Hippo Signaling Mediates Proliferation,Invasiveness, and Metastatic Potential of Clear Cell Renal Cell Carcinoma

Recent work has identified dysfunctional Hippo signaling to be involved in maintenance and progression of various human cancers, although data on clear cell renal cell carcinoma (ccRCC) have been limited. Here, we provide evidence implicating aberrant Hippo signaling in ccRCC proliferation, invasiveness, and metastatic potential. Nuclear overexpression of the Hippo target Yes-associated protein (YAP) was found in a subset of patients with ccRCC. Immunostaining was particularly prominent at the tumor margins and highlighted neoplastic cells invading the tumor-adjacent stroma. Short hairpin RNA-mediated knockdown of YAP significantly inhibited proliferation, migration, and anchorage-independent growth of ccRCC cells in soft agar and led to significantly reduced murine xenograft growth. Microarray analysis of YAP knockdown versus mock-transduced ccRCC cells revealed down-regulation of endothelin 1, endothelin 2, cysteine-rich, angiogenic inducer, 61 (CYR61), and c-Myc in ccRCC cells as well as up-regulation of the cell adhesion molecule cadherin 6. Signaling pathway impact analysis revealed activation of the p53 signaling and cell cycle pathways as well as inhibition of mitogen-activated protein kinase signaling on YAP down-regulation. Our data suggest CYR61 and c-Myc as well as signaling through the endothelin axis as bona fide downstream effectors of YAP and establish aberrant Hippo signaling as a potential therapeutic target in ccRCC.     

FGFR4 Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Mouth and Oropharynx

Background

Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis.

Patients and Methods

DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays.

Results

Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death.

Conclusion

Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.     

Circulating microRNA-200 Family as Diagnostic Marker in Hepatocellular Carcinoma

Goals

In this clinical study, we aimed to evaluate the role of circulating microRNA-200 family as a non-invasive tool to identify patients with cirrhosis-associated hepatocellular carcinoma (HCC).

Background

Prognosis of HCC remains poor with increasing incidence worldwide, mainly related to liver cirrhosis. So far, no reliable molecular targets exist for early detection of HCC at surgically manageable stages. Recently, we identified members of the microRNA-200 family as potential diagnostic markers of cirrhosis-associated HCC in patient tissue samples. Their value as circulating biomarkers for HCC remained undefined.

Methods

Blood samples and clinicopathological data of consecutive patients with liver diseases were collected prospectively. Expression of the microRNA-200 family was investigated by qRT-PCR in blood serum samples of 22 HCC patients with and without cirrhosis. Serum samples of patients with non-cancerous chronic liver cirrhosis (n = 22) and of healthy volunteers (n = 15) served as controls.

Results

MicroRNA-141 and microRNA-200a were significantly downregulated in blood serum of patients with HCC compared to liver cirrhosis (p<0.007) and healthy controls (p<0.002). MicroRNA-141 and microRNA-200a could well discriminate patients with cirrhosis-associated HCC from healthy volunteers with area under the receiver-operating characteristic curve (AUC) values of 0.85 and 0.82, respectively. Additionally, both microRNAs could differentiate between HCC and non-cancerous liver cirrhosis with a fair accuracy.

Conclusions

Circulating microRNA-200 family members are significantly deregulated in patients with HCC and liver cirrhosis. Further studies are necessary to confirm the diagnostic value of the microRNA-200 family as accurate serum marker for cirrhosis-associated HCC.     

Metastatic Renal Clear Cell Carcinoma In the Thyroid Gland Diagnosed By Fine Needle Aspiration Cytology

An 81-year-old woman presented with a mass in the right lobe of the thyroid. Fine needle aspiration of this lesion showed metastatic clear cell carcinoma. This was subsequently confirmed histologically. In patients with metastatic carcinoma, tumour deposits are frequently found in the thyroid. The most common metastatic tumour to masquerade as a primary thyroid tumour is a renal cell carcinoma. In patients with this tumour the possibility that a thyroid mass may be a metastatic deposit should always be considered.     

Serum MicroRNA-21 as a Diagnostic Marker for Lung Carcinoma: A Systematic Review and Meta-Analysis

Objectives

MicroRNA-21 in serum is a promising marker for the diagnosis of lung carcinoma. A meta-analysis was performed to assess the diagnostic accuracy and clinical value of serum microRNA-21 in patients with lung carcinoma.

Methods

PubMed, EMBASE, Web of Knowledge (ISI), the Cochrane Library, Scopus, BioMed Central, Science Direct, China National Knowledge Infrastructure (CNKI), Wan Fang data and Technology of Chongqing (VIP) databases were searched to identify studies in English and Chinese that assessed the diagnostic value of serum miR-21 for lung carcinoma, from inception to 9 April 2014. Two independent investigators identified and extracted the study characteristics from all articles according to defined inclusion and exclusion criteria. Quality assessment of diagnostic accuracy studies (QUADAS) was used to score the quality of the eligible studies. Stata12 and Meta-DiSc software were used to test the heterogeneity and to perform the meta-analysis.

Results

Our search returned 1008 articles, of which seven fulfilled the inclusion criteria, accounting for 500 patients and 386 controls. Using random-effect model analysis, the summary assessments revealed that the mean sensitivity was 0.71% (95%CI: 57–82%) and specificity was 0.84% (95%CI: 76–89%). The area under the receiver operating characteristic curve was 0.86 (95%CI: 0.83–0.89). In addition, heterogeneity was clearly apparent but was not caused by the threshold effect, as shown by Meta-DiSc analysis.

Conclusion

The current evidence suggests that serum miR-21 can be rapidly measured in lung carcinoma patients and has potential diagnostic value with moderate sensitivity and specificity. Further prospective studies to assess the early stage diagnostic value are needed in the future.     

High Expression of KCa3.1 in Patients with Clear Cell Renal Carcinoma Predicts High Metastatic Risk and Poor Survival

Background

Ca²⁺-activated K⁺ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca²⁺-activated K⁺ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival.

Methodology/Principal Findings

We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro.

Conclusions/Significance

Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.     

肾透明细胞癌相关微小RNA的研究进展

微小核糖核酸(miRNAs)是一类长约22个核苷酸的非编码单链小核糖核酸分子,miRNA通过与靶mRNA 3'端非翻译序列完全或部分互补结合,导致靶mRNA降解或转录后翻译抑制,从而调控靶基因的表达.最新研究显示人类血清/血浆中miRNA表达稳定,并在肿瘤患者血清中发现多种miRNA,其中的一些已经被证实与肾癌发生及发展相关,以往miRNA与肾癌的研究方.向多集中于肾癌组织,尽管发现很多有差异的miRNA,但不同研究者之间的结果常难以相互验证,而最近研究证实血清miRNA具有组织相关性和器官特异性,并对某些肿瘤具有高敏感性和特异性,因此其有望成为新的肿瘤标志物.肾癌是国内泌尿系统的第二常见恶性肿瘤,而且其近年来发病率和死亡率有逐年增高的趋势.由于肾透明细胞癌是肾癌的主要亚型,因此本文就血清miRNA在肾透明细胞癌的表达及其作用的研究进展作一综述.     

原发性肝透明细胞癌影像学及临床分析

目的:探讨原发性肝细胞癌(PCCCL)的影像学及临床病理等特点,提高对肝透明细胞癌的认识,早期确诊、早期治疗、改善预后等,以避免临床误诊,并评价PCCCL的预后比例及疗效。方法:回顾性分析了2009年至2010年我院肿瘤科收治的3例肝透明细胞癌患者的临床资料、影像学质料,以及临床治疗和随访情况。结果:3例均为男性,2例行氩氦刀靶向消融治疗,1例行CT引导下射频消融治疗,术后随访半年2例过世,1例生存。结论:原发性肝细胞癌透明细胞型早期诊断尤为重要,及时手术切除及消融治疗是取得较好疗效的关键。     

涎腺腺样囊性癌高低转移细胞系mRNA及蛋白质表达谱差异研究

转移性和侵袭性是恶性肿瘤的重要特征,与基因和蛋白质水平上一系列改变密切相关.应用mRNA抑制性消减杂交技术和双向电泳结合肽质量指纹分析技术,对涎腺腺样囊性癌高、低转移细胞系的mRNA和蛋白质表达谱的差异性进行比较研究.实验结果显示,在抑制性消减杂交中,分别以两个细胞系为测试子,共获得差异片段34个,其中高转移株细胞中高表达的基因序列有6个,低转移细胞系中有28个,其中包括两个新的表达序列标签（EST）.对这些基因序列,进一步以RNA 斑点杂交对这些基因的表达状况进行验证并排除假阳性结果,结果发现,32个基因在mRNA水平上有不同程度的表达量改变,改变趋势与消减杂交结果一致.以蛋白质等电聚焦结合SDS-聚丙烯酰胺凝胶电泳的双向电泳技术获得的总蛋白质分离,经PDQuest2DE软件分析结果表明, 高转移细胞系表达谱的平均蛋白质点数为(978±38), 低转移细胞系的平均蛋白质点数为(996±27).其中高转移细胞与低转移细胞相比,其蛋白质点有355个未被匹配 ,低转移细胞相比高转移细胞有222个未被匹配.对其中10个差异较明显的蛋白质点,进一步进行基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF-MS)测定肽质量指纹图谱,用Peptident软件对SWISS PROT数据库比较分析,结果显示,Stratifin、Symplekin和乳腺癌相关抗原NY-BR-20等蛋白质在高转移细胞中表达,在低转移中未被检测到,B7蛋白质在低转移细胞中高表达,在高转移细胞中低表达.结果表明涎腺腺样囊性癌高转移特性与多种基因、蛋白质相关,这些基因或蛋白质参与血管生成、蛋白质合成、信号传递、细胞周期调控、分子伴侣以及免疫共刺激等多种生理活动.同时在mRNA和蛋白质水平上进行表达谱分析使结果更全面,具有明显的互补性,这些结果为进一步探索腺样囊性癌肿瘤转移分子机理以及肿瘤控制和基因治疗提供了重要的依据.     

Tumor-infiltrating PD1-Positive Lymphocytes and FoxP3-Positive Regulatory T Cells Predict Distant Metastatic Relapse and Survival of Clear Cell Renal Cell Carcinoma

BACKGROUND: Clear cell renal cell carcinoma (CRCC) is the most common malignant tumor of the kidney, and the clinical outcome of CRCC is related with the metastatic potential of CRCC. A significant proportion of metastatic CRCC remains incurable. Recently, immunotherapy against specific targets such as programmed death 1 (PD1) has been adapted for fatal cases of CRCC. MATERIALS AND METHODS: In this study, we aimed to evaluate the potential of tumor-infiltrating PD1-positive lymphocytes or FoxP3-positive regulatory T cells (Tregs) as predictors of the metastatic potential or prognosis of CRCC and investigate possible correlations with Epstein-Barr virus (EBV) infection in 199 cases of CRCC. RESULTS: PD1 positivity, high Treg number, and EBV infection all predicted poor overall survival (OS) by univariate analysis. PD1 positivity and high Treg numbers were also significantly correlated with more distant metastatic relapse (DMR) and poor relapse-free survival (RFS) by univariate analysis. PD1 positivity and high Treg number were independent prognostic indicators for OS. In addition, PD1 positivity was an independent predictor of RFS and DMR. EBV infection was an independent predictor of OS of CRCC. CONCLUSION: This study demonstrates that intratumoral infiltration of PD1-positive or FoxP3-positive lymphocytes can be used as significant prognostic indicators of CRCC and PD1 positivity could be very helpful in the prediction of latent distant metastasis of CRCCs. Therefore, evaluation of the infiltration of PD-positive cells or Tregs in CRCC may be useful diagnostic tools for the selection of patients who could benefit from PD1- or Treg-based immunotherapy.     

Down-Regulated miR-30a in Clear Cell Renal Cell Carcinoma Correlated with Tumor Hematogenous Metastasis by Targeting Angiogenesis-Specific DLL4

Background

Endothelial DLL4 plays an important role in controlling of tumor angiogenesis, which is required for tumor invasive growth and metastasis. However, the regulation of DLL4 in clear cell renal cell carcinoma (ccRCC) has not yet been systematically elucidated.

Methodology

We performed bioinformatical analysis to explore miRNAs targeting DLL4. miR-30a was selected as a representative to validate its functional association in endothelial cell. Then, the expressions of DLL4 and mature miR-30a from 90 cases of ccRCC and 28 cases of nonmatched adjacent non-tumor tissues were measured by quantitative real-time PCR. Finally, the expression of miR-30a was correlated with DLL4 expression, tumor features (metastatic condition and microvessel density), and patient metastasis-free survival. The univariate and multivariate analyses were performed to select the risk factors associated with hematogenous metastasis, respectively.

Principal Findings

miR-30a negatively regulated DLL4 and inhibited the proliferation and migration of endothelial cells. DLL4 was up-regulated in ccRCC and further increased in hematogenous metastatic cases, while miR-30a was down-regulated in tumor tissues and further decreased in hematogenous metastatic ccRCC (student t test, all p<0.05). Additionally, expression of miR-30a was inversely correlated with expression of DLL4 and microvessel density (linear correlation analysis, both p<0.05). Low-level miR-30a also indicated a higher probability of developing metastasis (log-rank test, p = 0.010). Most importantly, miR-30a expression was an independent predictor of ccRCC hematogenous metastasis by the univariate analysis and binary logistic regression model (both p<0.05).

Conclusions

Down-regulated miR-30a in ccRCC was associated with tumor hematogenous metastasis through increasing microvessel density by targeting angiogenesis-specific DLL4.     

转移性肾癌生物治疗研究进展

转移性肾癌(mRCC)作为一种高度恶性的疾病,以进展快、病死率高为特点,且一直以来临床上对其治疗效果并不理想.联合化疗和(或)放疗也不能显著提高反应率或改善生存.在分子靶向药物诞生之前,临床上应用以细胞因子为基础的免疫治疗作为mRCC的一线治疗.分子靶向药物的问世,彻底打破了传统细胞因子免疫治疗mRCC的局面,使mRCC患者获得较好的临床治疗效果.本文将系统阐述mRCC的免疫治疗与靶向治疗的进展,详细介绍目前靶向治疗的临床应用情况,以期为mRCC治疗药物的合理选择提供参考.     

Metastatic Hurthle Cell Carcinoma of the thyroid presenting as a Breast Lump: A Case Report

Background

Hurthle cell carcinoma of the thyroid is a rare form of thyroid cancer. It may present as a low grade tumour or can present as a more aggressive metastatic carcinoma. Hurthle cell carcinoma has the highest incidence of metastasis among all differentiated thyroid cancers. Most commonly haematogenous spread to lungs, bones and brain, however spread to regional lymph nodes is not uncommon. The breast is a rare site for metastasis from extramammary sources. We present the first case of breast metastasis from Hurthle cell carcinoma of the thyroid.

Case presentation

We report a 77 year old lady who had total thyroidectomy and bilateral neck dissection followed by radiotherapy for a high grade metastatic Hurthle cell carcinoma of the thyroid. Ten months later she presented to the breast clinic with left breast lump and a lump at the left axilla. Fine needle aspiration cytology of the lumps and histology after wide local excision of the breast lump confirmed metastatic Hurthle cell carcinoma.

Conclusion

The presence of breast lumps in patients with history of extramammary cancer should raise the possibility of metastasis.

     

ICAM1 Is a Potential Cancer Stem Cell Marker of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer diagnosed in Asian countries, with its incidence on the rise. Cancer stem cell (CSC; also known as tumor-initiating cells, TIC) is inherently resistant to cytotoxic chemotherapy and radiation and associates with poor prognosis and therapy failure. Targeting therapy against cancer stem cell has emerged as a potential therapeutic approach to develop effective regimens. However, the suitable CSC marker of ESCC for identification and targeting is still limited. In this study, we screened the novel CSC membrane protein markers using two distinct stemness characteristics of cancer cell lines by a comparative approach. After the validation of RT-PCR, qPCR and western blot analyses, intercellular adhesion molecule 1 (ICAM1) was identified as a potential CSC marker of ESCC. ICAM1 promotes cancer cell migration, invasion as well as increasing mesenchymal marker expression and attenuating epithelial marker expression. In addition, ICAM1 contributes to CSC properties, including sphere formation, drug resistance, and tumorigenesis in mouse xenotransplantation model. Based on the analysis of ICAM1-regulated proteins, we speculated that ICAM1 regulates CSC properties partly through an ICAM1-PTTG1IP-p53-DNMT1 pathway. Moreover, we observed that ICAM1 and CD44 could have a compensation effect on maintaining the stemness characteristics of ESCC, suggesting that the combination of multi-targeting therapies should be under serious consideration to acquire a more potent therapeutic effect on CSC of ESCC.     

Identification of Potential Serum Proteomic Biomarkers for Clear Cell Renal Cell Carcinoma

Objective

To investigate discriminating protein patterns and serum biomarkers between clear cell renal cell carcinoma (ccRCC) patients and healthy controls, as well as between paired pre- and post-operative ccRCC patients.

Methods

We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with ccRCC. A total of 162 serum samples were analyzed in this study, among which there were 58 serum samples from ccRCC patients, 40 from additional paired pre- and post-operative ccRCC patients (n = 20), and 64 from healthy volunteers as healthy controls. ClinProTools software identified several distinct markers between ccRCC patients and healthy controls, as well as between pre- and post-operative patients.

Results

Patients with ccRCC could be identified with a mean sensitivity of 88.38% and a mean specificity of 91.67%. Of 67 m/z peaks that differed among the ccRCC, healthy controls, pre- and post-operative ccRCC patients, 24 were significantly different (P<0.05). Three candidate peaks, which were upregulated in ccRCC group and showed a tendency to return to healthy control values after surgery, were identified as peptide regions of RNA-binding protein 6 (RBP6), tubulin beta chain (TUBB), and zinc finger protein 3 (ZFP3) with the m/z values of 1466.98, 1618.22, and 5905.23, respectively.

Conclusion

MB-MALDI-TOF-MS method could generate serum peptidome profiles of ccRCC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

One-Carbon Metabolism Pathway Gene Variants and Risk of Clear Cell Renal Cell Carcinoma in a Chinese Population

Background

One-carbon metabolism is the basement of nucleotide synthesis and the methylation of DNA linked to cancer risk. Variations in one-carbon metabolism genes are reported to affect the risk of many cancers, including renal cancer, but little knowledge about this mechanism is known in Chinese population.

Methods

Each subject donated 5 mL venous blood after signing the agreement. The study was approved by the Institutional Review Board of the Nanjing Medical University, Nanjing, China. 18 SNPs in six one-carbon metabolism-related genes (CBS, MTHFR, MTR, MTRR, SHMT1, and TYMS) were genotyped in 859 clear cell renal cell carcinoma (ccRCC) patients and 1005 cancer-free controls by the Snapshot.

Results

Strong associations with ccRCC risk were observed for rs706209 (P = 0.006) in CBS and rs9332 (P = 0.027) in MTRR. Compared with those carrying none variant allele, individuals carrying one or more variant alleles in these two genes had a statistically significantly decreased risk of ccRCC [P = 0.001, adjusted odds ratio (OR) = 0.73, 95% confidence interval (CI) = 0.06–0.90]. In addition, patients carrying one or more variant alleles were more likely to develop localized stage disease (P = 0.002, adjusted OR = 1.37, 95%CI = 1.11–1.69) and well-differentiated ccRCC (P<0.001, adjusted OR = 1.42, 95%CI = 0.87–1.68). In the subgroup analysis, individuals carrying none variant allele in older group (P = 0.007, adjusted OR = 0.67, 95%CI = 0.49–0.91), male group (P = 0.007, adjusted OR = 0.71, 95%CI = 0.55–0.92), never smoking group (P = 0.002, adjusted OR = 0.68, 95%CI = 0.53–0.88) and never drinking group (P<0.001, adjusted OR = 0.68, 95%CI = 0.53–0.88) had an increased ccRCC risk.

Conclusions

Our results suggest that the polymorphisms of the one-carbon metabolism-related genes are associated with ccRCC risk in Chinese population. Future population-based prospective studies are required to confirm the results.