Genotypic and haplotype analysis of Interleukin-6 and -18 gene polymorphisms in association with clinicopathological factors in breast cancer.

Cytokines are multifunctional glycoproteins that play a vital role in the tumor microenvironment and progression of breast cancer. Genetic polymorphisms may influence the immune responses restrained by pro- and anti-inflammatory cytokine expression in tumors. Hence, the present study evaluated the contribution of Interleukin (IL) 6 (rs1800797, rs1800796, and rs1800795) and IL18 (rs1946518, rs187238, and rs549908) genotypes and their haplotypes to the risk, progression of breast cancer in South Indian population. The polymorphisms of IL6 -597G > A, -572C > G & -174G > C, and IL18 -607C > A, -137G > C, 105A > T were genotyped through PCR-RFLP and As-PCR assays in the blood DNA of 600 subjects. We have performed haplotype, LD, univariate, multivariate logistic regression, and Kaplan-Meier analyses for the obtained data. The frequency of AA genotype & A-allele of IL6 -597G > A, and CC genotype & C-allele of IL6 -174G > C polymorphism was higher in breast cancer patients and was found to be significantly associated with late (advanced) stage, metastasis, etc. Further, IL18 -607C > A, -137G > C, and 105A > T polymorphisms were found to be associated with lobular carcinoma subtype, PgR -ve, and HER2 +ve breast cancer patients. In survival analysis, we have observed that the C-allele of IL6 -174G > C polymorphism to be significantly associated with 5 years of overall survival in breast cancer subjects. All SNPs of the IL6 and IL18 genes showed perfect LD; the G-C-C, A-G-G, and A-C-C haplotype combinations of IL6 gene conferred 2.09, 2.25, and 4.72 folds risk for breast cancer respectively. Hence, our results suggest the importance of genotypic and haplotype analysis of IL6 and IL18 gene variants in the progression and risk prediction of breast cancer.

In silico identification of single nucleotide variations at CpG sites regulating CpG island existence and size.

Genetic and epigenetic modifications of genes involved in the key regulatory pathways play a significant role in the pathophysiology and progression of multifactorial diseases. The present study is an attempt to identify single nucleotide variations (SNVs) at CpG sites of promoters of ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1 and TIMP1 genes influencing CpG island (CGI) existence and size associated with the pathophysiology of Diabetes mellitus, Coronary artery disease and Cancers. Promoter sequences located between -2000 to + 2000 bp were retrieved from the EPDnew database and predicted the CpG island using MethPrimer. Further, SNVs at CpG sites were accessed from NCBI, Ensembl while transcription factor (TF) binding sites were accessed using AliBaba2.1. CGI existence and size were determined for each SNV at CpG site with respect to wild type and variant allele by MethPrimer. A total of 200 SNVs at CpG sites were analyzed from the promoters of ACAT1, APOB, APOE, CYBA, FAS, FLT1, KSR2, LDLR, MMP9, PCSK9, PHOX2A, REST, SH2B3, SORT1 and TIMP1 genes. Of these, only 17 (8.5%) SNVs were found to influence the loss of CGI while 70 (35%) SNVs were found to reduce the size of CGI. It has also been found that 59% (10) of CGI abolishing SNVs are showing differences in binding of TFs. The findings of the study suggest that the candidate SNVs at CpG sites regulating CGI existence and size might influence the DNA methylation status and expression of genes involved in molecular pathways associated with several diseases. The insights of the present study may pave the way for new experimental studies to undertake challenges in DNA methylation, gene expression and protein assays.

Hematopoietic PBX-interacting protein is a novel regulator of mammary epithelial cell differentiation.

Hematopoietic PBX-interacting protein (HPIP, also known as PBXIP1) is an estrogen receptor (ER) interacting protein that regulates estrogen-mediated breast cancer cell proliferation and tumorigenesis. However, its functional significance in the context of mammary gland development is unexplored. Here, we report that HPIP is required for prolactin (PRL)-induced lactogenic differentiation in vitro. Molecular analysis of HPIP expression in mice revealed its induced expression at pregnancy and lactation stages of mammary gland. Moreover, PRL is a lactogenic hormone that controls pregnancy as well as lactation and induces Hpip/Pbxip1 expression in a signal transducer and activator of transcription 5a-dependent manner. Using mammary epithelial and lactogenic-competent cell lines, we further show that HPIP plays a regulatory role in PRL-mediated mammary epithelial cell differentiation, which is measured by acini formation, ß-casein synthesis, and lipid droplet formation. Further mechanistic studies using pharmacological inhibitors revealed that HPIP modulates PRL-induced ß-casein synthesis via phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activation. This study also identified HPIP as a critical regulator of autocrine PRL signaling as treatment with the PRL receptor antagonist Δ1-9-G129R-hPRL restrained HPIP-mediated PRL synthesis, AKT activation, and ß-casein synthesis in cultured HC11 cells. Interestingly, we also uncovered that microRNA-148a (miR-148a) antagonizes HPIP-mediated mammary epithelial cell differentiation. Together, our study identified HPIP as a critical regulator of PRL signaling and revealed a novel molecular circuitry involving PRL, HPIP, PI3K/AKT, and miR-148a that controls mammary epithelial cell differentiation in vitro.

A reciprocal feedback loop between HIF-1α and HPIP controls phenotypic plasticity in breast cancer cells.

While phenotypic plasticity is a critical factor contributing to tumor heterogeneity, molecular mechanisms underlying this process are largely unknown. Here we report that breast cancer cells display phenotypic diversity in response to hypoxia or normoxia microenvironments by operating a reciprocal positive feedback regulation of HPIP and HIF-1α. We show that under hypoxia, HIF-1α induces HPIP expression that establishes cell survival, and also promotes cell migration/invasion, EMT and metastatic phenotypes in breast cancer cells. Mechanistic studies revealed that HPIP interacts with SRP14, a component of signal recognition particle, and stimulates MMP9 synthesis under hypoxic stress. Whereas, in normoxia, HPIP stabilizes HIF-1α, causing the Warburg effect to support cell growth. Concurrently, mathematical modelling corroborates this reciprocal feedback loop in enabling cell-state transitions in cancer cells. Clinical data indicate that elevated levels of HPIP and HIF-1α correlate with unfavorable prognosis and shorter survival rates in breast cancer subjects. Together, this data shows a reciprocal positive feedback loop between HPIP and HIF-1α that was unknown hitherto. It unveils how the tumor microenvironment influences phenotypic plasticity that has an impact on tumor growth and metastasis and, further signifies considering this pathway as a potential therapeutic target in breast cancer.

HPIP protooncogene differentially regulates metabolic adaptation and cell fate in breast cancer cells under glucose stress via AMPK and RNF2 dependent pathways.

While cancer cells rewire metabolic pathways to sustain growth and survival under metabolic stress in solid tumors, the molecular mechanisms underlying these processes remain largely unknown. In this study, cancer cells switched from survival to death during the early to late phases of metabolic stress by employing a novel signaling switch from AMP activated protein kinase (AMPK)-Forkhead box O3 (FOXO3a)-hematopoietic PBX1-interacting protein (HPIP) to the ring finger protein 2 (RNF2)-HPIP-ubiquitin (Ub) pathway. Acute metabolic stress induced proto-oncogene HPIP expression in an AMPK-FOXO3a-dependent manner in breast cancer (BC) cells. HPIP depletion reduced cell survival and tumor formation in mouse xenografts, which was accompanied by diminished intracellular ATP levels and increased apoptosis in BC cells in response to metabolic (glucose) stress. Glutamine flux (13C-labeled) analysis further suggested that HPIP rewired glutamine metabolism by controlling the expression of the solute carrier family 1 member 5 (SLC1A5) and glutaminase (GLS) genes by acting as a coactivator of MYC to ensure cell survival upon glucose deprivation. However, in response to chronic glucose stress, HPIP was ubiquitinated by the E3-Ub ligase, RNF2, and was concomitantly degraded by the proteasome-mediated pathway, ensuring apoptosis. In support of these data, clinical analyses further indicated that elevated levels of HPIP correlated with AMPK activation in BC. Taken together, these data suggest that HPIP is a signal coordinator during metabolic stress and thus serves as a potential therapeutic target in BC.

PCSK9 genetic (rs11591147) and epigenetic (DNA methylation) modifications associated with PCSK9 expression and serum proteins in CAD patients.

INTRODUCTION: Proprotein convertase subtilisin/kexin type 9 (PCSK9) genetic polymorphisms play a significant role in cholesterol homeostasis. Therefore, we aimed to investigate the association of PCSK9 genetic variations NM_174936.3:c.137G>T (R46L, rs11591147) and NM_174936.3:c.1120G>T (D374Y, rs137852912), as well as promoter DNA methylation status, with mRNA expression and circulating serum protein levels in coronary artery disease (CAD) patients. METHODS: The present study includes 300 CAD cases and 300 controls from South India. Biochemical assays were performed using commercially available kits. PCSK9 rs11591147 and rs137852912 polymorphisms were analyzed by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method, whereas promoter DNA methylation status and gene expression were determined using methylation specific PCR and quantitative PCR respectively. RESULTS: The genotypic distribution of PCSK9 rs11591147 revealed that individuals with the TT-genotype and T-allele have a reduced risk for CAD. Furthermore, patients with the PCSK9 rs11591147 TT genotype have a significantly lower total cholesterol and low-density lipoprotein-cholesterol levels and also higher high-density lipoprotein-cholesterol levels than individuals with the GG genotype. Logistic regression analysis has shown that the GG and GT (p = 1.51 × 10-8 , p = 1.47 × 10-9 ) genotypes predicted the risk for CAD with an odds ratio of 5.8 and 7.3 respectively. In addition, individuals with the TT genotype were hypermethylated at promoter DNA of PCSK9, resulting in lower mRNA expression and circulating serum proteins than in individuals with the GG genotype. In silico analyses revealed that rs11591147 T-allele has protein destabilizing capacity. CONCLUSIONS: In conclusion, the present study indicates that the PCSK9 gene expression and circulating serum protein levels are not only associated with rs11591147 genotype, but also with promoter DNA methylation. Furthermore, the findings with respect to both single nucleotide polymorphism and promoter DNA methylation may open avenues for novel treatment possibilities targeting PCSK9 for CAD management.

Association of CYBA gene (-930 A/G and 242 C/T) polymorphisms with oxidative stress in breast cancer: a case-control study.

BACKGROUND: Oxidative stress (OS) is a key characteristic feature in cancer initiation and progression. Among multiple cancers, NADPH oxidase (NOX) dependent free radical production is implicated in oxidative stress. P22phox, a subunit of NADPH oxidase encoded by the CYBA gene has functional polymorphisms associated with various complex diseases. The present study was aimed to examine the importance and association of the functional polymorphisms of CYBA gene (-930 A/G and 242 C/T) with the oxidative stress in breast cancer (BC) development and progression. MATERIALS AND METHODS: We have performed a case-control study on 300 breast cancer patients and 300 healthy individuals as controls to examine the role of CYBA gene -930 A/G and 242 C/T single nucleotide polymorphisms (SNPs) using As-PCR and PCR-RFLP assays and its association with OS as measured by plasma MDA levels. Linkage disequilibrium (LD) plots were generated using Haploviewtool and Multifactor dimensionality reduction (MDR) analysis was applied to assess high-order interactions between the SNPs. The Insilco analysis has been performed to predict the effect of SNPs on the gene regulation using online tools. RESULTS: We have found that genotype frequencies of CYBA gene -930 A/G and 242C/T polymorphism were significantly different between controls and BC patients (p < 0.05). The haplotype combination -930G/242C and -930G/242T were associated with 1.44 & 1.56 folds increased risk for breast cancer respectively. Further, the MDA levels were higher in the patients carrying -930G/242C and -930G/242T haplotype (p < 0.001). Our results have been substantiated by Insilco analysis. CONCLUSION: Results of the present study suggest that GG genotype of -930 A/G polymorphism, -930G/242C and -930G/242T haplotypes of CYBA gene polymorphisms have shown association with higher MDA levels in breast cancer patients, signify that elevated oxidative stress might aid in increased risk for breast cancer initiation and progression.

Collagenase-1 (-1607 1G/2G), Gelatinase-A (-1306 C/T), Stromelysin-1 (-1171 5A/6A) functional promoter polymorphisms in risk prediction of type 2 diabetic nephropathy.

Type 2 Diabetic Nephropathy (DN) is a common multifactorial disorder. Degradation of glomerular basement membrane (GBM) by matrix metalloproteases (MMPs) is a key event in the progression of renal disease. A functional polymorphism at position -1607 1G/2G, -1306 C/T and -1171 5A/6A has been shown to alter the transcriptional activity of MMP-1, MMP-2, and MMP-3 respectively, and also associated with several diseases contributing to inter-individual differences in susceptibility to type 2 DN. The study population consisted of 310 type 2 DN patients and 310 healthy controls. Genotypes of MMP-1, 2 and 3 were determined by PCR-RFLP assay. Gene interactions, Linkage disequilibrium, and haplotype analysis were carried out by MDR analysis and Haploview software respectively. The promoter binding sites of MMP genes were determined by using Alibaba 2.1 and the gene-gene interactions of MMPs were analyzed by GeneMania. The individuals carrying 2G allele of -1607, C allele of -1306 and 5A/6A genotype of -1171 were associated with type 2 DN susceptibility and progression from stage 1 to stage 5. 2G-5A haplotypes of MMP-1 (-1607 1G/2G) and MMP-3 (-1171 5A/6A) gene polymorphisms were found to be significantly predominant in the disease group. MDR analysis revealed a strong interaction between the genes under study. 2G allele of MMP-1, C allele of MMP-2 and 5A/6A genotype of MMP-3 were associated with susceptibility and disease progression of type 2 DN and might be used as potential markers for risk prediction and prognosis of type 2 DN.

Synergistic effect of collagenase-1 (MMP1), stromelysin-1 (MMP3) and gelatinase-B (MMP9) gene polymorphisms in breast cancer.

BACKGROUND: Extracellular matrix degradation by matrix metalloproteinases (MMPs) is an important mechanism involved in tumor invasion and metastasis. Genetic variations of MMPs have shown association with multiple cancers. The present study is focused to elucidate the association of MMP-1, 3 and 9 genetic variants with respect to epidemiological and clinicopathological variables by haplotype, LD, MDR, survival in silico analyses among South Indian women. MATERIAL AND METHODS: MMP3-1171 5A/6A and MMP9-1562 C/T SNPs were genotyped by Allele specific polymerase chain reaction and MMP1-1607 1G/2G polymorphism by restriction fragment length polymorphism assays respectively, in 300 BC patients and age-matched 300 healthy controls. Statistical analysis was performed using the SNPStats and SPSS software. Linkage disequilibrium and gene-gene interactions were performed using Haploview and MDR software respectively. Further, transcription factor binding sites in the promoter regions of SNPs under study were carried out using AliBaba2.1 software. RESULTS: We have observed an increased frequency of 2G-allele of MMP1, 6A-allele of MMP3 and T-allele of MMP9 (p<0.05) respectively in BC subjects. The 2G-6A haplotype (minor alleles of MMP-1 and MMP-3 respectively) has shown an increased susceptibility to BC. Further, MMP polymorphisms were associated with the clinical characteristics of BC patients such as steroid hormone receptor status, lymph node involvement and metastasis. SNP combinations were in perfect LD in controls. MDR analysis revealed a positive interaction between the SNPs. 5-years survival rate and cox-regression analysis showed a significant association with clinicopathological variables. CONCLUSION: Our results suggest that MMP1-1607 1G/2G, MMP3-1171 5A/6A and MMP9-1562 C/T gene polymorphisms have synergistic effect on breast cancer. The interactions of MMPs clinical risk factors such as lymph node involvement has shown a strong correlation and might influence the 5-years survival rate, suggesting their potential role in the breast carcinogenesis.

Polymorphic variants of Caspase genes (8 & 3) in the risk prediction of Coronary Artery Disease.

Apoptosis has been involved in a number of pathological conditions including coronary artery disease (CAD). Caspases (CASP) are important regulators and executioners in both extrinsic and intrinsic apoptotic pathways. The aim of the present study is to examine the role of Caspase 8 and 3 polymorphisms in the pathogenesis of CAD. CAD patients (n=300) and healthy controls (n=300) were genotyped for polymorphisms in CASP8 (-652 6N del/ins, IVS12-19G>A), CASP3 (rs4647601;G>T) by PCR-RFLP. Splicing defects were determined by HSF. Gene interactions, Linkage disequilibrium and haplotype analysis were carried out by MDR analysis and Haploview software respectively. Molecular analysis revealed that insertion genotype (II) of CASP8 -652 6N del/ins and TT genotype of CASP3 rs4647601;G>T polymorphism conferred risk for the development of CAD. HSF analysis showed that intronic cryptic donor site for CASP8 -652 6N del/ins and a new ESE site for CASP3 rs4647601;G>T polymorphisms. SNP combinations of Caspase 8 and 3 were in perfect LD (D'=1) in controls. D-A, I-G haplotypes of Caspase 8 polymorphisms (-652 6N del/ins & IVS12-19G>A) were found to be significantly predominant in the disease group. The present study suggests that CASP8 & 3 polymorphic variants might be used as markers for susceptibility to CAD.

MMP 1 circulating levels and promoter polymorphism in risk prediction of coronary artery disease in asymptomatic first degree relatives.

Coronary artery disease (CAD) remains to be the prominent health problem in India, and its incidence is growing in developing countries as well. Matrix metalloproteinase 1 (MMP 1) is highly expressed in disruption-prone shoulder regions of the fibrous plaques. The present study aims to investigate association of MMP 1 gene polymorphisms (-1607 1G/2G) and serum circulating levels with CAD. The study includes 300 CAD patients, 100 FDRS, and 300 controls. ELISA and PCR-RFLP were performed to determine MMP 1 serum levels and genotypes respectively. MMP1 levels were high in CAD patients, followed by FDRS compared to controls (2.15±1.2ng/ml; 1.46±1.04ng/ml and 0.96±0.53ng/ml) respectively. ROC analysis showed the AUC at 95% CI of serum MMP-1 to be 0.83 and 0.73-0.94, respectively. The optimal cut-off point (sensitivity; specificity) of serum MMP 1 was >1.5ng/ml (0.74; 0.90). The 2G/2G genotype was associated with high MMP 1 circulating levels in CAD patients, and a similar trend was observed in FDRS and controls. The pre-mRNA secondary structure of the 2G allele is much more stable than 1G allele. Our results suggest MMP 1 serum levels and polymorphism as potential independent prognostic markers for future cardiovascular events. These may also help to stratify CAD patients and to identify susceptibility for CAD in asymptomatic healthy FDRS.

Rrp1B gene polymorphism (1307T>C) in metastatic progression of breast cancer.

Rrp1B (ribosomal RNA processing1 homolog B) is a novel candidate metastasis modifier gene in breast cancer. Functional gene assays demonstrated that a physical and functional interaction existing between Rrp1b and metastasis modifier gene SIPA1 causes reduction in the tumor growth and metastatic potential. Ectopic expression of Rrp1B modulates various metastasis predictive extra cellular matrix (ECM) genes associated with tumor suppression. The aim of this study is to determine the functional significance of single nucleotide polymorphism (SNP) in human Rrp1B gene (1307 T>C; rs9306160) with breast cancer development and progression. The study consists of 493 breast cancer cases recruited from Nizam's Institute of Medical Sciences, Hyderabad, and 558 age-matched healthy female controls from rural and urban areas. Genomic DNA was isolated by non-enzymatic method. Genotyping was done by amplification refractory mutation system (ARMS-PCR) method. Genotypes were reconfirmed by sequencing and results were analyzed statistically. We have performed Insilco analysis to know the RNA secondary structure by using online tool m fold. The TT genotype and T allele frequencies of Rrp1B1307 T>C polymorphism were significantly elevated in breast cancer (χ (2); p = <0.008) cases compared to controls under different genetic models. The presence of T allele had conferred 1.75-fold risk for breast cancer development (OR = 1.75; 95% CI = 1.15-2.67). The frequency of TT genotype of Rrp1b 1307T>C polymorphism was significantly elevated in obese patients (χ (2); p = 0.008) and patients with advanced disease (χ (2); p = 0.01) and with increased tumor size (χ (2); p = 0.01). Moreover, elevated frequency of T allele was also associated with positive lymph node status (χ (2); p = 0.04) and Her2 negative receptor status (χ (2); p = 0.006). Presence of Rrp1b1307TT genotype and T allele confer strong risk for breast cancer development and progression.

Interleukin 6(-174G/C) variant and its circulating levels in coronary artery disease patients and their first degree relatives.

Interleukin-6 (IL-6) a pleiotropic cytokine is a central mediator of inflammation in the pathogenesis of coronary artery disease (CAD). Our aim is to evaluate the serum levels of IL-6 and C-reactive protein (CRP) and to analyze the IL-6 polymorphism in CAD patients and to identify the first-degree relatives (FDRs) at risk of the disease in comparison with healthy controls. Estimation of IL-6 levels by enzyme-linked immunosorbent assay (ELISA) and CRP by latex reagent kit method, and genotyping of IL6 gene variants -174 (G>C) was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 600 subjects. IL-6 and CRP levels were significantly high in patients followed by FDRs compared to controls. The frequency of the IL-6 genotype was significantly different between cases, FDRs and controls and association of serum IL-6 levels with genotype found to be significant in CC genotype compared to GC and GG at p < 0.01 in CAD patients and FDRs, while there is no significant difference observed in controls. The study shows the importance of inflammation in the pathogenesis of CAD and predicts the risk of future coronary events in healthy asymptomatic FDRs.