Gallbladder cancer-associated genetic variants rs1003349 and rs1004030 regulate MMP14 expression by altering SOX10- and MYB-binding sites.

The pathogenesis of gallbladder cancer is complex, involving environmental and genetic risk factors. The matrix metallopeptidase 14 (MMP14) alters the tumor microenvironment and promotes tumorigenesis. In this study, we have characterized the role of the MMP14 promoter variants rs1004030 and rs1003049 in gallbladder cancer pathogenesis. Previously, we have shown the association of rs1004030 and rs1003049 with GBC and allele-specific differential expression of MMP14 in GBC patients. These variants reside within the cis-regulatory element (CRE) with high DNase and H3K4me3 signals, suggesting an active regulatory role in MMP14 expression. The luciferase-based reporter assay showed the role of promoter variants on expression levels in two GBC cell lines. Deleting the 119 bp promoter region surrounding the variants rs1004030 and rs1003049 by CRISPR-Cas9 genome editing resulted in reduced MMP14 expression in G415 cells. Electrophoretic mobility shift assay shows the presence of risk allele 'C'/'G' at rs1004030 and rs1003049 and create binding sites for transcription factors SOX10 and MYB, respectively. Further, stable knockdown of these transcription factors in G415 and TGBC1TKB cells showed reduced expression of MMP14. However, in both GBC cells, ectopic expression of these transcription factors increased the expression of MMP14. Rescue of MYB and SOX10 expression levels showed a significant increase in luciferase activity only in risk allele-carrying constructs. In conclusion, our study unveils a mechanistic role of the MMP14 promoter variants rs1004030 and rs1003049 in gallbladder cancer.

EEF1A2 promotes HIF1A mediated breast cancer angiogenesis in normoxia and participates in a positive feedback loop with HIF1A in hypoxia.

BACKGROUND: The eukaryotic elongation factor, EEF1A2, has been identified as an oncogene in various solid tumors. Here, we have identified a novel function of EEF1A2 in angiogenesis. METHODS: Chick chorioallantoic membrane, tubulogenesis, aortic ring, Matrigel plug, and skin wound healing assays established EEF1A2's role in angiogenesis. RESULT: Higher EEF1A2 levels in breast cancer cells enhanced cell growth, movement, blood vessel function, and tubule formation in HUVECs, as confirmed by ex-ovo and in-vivo tests. The overexpression of EEF1A2 could be counteracted by Plitidepsin. Under normoxic conditions, EEF1A2 triggered HIF1A expression via ERK-Myc and mTOR signaling in TNBC and ER/PR positive cells. Hypoxia induced the expression of EEF1A2, leading to a positive feedback loop between EEF1A2 and HIF1A. Luciferase assay and EMSA confirmed HIF1A binding on the EEF1A2 promoter, which induced its transcription. RT-PCR and polysome profiling validated that EEF1A2 affected VEGF transcription and translation positively. This led to increased VEGF release from breast cancer cells, activating ERK and PI3K-AKT signaling in endothelial cells. Breast cancer tissues with elevated EEF1A2 showed higher microvessel density. CONCLUSION: EEF1A2 exhibits angiogenic potential in both normoxic and hypoxic conditions, underscoring its dual role in promoting EMT and angiogenesis, rendering it a promising target for cancer therapy.

Oncogenic activation of EEF1A2 expression: a journey from a putative to an established oncogene.

Protein synthesis via translation is a central process involving several essential proteins called translation factors. Although traditionally described as cellular "housekeepers," multiple studies have now supported that protein initiation and elongation factors regulate cell growth, apoptosis, and tumorigenesis. One such translation factor is eukaryotic elongation factor 1 alpha 2 (EEF1A2), a member of the eukaryotic elongation factor family, which has a canonical role in the delivery of aminoacyl-tRNA to the A-site of the ribosome in a guanosine 5'-triphosphate (GTP)-dependent manner. EEF1A2 differs from its closely related isoform, EEF1A1, in tissue distribution. While EEF1A1 is present ubiquitously, EEF1A2 replaces it in specialized tissues. The reason why certain specialized tissues need to essentially switch EEF1A1 expression altogether with EEF1A2 remains to be answered. Abnormal "switch on" of the EEF1A2 gene in normal tissues is witnessed and is seen as a cause of oncogenic transformation in a wide variety of solid tumors. This review presents the journey of finding increased expression of EEF1A2 in multiple cancers, establishing molecular mechanism, and exploring it as a target for cancer therapy. More precisely, we have compiled studies in seven types of cancers that have reported EEF1A2 overexpression. We have discussed the effect of aberrant EEF1A2 expression on the oncogenic properties of cells, signaling pathways, and interacting partners of EEF1A2. More importantly, in the last part, we have discussed the unique potential of EEF1A2 as a therapeutic target. This review article gives an up-to-date account of EEF1A2 as an oncogene and can draw the attention of the scientific community, attracting more research.

FRG1 is a direct transcriptional regulator of nonsense-mediated mRNA decay genes.

FRG1 is the primary candidate gene for Fascioscapulohumeral Muscular Dystrophy. So far, its role has been reported in muscle development, vasculogenesis, angiogenesis, and tumorigenesis. Mechanistically studies suggest FRG1's role in RNA biogenesis which may have implications in multiple physiological processes and diseases, including tumorigenesis. Its probable role as hnRNP and association with NMD-related genes prompted us to look into FRG1's effect on NMD gene expression and the mechanism. Using microarray profiling in cell lines, we found that FRG1 altered the mRNA surveillance pathway and associated pathways, such as RNA transport and spliceosome machinery molecules. Multiple sequence alignment of core factors, namely, UPF1, UPF3B, and SMG1, showed conserved stretches of nucleotide sequence 'CTGGG'. Structural modeling followed by EMSA, ChIP-qPCR, and luciferase reporter assays showed 'CTGGG' as a FRG1 binding site. Analysis of the publicly available datasets showed that the expression of FRG1 correlates with NMD genes in different tissue types. We validated the effect of FRG1 on NMD gene transcription by qRT-PCR. Overall, FRG1 might be a transcriptional regulator of NMD genes.

Genetic association of MMP14 promoter variants and their functional significance in gallbladder cancer pathogenesis.

Gallbladder cancer (GBC) is relatively rare but shows high frequency in certain geographical regions and ethnic groups, which include Northern and Eastern states of India. Previous studies in India have indicated the possible role of genetic predisposition in GBC pathogenesis. Although matrix metalloproteinase-14 (MMP14) is known modulator of tumour microenvironment and tumorigenesis and TCGA data also suggests its upregulation yet, its role in genetic predisposition for GBC is completely unknown. We explored MMP14 promoter genetic variants as risk factors and their implication in expression modulation and the pathogenesis of GBC. We genotyped all single nucleotide polymorphisms of MMP14 promoter by Sanger's sequencing in approximately 300 GBC and 300 control study subjects of Indian ethnicity and, in 26 GBC tissue samples. Protein expression of MMP14 in GBC tissue samples was checked by immunohistochemistry. In vitro luciferase reporter assay was carried out to elucidate role of promoter genetic variants on expression levels in two different cell lines. MMP14 promoter variants, rs1003349 (p value = 0.0008) and rs1004030 (p value = 0.0001) were significantly associated with GBC. Luciferase reporter assay showed high expression for risk alleles of both the SNPs. Genotype-phenotype correlation for rs1003349 and rs1004030, in patient sample, confirmed that risk allele carriers had higher expression levels of MMP14; moreover, the correlation pattern matched with genetic association models. Overall, this study unravels the association of MMP14 promoter SNPs with GBC which contribute to pathogenesis by increasing its expression.

The 2'-caged-tethered-siRNA shows light-dependent temporal controlled RNAi activity for GFP gene into HEK293T cells.

Small interfering RNA (siRNA) exhibits gene-specific RNAi activity by the formation of RISC complex with mRNA of gene. The structural modification of siRNA at appropriate positions affects the structure of RISC complex and then RNAi activity. The modified siRNA are mostly prepared from the incorporation of sugar ring modified, and nucleobase modified RNA nucleotides. It is learned that the introduction of the sterically hindered nucleoside at the specific position of siRNA, severely affects siRNA-RISC complex formation. This report describes the syntheses of bulkier siRNA from 2'-caged-tethered-siRNAs, containing bulkier photolabile protecting group (o-nitrobenzyl) at 2'-position of ribose nucleoside. Importantly, these 2'-caged-siRNAs exhibit the light-dependent RNA interference (RNAi) activity into HEK293T cells for the GFP gene expression. The 2'-caged-siRNAs are synthesized by caging the sense and antisense strand of siRNA. The biochemical evaluations of these caged-siRNAs show that antisense-strand caged-siRNAs decrease RNAi activity temporarily in dark while enhancing RNAi activity, almost like control, after exposure withUV- light. However, 2'-caged sense strand siRNA increase RNAi activity temporarily while decreasing RNAi activity after exposure with light. These caged-siRNAs are also stable in the serum (fetal bovine serum) as like native siRNA. Hence these results strongly support that 2'-caged-tethered-siRNAs are promising analogues to control RNAi activity by UV-light.

Reduced FRG1 expression promotes prostate cancer progression and affects prostate cancer cell migration and invasion.

BACKGROUND: Prostate cancer is the most common form of cancer in males and accounts for high cancer related deaths. Therapeutic advancement in prostate cancer has not been able to reduce the mortality burden of prostate cancer, which warrants further research. FRG1 which affects angiogenesis and cell migration in Xenopus, can be a potential player in tumorigenesis. In this study, we investigated the role of FRG1 in prostate cancer progression. METHODS: Immunohistochemistry was performed to determine FRG1 expression in patient samples. FRG1 expression perturbation was done to investigate the effect of FRG1 on cell proliferation, migration and invasion, in DU145, PC3 and LNCaP cells. To understand the mechanism, we checked expression of various cytokines and MMPs by q-RT PCR, signaling molecules by western blot, in FRG1 perturbation sets. Results were validated by use of pharmacological inhibitor and activator and, western blot. RESULTS: In prostate cancer tissue, FRG1 levels were significantly reduced, compared to the uninvolved counterpart. FRG1 expression showed variable effect on PC3 and DU145 cell proliferation. FRG1 levels consistently affected cell migration and invasion, in both DU145 and PC3 cells. Ectopic expression of FRG1 led to significant reduction in cell migration and invasion in both DU145 and PC3 cells, reverse trends were observed with FRG1 knockdown. In androgen receptor positive cell line LNCaP, FRG1 doesn't affect any of the cell properties. FRG1 knockdown led to significantly enhanced expression of GM-CSF, MMP1, PDGFA and CXCL1, in PC3 cells and, in DU145, it led to higher expression of GM-CSF, MMP1 and PLGF. Interestingly, FRG1 knockdown in both the cell lines led to activation of p38 MAPK. Pharmacological activation of p38 MAPK led to increase in the expression of GM-CSF and PLGF in DU145 whereas in PC3 it led to enhanced expression of GM-CSF, MMP1 and CXCL1. On the other hand, inhibition of p38 MAPK led to reduction in the expression of above mentioned cytokines. CONCLUSION: FRG1 expression is reduced in prostate adenocarcinoma tissue. FRG1 expression affects migration and invasion in AR negative prostate cancer cells through known MMPs and cytokines, which may be mediated primarily via p38 MAPK activation.

Dideoxy nucleoside triphosphate (ddNTP) analogues: Synthesis and polymerase substrate activities of pyrrolidinyl nucleoside triphosphates (prNTPs).

The dideoxynucleoside triphosphates (ddNTPs) terminate the bio-polymerization of DNA and become essential chemical component of DNA sequencing technology which is now basic tool for molecular biology research. In this method the radiolabeled or fluorescent dye labeled ddNTP analogues are being used for DNA sequencing by detection of the terminated DNA fragment after single labeled ddNTP incorporation into DNA under PCR conditions. This report describes the syntheses of rationally designed novel amino-functionalized ddNTP analogue such as Pyrrolidine nucleoside triphosphates (prNTPs), and their polymerase activities with DNA polymerase by LC-MS and Gel-electrophoretic techniques. The Mass and PAGE analyses strongly support the incorporation of prNTPs into DNA oligonucleotide with Therminator DNA polymerase as like control substrate ddNTP. As resultant the DNA oligonucleotide are functionalized as amine group by prNTP incorporation with polymerase. Hence prNTPs provide opportunities to prepare demandable conjugated DNA with other biomolecules/dyes/fluorescence molecule without modifying nucleobase structure.

Distinctive mutation spectrum of the HBB gene in an urban eastern Indian population.

ABSTRACT Hemoglobinopathies such as ß-thalassemia (ß-thal) and sickle cell anemia (or Hb S [ß6(A3)Glu→Val]) impose a major health burden in the Indian population. To determine the frequencies of the HBB gene mutations in eastern Indian populations and to compare with the available data, a comprehensive molecular analysis of the HBB gene was done in the normal Odisha State population. Using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), amplification refractory mutation system (ARMS) and DNA sequencing techniques, ß-thal and sickle cell anemia mutations were characterized in 267 healthy individuals. Entire HBB gene sequencing showed 63 different mutations including 11 new ones. The predominant mutation HBB: c.9T > C was observed at a high frequency (19.57%) in the normal population. In the urban population of Odisha State, India, carrier frequency of hemoglobinopathies was found to be 18.48%, and for ß-thal, the carrier rate was 14.13%, which is very high indeed. In the absence of a complete cure by any expensive treatment and drug administration, this information would be helpful for planning a population screening program and establishing prenatal diagnosis of ß-thal in order to reduce the burden of such a genetic disease.

Functional relevance of MMP2 promoter variants in gallbladder cancer: A case-control study in an Eastern Indian Population.

Gallbladder cancer (GBC) is a prevalent and deadly form of bile duct cancer, associated with poor prognosis. This study aimed to investigate the genetic factors contributing to the high incidence of GBC in certain geographical regions, particularly in the Northern and Eastern parts of India. The present case-control study focused on MMP2, a gene involved in tumor progression and metastasis, as a potential candidate in GBC pathogenesis. We scanned MMP2 promoter for twelve SNPs using Sanger's sequencing and carried out a case-control study in 300 cases and 300 control samples. We found five rare variants (rs1961998763, rs1961996235, rs1391392808, rs1488656253, and rs17859816) and one nonpolymorphic SNP (rs17859817). Our results revealed a significant association between GBC and MMP2 promoter SNPs, rs243865 (Allelic-Padjusted = 0.0353) and g.55477735G > A (Allelic-Padjusted = 9.22E-05). Moreover, the haplotype "C-C-A-C-C" exhibited a significant association with GBC (P = 4.23E-05). Genotype-phenotype correlation for variant rs243865, in the GBC patient tissue samples, established that 'T' risk allele carriers had higher expression levels of MMP2. Additionally, luciferase reporter assay in HEK293T cells revealed the probable regulatory role of rs243865 variant allele 'T' in MMP2 expression. Our study uncovers the association of MMP2 promoter SNPs with GBC and their role in regulating its expression.

Reduced FRG1 expression promotes angiogenesis via activation of the FGF2-mediated ERK/AKT pathway.

Identifying novel targets that control both tumorigenesis and angiogenesis can aid in developing a more potent anti-angiogenic therapeutic strategy. We previously reported that reduction of FRG1 is associated with increased p38-MAPK signaling in prostate cancer and with elevated MEK-ERK signaling in breast cancer. Here, we reveal the role of FRG1 in tumor angiogenesis. Our findings demonstrate that depleted FRG1 levels enhance the proliferation, migration, and tubule formation of HUVECs in a paracrine manner, and this was further substantiated in multiple animal models. Mechanistically, FRG1 depletion activated the expression of FGF2 in breast cancer cells, which triggered the ERK/AKT cascade in endothelial cells. As FRG1 affects multiple tumorigenic properties and it is upstream of FGF2, it can be explored as a therapeutic target that is less prone to resistance.

N-Salicyl-AAn-picolamide Foldameric Peptides Exhibit Quorum Sensing Inhibition of Pseudomonas aeruginosa (PA14).

An organic acid, salicylic acid, and its derivatives are constituents of various natural products possessing remarkable bioactivity. O-Acetyl salicylate (aspirin) is a well-known life-saving drug. Its peptide derivative salicylamide has also been explored in the designing of peptide-based therapeutic drugs. An organic base, picolylamine has been recently explored for designing diagnostic probes. However, both the acid and base have common features as metal chelating with coordinating metals. Thus, these scaffolds could be used for designing inhibitors of various metalloenzymes. Their characteristic properties encourage us to design peptides containing both scaffolds (salicylic acid and picolylamine) at opposite terminals. So far there is no report available on such conjugated peptides. This report describes the synthesis, conformational analysis, and biochemical assessment of rationally designed N-salicyl-AAn-picolamide peptides. Pleasantly, we have obtained the crystal structures of representative peptides that confirm their roles in conformational changes. Our biological assessment as quorum sensing inhibitors has revealed that their di/tripeptides inhibit quorum sensing of the pathogenic bacterium PA14 strain. Hence, these peptides have promising foldameric and therapeutic values.

IQ Motif Containing GTPase Activating Proteins (IQGAPs), A-Kinase Anchoring Proteins (AKAPs) and Kinase Suppressor of Ras Proteins (KSRs) in Scaffolding Oncogenic Pathways and Their Therapeutic Potential.

Scaffolding proteins colocalize interacting partners on their surface and facilitate complex formation. They have multiple domains and motifs, which provide binding sites for various molecules. This property of scaffolding proteins helps in the orderly transduction of signals. Abnormal signal transduction is frequently observed in cancers, which can also be attributed to the altered functionality of scaffolding proteins. IQ motif containing GTPase activating proteins (IQGAPs), kinase suppressor of Ras (KSR), and A-kinase anchoring proteins (AKAPs) tether oncogenic pathways RAS/RAF/MEK/ERK, PI3K/AKT, Hippo, Wnt, and CDC42/RAC to them. Scaffolding proteins are attractive drug targets as they are the controlling hub for multiple pathways and regulate crosstalk between them. The first part of this review describes the human scaffolding proteins known to play a role in oncogenesis, pathways altered by them, and the impact on oncogenic processes. The second part provides information on the therapeutic potential of scaffolding proteins and future possibilities. The information on the explored and unexplored areas of the therapeutic potential of scaffolding proteins will be equally helpful for biologists and chemists.

Cu-Catalyzed Synthesis of Alkylaminotroponyl Sulfones as Pseudomonas Aeruginosa Quorum Sensing Inhibitors Targeting lasI/R QS Circuitry.

The scarcity of novel bioactive molecules against multidrug-resistant (MDR) bacterial strains like Pseudomonas aeruginosa is alarming. This bacterial virulence is regulated via Quorum sensing (QS), a cell-cell communication process. Disabling QS circuits (las, pqs, rhl) with small molecules has been proposed as a potential strategy to prevent bacterial pathogenicity. This strategy focuses on interruption of bacterial virulence, rather than killing them to tackle the drug resistance problem. Herein, we describe the synthesis of rationally designed Alklyamionotroponyl Sulfone (ATS) derivatives by Cu-catalyzed C(sp2 )-H functionalization at tropone ring and the screening of their anti-QS activity against P. aeruginosa. Importantly, two sulfones (∼20 µM) remarkably exhibit the down regulation of the lasI/R QS genes. These molecules also inhibit swarming motility, biofilm formation and pyocyanin production, which reduce P. aeruginosa virulence in cells. Hence, ATS derivatives could be considered as potential therapeutic candidates for the treatment of P. aeruginosa infections.

One-pot synthesis of cyclic-aminotropiminium carboxylate derivatives with DNA binding and anticancer properties.

Tropolone, a nonbenzenoid aromatic molecule, is a constituent of troponoid natural products possessing a wide range of bioactivities, including anticancer. This report describes the one-pot synthesis and mechanistic studies of fifteen fluorescent Caryl-Nalkyl-substituted cyclic-aminotroponiminium carboxylate (cATC) derivatives by unusual cycloaddition and rearrangement reactions. Herein, the biochemical studies of four cATC derivatives reveal a non-intercalative binding affinity with DNA duplex. In vitro/in vivo studies show strong anti-tumor activity in three cATC derivatives. These derivatives enter the cells and localize to the nucleus and cytoplasm, which are easily traceable due to their inherent fluorescence properties. These three cATC derivatives reduce the proliferation and migration of HeLa cells more than the non-cancer cell line. They induce p38-p53-mediated apoptosis and inhibit EMT. In xenograft-based mouse models, these cATC derivatives reduce tumor size. Overall, this study reports the synthesis of DNA binding fluorescent Caryl-Nalkyl-cyclic-aminotroponiminium derivatives which show anti-tumor activity with the minimum side effect.

Nonbenzenoid BODIPY Analogues: Synthesis, Structural Organization, Photophysical Studies, and Cell Internalization of Biocompatible N-Alkyl-Aminotroponyl Difluoroboron (Alkyl-ATB) Complexes.

The alkyl-BODIPY derivatives are lipid types of fluorescent molecules that exhibit a unique structure and functions including sensing of hydrophobic microenvironments in living cells. Their synthesis involves multisteps from the core structure dipyrromethene scaffold. The alkyl-BODIPY analogues are sought to derivatize with minimal synthetic steps even by altering the core structures derived from benzenoid aromatic moiety. Recently, the nonbenzenoid scaffold (aminotropone) has been explored to synthesize troponyl-BODIPY analogues, which are fluorescent. In the repertoire of nonbenzenoid analogue, N-alkyl-aminotroponyl difluoroboron (alkyl-ATB) is rationally designed comprising long-chain hydrocarbons to explore the lipid type of fluorescent molecules. This report describes the synthesis, photophysical studies, structural organization, and biocompatibilities of ATB derivatives containing different lengths of alkyl chain at 2-aminotropone scaffold. The photophysical studies of ATB derivatives reveal their fluorescence behaviors in organic solvents (CH3OH/CH3CN) with a quantum yield of ∼10 to 15%. These ATB derivatives also exhibit fluorescence characters in the solid state though their quantum yield is relatively low. Cell permeability and cytotoxicity studies reveal that alkyl-ATB derivatives are permeable to HeLa/HEK293T cell lines and show negligible cytotoxicity. The biocompatibility of alkyl-ATB derivatives is studied and confirmed by cell viability (MTT) assay to the HeLa/HEK293T cell lines. Importantly, the cell internalization studies of the representative alkyl-ATB molecule by fluorescence microscopy show that octyl-ATB is efficiently detectable at the cytoplasmic membrane and cellular nucleus in HeLa cells. Hence, alkyl-ATB derivatives are potential fluorescent molecules for developing probes to visualize cellular components under a fluorescence microscope.

IQ Motif-Containing GTPase-Activating Protein 2 Inhibits Breast Cancer Angiogenesis by Suppressing VEGFR2-AKT Signaling.

Antiangiogenesis cancer therapies are facing setbacks due to side effects and resistance. Parallel targeting of multiple pathways can help in the development of more effective therapies. This requires the discovery of new molecules that can regulate multiple cellular processes. Our study has recently established the association of reduced IQGAP2 expression in breast cancer with EMT and poor prognosis of the patient. Existing literature indirectly suggests the role of IQGAP2 in angiogenesis that is still unexplored. In this study, we searched the role of IQGAP2 in tumor angiogenesis in a comprehensive manner using cell culture, patients, and animal models. Depletion of IQGAP2 in breast cancer cells increased proliferation, migration, and tubulogenesis of HUVECs. Findings were validated in ex ovo CAM, Matrigel plug and skin wound-healing assays in mouse model, showing that the reduction of IQGAP2 significantly increased angiogenesis. As a confirmation, IHC analysis of the patient's tissues showed a negative correlation of IQGAP2 expression with the microvessel density. Mechanistically, loss of IQGAP2 appeared to activate VEGF-A via ERK activation in tumor cells, which activated the VEGFR2-AKT axis in HUVECs. IMPLICATIONS: The findings of this study suggest the antiangiogenic properties of IQGAP2 in breast cancer. The Dual effect of IQGAP2 on EMT and angiogenesis makes it a potential target for anticancer therapy.

Reduced expression of FRG1 facilitates breast cancer progression via GM-CSF/MEK-ERK axis by abating FRG1 mediated transcriptional repression of GM-CSF.

Multiple molecular subtypes and distinct clinical outcomes in breast cancer, necessitate specific therapy. Moreover, despite the improvements in breast cancer therapy, it remains the fifth cause of cancer-related deaths, indicating the involvement of unknown genes. To identify novel contributors and molecular subtype independent therapeutic options, we report reduced expression of FRG1 in breast cancer patients, which regulates GM-CSF expression via direct binding to its promoter. Reduction in FRG1 expression enhanced EMT and increased cell proliferation, migration, and invasion, in breast cancer cell lines. Loss of FRG1 increased GM-CSF levels which activated MEK/ERK axis and prevented apoptosis by inhibiting p53 in an ERK-dependent manner. FRG1 depletion in the mouse model increased tumor volume, phospho-ERK, and EMT marker levels. The therapeutic potential of anti-GM-CSF therapy was evident by reduced tumor size, when tumors with decreased FRG1 were treated with anti-GM-CSF mAb. We found an inverse expression pattern of FRG1 and phospho-ERK levels in breast cancer patient tissues, corroborating the in vitro and mouse model-based findings. Our findings first time elucidate the role of FRG1 as a metastatic suppressor of breast cancer by regulating the GM-CSF/MEK-ERK axis.

Association and functional significance of genetic variants present in regulatory elements of SERPINB5 gene in gallbladder cancer.

SERPINB5 is a mammary serine protease inhibitor, which is involved in various cellular functions. The aberrant expression of SERPINB5 is reported in many cancers along with GBC but limited information is available about its role in genetic predisposition for GBC. We carried out case-control study in 206 cases and 219 controls. Promoter SNPs were genotyped by Sanger's sequencing. In-silico promoter analysis and luciferase reporter assay were done to elucidate the role of promoter variants in regulation of SERPINB5 expression. Out of four SNPs, three SERPINB5 promoter variants showed association with GBC in different models. The 'C' allele of variant rs17071138 was found to be significantly associated with GBC (p = 0.017). The 'T' allele of rs3744940 significantly increased the risk for GBC in dominant (p = 0.035) and additive models (p = 0.005). Also, rs3744941 'T' allele increased the risk for GBC by dominant (p = 0.042) as well as additive models (p = 0.016). In-silico promoter analysis and luciferase reporter assay revealed the probable regulatory role of the SERPINB5 promoter variant rs17071138 on the expression. Overall, our study reveals the genetic association of SERPINB5 promoter variants with GBC and possible role of rs17071138 in the regulation of expression.

Role of FRG1 in predicting the overall survivability in cancers using multivariate based optimal model.

FRG1 has a role in tumorigenesis and angiogenesis. Our preliminary analysis showed that FRG1 mRNA expression is associated with overall survival (OS) in certain cancers, but the effect varies. In cervix and gastric cancers, we found a clear difference in the OS between the low and high FRG1 mRNA expression groups, but the difference was not prominent in breast, lung, and liver cancers. We hypothesized that FRG1 expression level could affect the functionality of the correlated genes or vice versa, which might mask the effect of a single gene on the OS analysis in cancer patients. We used the multivariate Cox regression, risk score, and Kaplan Meier analyses to determine OS in a multigene model. STRING, Cytoscape, HIPPIE, Gene Ontology, and DAVID (KEGG) were used to deduce FRG1 associated pathways. In breast, lung, and liver cancers, we found a distinct difference in the OS between the low and high FRG1 mRNA expression groups in the multigene model, suggesting an independent role of FRG1 in survival. Risk scores were calculated based upon regression coefficients in the multigene model. Low and high-risk score groups showed a significant difference in the FRG1 mRNA expression level and OS. HPF1, RPL34, and EXOSC9 were the most common genes present in FRG1 associated pathways across the cancer types. Validation of the effect of FRG1 mRNA expression level on these genes by qRT-PCR supports that FRG1 might be an upstream regulator of their expression. These genes may have multiple regulators, which also affect their expression, leading to the masking effect in the survival analysis. In conclusion, our study highlights the role of FRG1 in the survivability of cancer patients in tissue-specific manner and the use of multigene models in prognosis.