Differential association of hedgehog pathway in development of cervical carcinoma and its chemo-tolerance.

Cervical carcinoma (CACX) is still a dreadful threat to women in developing countries. Available conventional chemo-radiation therapies are not sufficient to restrict the disease recurrence. To unravel the mechanism of the disease recurrence, alteration of hedgehog self-renewal pathway was evaluated during development of CACX and in chemo-tolerance of the tumor. We have analyzed the alterations (expression/methylation/deletion) of some key regulatory genes (HHIP/SUFU/SHH/ SMO/GLI1) of hedgehog self-renewal pathway in cervical lesions at different clinical stages and compared with different datasets, followed by their clinico-pathological correlations. The changes in expression/methylation of the genes were then evaluated in two CACX cell lines (SiHa/HeLa) after treatment with chemotherapeutic drug cisplatin at different concentrations. Down regulation (mRNA/protein) of the antagonists HHIP and SUFU due to promoter methylation and/or deletion along with upregulation (protein) of agonists SHH, SMO and GLI1 was seen in early invasive lesions and subsequent clinical stages. Reduced protein expression of HHIP and SUFU showed significant association with high/intermediate expression of agonists SHH, SMO, GLI1 in the tumors and also poor prognosis of the patients. It was evident that cisplatin could restrict the growth of HeLa and SiHa cells through significant upregulation of antagonists HHIP and SUFU due to their promoter hypomethylation and down regulation of SHH in a concentration dependent manner without any significant changes in expression of SMO and GLI1, leading to the tumor cells in a dormant state. Thus, interplay of the agonists and antagonists has important role in activation of hedgehog pathway during development of CACX, whereas inactivation of the pathway due to upregulation of the antagonists is an important phenomenon in chemo-tolerance of the tumor. This suggests importance of epigenetic modification in chemo-resistance of CACX.

Promoter methylation and enhanced SKP2 are associated with the downregulation of CDKN1C in cervical squamous cell carcinoma.

PURPOSE: Cervical Squamous Cell Carcinoma (CSCC) is one of the significant causes of cancer deaths among women. Distinct genetic and epigenetic-altered loci, including chromosomal 11p15.5-15.4, have been identified. CDKN1C (Cyclin-Dependent Kinase Inhibitor 1C, p57KIP2), a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs), located at 11p15.4, is a putative tumor suppressor. Apart from transcriptional control, S-Phase Kinase Associated Protein 2 (SKP2), an oncogenic E3 ubiquitin ligase, regulates the protein turnover of CDKN1C. But the molecular status of CDKN1C in CSCC and the underlying mechanistic underpinnings have yet to be explored. METHODS: TCGA and other publicly available datasets were analyzed to evaluate the expression of CDKN1C and SKP2. The expression (transcript/protein) was validated in independent CSCC tumors (n = 155). Copy number alteration and promoter methylation were correlated with the expression. Finally, in vitro functional validation was performed. RESULTS: CDKN1C was down-regulated, and SKP2 was up-regulated at the transcript and protein levels in CSCC tumors and the SiHa cell line. Notably, promoter methylation (50%) was associated with the downregulation of the CDKN1C transcript. However, high expression of SKP2 was found to be associated with the decreased expression of CDKN1C protein. Independent treatments with 5-aza-dC, MG132, and SKP2i (SKPin C1) in SiHa cells led to an enhanced expression of CDKN1C protein, validating the mechanism of down-regulation in CSCC. CONCLUSION: Collectively, CDKN1C was down-regulated due to the synergistic effect of promoter hyper-methylation and SKP2 over-expression in CSCC tumors, paving the way for further studies of its role in the pathogenesis of the disease.

Novel eIF4A1 inhibitors with anti-tumor activity in lymphoma.

BACKGROUND: Deregulated translation initiation is implicated extensively in cancer initiation and progression. It is actively pursued as a viable target that circumvents the dependency on oncogenic signaling, a significant factor in current strategies. Eukaryotic translation initiation factor (eIF) 4A plays an essential role in translation initiation by unwinding the secondary structure of messenger RNA (mRNA) upstream of the start codon, enabling active ribosomal recruitment on the downstream genes. Several natural product molecules with similar scaffolds, such as Rocaglamide A (RocA), targeting eIF4A have been reported in the last decade. However, their clinical utilization is still elusive due to several pharmacological limitations. In this study we identified new eIF4A1 inhibitors and their possible mechanisms. METHODS: In this report, we conducted a pharmacophore-based virtual screen of RocA complexed with eIF4A and a polypurine RNA strand for novel eIF4A inhibitors from commercially available compounds in the MolPort Database. We performed target-based screening and optimization of active pharmacophores. We assessed the effects of novel compounds on biochemical and cell-based assays for efficacy and mechanistic evaluation. RESULTS: We validated three new potent eIF4A inhibitors, RBF197, RBF 203, and RBF 208, which decreased diffuse large B-cell lymphoma (DLBCL) cell viability. Biochemical and cellular studies, molecular docking, and functional assays revealed that thosenovel compounds clamp eIF4A into mRNA in an ATP-independent manner. Moreover, we found that RBF197 and RBF208 significantly depressed eIF4A-dependent oncogene expression as well as the colony formation capacity of DLBCL. Interestingly, exposure of these compounds to non-malignant cells had only minimal impact on their growth and viability. CONCLUSIONS: Identified compounds suggest a new strategy for designing novel eIF4A inhibitors.

PTPRJ is downregulated in cervical squamous cell carcinoma.

Squamous cell carcinoma of the uterine cervix (CSCC) is one of the leading causes of death in Indian women. Protein tyrosine phosphatase receptor (PTPR) type J (also known as DEP1) is a recently reported tumour suppressor receptor phosphatase. Critical molecular analysis of PTPRJ/DEP1 (11p11.2) has not performed in CSCC to date. Here, we observed frequent downregulation of cancer samples (n=31) at the transcriptional level. Immunohistochemistry revealed concordant low expression of PTPRJ protein with a few samples showing intermediate expression. To probe for the cause of such downregulation of the gene in CSCC (n=155), we analysed the copy number and promoter methylation of PTPRJ. The genetic locus showed deletion (14.8%) and the promoter showed methylation (33.5%) of PTPRJ. To the best of our knowledge, for the first time we explored the molecular status of PTPRJ although we observed no statistically significant association with the prognosis of Indian CSCC patients (n=76). However, we observed enhanced expression of PTPRJ protein levels that contributes to effective cisplatin chemotherapy in the SiHa cell line. Thus, the present study paves the way for further research into the plausible mechanisms of downregulation of PTPRJ in cervical cancer.

Specific dopaminergic genetic variants influence impulsivity, cognitive deficit, and disease severity of Indian ADHD probands.

BACKGROUND: Impulsivity (Imp), being one of the cardinal symptoms of Attention Deficit Hyperactivity Disorder (ADHD), often leads to inappropriate responses to stimuli. Since the dopaminergic system is the primary target for pharmaceutical intervention in ADHD, we investigated the association between ADHD-related Imp and functional gene variants of the dopamine transporter (SLC6A3) and catechol-O-methyltransferase involved in dopamine clearance. METHODS AND RESULTS: Indo-Caucasoid families with ADHD probands (N = 217) were recruited based on the Diagnostic and Statistical Manual of Mental Disorders (DSM). Imp of the probands was assessed using the Domain Specific Imp Scale for Children and DSM. Peripheral blood was collected after obtaining informed written consent for participation, genomic DNA was isolated, and target sites were genotyped by DNA sequencing. The association of genetic variants with Imp was examined by the Quantitative trait analysis (QTA) and Analysis of variance (ANOVA). Post-Hoc analysis following QTA and ANOVA showed significant associations of rs2254408, rs2981359, and rs2239393 with different domains of Imp (P < 0.05). Various haplotypic combinations also showed statistically significant associations with Imp (P < 0.05). Multifactor dimensionality reduction models revealed strong effects of the variants on Imp. ADHD probands harboring the risk alleles exhibited a deficit in performance during cognitive assessment. Longitudinal follow-up revealed a significant association of rs2254408 with trait persistence. CONCLUSION: The present study indicates the influence of the studied genetic variants on ADHD-associated imp, executive deficit, and disease persistence. Thus, these variants may be helpful as predictors for the success of individual therapeutic sessions during cognitive training.

PARK2 regulates eIF4B-driven lymphomagenesis.

Patients with high-risk diffuse large B-cell lymphoma (DLBCL) have poor outcomes following first-line cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab (R-CHOP); thus, treatment of this fatal disease remains an area of unmet medical need and requires identification of novel therapeutic approaches. Dysregulation of protein translation initiation has emerged as a common downstream node in several malignancies, including lymphoma. Ubiquitination, a prominent post-translational modification associated with substrate degradation, has recently been shown to be a key modulator of nascent peptide synthesis by limiting several translational initiation factors. While a few deubiquitinases have been identified, the E3-ligase responsible for the critical ubiquitination of these translational initiation factors is still unknown. In this study, using complementary cellular models along with clinical readouts, we establish that PARK2 ubiquitinates eIF4B and consequently regulates overall protein translational activity. The formation of this interaction depends on upstream signaling, which is negatively regulated at the protein level of PARK2. Through biochemical, mutational, and genetic studies, we identified PARK2 as a mTORC1 substrate. mTORC1 phosphorylates PARK2 at Ser127, which blocks its cellular ubiquitination activity, thereby hindering its tumor suppressor effect on eIF4B's stability. This resultant increase of eIF4B protein level helps drive enhanced overall protein translation. These data support a novel paradigm in which PARK2-generated eIF4B ubiquitination serves as an anti-oncogenic intracellular inhibitor of protein translation, attenuated by mTORC1 signaling. Implications: Our data implicates the FASN/mTOR-PARK2-eIF4B axis as a critical driver of enhanced oncogene expression contributing to lymphomagenesis.

PARK2 regulates eIF4B-driven lymphomagenesis.

Patients with high-risk diffuse large B-cell lymphoma (DLBCL) have poor outcomes following first-line cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab (R-CHOP); thus, treatment of this fatal disease remains an area of unmet medical need and requires identification of novel therapeutic approaches. Dysregulation of protein translation initiation has emerged as a common downstream node in several malignancies, including lymphoma. Ubiquitination, a prominent post-translational modification associated with substrate degradation, has recently been shown to be a key modulator of nascent peptide synthesis by limiting several translational initiation factors. While a few deubiquitinases have been identified, the E3-ligase responsible for the critical ubiquitination of these translational initiation factors is still unknown. In this study, using complementary cellular models along with clinical readouts, we establish that PARK2 ubiquitinates eIF4B and consequently regulates overall protein translational activity. The formation of this interaction depends on upstream signaling, which is negatively regulated at the protein level of PARK2. Through biochemical, mutational, and genetic studies, we identified PARK2 as a mTORC1 substrate. mTORC1 phosphorylates PARK2 at Ser127, which blocks its cellular ubiquitination activity, thereby hindering its tumor suppressor effect on eIF4B's stability. This resultant increase of eIF4B protein level helps drive enhanced overall protein translation. These data support a novel paradigm in which PARK2-generated eIF4B ubiquitination serves as an anti-oncogenic intracellular inhibitor of protein translation, attenuated by mTORC1 signaling. Implications: Our data implicates the FASN/mTOR-PARK2-eIF4B axis as a critical driver of enhanced oncogene expression contributing to lymphomagenesis.

Landscape of toll-like receptors expression in tumor microenvironment of triple negative breast cancer (TNBC): Distinct roles of TLR4 and TLR8.

TNBC is the most aggressive and hormone receptor-negative subtype of breast cancer with molecular heterogeneity in bulk tumors hindering effective treatment. Toll-like receptors (TLRs) have the potential to ignite diverse immune responses in the tumor microenvironment (TME). This encouraged us to screen their transcript expression in the publically available TCGA datasets. Reported molecular subtypes of TNBC may represent different TMEs and we observed differentially expressed TLRs (DETs) i.e. TLR3/4/6/8/9 have unique expression pattern in the TNBC subtypes, particularly in Immunomodulatory (IM) TNBC subtype. We then dissected expression of the DETs in immune and other components of the TME. TLR4 and TLR8 showed significant (p-value ≤ 0.05) negative partial correlation with tumor purity compared to other DETs. Interestingly, TLR4 and TLR8 expression showed a significant (adjusted p-value ≤ 0.05) correlation with different subsets of immune infiltrating cells having the highest correlation with monocytes/macrophage/dendritic cell populations mediating both innate and adaptive response in TNBC. The co-expression network identified genes correlated with these immune cells. Further, GSEA analysis of co-expressed genes showed a significant association of TLR8 partners with 'Peptide ligand binding', 'Gά-signaling', and 'Cytokine-cytokine interaction' while TLR4 associated genes correlated with 'Adaptive immune system' and 'Systemic lupus erythematosus' interactome. Finally, the expression of TLR4 protein was validated in a panel of TNBC cell lines. TLR4 expression in chemoresponsive TNBC was also validated in TNBC cell lines upon Paclitaxel (PTX) treatment. Collectively, the present study identified specific DETs in TNBC and discovered a prospective role of TLR4 and TLR8 in the maintenance of tumor-immune-microenvironment.

Differential Wnt-ß- catenin pathway activation in HPV positive and negative oral epithelium is transmitted during head and neck tumorigenesis: clinical implications.

The aim of this study is to understand the association of HPV infection and wnt-ß-catenin self-renewal pathway in development of head and neck squamous cell carcinoma (HNSCC). For this reason, the molecular profiles (methylation/deletion/expression) of antagonists (SFRP1/2 and DKK1), agonists (FZD7 and LRP6) and effector protein ß-catenin of the pathway were analyzed in HPV positive/negative oral epithelium at first, followed by its changes during development of the tumor along with correlations with different clinico-pathological parameters. HPV infection alone or in combination with tobacco habit could activate p- ß-catenin expression in basal/parabasal layers of oral epithelium through high expression of FZD7 and significant down regulation of SFRP1/2 through promoter hypermethylation due to over expression of DNMT1 with ubiquitous down regulation of DKK1 and up-regulation of LRP6. This phenomenon has been seen in respective HPV positive and negative HNSCC tumors with additional deletion/microsatellite size alterations in the antagonists. Overall alterations (methylation/deletion) of SFRP1/2, DKK1 gradually increased from Group I (HPV-/Tobacco-) to Group IV(HPV+/Tobacco+) tumors, leading to the worst prognosis of the patients. Thus, the transmission of differentially activated wnt-ß-catenin pathway from HPV positive/negative basal/parabasal layers of oral epithelium to HNSCC tumors determines differences in molecular pathogenesis of the disease.

Integrative genomics and pathway analysis identified prevalent FA-BRCA pathway alterations in arsenic-associated urinary bladder carcinoma: Chronic arsenic accumulation in cancer tissues hampers the FA-BRCA pathway.

Arsenic in drinking water is one of the major etiological factors in urinary bladder carcinoma (BlCa). Here, high-resolution CGH-SNP microarray analysis in arsenic accumulated BlCa tissues showed significant (p < 0.05) association of chromosomal alterations with high arsenic (≥112 ng/g) accumulation, further corroborated by high γH2AX nuclear expression. Cytobands 5q11-35, 9p24.3-21.5, 18q11.1-25, etc. showed deletion, whereas 12q was amplified in high arsenic samples (AsH). Consecutively, IPA® found FA-BRCA pathway to be exclusively altered in AsH group. Validation of several key regulatory genes (RAD50, BRIP1, UIMC1, FANCD2, BRCA2 and BRCA1) of the pathway, were performed in independent BlCa cases (n = 81). UIMC1, RAD50 and BRIP1 were differentially deleted and associated with poor survival of AsH samples. Moreover, reduced nuclear expression with diffused cytoplasmic expression of FANCD2 was higher in AsH samples. Collectively, frequent deregulation of RAD50, UIMC1 and BRIP1 may result in reduced nuclear translocation of FANCD2, which may cause more chromosomal aberrations among AsH samples.

sigFeature: Novel Significant Feature Selection Method for Classification of Gene Expression Data Using Support Vector Machine and t Statistic.

Biological data are accumulating at a faster rate, but interpreting them still remains a problem. Classifying biological data into distinct groups is the first step in understanding them. Data classification in response to a certain treatment is an extremely important aspect for differentially expressed genes in making present/absent calls. Many feature selection algorithms have been developed including the support vector machine recursive feature elimination procedure (SVM-RFE) and its variants. Support vector machine RFEs are greedy methods that attempt to find superlative possible combinations leading to binary classification, which may not be biologically significant. To overcome this limitation of SVM-RFE, we propose a novel feature selection algorithm, termed as "sigFeature" (https://bioconductor.org/packages/sigFeature/), based on SVM and t statistic to discover the differentially significant features along with good performance in classification. The "sigFeature" R package is centered around a function called "sigFeature," which provides automatic selection of features for the binary classification. Using six publicly available microarray data sets (downloaded from Gene Expression Omnibus) with different biological attributes, we further compared the performance of "sigFeature" to three other feature selection algorithms. A small number of selected features (by "sigFeature") also show higher classification accuracy. For further downstream evaluation of its biological signature, we conducted gene set enrichment analysis with the selected features (genes) from "sigFeature" and compared it with the outputs of other algorithms. We observed that "sigFeature" is able to predict the signature of four out of six microarray data sets accurately, whereas the other algorithms predict less data set signatures. Thus, "sigFeature" is considerably better than related algorithms in discovering differentially significant features from microarray data sets.

Deregulation of H19 is associated with cervical carcinoma.

CACX is one of the most common cancer affecting women world-wide. Here, expression microarray analysis revealed 8 over-expressed transcribed pseudogenes (GBP1P1, HLA-DRB6, HLA-H, SLC6A10P, NAPSB, KRT16P2, PTTG3P and RNF126P1), down-regulated 7 lincRNAs (H19, MIR100HG, MEG3, DIO3OS, HOXA11-AS, CD27-AS1 and EPB41L4A-AS) and 6 snoRNAs (SNORD97, SNORD3A, SNORD3C, SNORD3D, SNORA12 and SCARNA9) as DEncGs (log2 fold-change ≥ ±1.0) in CACX. Consequently, down-regulation of lincRNA MEG3 and over-expression of pseudogenes, GBP1P1 and PTTG3P in the microarray analysis were found concordant with the real-time quantitative PCR results upon validation. Then, Ingenuity® Pathway analysis (IPA®) analysis with deregulated DEncGs identified functionally important gene, H19. Further, validation (n = 52) of expression confirmed frequent downregulation of H19 with significant association with its deletion (LOH) and promoter methylation (n = 128) in CACX. Moreover, clinicopathological analysis found Indian CACX patients (n = 26) with alterations of H19 by deletion or, promoter methylation with concomitant low expression have poor prognosis.

Sonochemically Synthesized Spin-Canted CuFe2O4 Nanoparticles for Heterogeneous Green Catalytic Click Chemistry.

Heterogeneous green catalysis by using magnetically separable nanometal-oxide catalysts has become a subject of prime focus recently. PXRD (powder X-ray diffraction), FESEM (field emission scanning electron microscopy), and HRTEM (high-resolution tunneling electron microscopy) with IR and Raman spectroscopy are applied to analyze the structural and microstructural properties of nanosized (∼15.3 nm) CuFe2O4 synthesized by both sonochemical and mechanochemical processes. The sonochemical process provides a better uniformity of sizes of the nanoparticles (NPs). Rietveld refinement with the PXRD pattern reveals the inverse spinel-like architecture of CuFe2O4 NPs. The Raman spectra also indicate the phase purity of the synthesized material. The static magnetic measurements are performed at different magnetic fields and temperature ranges from 300 to 5 K, which confirms the existence of the ferrimagnetic phase mixed with some finer superparamagnetic (SPM) nanophases within the sample. Unsaturated magnetization is observed even at an applied 5 T magnetic field for the presence of spin-canting nature in the partially inverted copper ferrite phases at the surfaces of the nanoparticles. Now, these coupled magnetic CuFe2O4 NPs are used as a heterogeneous catalyst for three-component Huisgen 1,3-dipolar cycloaddition click reaction in aqueous media. By this catalyst system, we were able to couple alkyl halide, epoxide, or boronic acid with alkynes efficiently to furnish 1,4-disubstituted 1,2,3-triazoles in excellent yields within very short reaction time. The test for heterogeneity, reusability, and reproducibility of the catalyst has also been performed successfully without prominent decrease in yield up to the fifth cycle.

IGF2 is Deregulated During the Development of Uterine Cervical Carcinoma in Indian Patients.

Uterine cervical carcinoma (CACX) is one of the leading causes of deaths in Indian women. Chromosomal alterations including 11p15.5 locus were reported in CACX. Consequently, we strived for the first time to understand the molecular status of the candidate gene Insulin-like growth factor 2, IGF2 (11p15.5) in Indian CACX patients (n = 128). DNA copy number (CN) analysis using CGH-SNP analysis showed no genetic alteration and it was further validated by comparison with publicly available CN datasets. But promoter hypo-methylation during the progression of CACX was observed and also found to be concordant with publicly available DNA methylation datasets. Interestingly, we found diverse expression of IGF2 transcript in both normal cervical epithelium (NCE) and CACX tumors. Similar heterogeneous expression pattern was seen in publicly available expression datasets as well. Finally, protein expression analysis in NCE showed concordance with transcript expression but tumors showed frequent low expression. Log-rank test showed a difference (p-value = 0.057) in overall survival between cases with and without alteration for IGF2 in Indian CACX patients. Collectively, our study proposes that regulation of IGF2 expression in NCE appeared to be multifaceted and deregulation during the development of CACX resulted in the differential expression.

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

Activation of Wnt-ß-catenin pathway in basal-parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma.

In this study, importance of Wnt-ß-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of ß-catenin, p-ß-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74-95%) expression of ß-catenin/p-ß-catenin (Y654) and Wnt3a and low expression (23-26%) of APC in proliferating basal-parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal-parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal-parabasal layers and the cervical lesions (42-69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/ß-catenin/p-ß-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of ß-catenin during the development of CACX.

Phylogenetic analysis of Human papillomavirus 16 variants isolated from Indian Breast cancer patients showed difference in genetic diversity with that of cervical cancer isolates.

The genetic variations of HPV16 in Breast Cancer (BC) are not well studied unlike HPV16 in Cervical Cancer (CACX). In this study, the genetic variations of HPV16 in BC were compared with HPV16 in CACX. In sequencing analysis of LCR, E6 and E7 regions of HPV16 in BC and CACX the A lineage was seen to be 64.2% and 66.6% respectively. The other lineages showed differential frequency in BC and CACX. The mutation frequency index of the regions in BC and CACX was in the following order: LCR>E6>E7. However, the inter-patient genetic diversity in LCR and E6/E7 regions was high in BC than CACX. The LCR region showed more variations than the E6/E7 region in BC. Apart from some common variations, some unique tissue specific variants in LCR and E6/E7 region were seen in BC and in CACX. Besides the selection of some common variants in both BC and CACX, some unique variants in BC (D98Y; 395 G>T) and CACX (R48W; 245 G>T) were observed. The 7521 G>A variant of LCR showed association with Luminal B subtype of BC and progression of CACX. Whereas, 145 G>T (Q14H) and 335 C>T (H78Y) variants of E6 showed association with either early invasiveness of BC and/or poor outcome of the patients. Thus, this study indicates that there may be a difference in the genetic variation of HPV16 in BC and in CACX.

Pilot study indicate role of preferentially transmitted monoamine oxidase gene variants in behavioral problems of male ADHD probands.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is an etiologically complex childhood onset neurobehavioral disorder characterized by age-inappropriate inattention, hyperactivity, and impulsivity. Symptom severity varies widely and boys are diagnosed more frequently than girls. ADHD probands were reported to have abnormal transmissions of dopamine, serotonin, and/or noradrenaline. Monoamine oxidase A (MAOA) and B (MAOB), mitochondrial outer membrane bound two isoenzymes, mediate degradation of these neurotransmitters and thus regulating their circulating levels. Case-control analyses in different populations, including Indians, suggested involvement of MAOA and MAOB genes in the etiology of ADHD. Due to high heritability rate of ADHD, we tested familial transmission of MAOA and MAOB variants to ADHD probands in 190 nuclear families having ADHD probands from Indo-Caucasoid ethnicity. METHODS: Subjects were recruited following the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV). Appropriate scales were used for measuring the behavioral traits in probands. Genotyping was performed through PCR-based amplification of target sites followed by DNA-sequencing and/or gel-electrophoresis. Data obtained were analyzed by family based statistical methods. RESULTS: Out of 58 variants present in the analyzed sites only 15 were found to be polymorphic (30 bp-uVNTR, rs5906883, rs1465107, rs1465108, rs5905809, rs5906957, rs6323, rs1137070 from MAOA and rs4824562, rs56220155, rs2283728, rs2283727, rs3027441, rs6324, rs3027440 from MAOB). Statistically significant maternal transmission of alleles to male probands was observed for MAOA rs5905809 'G' (p = 0.04), rs5906957 'A' (p = 0.04), rs6323 'G' (p = 0.0001) and MAOB rs56220155 'A' (p = 0.002), rs2283728 'C' (p = 0.0008), rs2283727 'C' (p = 0.0008), rs3027441 'T' (p = 0.003), rs6324 'C' (p = 0.003), rs3027440 'T' (p = 0.0002). Significantly preferential maternal transmissions of different haplotype combinations to male probands were also noticed (p < 0.05), while female probands did not reveal such transmission bias. Behavioral traits of male probands exhibited significant association with gene variants. Age of the mother at pregnancy also revealed association with risk variants of male probands. CONCLUSIONS: It may be inferred that the MAOA and MAOB variants may contribute to the etiology of ADHD in the Indo-Caucasoid population and could be responsible for higher occurrence of ADHD in the boys.

p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

Study of association and molecular analysis of human papillomavirus in breast cancer of Indian patients: Clinical and prognostic implication.

OBJECTIVES: Human papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile. METHODS: HPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done. RESULTS: High prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20-45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants. CONCLUSION: Thus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.