Identification and validation of plasma biomarkers for diagnosis of breast cancer in South Asian women.

Breast cancer is the most common malignancy among women globally. Development of a reliable plasma biomarker panel might serve as a non-invasive and cost-effective means for population-based screening of the disease. Transcriptomic profiling of breast tumour, paired normal and apparently normal tissues, followed by validation of the shortlisted genes using TaqMan® Low density arrays and Quantitative real-time PCR was performed in South Asian women. Fifteen candidate protein markers and 3 candidate epigenetic markers were validated first in primary breast tumours and then in plasma samples of cases [N = 202 invasive, 16 DCIS] and controls [N = 203 healthy, 37 benign] using antibody array and methylation specific PCR. Diagnostic efficiency of single and combined markers was assessed. Combination of 6 protein markers (Adipsin, Leptin, Syndecan-1, Basic fibroblast growth factor, Interleukin 17B and Dickopff-3) resulted in 65% sensitivity and 80% specificity in detecting breast cancer. Multivariate diagnostic analysis of methylation status of SOSTDC1, DACT2, WIF1 showed 100% sensitivity and up to 91% specificity in discriminating BC from benign and controls. Hence, combination of SOSTDC1, DACT2 and WIF1 was effective in differentiating breast cancer [non-invasive and invasive] from benign diseases of the breast and healthy individuals and could help as a complementary diagnostic tool for breast cancer.

Identification of small molecule inhibitors for differentially expressed miRNAs in gastric cancer.

Gastric cancer is a common cancer in the world with higher incidence rates. Micro RNAs (miRNAs) are known to control biological processes and are also involved in the initiation and progression of numerous cancers. The aim of this study was to identify small molecule inhibitors using bioinformatics methods for over-expressed miRNAs that are known to play a major role in the genesis of gastric cancer. Our study included three datasets from Gene expression omnibus (GEO) database and we used bioinformatics tool to identify differentially expressed miRNAs in all the three datasets. Gene ontology and pathway enrichment analysis of the experimentally validated miRNA target genes and their linkage to oncogenesis were analyzed. Target genes thus identified were found to be linked with the major pathways of cancer. We were interested in identifying small molecules which are specific to over-expressed miRNAs which can arrest oncogenesis. Thus, the computer aided high throughput virtual screening was carried out to identify potent small molecules for over expressed miRNAs in gastric cancer using Glide. Based on docking score top five ligands specific for each miRNAs were identified.

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

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.

Nilotinib based pharmacophore models for BCRABL.

UNLABELLED: Tyrosine kinase inhibitors have revolutionized the treatment of several malignancies, converting lethal diseases in a manageable aspect. Imitanib, a small molecule ABL kinase inhibitor is a highly effective therapy for early phase chronic myeloid leukemia (CML), which has constitutively active ABL kinase activity owing to the over expression of the BCR-ABL fusion protein. But some patients develop imatinib resistance, particularly in the advanced phases of CML.The discovery of resistance mechanisms of imitanib; urge forward the development of second generation drugs. Nilotinib, a second generation drug is more potent inhibitor of BCR-ABL than imatinib. But nilotinib also develops dermatologic events and headache in patients. Large information about BCR-ABL structure and its inhibitors are now available. Based on the pharmacophore modeling approaches, it is possible to decipher the molecular determinants to inhibit BCR-ABL. We conducted a structure based and ligand based study to identify potent natural compounds as BCR-ABL inhibitor. First kinase inhibitors were docked with the receptor (BCR-ABL) and nilotinib was selected as a pharmacophore due its high binding efficiency. Eleven compounds were selected out of 1457 substances which have mutual pharmacopohre features with nilotinib. These eleven compounds were validated and used for docking study to find the drug like molecules. The best molecules from the final set of screening candidates can be evaluated in cell lines and may represent a novel class of BCR-ABL inhibitors. ABBREVIATIONS: CML - Chronic myeloid leukemia, PDGFR - Platelet derived growth factor receptor, TKI - Tyrosine kinase inhibitors.

Identification of small molecule inhibitors against UBE2C by using docking studies.

An increased expression of UBE2C (Ubiquitin-conjugating enzyme E2C) has been associated with high tumor grade and cancer progression. It is an essential indicator of the mitotic destruction events. Our microarray study on cervical cancers showed UBE2C to be over expressed in cervical cancer. Subsequent studies from our laboratory, showed that inhibition of UBE2C can enhance radiation and chemosensitivity. Therefore it can be an appropriate target for drug development to identify potential and specific inhibitor of cancer. To identify small molecule inhibitors, a computational approach was used to model UBE2C and further docking studies were carried out. Different ligand subsets such as ChemBank, PDB, KEGG, Drug-likeness NCI, Not annotated NCI of ligand library ligands were downloaded and docked with UBE2C. Schrodinger tools were used for identifying active sites and docking studies of ligands with UBE2C. Based on glide score, the potential ligands were screened and its interaction with UBE2C was identified. We also analyzed the drug like properties such as absorption, distribution, metabolism, excretion and toxicity (ADME/T) of docked compounds. Our results suggest that 2,4-diimino-1-methyl-1,3,5-triazepan-6-one, sulfuric acid compound with 5,6-diamino-2,4-pyrimidinediol (1:1) and 7-alpha-d-ribofuranosyl-2-aminopurine-5'-phosphate may act as best inhibitors and further in vitro studies, may lead to development of novel and best inhibitor of UBE2C.

Identification and validation of genes involved in cervical tumourigenesis.

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.

Identification and validation of genes involved in gastric tumorigenesis.

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-1ß (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.

A 7 gene expression score predicts for radiation response in cancer cervix.

BACKGROUND: Cervical cancer is the most common cancer among Indian women. The current recommendations are to treat the stage IIB, IIIA, IIIB and IVA with radical radiotherapy and weekly cisplatin based chemotherapy. However, Radiotherapy alone can help cure more than 60% of stage IIB and up to 40% of stage IIIB patients. METHODS: Archival RNA samples from 15 patients who had achieved complete remission and stayed disease free for more than 36 months (No Evidence of Disease or NED group) and 10 patients who had failed radical radiotherapy (Failed group) were included in the study. The RNA were amplified, labelled and hybridized to Stanford microarray chips and analyzed using BRB Array Tools software and Significance Analysis of Microarray (SAM) analysis. 20 genes were selected for further validation using Relative Quantitation (RQ) Taqman assay in a Taqman Low-Density Array (TLDA) format. The RQ value was calculated, using each of the NED sample once as a calibrator. A scoring system was developed based on the RQ value for the genes. RESULTS: Using a seven gene based scoring system, it was possible to distinguish between the tumours which were likely to respond to the radiotherapy and those likely to fail. The mean score +/- 2 SE (standard error of mean) was used and at a cut-off score of greater than 5.60, the sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) were 0.64, 1.0, 1.0, 0.67, respectively, for the low risk group. CONCLUSION: We have identified a 7 gene signature which could help identify patients with cervical cancer who can be treated with radiotherapy alone. However, this needs to be validated in a larger patient population.