Interaction of Gut Microbiome and Host microRNAs with the Occurrence of Colorectal and Breast Cancer and Their Impact on Patient Immunity.

Breast and colorectal cancers are two primary malignancies on which most of the research done worldwide investigates the potential genetic and environmental risk factors and thereby tries to develop therapeutic methods to improve prognosis. Breast cancer is the most diagnosed cancer type in women, while colorectal cancer is diagnosed in males as the third most and females as the second most cancer type. Though these two cancer types are predominantly seen in adult patients worldwide, in the current context, these malignancies are diagnosed at a younger age with a significant rate of incidents than previous. Such early-onset cancers are generally present at an advanced stage of the most aggressive type with a poor prognosis. In the past, the focus of the research was mainly on studying possible candidate genes to understand the onset. However, it is now recognized that genetics, epigenetics, and other environmental factors play a pivotal role in cancer susceptibility. Thus, most studies were diversified to study the behavior of host microRNAs, and the involvement of gut microbiota and good communication between them surfaced in the occurrence and state of the disease. It is understood that the impact of these factors affects the outcome of the disease. Out of the adverse outcomes identified relating to the disease, immunosuppression is one of the most concerning outcomes in the current world, where such individuals remain vulnerable to infections. Recent studies revealed that microbiome and microRNA could create a considerable impact on immunosuppression. This review focused on the behavior of host microRNAs and gut microbiome for the onset of the disease and progression, thereby influencing an individual's immunosuppression. Understanding the interactions among microRNA, microbiome, presentation of the disease, and impact on the immune system will be immensely useful for developing future therapeutic strategies based on targeting host microRNA and the patient's gut microbiome. Therapies such as inhibitory-miRNA therapies, miRNA mimic-based therapeutics, immune checkpoint blockade therapies, and bacteria-assisted tumor-targeted therapies help modulate cancer. At the same time, it paid equal attention to potential noninvasive biomarkers in diagnosis, prognosis, and therapeutics in both cancers.

Pattern of nucleotide variants of TP53 and their correlation with the expression of p53 and its downstream proteins in a Sri Lankan cohort of breast and colorectal cancer patients.

BACKGROUND: Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. METHODS: 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. RESULTS: Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS; three of them were on codon 12 and one was on codon 61. CONCLUSION: The prevalence of pathogenic somatic TP53 variants was 31 and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5-8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

Nucleotide variants and protein expression of TP53 in a Sri Lankan cohort of patients with head and neck cancer.

Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cancer among Sri Lankan females. This is the first study, to the best of our knowledge, that has focused on investigating the association between TP53 somatic DNA variants, with p53 protein expression and risk factors in a cohort of Sri Lankan patients with HNC. A total of 44 patients with cancer and 20 healthy controls were studied. In total, 36 genomic DNA sequence variants were found, including several novel variants (two deletions in exons 4 and 6, two in the 3' untranslated region and several intronic variants). A total of 14 tumour samples carried pathogenic TP53 mutations. A random selection of 24 samples was analysed immunohistochemically for p53 protein expression. All the samples with point missense variants were strongly immuno­positive, whereas, samples with nonsense and frameshift TP53 variants were immuno­negative for p53 immunohistochemical staining. Although, the human papilloma virus is a known risk factor for HNC, results from the present study identified an absence or lower level of infection in the Sri Lankan cohort.

Overview of the genetic basis toward early detection of breast cancer.

Cancer is a socioeconomical burden in any nation. Out of that, breast cancer is identified as the most common malignancy worldwide among women irrespective of age. As women are an important segment in a community, the weakening of their strength toward the development of a nation is a critical problem in each nation. In this review, it was aimed to discuss the characteristics of cancer genome, cancer genetics, and cancer epigenetics in general and then focus on discussing both genetic and nongenetic factors responsible for the predisposition of breast cancer in humans. More emphasis was placed on genes responsible for the early onset of the disease and which can be used as genetic tools in the identification of the disease at an early stage. Then the context of genetic involvement toward the breast cancer occurrence before age of 40 years was highlighted accordingly. In addition to genetic testing, the review paid adequate attention to mention novel liquid biopsy techniques and other clinical, laboratory, and radiologic assessments. These techniques can be used in early detection and recurrence as well as the surveillance of the patients after primary therapies.

Circulating leptin, soluble leptin receptor, free leptin index, visfatin and selected leptin and leptin receptor gene polymorphisms in sporadic breast cancer.

Leptin and visfatin are implicated in breast cancer risk but studies accounting for bioavailability of leptin are sparse. Reports on the association of leptin gene (LEP) and leptin receptor gene (LEPR) polymorphisms with breast cancer are also inconsistent. Only a very few studies have examined biochemical and genetic variables concomitantly in the same cohort. A matched pairs study was carried out to ascertain whether plasma leptin, soluble leptin receptor, free leptin index (leptin/soluble leptin receptor), serum visfatin and selected LEP and LEPR polymorphisms are risk factors for sporadic breast cancer. Newly diagnosed sporadic breast cancer patients (N=80) were matched for age, body mass index (BMI) and menopausal status with healthy controls. Plasma leptin, soluble leptin receptor and serum visfatin were measured by enzyme-immunoassay. LEP -2548 A/G and LEPR K109R, LEPR Q223R polymorphisms were determined by genotyping. Leptin (p=0.0234), leptin/BMI (p=0.0468), free leptin index (p<0.0001) and visfatin (p=0.0002) were significantly higher and soluble leptin receptor (p<0.0001) was significantly lower in patients. LEPR gene K109R A/G polymorphism increased breast cancer risk (odds ratio: 4.125). Multivariate analysis confirmed that leptin, soluble leptin receptor, free leptin index and G109 (R109) allele of the LEPR gene K109R polymorphism are risk factors for breast cancer. When stratified by menopausal status free leptin index and soluble leptin receptor remained as risk factors irrespective of menopausal status while LEPR gene K109R A/G polymorphism remained as a risk factor only in the postmenopausal group.

In silico characterization of a RNA binding protein of cattle filarial parasite Setaria digitata.

Human lymphatic filariasis (HLF) is a neglected tropical disease which threatens nearly 1.4 billion people in 73 countries worldwide. Wuchereria bancrofti is the major causative agent of HLF and it closely resembles cattle filarial parasite Setaria digitata. Due to difficulties in procuring W. bancrofti parasite material, S. digitata cDNA library has been constructed to identify novel drug targets against HLF and many of the cDNA sequences are yet to be assigned structure and function. In this study, a 549 bp long cDNA (sdrbp) has been sequenced and characterized in silico. The shortest ORF of 249 bp from the isolated cDNA encodes a polypeptide of 82 amino acids and shows an amino acid identity of 54% with the RRM domain of human cleavage stimulation factor-64 kDa subunit (CstF-64). Structure of the protein (sdRBP) obtained by homology modelling using RRM of CstF-64 as template adopts classical RRM topology (ß1α1ß2ß3α2ß4). sdRBP model built was validated by superimposition tools and Ramachandran plot analysis. CstF-64 plays an important role in pre-mRNA polyadenylation by interacting with specific GU-rich downstream sequence element. Molecular docking studies of sdRBP with different RNA molecules revealed that sdRBP has greater binding affinity to GU-rich RNA and comparable results were obtained upon similar docking of RRM of CstF-64 with the same RNA molecules. Therefore, sdRBP is likely to perform homologous function in S. digitata. This study brings new dimensions to the functional analysis of RNA binding proteins of S. digitata and their evaluation as new drug targets against HLF.

Analysis of BRCA1and BRCA2 large genomic rearrangements in Sri Lankan familial breast cancer patients and at risk individuals.

BACKGROUND: Majority of mutations found to date in the BRCA1/BRCA2 genes in breast and/or ovarian cancer families are point mutations or small insertions and deletions scattered over the coding sequence and splice junctions. Such mutations and sequence variants of BRCA1 and BRCA2 genes were previously identified in a group of Sri Lankan breast cancer patients. Large genomic rearrangements have been characterized in BRCA1 and BRCA2 genes in several populations but these have not been characterized in Sri Lankan breast cancer patients. FINDINGS: A cohort of familial breast cancer patients (N = 57), at risk individuals (N = 25) and healthy controls (N = 23) were analyzed using multiplex ligation-dependent probe amplification method to detect BRCA1 and BRCA2 large genomic rearrangements. One familial breast cancer patient showed an ambiguous deletion in exon 6 of BRCA1 gene. Full sequencing of the ambiguous region was used to confirm MLPA results. Ambiguous deletion detected by MLPA was found to be a false positive result confirming that BRCA1 large genomic rearrangements were absent in the subjects studied. No BRCA2 rearrangement was also identified in the cohort. CONCLUSION: Thus this study demonstrates that BRCA1 and BRCA2 large genomic rearrangements are unlikely to make a significant contribution to aetiology of breast cancer in Sri Lanka.

LEPR c.668A>G polymorphism in a cohort of Sri Lankan women with pre-eclampsia / pregnancy induced hypertension: a case control study.

BACKGROUND: Leptin is known to be elevated in pre-eclampsia/ pregnancy induced hypertension (PE/PIH). However the reports on the association of leptin receptor (LEPR) c.668A>G polymorphism with PE/PIH are inconsistent. FINDINGS: LEPR c.668A>G polymorphism was studied in a cohort of women with PE/PIH (N = 61) and normotensive pregnancies (N = 40) by polymerase chain reaction / restriction fragment length polymorphism. Genotype and allele frequencies were in Hardy-Weinberg equilibrium within both groups (Chi square test). Allele and genotype frequencies were not significantly different between PE/PIH and normotensive pregnancies (Chi square test). Leptin levels (Kruskal Wallis analysis of variance) and leptin/body mass index (one way analysis of variance) were not significantly different between genotypes within each group. However, leptin (Mann Whitney U test) and leptin normalised to body mass index (unpaired t test) were significantly higher in PE/PIH women homozygous and heterozygous for the G668 allele than in respective normotensives. CONCLUSIONS: Whether the leptin receptor c.668A>G polymorphism increases the risk of developing PE/PIH in Sri Lankan women remains inconclusive in view of the smaller sample studied. However leptin levels in PE/PIH appeared to be modulated by this polymorphism.

A putative nuclear growth factor-like globular nematode specific protein.

Expressed sequence tags (ESTs) are an effective approach for discovery of novel genes. In the current study, approximately 250 ESTs of the cattle parasitic nematode Setaria digitata were examined and a cDNA clone identified whose coding sequence could not be functionally annotated by searching over publicly available genome, protein, EST and STS databases. Here, we report the extensive characterization of this ORF (UP) and its homologues using a bioinformatic approach. Uncharacterized protein (SDUP) of S. digitata consists of 204 amino acids with a predicted molecular weight and isoelectric point of 22.8KDa and 9.94, respectively. A search carried out using SDUP over nucleotide, EST and protein databases at NCBI, NEMBASE3 and Parasite Genome Database (PGD) identified homologous counterparts from the human parasitic nematodes Wuchereria bancrofti (WB), Brugia malayi (BM), Onchocerca volvulus (OV), the mouse filarial worm Litomosoides sigmodontis (LS), swine parasitic nematodes Ascaris suum (AS) and diverged counterparts from the plant parasitic nematode Meloidogyne hapla (MH) and free living nematodes Caenorhabditis elegans (CE) and Caenorhabditis briggsae (CB). Phylogenetic analyses revealed the UPs to be undergoing divergent evolution. A search of the ESTs at PGD showed that UP is expressed in all the stages of BM. Secondary structure analyses of multiply-aligned sequences of homologues using Jpred server indicated UPs to be rich in beta-pleated structures. TMMHH server and beta barrel finder programme indicated, UPs to be neither transmembrane or beta barrels proteins but are likely to be globular proteins. Further, the Motif discovery tool of MEME identified three novel potential motifs for UPS, of which only two are present in CE, CB & MH. Analyses of UPs using Signal IP, TargetP, Psort servers predicted this group of proteins to be devoid of signal peptide cleavage sites, are not mitochondrial targeting peptides but appear to be localized to the nucleus, respectively. Further analyses of the UPs using ScanProsite server for phosphorylation revealed potential sites for cAMP- and cGMP-dependent protein kinase, Protein kinase C and Casein kinase II. Putative functional analysis using ProtFun 2.1 Server indicated UPs to be nonenzymatic, growth factor like protein. Finally, collating all the information derived from bioinformatic analyses, we conclude that the UPs of nematodes are most likely to be expressed at all stages in the life cycle, localized to the nucleus, regulated by phosphorylation, rich in beta-pleated strands and are growth factor like nematode specific proteins.