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Post-translational modification of some mitoribosomal proteins has been found to regulate their functions. MRPS23 has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. Furthermore, MRPS23 is a driver of luminal subtype breast cancer. However, its exact role and function in cancer remains unknown. METHODS AND RESULTS: Our previous study identified protein-protein interactions involving MRPS23 and CDK11A. In this study, we confirmed the interaction of MRPS23 with the p110 and p58 isoforms of CDK11A. Phosphoprotein enrichment studies and in vitro kinase assay using CDK11A/cyclin D3 followed by MALDI-ToF/ToF analysis confirmed the phosphorylation of MRPS23 at N-terminal serine 11 residue. Breast cancer cells expressing the MRPS23 (S11G) mutant showed increased cell proliferation, increased expression of PI3-AKT pathway proteins [p-AKT (Ser47), p-AKT (Thr308), p-PDK (Ser241) and p-GSK-3β (Ser9)] and increased antiapoptotic pathway protein expression [Bcl-2, Bcl-xL, p-Bcl2 (Ser70) and MCL-1] when compared with the MRPS23 (S11A) mutant-overexpressing cells. This finding indicated the role of MRPS23 phosphorylation in the proliferation and survival of breast cancer cells. The correlation of inconsistent MRPS23 phosphoserine 11 protein expression with CDK11A in the breast cancer cells suggested phosphorylation by other kinases. In vitro kinase assay showed that CDK1 kinase also phosphorylated MRPS23 and that inhibition using CDK1 inhibitors lowered phospho-MRPS23 (Ser11) levels. Additionally, modulating the expression of MRPS23 altered the sensitivity of the cells to CDK1 inhibitors.In conclusion, phosphorylation of MRPS23 by mitotic kinases might potentially be involved in the proliferation of breast cancer cells. Furthermore, MRPS23 can be targeted for sensitizing the breast cancer cells to CDK1 inhibitors.

Human Mitoribosomal Small Subunit unit (MRPS) family of genes appears to have role in cancer. Gene expression analysis of select MRPS genes (n = 9) in 15 cancer cell lines showed altered expression in cancer cells. Protein levels of MRPS6, MRPS23 showed significant overexpression in breast cancer cells and tissues. Interestingly, their overexpression did not correlate with mitochondrial ribosome translated COX2 protein levels in breast cancer. Subcellular fractionation analysis showed a distinct presence of MRPS23 in the nuclear fraction. GST/MRP6 and GST/MRPS23 pulldown assays identified 32 novel protein–protein interactions (PPIs) and MRPS23-RIPK3 interaction was validated. Co-expression module identification tool (CEMi) analysis of breast cancer gene expression and MRPS6 and MRPS23 interactions revealed hub interactions in gene expression modules having functional roles in cancer-associated cellular processes. Based on PPI network analysis a novel interaction MRPS23-p53 was validated. Knockdown of MRPS6 and MRPS23 decreased proliferation, expression of select mesenchymal markers, oncogenes, and increased expression of tumor suppressor genes. Taken together present study has revealed that MRPS6 and MRPS23 genes have pro-tumorigenic functions in breast cancer.

Our previous studies on gastric cancer tissue and patient plasma samples identified several cytokines/chemokines/growth factors to be differentially expressed, compared to normal samples. In this study our aim was to understand the localization patterns of the markers in gastric tissues. We investigated the expression of PDGFRB, CCL3, MMP3, CXCL8, CXCL10, CCL20, IGFBP3, CXCL9, SPP1, CCL18, TIMP1, CCL15, CXCL5 and CCL4 in gastric tissues using Immunohistochemistry (IHC) on Tissue Microarrays (TMA). The TMA comprised of 25 apparently normal (AN), 87 paired normal (PN) and 134 gastric cancer (T) tissues. The epithelial and stromal expression of markers and their correlation with patient characteristics and outcome were analyzed. Several of the markers [PDGFRB (p

The aim of the current study is to comprehend the prognostic and therapeutic value of matrix metalloproteinase (MMP11) in breast cancer using computational analyses. Gene expression-based Outcome for Breast cancer Online (GOBO) analysis revealed that MMP11 expression was increased in tumor when compared to normal. Among the subtypes, human epidermal growth factor receptor 2 (HER2) enriched and estrogen receptor (ER) positive showed higher expression of MMP11. The results were independently validated with cBioPortal and Oncomine databases. Tumors with high MMP11 expression showed poor overall survival and distant metastasis free survival. Further, protein-protein interactome network was constructed and molecular pathway analysis using Reactome database identified its putative role in diverse cellular processes. In addition, UALCAN web portal revealed hypomethylation in the promoter region of MMP11 in breast tumors. Molecular docking using iGEMDOCK and Autodock Vina were performed to identify natural compounds and repurposed drugs interaction with MMP11. Our study confirms the prognostic significance of MMP11 in breast cancer and their intrinsic subtypes. Twelve bio-compounds which were predominantly flavonoids and anti-psychotic drug pimozide showed significant docking scores with MMP11. Further, potential of these compounds in inhibiting MMP11 related molecular pathways and in targeting breast cancer cells are yet to be studied.

Background Gastric cancer is worldwide the third major cause of cancer related death. Risk factors for gastric cancer includes Helicobacter pylori infection, gastric ulcer, less hygienic condition, use of tobacco, alcohol consumption, use of salted, smoked food, genetic alterations etc. In order to identify the risk factors associated with gastric cancer in South Indian population a case-control study involving 200 proven gastric cancer cases and 400 controls was conducted. Methods A structured questionnaire was used to interview all the subjects who participated in our study. Genotyping assay was performed using Taqman allelic discrimination assay for 5 Single Nucleotide Polymorphisms (SNPs)-TGFβ C-509T, TGFβ T869C, XRCC1 Arg194Trp, IkBα C642T and IL4C-590T. Results Odds Ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression. Statistical analysis on socio-economic factors, lifestyle factors had showed that subjects from low socio economic status, use of tobacco and consumption of non-vegetarian food had increased risk of developing gastric cancer. Multi-factorial analysis for the SNPs adjusting for the risk factors obtained in this study showed that TGFΒ C-509T TT genotypes had four fold increased risk of gastric cancer (OR = 4.11, CI = 1.02–16.56) and TGFβ T869C CC genotype had a decreased risk of gastric cancer (OR = 0.21, CI = 0.05–0.85). Conclusion Economic status, tobacco use and food habits play a significant role in gastric cancer development. TT genotype for TGFβ C-509T had an increased risk and CC genotype for TGFβ T869C had a decreased risk of gastric cancer in south Indian population after adjusting for socio-economic factors and lifestyle factors.

The objective of this study was to determine radiation, doxorubicin, tamoxifen and letrozole sensitivity of breast cancer cells in response to functional inhibition of the ubiquitin conjugating enzyme UBE2C. Taqman Real time PCR was performed to measure UBE2C levels in breast cancer cell lines and control HBL100 and HEK293T cells. A dominant negative form of UBE2C (DN-UBE2C) was used to functionally inhibit wild type UBE2C. Cell proliferation and anchorage independent growth were measured by colorimetric and soft agar assays, respectively. Radiation, doxorubicin, tamoxifen and letrozole responses of the cell lines were assessed by colorimetric and clonogenic assays. Overexpression of UBE2C was observed in all breast cancer cell lines tested using quantitative real time PCR. UBE2C expression was found to be highest in MDAMB231 and relatively lowest in MCF7 cells, compared to control cells. Both the growth rate and the anchorage independent growth of MCF7 and MDAMB231 cells transfected with DN-UBE2C were significantly reduced compared to cells transfected with vector alone. MCF7 and MDAMB231 cells expressing DN-UBE2C were significantly more sensitive to different doses of radiation and doxorubicin compared to both wild type and vector alone transfected cells. In addition, DN-UBE2C transfected MCF7 cells were more sensitive to inhibition by tamoxifen and letrozole compared to wild type and vector alone transfected cells. Our results show that inhibition of UBE2C sensitizes breast cancer cells to radiation, doxorubicin and hormone blocking agents. UBE2C may, therefore, serve as a potential therapeutic target aimed at inducing radiation and chemo sensitization.

Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA down- regulation in gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms could be responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3 was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5'-AzaDC and methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysis was on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulation of ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with 5'-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did not indicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR (MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancer cell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples, 15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulation and methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA from gastric cancer patient's plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylation and methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.

Sclerostin domain containing 1 (SOSTDC1) is reportedly down-regulated in various cancers. Our purpose was to study whether epigenetic mechanisms were involved in the down-regulation of expression in gastric cancer. Expression analysis of SOSTDC1 in gastric cancer cell lines indicated mRNA down-regulation. Our reporter assays and gene reactivation studies using 5-aza-2'-deoxycytidine, a DNA demethylating agent, and trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, demonstrated that epigenetic mechanisms are involved in the down-regulation of SOSTC1 expression. Methylation analysis of the SOSTDC1 promoter CpGs using methylation-specific polymerase chain reaction analysis revealed methylation in gastric cancer cell lines and tissue samples. A majority of tumors (17 of 18) with observed methylation exhibited down-regulation of mRNA expression relative to apparently normal gastric tissues. Immunoreactivity for SOSTDC1 in gastric tumors (24 of 46, 52.1%) was down-regulated relative to normal tissues (36 of 38, 94.7%) (P = 0.00001). The difference in expression between gastric tumor subtypes, intestinal and diffuse, was significant (P = 0.040). Expression of SOSTDC1 in gastric tumors increased the probability of both overall and disease-free survival. When overexpressed in AGS cells, cell proliferation, cell cycle progression, and anchorage-independent growth was repressed. The present findings indicate SOSTDC1 down-regulation involves methylation; SOSTDC1 expression is a potential prognostic factor and tumor suppressor in gastric cancer.

Background Ewing’s sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. Methods In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251–343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251–280 ectopically in Ewing’s sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Results Our modelling analysis indicated that Junction region (aa 251–343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing’s sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Conclusions Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing’s Sarcoma.

In our recent report on gene expression in gastric cancer we identified the endo-sulfatase Sulf-1 gene to be up-regulated in gastric tumors relative to apparently normal (AN), and paired normal (PN) gastric tissue samples. In the present report we investigate the protein expression levels of Sulf-1 gene in gastric tumors, AN and PN samples using tissue microarray (TMA) and immunohistochemistry. Expression data was collected from two sets of TMA's containing replicate sections of tissue samples. Scoring data from TMA set-1 revealed a significant difference in Sulf-1 immunoreactivity between tumors and "normals" (PN and AN) (p-value = 0.001928). Also, Sulf-1 expression in tumors was also significantly different from either PN (p-value = 0.019) or AN (p-value = 0.006) samples. Similar results were obtained from analysis of scoring data from the second set of arrays. Comparison of mRNA expression and protein expression in gastric tumor tissues revealed that in 6/20 (30%) tumor samples showed up-regulated protein expression concordant with over-expression of mRNA. However, a discord with mRNA being over-expressed relative to down regulated protein expression was observed in majority 14/20 (70%) of tumor samples. Our study indicates down regulation of Sulf-1 protein expression in gastric tumors relative to PN and AN samples which is discordant with mRNA over-expression seen in tumors.

Background Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer. Materials and methods A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay. Results Our study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells. Conclusions Several novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a therapeutic target in cervical cancer.

Background Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets. Materials and methods We used 24 gastric cancers, 20 Paired normal (PN) and 5 apparently normal gastric tissues obtained from patients with non-gastric cancers (Apparently normal - AN) for the microarray study followed by validation of the significant genes (n = 63) by relative quantitation using Taqman Low Density Array Real Time PCR. We then used a custom made Quantibody protein array to validate the expression of 15 proteins in gastric tissues (4 AN, 9 PN and 9 gastric cancers). The same array format was used to study the plasma levels of these proteins in 58 patients with gastric cancers and 18 from patients with normal/non-malignant gastric conditions. Results Seventeen genes (ASPN, CCL15/MIP-1δ, MMP3, SPON2, PRSS2, CCL3, TMEPAI/PMEPAI, SIX3, MFNG, SOSTDC1, SGNE1, SST, IGHA1, AKR1B10, FCGBP, ATP4B, NCAPH2) were shown to be differentially expressed between the tumours and the paired normal, for the first time. EpCAM (p = 0.0001), IL8 (p = 0.0003), CCL4/MIP-1β (p = 0.0026), CCL20/MIP-3α (p = 0.039) and TIMP1 (p = 0.0017) tissue protein levels were significantly different (Mann Whitney U test) between tumours versus AN & PN. In addition, median plasma levels of IL8, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, PDGFR-B and TIMP1 proteins were significantly different between the non-malignant group and the gastric cancer group. The post-surgical levels of EpCAM, IGFBP3, IL8, CXCL10/IP10, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, SPP1/OPN and PDGFR-B showed a uniform drop in all the samples studied. Conclusions Our study has identified several genes differentially expressed in gastric cancers, some for the first time. Some of these have been confirmed at the protein level, as well. Some of these proteins will need to be evaluated further for their potential as diagnostic biomarkers in gastric cancers and some could be useful as follow-up markers in gastric cancer.