stage IV disease in terms of survival outcome.

A Comparative Study of Stone Re-Treatment after Lithotripsy.

The high recurrence rate has always been a problem associated with urolithiasis. This study aimed to explore the effectiveness of single interventions, combined therapies, and surgical and nonsurgical interventions. Herein, three lithotripsy procedures­extracorporeal shock wave lithotripsy (ESWL), percutaneous nephrolithotomy (PCNL), and ureteroscopic lithotripsy (URSL)­were assessed and a retrospective cohort was selected in order to further analyze the association with several risk factors. Firstly, a population-based cohort from the Taiwan National Health Insurance Research Database (NHIRD) from 1997 to 2010 was selected. In this study, 350 lithotripsy patients who underwent re-treatment were followed up for at least six years to compare re-treatment rates, with 1400 patients without any lithotripsy treatment being used as the comparison cohort. A Cox proportional hazards regression model was applied. Our results indicate that the risk of repeat urolithiasis treatment was 1.71-fold higher in patients that received lithotripsy when compared to patients that were not treated with lithotripsy (hazard ratio (HR) 1.71; 95% confidence interval (CI) = 1.427−2.048; p < 0.001). Furthermore, a high percentage of repeated treatment was observed in the ESWL group (HR 1.60; 95% CI = 1.292−1.978; p < 0.001). Similarly, the PCNL group was also independently associated with a high chance of repeated treatment (HR 2.32; 95% CI = 1.616−3.329; p < 0.001). Furthermore, age, season, level of care, and Charlson comorbidities index (CCI) should always be taken into consideration as effect factors that are highly correlated with repeated treatment rates.

A Highly Sensitive Urinary Exosomal miRNAs Biosensor Applied to Evaluation of Prostate Cancer Progression.

Prostate cancer is the most common cancer in the male population, carrying a significant disease burden. PSA is a widely available screening tools for this disease. Current screen-printed carbon electrode (SPCE)-based biosensors use a two-pronged probe approach to capture urinary miRNA. We were able to successfully detect specific exosomal miRNAs (exomiRs) in the urine of patients with prostate cancer, including exomiR-451 and exomiR-21, and used electrochemistry for measurement and analysis. Our results significantly reaffirmed the presence of exomiR-451 in urine and that a CV value higher than 220 nA is capable of identifying the presence of disease (p-value = 0.005). Similar results were further proven by a PAS greater than 4 (p-value = 0.001). Moreover, a higher urinary exomiR-21 was observed in the high-T3b stage; this significantly decreased following tumor removal (p-values were 0.016 and 0.907, respectively). According to analysis of the correlation with tumor metastasis, a higher exomiR-21 was associated with lymphatic metastasis (p-value 0.042), and higher exomiR-461 expression was correlated with tumor stage (p-value 0.031), demonstrating that the present exomiR biosensor can usefully predict tumor progression. In conclusion, this biosensor represents an easy-to-use, non-invasive screening tool that is both sensitive and specific. We strongly believe that this can be used in conjunction with PSA for the screening of prostate cancer.

Urinary MicroRNA Sensing Using Electrochemical Biosensor to Evaluate Colorectal Cancer Progression.

Research in cancer diagnostics has recently established its footing and significance in the biosensor sphere, emphasizing the idea of a unique probe design used as a sensor and actuator, to identify the presence of protein, DNA, RNA, or miRNA. The fluorescein isothiocyanate (FITC) probe and biotinylated probe are designed for a two-pronged approach to the detection of the urinary miR-21 and miR-141, both of which have demonstrated significance in the development and progression of colorectal cancer, a leading cause of mortality and morbidity. The remainder of the apparatus is composed of a modified screen-printed carbon electrode (SPCE), to which the probes adhere, that transduces signals via the redox reaction between H2O2 and HRP, measured with chronoamperometry and cyclic voltammetry. The precise nature of our ultra-non-invasive biosensor makes for a highly sensitive and practical cancer detector, concluded by the significance when establishing disease presence (miR-21 p-value = 0.0176, miR-141 p-value = 0.0032), disease follow-up (miR-21 p-value = 0.00154, miR141 p-value < 0.0005), and even disease severity. This article hopes to emphasize the potential of an additional clinical tool for the management of colorectal cancer.

EPA Modulates KLK Genes via miR-378: A Potential Therapy in Prostate Cancer.

It is known that miRNA-378a-3p (miR-378) could be induced by eicosapentaenoic acid (EPA), an omega-3 fatty acid. Herein, we first demonstrated how miR-378 exerts anti-prostate cancer (PCa) actions by influencing multiple target genes, including KLK2, KLK4, KLK6, and KLK14, which are implicated in PCa development, cell proliferation, and cell survival. Furthermore, these genes also correlate with androgen and mTOR signaling transduction, and are considered pivotal pathways for the onset and progression of PCa. In total, four PCa cell lines and eight pairing tissues (tumor vs. normal) from clinical PCa patients were included in the current study. The results showed high significance after EPA induced tumor cells containing higher expression levels of miR-378, and led the PCa cells having low cell viabilities, and they progressed to apoptosis when compared with normal prostate cells (p < 0.001). The findings indicated that EPA might become a potential therapy for PCa, especially because it is derived from the components of natural fish oil; it may prove to be a great help for solving the problem of castration-resistant prostate cancer (CRPC).

Gold Nanocluster-Based Fluorometric Banoxantrone Assay Enabled by Photoinduced Electron Transfer.

Monitoring the blood concentration of banoxantrone (AQ4N) is important to evaluate the therapeutic efficacy and side effects of this new anticancer prodrug during its clinical applications. Herein, we report a fluorescence method for AQ4N detection through the modulation of the molecule-like photoinduced electron transfer (PET) behavior of gold nanoclusters (AuNCs). AQ4N can electrostatically bind to the surface of carboxylated chitosan (CC) and dithiothreitol (DTT) co-stabilized AuNCs and quench their fluorescence via a Coulomb interaction-accelerated PET process. Under optimized experimental conditions, the linear range of AQ4N is from 25 to 200 nM and the limit of detection is as low as 5 nM. In addition, this assay is confirmed to be reliable based on its successful use in AQ4N determination in mouse plasma samples. This work offers an effective strategy for AQ4N sensing based on fluorescent AuNCs and widens the application of AuNCs in clinical diagnosis and pharmaceutical analysis.

Micro-RNA378a-3p Induces Apoptosis in Sarcomatoid Renal Cell Carcinoma and Regulates POLR2A and RUNX2 Expression.

BACKGROUND/AIM: Sarcomatoid renal cell carcinoma (sRCC) is an aggressive subtype of RCC. In the current study, we investigated whether the POLR2A and RUNX2 genes are involved in RCC-related signaling pathway and associated with miR-378. MATERIALS AND METHODS: Thirteen formalin-fixed and paraffin-embedded RCC samples were collected. Three software programs were used to predict the potential gene regulation in RCC by miR-378 and immunohistochemistry analysis was applied to confirm the expression of targeted proteins in FFPE samples. MicroRNA-378 transfection, analysis of mRNA and protein expression via Western Blotting and cell apoptosis analysis via flow cytometry for POLR2A and RUNX2 were further studied in four renal cell carcinoma cell lines. RESULTS: Both the mRNA and protein expression levels of POLR2A and RUNX2 in sRCC cell lines and were significantly higher than those in other subtypes of RCC, and similar results were obtained in clinical samples (p<0.01). Second, overexpression of miR-378 significantly suppressed the expression of POLR2A and RUNX2 in sRCC cells (p<0.01) and enhanced apoptosis (p<0.05). CONCLUSION: miR-378 significantly inhibits the expression of POLR2A and RUNX2 in sRCC and further promotes apoptosis of sRCC cells. We speculated that the apoptosis mechanism in sRCC occurs via regulation of the ERK2 and PI3K/AKT pathways, which might distinguish it from other subtypes of RCC.

An Effective SARS-CoV-2 Electrochemical Biosensor with Modifiable Dual Probes Using a Modified Screen-Printed Carbon Electrode.

Due to the severe acute respiratory syndrome coronavirus (SARS-CoV-2, also called coronavirus disease 2019 (COVID-19)) pandemic starting in early 2020, all social activities ceased in order to combat its high transmission rate. Since vaccination combats one aspect for halting the spread of the virus, the biosensor community has looked at another aspect of reducing the burden of the COVID-19 pandemic on society by developing biosensors that incorporate point-of-care (POC) testing and the rapid identification of those affected in order to deploy appropriate measures. In this study, we aim first to propose a screen-printed carbon electrode (SPCE)-based electrochemical biosensor that meets the ASSURED criteria (i.e., affordable, sensitive, specific, user-friendly, rapid, equipment-free, and deliverable) for POC testing, but more importantly, we describe the novelty of our biosensor's modifiability that uses custom dual probes made from target nucleic acid sequences. Additionally, regarding the sensitivity of the biosensor, the lowest sample concentration was 10 pM (p = 0.0257) without amplification, which might challenge the traditional technique of reverse transcriptase-polymerase chain reaction (RT-PCR). The purpose of this study is to develop a means of diagnostics for the current pandemic as well as to provide an established POC platform for future epidemics.

Gain of TPPP as a predictor of progression in patients with bladder cancer.

The present study investigated the role of tubulin polymerization promoting protein (TPPP) in the regulation of bladder cancer (BC) cell proliferation and migration, in addition to the association between TPPP gene copy number amplification and clinicopathological characteristics of BC. TPPP gene amplification was measured in human BC epithelial cells and samples obtained from 52 patients with BC via fluorescence in situ hybridization. TPPP gain was defined as mean TPPP copy number >2.2 per nucleus (cutoff). The neutrophil-to-lymphocyte ratio (NLR) was also obtained from the preoperative data of the patients. For in vitro assays, BC cell lines were transfected with either TPPP small interfering RNAs or scrambled control, following which cell proliferation and migration were determined using Cell Counting Kit-8 and Transwell migration assays, respectively. The percentage of cells with TPPP copy number amplification in the four BC epithelial cell lines (MGH-U1, -U1R, -U3, -U4) examined (86.0-100.0%) was found to be higher compared with that in the normal human uroepithelial cell lines (3.0 and 9.0%). Patients were divided into one- (1.9%), two- (55.8%), three- (7.7%), four- (26.9%) and five-copy (7.7%) types. Results calculated using Fisher's exact test indicated that the gain of TPPP in patients with BC associated significantly with age (P<0.05), advanced histological grade (P<0.001), tumor stage (P<0.05), histological type (P<0.001) and NLR (P<0.05). In MGH-U1R and MGH-U4 cells, cell proliferation and migration were revealed to be significantly lower following TPPP knockdown compared with those in cells transfected with the scrambled control. In conclusion, findings from the present study suggest that TPPP is important for cell proliferation, cell migration and BC progression, such that TPPP copy number assessment would be advised for preoperative urine cytology for urothelial neoplasia diagnosis.

High-Sensitivity Dual-Probe Detection of Urinary miR-141 in Cancer Patients via a Modified Screen-Printed Carbon Electrode-Based Electrochemical Biosensor.

The screening and diagnosis of cancer are hallmarks of medicine in the aging population. Recently, microRNAs have shown potential for use as biomarkers, which could advance the field of diagnostics. The presence of miRNA-141 in the serum has been well described in several malignancies. However, the invasive approach used for sampling represents the major limitation for its practical application and, hence, its notable absence as a method for screening the general population. In light of this, we aimed to develop a high-sensitivity microRNA (miR) biosensor for application in the diagnosis of all miR-141-associated cancers, such as colorectal cancer (CRC) and breast cancer (BC). The novelty lies in our dual-probe design, which is reliant on the hybridization of the fluorescein isothiocyanate (FITC) targeting probe onto an existing sample of urinary miR-141 in the first step, followed by complementary binding with a biotinylated probe that has been coated on a modified screen-printed carbon electrode (SPCE). The hybridization of the probe and sensor produces signals via the catalytic reduction of H2O2 at HRP-modified SPCEs in the presence of H2O, which was measured by either cyclic voltammetry or chronoamperometry (CA) currents. In our study, the detection and expression of miR-141 in a cohort of colorectal cancer (n = 6) and breast cancer (n = 4) samples showed that its levels were significantly higher than in a healthy cohort (n = 9) (p < 0.004). Moreover, our miR sensor demonstrated high stability, reliability, and sensitivity (p < 0.0001). This work hopefully provides new information for the detection and monitoring of de novo and existing cancers.

Glucose/Glutathione Co-triggered Tumor Hypoxia Relief and Chemodynamic Therapy to Enhance Photothermal Therapy in Bladder Cancer.

Photothermal therapy (PTT) is a potential treatment for cancer that makes use of near-infrared (NIR) laser irradiation and is expected to assist traditional anti-cancer drug therapies; however, the therapeutic efficacy of PTT is restricted by thermal resistance due to the overexpression of heat shock proteins and insufficient penetration depth of lasers. Thus, PTT needs to be combined with additional therapeutic methods to obtain the optimal therapeutic efficacy for cancer. Herein, a multifunctional therapeutic platform combining PTT with glucose-triggered chemodynamic therapy (CDT) and glutathione (GSH)-triggered hypoxia relief was developed via GOx@MBSA-PPy-MnO2 NPs (GOx for glucose oxidase, M for Fe3O4, BSA for bovine serum albumin, and PPy for polypyrrole). GOx@MBSA-PPy-MnO2 NPs have excellent photothermal efficiency and can release Mn2+, which catalyzes the transformation of H2O2 into hydroxyl radicals (·OH) and O2 via a Fenton-like reaction, effectively destroying cancer cells and relieving tumor hypoxia. Meanwhile, a high content of H2O2 was produced via GOx catalysis of glucose, further enhancing the CDT efficiency. In addition, in vitro and in vivo experiments showed that the inhibition of cancer cell proliferation and effective inhibition of tumors could be caused by the combined PTT/glucose-triggered CDT effects and hypoxia relief of the GOx@MBSA-PPy-MnO2 NPs. Overall, this work provides evidence of a synergistic therapy that remarkably improves therapeutic efficacy and significantly prolongs the lifetime of mice compared with controls.

Low PTEN expression and overexpression of phosphorylated AktSer473 and AktThr308 are associated with poor overall survival in upper tract urothelial carcinoma.

The PI3K/Akt signaling pathway serves an essential role in various cellular processes, including cell growth, survival, cell motility, angiogenesis and cell metabolism. Loss of PTEN expression and hyperactivation of Akt can result in tumorigenesis. Previous studies observed expression of the Akt protein and absence of the PTEN protein in bladder cancer and non-small cell lung carcinoma tissues. The aim of the present study was to evaluate the expression status and prognostic value of PTEN and the PI3K/Akt signaling pathway in Taiwanese patients with upper tract urothelial carcinoma (UTUC). Archival formalin-fixed, paraffin-embedded (FFPE) tissues from 65 UTUC cases were stained via immunohistochemistry for PTEN, phosphorylated (p)Akt serine (Ser)473 and pAkt threonine (Thr)308. The expression levels of each protein were significantly correlated with clinicopathological parameters. PTEN, pAktSer473 and pAktThr308 protein expression levels were higher in adjacent normal tissues compared with those in tumor tissues. Cytoplasmic PTEN protein expression levels were lower in high-stage tumors compared with those in low-stage tumors, and nuclear and cytoplasmic pAktThr308 protein expression levels were higher in high-grade tumors compared with those in low-grade tumors. Univariate analysis showed that high pathological tumor stage (pT2-4) [P=0.01; hazard ratio (HR)=3.40; 95% confidence interval (CI), 1.34-8.60], metastatic status (P=0.003; HR=3.55, 95% CI, 1.55-8.11), low cytoplasmic PTEN protein expression levels (P=0.016; HR=3.14; 95% CI, 1.24-7.95) and high cytoplasmic pAktSer473 protein expression levels (P=0.019, HR=2.71, 95% CI, 1.18-6.21) were predictive of poor overall survival. However, only metastatic status (P=0.031; HR=2.73; 95% CI, 1.10-6.78), low cytoplasmic PTEN protein expression levels (P=0.017; HR=3.29; 95% CI, 1.24-8.73) and high cytoplasmic pAktSer473 protein expression levels (P=0.027; HR=2.64; 95% CI, 1.12-6.23) remained significant in the multivariate analysis. Kaplan-Meier survival analysis showed that high T stage, metastasis, low expression levels of cytoplasmic PTEN protein and high expression levels of cytoplasmic pAktSer473 protein were significantly associated with poor survival (P=0.006, 0.001, 0.011 and 0.014, respectively). Co-expression of PTENlow/pAktSer473/high and pAktThr308/high phenotypes was associated with a less favorable overall survival (P=0.001). Overall, the present findings demonstrated that low expression levels of PTEN and high expression levels of pAktSer473 and pAktThr308 were predictors for poor overall survival in patients with UTUC.

Over-expression of TNNI3K is associated with early-stage carcinogenesis of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a devastating disease with very poor prognosis due to late diagnosis and resistance to traditional chemotherapies and radiotherapies. Herein, thioacetamide (TAA)-induced rat CCA model and CGCCA cell line were used; we aim to study the cytogenetic features during tumoral development of CCA and uncover the mystery regarding carcinogenesis of CCA. The Array comparative genomic hybridization analysis, in silico method, gene knockdown, Western blot, cell count proliferation assay, clonogenecity assay, and IHC staining were applied in this study. Array comparative genomic hybridization analysis was performed on all different TAA-induced phases of rat tissues to reveal the certain pattern, +2q45, +Xq22, -12p12, have been identified for the tumor early stage, where involve the gene TNNI3K. In addition, 16 genes and 3 loci were associated with rapid tumor progression; JAK-STAT signaling pathway was highly correlated to late stage of CCA. In silico database was used to observe TNNI3K was highly express at tumor part compared with normal adjacent tissue in CCA patients from TCGA dataset. Furthermore, the growth of TNNI3K-knockdown SNU308 and HuCCT1 cells decreased when compared with cells transfected with an empty vector cell demonstrated by proliferation and colonogenecity assay. Besides, over expression of TNNI3K was especially confirmed on human CCA tumors and compared with the intrahepatic duct stone bile duct tissues and normal bile duct tissues (P < 0.001). Our findings might uncover the mystery regarding carcinogenesis of CCA, and provide the potential genetic mechanism to the clinicians some ideas for the patients' treatment.

EPA significantly improves anti-EGFR targeted therapy by regulating miR-378 expression in colorectal cancer.

It is known that colorectal cancer (CRC) cells containing mutations of the genes KRAS and BRAF are predominate mechanisms causing resistance to epidermal growth factor receptor (EGFR) inhibitors, and commonly exhibit a lower expression of microRNA-378 (miR-378) when compared with the wild type. In the present study, the aim was to determine the possible mechanism which associates miR-378 with the mitogen-activated protein kinase pathway, and to determine the efficiency of eicosapentaenoic acid ethyl ester (EPA) in its ability to restore sensitivity towards cetuximab, an EGFR inhibitor. The results demonstrated that a combined treatment of 40 µM EPA with 0.2 µM cetuximab can significantly suppress the cell growth in KRAS-mutant and control wild-type cells. Furthermore, the higher phosphorylated protein level of extracellular-signal-regulated kinase 1/2 was notable in KRAS EPA-treated cells (P=0.006-0.047) and resulted in significantly increased cell death; however, inconsistent results were indicated in EPA-treated BRAF-mutant cells, compared with the original cells (without treatment). KRAS-mutant and wild-type Caco-2 cells treated with EPA exhibited increased cetuximab response rates, but these response rates were reduced in the BRAF-mutant cells. In conclusion, upregulation of miR-378 induced by EPA may result in the significant restoration of sensitivity to cetuximab in the KRAS-mutant cells. The present data will contribute to a notable potential therapeutic solution for future clinical CRC treatments.

Real-time circulating tumor cells detection via highly sensitive needle-like cytosensor-demonstrated by a blood flow simulation.

The concept of rapid detection of circulating tumor cells (CTCs) has always been the focal point of modern and future medicine. However, the dispersity and rarity of CTCs in the bloodstream makes it hard to detect metastasis. Herein, our newly designed needle-like cytosensor demonstrates that the capture and analysis of CTCs are a much less laborious process and have more potential than ever. Our aim is to detect and capture CTCs directly in the bloodstream without altering the genetic information; further benefit of current cytosensor is allows for the whole circulation of blood to run through the cytosensor, giving a much better sensitivity and chance of detecting CTCs. Our functionalized needle-like cytosensor has been modified with 3-aminopropyltriethoxysilane, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and conjugated streptavidin to allow the binding of the biotinylated-antibody of epithelial cell adhesion molecules, which captures targeted colon cancer CTC. The capability of our needle-like cytosensor to detect CTCs spanned from 102 to 106 cells/mL. Beyond this, the needle-like cytosensor avoids the distortion of the cell information. In addition, we constructed a blood flow simulation that mimics human circulating system about 10 mL/min speed; by using cyclic voltammetry we could detect significant signals from captured cancer CTCs more than 21 cells/mL without delay; the fluorescence dye detection was further performed for data confirmation. The future of biosensors begins with this, by providing early monitoring quality care in cancer therapy.

Cabozantinib-induced osteoblast secretome promotes survival and migration of metastatic prostate cancer cells in bone.

Therapies that target cancer cells may have unexpected effects on the tumor microenvironment that affects therapy outcomes or render therapy resistance. Prostate cancer (PCa) bone metastasis is uniquely associated with osteoblastic bone lesions and treatment with cabozantinib, a VEGFR-2 and MET inhibitor, leads to a reduction in number and/or intensity of lesions on bone scans. However, resistance to cabozantinib therapy inevitably occurs. We examined the effect of cabozantinib on osteoblast differentiation and secretion in the context of therapy resistance. We showed that primary mouse osteoblasts express VEGFR2 and MET and cabozantinib treatment decreased osteoblast proliferation but enhanced their differentiation. A genome-wide analysis of transcriptional responses of osteoblasts to cabozantinib identified a set of genes accounting for inhibition of proliferation and stimulation of differentiation, and a spectrum of secreted proteins induced by cabozantinib, including pappalysin, IGFBP2, WNT 16, and DKK1. We determined that these proteins were upregulated in the conditioned medium of cabozantinib-treated osteoblasts (CBZ-CM) compared to control CM. Treatment of C4-2B4 or PC3-mm2 PCa cells with CBZ-CM increased the anchorage-independent growth and migration of these PCa cells compared to cells treated with control CM. These results suggest that the effect of cabozantinib on the tumor microenvironment may increase tumor cell survival and cause therapy resistance.

Loss of tumor suppressor KDM6A amplifies PRC2-regulated transcriptional repression in bladder cancer and can be targeted through inhibition of EZH2.

Trithorax-like group complex containing KDM6A acts antagonistically to Polycomb-repressive complex 2 (PRC2) containing EZH2 in maintaining the dynamics of the repression and activation of gene expression through H3K27 methylation. In urothelial bladder carcinoma, KDM6A (a H3K27 demethylase) is frequently mutated, but its functional consequences and therapeutic targetability remain unknown. About 70% of KDM6A mutations resulted in a total loss of expression and a consequent loss of demethylase function in this cancer type. Further transcriptome analysis found multiple deregulated pathways, especially PRC2/EZH2, in KDM6A-mutated urothelial bladder carcinoma. Chromatin immunoprecipitation sequencing analysis revealed enrichment of H3K27me3 at specific loci in KDM6A-null cells, including PRC2/EZH2 and their downstream targets. Consequently, we targeted EZH2 (an H3K27 methylase) and demonstrated that KDM6A-null urothelial bladder carcinoma cell lines were sensitive to EZH2 inhibition. Loss- and gain-of-function assays confirmed that cells with loss of KDM6A are vulnerable to EZH2. IGFBP3, a direct KDM6A/EZH2/H3K27me3 target, was up-regulated by EZH2 inhibition and contributed to the observed EZH2-dependent growth suppression in KDM6A-null cell lines. EZH2 inhibition delayed tumor onset in KDM6A-null cells and caused regression of KDM6A-null bladder tumors in both patient-derived and cell line xenograft models. In summary, our study demonstrates that inactivating mutations of KDM6A, which are common in urothelial bladder carcinoma, are potentially targetable by inhibiting EZH2.

Genetic alterations of HER genes in chromophobe renal cell carcinoma.

Chromophobe (ch) renal cell carcinoma (RCC) is the 3rd most common subtype of RCC and occurs in 5% of all RCCs. Although chRCC generally demonstrates more favorable outcomes compared with other subtypes of RCC, there is a 6-7% probability of tumor progression and metastasis in this disease. The subclassification of a more aggressive subtype of chRCC may be useful for the management of this cancer. The Erb-B2 receptor tyrosine kinase 2 [also known as human epidermal growth factor receptor (HER) 2] gene has been reported to be important in chRCC. The present study aimed to further investigate the abnormalities of the HER family genes and their potential association with chRCC. Fluorescence in situ hybridization was performed on 11 chRCC tissue specimens, and the Spearman's rank correlation coefficient analysis was used to assess the results. The loss of one copy of the HER2 and HER4 genes was observed to be the major alteration of the tumor cells in all chRCC cases. Statistical data indicated that loss of the HER2 gene was strongly correlated with loss of the HER4 gene (P=0.019). The findings of previous studies were also combined for analysis, and were consistent with those of the present study. In addition, the amplification of HER1 was also strongly correlated with the amplification of HER4 (P=0.004). Furthermore, a high percentage of genetic structural rearrangements was observed in HER3 genes, which was significantly associated with amplification of HER2 (P=0.005). Certain alterations in the HER gene family were also noted as a phenomenom in chRCC. Therefore, the characterization of the underlying aberrant functions of HER genes may be of interest for additional studies in the context of using HER genes to distinguish between RCC subtypes in order to establish improved treatment guidelines.

Lauric acid can improve the sensitization of Cetuximab in KRAS/BRAF mutated colorectal cancer cells by retrievable microRNA-378 expression.

EGFR-inhibitor (Cetuximab) is one of the main targeted drugs used for metastatic colorectal carcinoma (CRC). The benefit from Cetuximab appears to be limited to a subtype of patients, not for the patients with tumors harboring mutated BRAF or KRAS genes; unfortunately, it accounts for ~40-50% of CRC cases. Previous studies have connected higher expression levels of miR-378 to be commonly presented in patients without BRAF or KRAS mutants than in mutated CRCs. The microRNA-378 (miR-378) is coexpressed with PGC-1ß and can be easily induced by fatty acid, for example lauric acid. Therefore, we hypothesized that elevation of miR-378 expression in mutated CRCs may stimulate the cell response to Cetuximab. Herein, seven CRC cell lines with confirmed mutation status were involved in two parallel experiments; directly in vitro transfected miR-378 mimics, and using lauric acid to indirectly induce the level of miR-378 in cells. After the increase of miR-378 in cells by either direct or indirect approaches, sensitivity to Cetuximab was restored in all BRAF mutants (p-value <0.0001-0.0003), and half of KRAS mutants CRC (p-value 0.039-0.007). Further evidence was gained by decreasing expression of MEK and ERK2 proteins after transfection with miR-378; it was similar to the indirect induction by lauric acid approach. In conclusion, the present study demonstrated that lauric acid may efficiently induce miR-378 expression in CRC mutants, and both BRAF and a subtype of KRAS mutants presented significantly improved sensitivity to Cetuximab. Notably, BRAF mutants could even be inhibited in cell proliferation after elevated concentration of miR-378 in cells without combining with targeted therapy. This new approach may shed new light on BRAF or KRAS mutation in CRC patients for clinical trial, since lauric acid may easily be obtain from natural food, and it is supposed to be harmless to the cardiovascular system.

Mutation signatures implicate aristolochic acid in bladder cancer development.

BACKGROUND: Aristolochic acid (AA) is a natural compound found in many plants of the Aristolochia genus, and these plants are widely used in traditional medicines for numerous conditions and for weight loss. Previous work has connected AA-mutagenesis to upper-tract urothelial cell carcinomas and hepatocellular carcinomas. We hypothesize that AA may also contribute to bladder cancer. METHODS: Here, we investigated the involvement of AA-mutagenesis in bladder cancer by sequencing bladder tumor genomes from two patients with known exposure to AA. After detecting strong mutational signatures of AA exposure in these tumors, we exome-sequenced and analyzed an additional 11 bladder tumors and analyzed publicly available somatic mutation data from a further 336 bladder tumors. RESULTS: The somatic mutations in the bladder tumors from the two patients with known AA exposure showed overwhelming AA signatures. We also detected evidence of AA exposure in 1 out of 11 bladder tumors from Singapore and in 3 out of 99 bladder tumors from China. In addition, 1 out of 194 bladder tumors from North America showed a pattern of mutations that might have resulted from exposure to an unknown mutagen with a heretofore undescribed pattern of A > T mutations. Besides the signature of AA exposure, the bladder tumors also showed the CpG > TpG and activated-APOBEC signatures, which have been previously reported in bladder cancer. CONCLUSIONS: This study demonstrates the utility of inferring mutagenic exposures from somatic mutation spectra. Moreover, AA exposure in bladder cancer appears to be more pervasive in the East, where traditional herbal medicine is more widely used. More broadly, our results suggest that AA exposure is more extensive than previously thought both in terms of populations at risk and in terms of types of cancers involved. This appears to be an important public health issue that should be addressed by further investigation and by primary prevention through regulation and education. In addition to opportunities for primary prevention, knowledge of AA exposure would provide opportunities for secondary prevention in the form of intensified screening of patients with known or suspected AA exposure.