The Utility of ERBB4 and RB1 Immunohistochemistry in Distinguishing Chromophobe Renal Cell Carcinoma From Renal Oncocytoma.

Objectives. Differentiating renal oncocytoma (RO) from chromophobe renal cell carcinoma (ChRCC) can occasionally be challenging. We evaluated the expression of RB1 and ERBB4 in RO and ChRCC, and compared the immunohistochemistry (IHC) results to RB1 and ERBB4 gene abnormalities detected by fluorescence in situ hybridization (FISH). Materials and Methods. Fifty-three kidney resections (ChRCC, n=28; RO, n=25) were stained for RB1 and ERBB4 IHC and FISH was performed to evaluate gene copy number analysis. Results. A loss of RB1 staining was identified in 64% (18/28) of ChRCCs, which was not found in any ROs (0/25; P <.001). FISH analysis revealed 36% (10/28) of ChRCCs contained a RB1 hemizygous deletion with a concordance of 56% (10/18) between the IHC and FISH findings. No RB1 gene copy number variations were detected in any of the ROs (0/25; P <.001) and retained expression of RB1 by IHC. ERBB4 showed cytoplasmic/membranous staining in all ROs and ChRCCs. However, 75% (21/28) of ChRCCs also contained nuclear positivity for ERBB4, which was uncommonly seen in ROs (3/25, 12%; P < .001). A hemizygous ERBB4 gene deletion was detected in 46% of ChRCCs (13/28), but none of the ROs (0/25; 0%). Loss of labeling by RB1 or nuclear staining for ERBB4 IHC identified 25 of 28 (89%) of ChRCCs. Conclusion. In summary, the loss of RB1 expression is a highly specific diagnostic biomarker in distinguishing ChRCC from RO. Nuclear ERBB4 expression also appears to be a sensitive diagnostic biomarker for ChRCC, albeit the mechanism is unknown.

Nicotine upregulates FGFR3 and RB1 expression and promotes non-small cell lung cancer cell proliferation and epithelial-to-mesenchymal transition via downregulation of miR-99b and miR-192.

BACKGROUND: Tobacco smoke is by far the greatest risk factor for non-small-cell lung cancer (NSCLC). Nicotine, an active alkaloid in tobacco, is unable to initiate tumorigenesis in humans and rodents, but can promote the growth and metastasis of various tumors, including NSCLC, initiated by tobacco carcinogens. Recently, cigarette smoke is reported to downregulate 24 miRNAs more than 3-fold in the lungs of rats, and most of these downregulated miRNAs are associated with NSCLC initiation and development. Nicotine as the major tobacco component might be associated with the expression changes of some miRNAs. METHODS: qRT-PCR was performed to determine the miRNA and mRNA expression, and western blot was conducted to measure protein expression. MTT assay was used to detect cell proliferation. RESULTS: The effects of nicotine on the expression of 24 miRNAs in NSCLC cell lines were determined, and the results showed that nicotine treatment decreased miR-99b and miR-192 expression. Cell proliferation and epithelial-to-mesenchymal transition (EMT) detection showed that nicotine promoted NSCLC cell proliferation and EMT, and restoration of miR-99b or miR-192 expression relieved the effects of nicotine on NSCLC cell proliferation and EMT. Subsequently, fibroblast growth factor receptor 3 (FGFR3) and retinoblastoma 1 (RB1) were confirmed to be the targets of miR-99b and miR-192, respectively, and were upregulated by nicotine in NSCLC cells. In addition, FGFR3 or RB1 knockdown inhibited NSCLC cell proliferation and EMT. CONCLUSION: This study, for the first time, elucidates nicotine-miR-99b/miR-192-FGFR3/RB1 regulatory network that nicotine promotes NSCLC cell proliferation and EMT by downregulating miR-99b and miR-192, and upregulating their targets FGFR3 and RB1. These findings offer novel insights into the understanding of underlying molecular mechanisms of NSCLC related with the nicotine effects.

[Differential expression of an ensemble of the key genes involved in cell-cycle regulation in lung cancer].

Targeted cancer therapy directed at individual targets is often accompanied by the rapid development of drug resistance. The development of a new generation of antitumor drugs involves the search for many targets simultaneously to block or, conversely, restore their activity. In this regard, simultaneous analysis of gene expression in a complex network of interactions, primarily cell cycle control elements, is relevant for the search of specific molecular markers for the differential diagnosis of adenocarcinoma (ADC) and squamous cell lung cancer (SCC), as well as new targets for therapy. In this paper we performed an extended quantitative analysis of the expression of two suppressor genes, CTDSPL and its target RB1, as well as 84 genes of the main participants of the p16^(INK4A)-Cdk/cyclin D1-Rb and p53/p21^(Waf1) signaling pathways in the histological types of non-small-cell lung cancer (NSCLC), i.e., ADC and SCC, using the special panel of the Human Cell Cycle Regulation Panel. The expression profile of some genes shows the specificity to the histological type of NSCLC and the presence of metastases. The genes with a significantly increased expression that affect the activity of Rb (cyclins, cyclin-dependent kinases, their activators, inhibitors, etc.) can serve as potential targets for combined therapy of both ADC and SCC.

miR-215 promotes cell migration and invasion of gastric cancer by targeting Retinoblastoma tumor suppressor gene 1.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumor and has high mortality worldwide. microRNAs (miRNAs) play critical roles in carcinogenesis. Previous studied showed that miR-215 was involved in tumorigenesis and progression. This study was designed to clarify the biological function of miR-215 in GC. METHODS: qRT-PCR was used to detect the miR-215 expression in GC tissues and 6 human GC cell lines (AGS, SGC-7901, NCI-N87, GES-1, MKN-45 and BGC-823) as well. Transwell assay was used to investigate the biological function of miR-215 in GC. Luciferase reporter assay was used to confirm its effect on the regulation of the target gene Retinoblastoma tumor suppressor gene 1 (RB1). RESULTS: miR-215 was frequently up-regulated in GC tissues compared to adjacent non-tumor tissues and GC cell lines. miR-215 expression level was correlated with the progression of tumor invasion and tumor-node-metastasis (TNM) stage. Over-expression miR-215 in GC cell lines promoted cell migration and invasion. Besides, miR-215 could down-regulate the expression of RB1 in vitro via directly binding to its 3'-untranslated region (UTR), while the expression of RB1 would suppress the miR-215-indueced GC cell migration and invasion. CONCLUSIONS: miR-215 promoted cell migration and invasion of gastric cancer by directly targeting RB1.

MiR-26a downregulates retinoblastoma in colorectal cancer.

MicroRNAs are non-coding short RNAs that target the 3' untranslated region of messenger RNAs (mRNAs) and lead to their degradation or to translational repression. Several microRNAs have been designated as oncomirs, owing to their regulating tumor suppressor genes. Interestingly, a few of them have been found to target multiple genes whose simultaneous suppression contributes to the development of a tumoral phenotype. Here, we have showed that miR-26a is overexpressed in colorectal cancer data obtained from TCGA Research Network and in human colon cancer pathological specimens; moreover, an orthotopic in vivo model of colon cancer showed overexpression of miR-26a, while Rb1 expression inversely correlated to miR-26a in TCGA Research Network data, pathological samples, and the in vivo model. Then, by means of luciferase assay, we demonstrated that miR-26a targets the 3' untranslated region of Rb1 mRNA directly. This is, to our knowledge, the first report of miR-26a targeting Rb1 in colon cancer. The results of this study suggested that miR-26a could serve as a progression biomarker in colorectal cancer. Further validation studies are still needed to confirm our findings.

Retinoblastoma binding protein 6 and crystallin lambda 1 are cadmium-responsive genes in zebrafish embryos and adults retinae.

Nonessential metal cadmium is widely used and released in the environment, causing cell toxicity and posing a severe threat to wildlife. Zebrafish (Danio rerio) is one of the most commonly used animals in the investigation of environmental cadmium toxicity in vertebrates. In this study, we identified two cadmium-responsive genes, RBBP6 and CRYL1, in the early phases of zebrafish development, at the gastrula stage. The retinoblastoma binding protein 6 is associated with increased protein degradation and cell proliferation; crystallin-lambda 1 is a lens protein with redox activity. In situ hybridization analysis performed on adult zebrafish exposed to 1.5-40 µM cadmium for 30 days confirmed the ability of cadmium to up-regulate the expression of both genes in retinal cells in a dose-dependent manner. The over-expression was transient, being switched off when cadmium was removed. The involvement of RBBP6 and CRYL1 in the onset of cadmium-induced morphological alterations in adult zebrafish retina is discussed.

SOX2 and Rb1 in esophageal small-cell carcinoma: their possible involvement in pathogenesis.

Clinicopathological features and pathogenesis of esophageal small-cell carcinoma remain unclear. We hypothesized common cellular origin and pathogenesis in small-cell carcinoma of esophagus and lung associated with SOX2 overexpression and loss of Rb1. Expression of squamous-basal markers (CK5/6 and p40), glandular markers (CK18 and CEA), SOX2, and Rb1 were evaluated in 15 esophageal small-cell carcinomas, 46 poorly differentiated squamous cell carcinomas, and 88 small-cell lung carcinoma, as well as 16 embryonic esophagus. Esophageal small-cell carcinoma expressed higher levels of glandular markers and lower levels of squamous-basal markers than poorly differentiated squamous cell carcinoma. No significant differences were observed in immunohistochemistry profiles between small-cell carcinoma of the esophagus and the lung. SOX2 expression was high in esophageal small-cell carcinoma (70%±33% of nuclei), small-cell lung carcinoma (70%±26%), and the embryonic esophagus (75%±4%), and it was significantly lower in poorly differentiated squamous cell carcinoma (29%±28%). Rb1 expression was significantly lower in esophageal small-cell carcinoma (0.3%±1%), small-cell lung carcinoma (2%±6%), and the embryonic esophagus (7%±5%), and it was significantly higher in poorly differentiated squamous cell carcinoma (51%±24%). The immunohistochemistry profiles of small-cell carcinoma of the esophagus and the lung are highly similar. The loss of Rb1 function is a key contributor to the pathogenesis of both neoplasms. In addition, SOX2 overexpression observed in esophageal and lung small-cell carcinoma as well as in the embryonic esophagus indicated that esophageal small-cell carcinoma may arise from embryonic-like stem cells in the esophageal epithelium. The two distinct differentiation patterns (neuroendocrine and glandular) of esophageal small-cell carcinoma further support the fact that SOX2 has a pivotal role in the differentiation of pluripotent stem cells into esophageal small-cell carcinoma cells.

Altered expression of retinoblastoma 1 in Hirschsprung's disease.

PURPOSE: The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development, and has been widely documented to be inactivated in human cancer. A recent study using RB1 knockout mice suggested a new role for RB1 in the normal regulation of the enteric nervous system (ENS), because of knockout mice showing ENS abnormalities and severe intestinal dysmotility. The aim of our study was to investigate the expression of RB1 in the normal human colon and in Hirschsprung's disease (HD). MATERIALS AND METHODS: HD tissue specimens (n=10) were collected at the time of pull-through surgery, while colonic control samples were obtained at the time of colostomy closure in patients with imperforate anus (n=10). Immunolabeling of RB1 was visualized using confocal microscopy to assess protein distribution, while western blot analysis was undertaken to quantify RB1 protein expression. RESULTS: Immunohistochemistry revealed RB1 co-localized with platelet derived growth factor receptor alpha-positive (PDGFRα+) cells, nitrergic neurons and glia in controls and the ganglionic region of HD, with a marked reduction in the aganglionic HD specimens. Western blotting revealed a marked decrease in RB1 protein expression in the aganglionic region of HD colon compared to ganglionic and normal controls. CONCLUSION: We provide evidence of the presence of RB1 expression in the human colon in HD. As RB1 is known to colocalize with nitrergic neurons, the decreased expression of RB1 in the aganglionic bowel is most likely a secondary phenomenon because of the deficient nitrergic innervation in HD.