Manish Mani Subramaniam

MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer

AIMnAccumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3.nnnMETHODSnFour bioinformatic algorithms were used to predict miRNA binding to RUNX3. The correlation between candidate miRNAs and RUNX3 expression in cell lines was determined by real-time reverse transcriptase quantitative PCR (RT-qPCR) and Western blot. Candidate miRNAs were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability, apoptosis and Bim expression. miRNA and RUNX3 expression, RUNX3 methylation and RUNX3 protein levels were assessed in gastric tissue by RT-qPCR, Methylight analysis and immunohistochemistry, respectively.nnnRESULTSnBioinformatics, gene and protein expression analysis in eight gastric cell lines identified miR-130b as the top candidate miRNA for RUNX3 binding. Overexpression of miR-130b increased cell viability, reduced cell death and decreased expression of Bim in TGF-beta mediated apoptosis, subsequent to the downregulation of RUNX3 protein expression. In 15 gastric tumours, miR-130b expression was significantly higher compared to matched normal tissue, and was inversely associated with RUNX3 hypermethylation.nnnCONCLUSIONnAttenuation of RUNX3 protein levels by miRNA may reduce the growth suppressive potential of RUNX3 and contribute to tumourigenesis.

Molecular pathology of RUNX3 in human carcinogenesis

A major goal of molecular biology is to elucidate the mechanisms underlying cancer development and progression in order to achieve early detection, better diagnosis and staging and novel preventive and therapeutic strategies. We feel that an understanding of Runt-related transcription factor 3 (RUNX3)-regulated biological pathways will directly impact our knowledge of these areas of human carcinogenesis. The RUNX3 transcription factor is a downstream effector of the transforming growth factor-beta (TGF-beta) signaling pathway, and has a critical role in the regulation of cell proliferation and cell death by apoptosis, and in angiogenesis, cell adhesion and invasion. We previously identified RUNX3 as a major gastric tumor suppressor by establishing a causal relationship between loss of function and gastric carcinogenesis. More recently, we showed that RUNX3 functions as a bona fide initiator of colonic carcinogenesis by linking the Wnt oncogenic and TGF-beta tumor suppressive pathways. Apart from gastric and colorectal cancers, a multitude of epithelial cancers exhibit inactivation of RUNX3, thereby making it a putative tumor suppressor in human neoplasia. This review highlights our current understanding of the molecular mechanisms of RUNX3 inactivation in the context of cancer development and progression.

RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression

AbstractBackground We had previously established that inactivation of RUNX3 occurs by frequent promoter hypermethylation and protein mislocalization in invasive ductal carcinomas (IDC) of breast. Here, we hypothesize that inactivation of RUNX3 occurring in ductal carcinoma inxa0situ (DCIS) represent early event in breast carcinogenesis. nMethods The study cohort of 40 patients included 17 pure DCIS cases and 23 cases of DCIS with associated IDC (DCIS-IDC). The DCIS and IDC components of mixed cases were manually microdissected to permit separate evaluation. All the 63 samples including 17 pure DCIS, 23 samples each of DCIS and IDC of DCIS-IDC cases were analyzed for RUNX3 protein expression using R3-6E9 monoclonal antibody as well as promoter methylation status by methylation specific PCR. nResults Compared to matched normal breast samples (4 of 40, 10%), DCIS (35 of 40, 88%) and IDC (21 of 23, 91%) exhibited significant RUNX3 inactivation (Pxa0<xa00.001) in the form of negative or weak nuclear staining. In contrast to normal breast tissues (1/10, 10%), promoter hypermethylation of RUNX3 was significantly higher in the neoplastic breast samples (46 of total 61, 75%) including 30 of 40 (75%) DCIS and 16 of 21 (76%) IDC samples (Pxa0=xa00.009). Overall, promoter hypermethylation correlated with RUNX3 inactivation in 42 of 46 (91%) methylated samples (Pxa0=xa00.03). Mislocalized cytoplasmic expression also accounted for RUNX3 inactivation in majority of DCIS (33/40, 83%) and IDC (20/23, 87%) samples independent of promoter hypermethylation. nConclusion Our data suggest that RUNX3 inactivation by promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression.

RUNX3 Inactivation in Colorectal Polyps Arising Through Different Pathways of Colonic Carcinogenesis

OBJECTIVES:We hypothesized that RUNX3 inactivation by promoter hypermethylation in colorectal polyps is an early molecular event in colorectal carcinogenesis.METHODS:RUNX3 protein expression was analyzed immunohistochemically in 50 sporadic colorectal polyps comprising 19 hyperplastic polyps (HPs), 14 traditional serrated adenomas (TSAs), and 17 sporadic traditional adenomas (sTAs) as well as in 19 familial adenomatous polyposis (FAP) samples from 10 patients showing aberrant crypt foci (ACF) (n=91), small adenomas (SmAds) (n=40), and large adenomas (LAds) (n=13). In addition, we assessed the frequency of promoter hypermethylation of RUNX3 by methylation-specific PCR (MSP) in all the 50 sporadic polyps as well as 38 microdissected FAP polyps comprising ACF, SmAds, and LAds obtained from 7 FAP samples. A total of 12 normal colon samples were also included for RUNX3 MSP analysis.RESULTS:Compared to normal colon (2 of 12, 16%) and sTAs (3 of 17, 18%), HPs (15 of 19, 79%) and TSAs (8 of 14, 57%) displayed significant inactivation of RUNX3 (P<0.05). In FAP, RUNX3 inactivation was more frequently seen in ACF (78 of 91, 86%), SmAds (25 of 40, 62%), and LAds (6 of 13, 46%) compared to normal mucosa (0 of 19, 0%) in the same samples (all P<0.05). Promoter hypermethylation of RUNX3 was significantly higher in colorectal polyps (64 of 87, 74%) compared to normal colon (2 of 12, 16%) (P=0.001). Serrated polyps such as HPs (17 of 19, 89%) and TSAs (12 of 14, 86%) were significantly more methylated than sTAs (7 of 17, 44%) (P=0.004). RUNX3 hypermethylation was observed in 28 of the total 38 (74%) FAP polyps. Overall, RUNX3 promoter methylation correlated with inactivation of RUNX3 expression in sporadic (27 of 36, 75%) (P=0.022) and FAP (21 of 28, 75%) (P=0.021) polyps.CONCLUSIONS:Our data suggest that RUNX3 inactivation due to promoter hypermethylation in colorectal polyps represents an early event in colorectal cancer (CRC) progression. In addition, epigenetic RUNX3 inactivation is a frequent event in the serrated colonic polyps as well as in the ACF of FAP polyps.

Clonal characterization of sporadic cribriform-morular variant of papillary thyroid carcinoma by laser microdissection-based APC mutation analysis.

Cribriform-morular variant (C-MV) of papillary thyroid carcinoma (PTC) is a rare and unusual neoplasm composed of multiple histologic components, including cribriform, papillary, solid, tall columnar, and morular patterns. Analyses of gross C-MV of PTC lesions has linked adenomatous polyposis coli (APC) mutations to its pathogenesis; however, the extent of involvement of mutations in the development of individual components is unclear. We report on bidirectional sequencing of the mutation cluster region (codons 1032-1565) of the APC gene in individually laser-microdissected components of a previously unreported C-MV of PTC. A silent Thr1493Thr gene variant was found in all tumoral components, whereas a 5-base-pair frameshift deletion at codon 1309 was identified only in the morules. Neither variant was observed in matched normal thyroid tissue. These results show the histologic components of C-MV of PTC to have some common mutational background, although additional somatic mutations may be involved in the development of morular structures.

Molecular characterization of dedifferentiated mucoepidermoid carcinoma of the trachea using laser microdissection-based TP53 mutation analysis.

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Dedifferentiated solitary fibrous tumour of the nasal cavity: the first case reported with molecular characterization of a TP53 mutation

invasiveness. At ameloblastoma invasion sites cortactin would recruit MT-MMP, inducing focal degradation in stroma. Correlating cortactin and MT1-MMP presence in ameloblastoma with our previous findings on MMPs and growth factors, we suggest that cortactin, MT1MMP, proteases and growth factors would combine forces to regulate ameloblastoma invasiveness. We demonstrated cortactin and MT1-MMP expression in ameloblastoma. These proteins were increased significantly compared to CCOT, a non-invasive odontogenic tumour. Our results indicate that invadopodia proteins cooperate to regulate ameloblastoma invasion.

Poorly differentiated synovial sarcoma of the sphenoid sinus: report of the first case and review of synovial sarcomas of the sinonasal tract

cyst wall were mostly positive for lambda light chain and IgA, which are diagnostic findings of thymic MALT lymphoma (Figure 2A,B). In addition, Hodgkin ⁄ Reed–Sternberg (HRS)-like large cells (Figure 1C) were scattered in minor areas where lymph follicles were inconspicuous. In those areas, up to 100 EBV-encoded small RNA (EBER) in-situ hybridization-positive lymphocytes per high-power field were observed, admixed with EBER-negative small lymphocytes and plasma cells (Figure 2C). Although classical Hodgkin’s lymphoma (cHL) within MALT lymphoma was considered as a differential diagnosis, variation in the size of EBERpositive cells and the immunophenotype of EBERpositive HRS-like cells (CD15-negative, CD20-positive, and CD30-positive) were against the diagnosis of cHL. We made a pathological diagnosis of composite MALT lymphoma and EBV-positive polymorphic LPD associated with MTX. At 4 years after the surgery, without any additional treatment, no recurrence was observed. From the study of serial cases of composite MALT lymphoma and cHL, MALT lymphoma is recognized to have the potential to transform to cHL, and EBV infection is considered to be a key factor in its transformation. The present case suggests that composite MALT lymphoma and MTX-associated EBV-positive polymorphic LPD can also occur. The pathological differentiation between EBV-positive polymorphic LPD and cHL is important. Reduction or cessation of immunosuppressive drugs is generally the first-line treatment for iatrogenic LPD. In contrast, postoperative chemotherapy is usually considered for thymic cHL. Although cases like that in the present study may be rare, the combination of MALT lymphoma and iatrogenic LPD should be considered in the differential diagnosis of thymic lymphoma, especially in patients with autoimmune disease.

The Topography of DNA Methylation in the Non-Neoplastic Colonic Mucosa Surrounding Colorectal Cancers

The degree of gene hypermethylation in non‐neoplastic colonic mucosa (NNCM) is a potentially important event in the development of colorectal cancer (CRC), particularly for the subgroup with a CpG island methylator phenotype (CIMP). In this study, we aimed to use an unbiased and high‐throughput approach to evaluate the topography of DNA methylation in the non‐neoplastic colonic mucosa (NNCM) surrounding colorectal cancer (CRC). A total of 61 tissue samples comprising 53 NNCM and 8 tumor samples were obtained from hemicolectomy specimens of two CRC patients (Cases 1 and 2). NNCM was stripped from the underlying colonic wall and samples taken at varying distances from the tumor. The level of DNA methylation in NNCM and tumor tissues was assessed at 1,505 CpG sites in 807 cancer‐related genes using Illumina GoldenGate® methylation arrays. Case 1 tumor showed significantly higher levels of methylation compared to surrounding NNCM samples (Pu2009<u20090.001). The average level of methylation in NNCM decreased with increasing distance from the tumor (ru2009=u2009−0.418; Pu2009=u20090.017), however this was not continuous and “patches” with higher levels of methylation were observed. Case 2 tumor was less methylated than Case 1 tumor (average β‐value 0.181 vs. 0.415) and no significant difference in the level of methylation was observed in comparison to the surrounding NNCM. No evidence was found for a diminishing gradient of methylation in the NNCM surrounding CRC with a high level of methylation. Further work is required to determine whether CIMP+ CRC develop from within “patches” of NCCM that display high levels of methylation.

Lack of RUNX3 inactivation in columnar cell lesions of breast

Subramaniam M M, Chan J Y, Omar M F M, Ito K, Ito Y, Yeoh K G, Salto‐Tellez M & Putti T Cu2028(2010) Histopathology57, 555–563u2028Lack of RUNX3 inactivation in columnar cell lesions of breast