High-throughput sequencing and in-silico analysis confirm pathogenicity of novel MSH3 variants in African American colorectal cancer.

The maintenance of DNA sequence integrity is critical to avoid accumulation of cancer-causing mutations. Inactivation of DNA Mismatch Repair (MMR) genes (e.g., MLH1 and MSH2) is common among many cancers, including colorectal cancer (CRC) and is the driver of classic microsatellite instability (MSI) in tumors. Somatic MSH3 alterations have been linked to a specific form of MSI called elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that is associated with patient poor prognosis and elevated among African American (AA) rectal cancer patients. Genetic variants of MSH3 and their pathogenicity vary among different populations, such as among AA, which are not well-represented in publicly available databases. Targeted exome sequencing of MSH3 among AA CRC samples followed by computational bioinformatic pipeline and molecular dynamic simulation analysis approach confirmed six identified MSH3 variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) that corresponded to MSH3 amino-acid changes (p.E413K; p.S466N; p.S920F; p.E976K; p.H1010Y; p.E1081K). All identified MSH3 variants were non-synonymous, novel, pathogenic, and show loss or gain of hydrogen bonding, ionic bonding, hydrophobic bonding, and disulfide bonding and have a deleterious effect on the structure of MSH3 protein. Some variants were located within the ATPase site of MSH3, affecting ATP hydrolysis that is critical for MSH3's function. Other variants were in the MSH3-MSH2 interacting domain, important for MSH3's binding to MSH2. Overall, our data suggest that these variants among AA CRC patients affect the function of MSH3 making them pathogenic and likely contributing to the development or advancement of CRC among AA. Further clarifying functional studies will be necessary to fully understand the impact of these variants on MSH3 function and CRC development in AA patients.

Exploring the Incidence and Associated Risk Factors of Barrett's Esophagus in African Americans: A Retrospective Study.

Background: The prevalence of Barrett's esophagus (BE) in African Americans (AA) is uncertain. However, several potential risk factors, includes family history, male sex, ethnicity, chronic heartburn and acid reflux, age over 60, current or past smoking, and obesity are associated with BE. The aim of this study is to determine the incidence of BE in AA patients who underwent Esophageal Gastro Endoscopy (EGD). Methods: Medical and demographic data of 1,253 AA patients with various symptoms, including BE, Esophageal adenocarcinoma (EAC), esophageal squamous adenocarcinoma (SCC), hiatal hernia, H. pylori infection, Gastro-Esophageal Reflux Disease (GERD), Gastritis, and esophagitis, were collected from January 2004 to December 2014 at Howard University Hospital. Results: Among the 1,253 patients, the median age was 61 and 49% were male out of the total. The frequencies of EAC (p= 0.05), and SCC (p= 0.002) were significantly high in males, along with SCC frequency significantly increased with older age (p<0.001). Furthermore, esophageal polyps with hiatal hernia (p=0.008) and H. pylori (p=<0.001) were found to be associated with esophagitis, and its presence may contribute to the development of BE. Conclusion: The findings highlighted the high prevalence of GERD symptoms and pathologic gastritis along with EAC was less common than SCC and both types of esophageal cancer were associated with male gender and older age whereas, H. pylori infection was identified as a risk factor for pathologic gastritis in AA. Overall data emphasize the need for extensive research, increased awareness, diagnosis, and management of GERD, gastritis, and related conditions to uncover the underlying mechanisms and factors contributing to these conditions in the AA population.

Microbiomic and Metabolomic Analyses Unveil the Protective Effect of Saffron in a Mouse Colitis Model.

Despite the existence of effective drugs used to treat inflammatory bowel disease (IBD), many patients fail to respond or lose response over time. Further, many drugs can carry serious adverse effects, including increased risk of infections and malignancies. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD and how the microbiome and metabolome play a role has not been explored extensively. We aimed to establish whether saffron treatment modulates the host microbiome and metabolic profile in experimental colitis. Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron in a dose of 20 mg/kg body weight or vehicle through daily gavage. On day 10, stool pellets from mice were collected and analyzed to assess saffron's effect on fecal microbiota and metabolites through 16S rRNA sequencing and untargeted primary metabolite analysis. Saffron treatment maintained gut microbiota homeostasis by counter-selecting pro-inflammatory bacteria and maintained Firmicutes/Bacteroides ratio, which was otherwise disturbed by DSS treatment. Several metabolites (uric acid, cholesterol, 2 hydroxyglutaric acid, allantoic acid, 2 hydroxyhexanoic acid) were altered significantly with saffron treatment in DSS-treated mice, and this might play a role in mediating saffron's colitis-mitigating effects. These data demonstrate saffron's therapeutic potential, and its protective role is modulated by gut microbiota, potentially acting through changes in metabolites.

Mechanism of Antitumor Effects of Saffron in Human Prostate Cancer Cells.

Prostate cancer is the most common cancer and the second leading cause of cancer deaths among men in the USA. Several studies have demonstrated the antitumor properties of saffron in different types of cancers, including prostate cancer. The oral administration of saffron extract has been reported to have antitumor effects on aggressive prostate-cancer-cell-line-derived xenografts in nude male mice. The objective of this study was to carry out in vitro studies of saffron-treated prostate cancer cells to ascertain the effects of saffron on key intermediates in prostate carcinogenesis. Our studies demonstrated the significant inhibition of cell proliferation for androgen-sensitive prostate cancer cell lines via apoptotic pathways. We also demonstrate the statistically significant down-regulation of DNA methyltransferases (COMT, MGMT, EHMT2, and SIRT1 deacetylase) in saffron-treated prostate cancer cells. In addition, saffron-treated prostate cancer cells displayed a statistically significant dysregulation of DNA repair intermediates (WRN, p53, RECQ5, MST1R, and WDR70) in a time-dependent manner. Furthermore, Western blot analysis demonstrated that saffron treatment induced changes in the expression of other key genes (DNMT1, DNMT3b, MBD2, CD44, HDAC3, c-Myc, NF-kB, TNFα, AR, N-RAS, and PTEN) in prostate cancer cells. Collectively, our findings demonstrate the important mechanisms by which saffron mediates anti-tumor properties in prostate cancer. These findings suggest that the use of saffron supplements alongside standard treatment protocols may yield beneficial effects for individuals with prostate cancer.

Saffron, Its Active Components, and Their Association with DNA and Histone Modification: A Narrative Review of Current Knowledge.

Intensive screening for better and safer medications to treat diseases such as cancer and inflammatory diseases continue, and some phytochemicals have been discovered to have anti-cancer and many therapeutical activities. Among the traditionally used spices, Crocus sativus (saffron) and its principal bioactive constituents have anti-inflammatory, antioxidant, and chemopreventive properties against multiple malignancies. Early reports have shown that the epigenetic profiles of healthy and tumor cells vary significantly in the context of different epigenetic factors. Multiple components, such as carotenoids as bioactive dietary phytochemicals, can directly or indirectly regulate epigenetic factors and alter gene expression profiles. Previous reports have shown the interaction between active saffron compounds with linker histone H1. Other reports have shown that high concentrations of saffron bind to the minor groove of calf thymus DNA, resulting in specific structural changes from B- to C-form of DNA. Moreover, the interaction of crocin G-quadruplex was reported. A recent in silico study has shown that residues of SIRT1 interact with saffron bio-active compounds and might enhance SIRT1 activation. Other reports have shown that the treatment of Saffron bio-active compounds increases γH2AX, decreases HDAC1 and phosphorylated histone H3 (p-H3). However, the question that still remains to be addressed how saffron triggers various epigenetic changes? Therefore, this review discusses the literature published till 2022 regarding saffron as dietary components and its impact on epigenetic mechanisms. Novel bioactive compounds such as saffron components that lead to epigenetic alterations might be a valuable strategy as an adjuvant therapeutic drug.

DNA methylation mediated downregulation of histone H3 variant H3.3 affects cell proliferation contributing to the development of HCC.

Chromatin alterations brought by histone variants and modifications potentially regulate gene transcription from tumor initiation to progression. Histone H3.3 variant is one such epigenetic player important for disease progression and development. Though many studies have implicated H3.3 role in cancer progression and metastasis, its regulation, importance of specific modifications and chaperones have been not understood yet. We report DNA methylation mediated downregulation of histone H3 variant H3.3 in HCC and a concomitant increase in the level of the H3.2 variant. The loss of H3.3 in cancer tissues correlates with a decrease in the histone modifications associated with active transcription like H3K9/K14/K27Ac and H3K4Me3. The ectopic overexpression of H3.3 and H3.2 did not affect global PTMs and cell physiology, probably owing to the deregulation of specific histone chaperones CAF-1 (for H3.2) and HIRA (for H3.3) as observed in HCC tissues. Notably, knockdown of P150, a subunit of CAF-1 leads to a cell cycle arrest in S-phase in a neoplastic rat liver cell line, possibly due to the decrease in the histone levels necessary for DNA packaging. Remarkably, modulation of H3.3 in pre-neoplastic rat liver cells lead to an increase in cell proliferation and a decreased transcription of tumor suppressor genes, recapitulating the tumor cell phenotype. Our data suggests, inhibition of DNA methylation and histone deacetylation leads to the restoration of histone H3 variant expression in tumor cells.

RPS13, a potential universal reference gene for normalisation of gene expression in multiple human normal and cancer tissue samples.

BACKGROUND: Reference genes are considered stable genes and are used for normalizing the gene expression profile across different cell types; as well as, in normal and diseased samples. However, these gene associates with different biological processes, and hence expression vary in different pathological conditions. Therefore, in the present study, eight different reference genes were used and compared to identify common reference gene usable for an array of different cell types and human cancers. METHODS AND RESULTS: The expression stability of the eight reference genes across eleven normal and cancerous tissues was confirmed through real time-qPCR. Ribosomal protein S13 (RPS13) was found to be a common and stable reference gene across intra- and inter-comparison between various normal and tumor tissue types. Further, TCGA data analysis across and between normal and tumor tissue types also showed minimum deviation in expression of RPS13 gene out of eight routinely used reference genes. CONCLUSION: RPS13 is the common stable reference gene in normalization for gene expression based analysis in cancer research.

In-Silico Analysis of Chromatin Modifiers and Profiling of Histone Deacetylases (HDAC's) in Human Oral Cancer.

Histone modifications have been demonstrated to play a significant role in oral squamous cell carcinoma (OSCC) epigenetic regulation. An in-silico analysis of The Cancer Genome Atlas (TCGA) of various histone acetyl transferases (HATs) and histone deacetylases (HDACs) suggested that HATs do not differ between normal and tumor samples whereas HDAC2 and HDAC1 change maximally and marginally respectively between normal and tumor patients with no change being noted in HDAC6 expression. Hence, this investigation was carried out to validate the expression states of HDAC 1, 2 and 6 mRNAs in buccal mucosa and tongue SCC samples in an Indian cohort. Buccal mucosa and tongue squamous cell carcinoma tissues with intact histopathology were processed for RNA isolation followed by cDNA synthesis which was then subjected to q-PCR for HDACs. The average RNA yield of the tongue tissue sample was ∼2 µg/mg of tissue and the A260/280 ratios were between 2.03 and 2.06. The average RNA yield of buccal mucosa tissue sample was ∼1 µg/mg of tissue and the A260/280 ratio were between 2.00 and 2.08. We have demonstrated that HDAC2 was overexpressed in tongue and buccal mucosa samples. Over-expression of HDAC2 imply potential use of HDACi along with standard chemotherapeutic drug in oral cancer treatment.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis.

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

Histone deacetylase inhibitor pre-treatment enhances the efficacy of DNA-interacting chemotherapeutic drugs in gastric cancer.

BACKGROUND: The prognosis of gastric cancer continues to remain poor, and epigenetic drugs like histone deacetylase inhibitors (HDACi) have been envisaged as potential therapeutic agents. Nevertheless, clinical trials are facing issues with toxicity and efficacy against solid tumors, which may be partly due to the lack of patient stratification for effective treatments. AIM: To study the need of patient stratification before HDACi treatment, and the efficacy of pre-treatment of HDACi as a chemotherapeutic drug sensitizer. METHODS: The expression activity of class 1 HDACs and histone acetylation was examined in human gastric cancer cells and tissues. The potential combinatorial regime of HDACi and chemotherapy drugs was defined on the basis of observed drug binding assays, chromatin remodeling and cell death. RESULTS: In the present study, the data suggest that the differential increase in HDAC activity and the expression of class 1 HDACs are associated with hypo-acetylation of histone proteins in tumors compared to normal adjacent mucosa tissue samples of gastric cancer. The data highlights for the first time that pre-treatment of HDACi results in an increased amount of DNA-bound drugs associated with enhanced histone acetylation, chromatin relaxation and cell cycle arrest. Fraction-affected plots and combination index-based analysis show that pre-HDACi chemo drug combinatorial regimes, including valproic acid with cisplatin or oxaliplatin and trichostatin A with epirubicin, exhibit synergism with maximum cytotoxic potential due to higher cell death at low combined doses in gastric cancer cell lines. CONCLUSION: Expression or activity of class 1 HDACs among gastric cancer patients present an effective approach for patient stratification. Furthermore, HDACi therapy in pre-treatment regimes is more effective with chemotherapy drugs, and may aid in predicting individual patient prognosis.

Elevated HDAC activity and altered histone phospho-acetylation confer acquired radio-resistant phenotype to breast cancer cells.

BACKGROUND: Poor-responsiveness of tumors to radiotherapy is a major clinical problem. Owing to the dynamic nature of the epigenome, the identification and targeting of potential epigenetic modifiers may be helpful to curb radio-resistance. This requires a detailed exploration of the epigenetic changes that occur during the acquirement of radio-resistance. Such an understanding can be applied for effective utilization of treatment adjuncts to enhance the efficacy of radiotherapy and reduce the incidence of tumor recurrence. RESULTS: This study explored the epigenetic alterations that occur during the acquirement of radio-resistance. Sequential irradiation of MCF7 breast cancer cell line up to 20 Gy generated a radio-resistant model. Micrococcal nuclease digestion demonstrated the presence of compact chromatin architecture coupled with decreased levels of histone PTMs H3K9ac, H3K27 ac, and H3S10pK14ac in the G0/G1 and mitotic cell cycle phases of the radio-resistant cells. Further investigation revealed that the radio-resistant population possessed high HDAC and low HAT activity, thus making them suitable candidates for HDAC inhibitor-based radio-sensitization. Treatment of radio-resistant cells with HDAC inhibitor valproic acid led to the retention of γH2AX and decreased H3S10p after irradiation. Additionally, an analysis of 38 human patient samples obtained from 8 different tumor types showed variable tumor HDAC activity, thus demonstrating inter-tumoral epigenetic heterogeneity in a patient population. CONCLUSION: The study revealed that an imbalance of HAT and HDAC activities led to the loss of site-specific histone acetylation and chromatin compaction as breast cancer cells acquired radio-resistance. Due to variation in the tumor HDAC activity among patients, our report suggests performing a prior assessment of the tumor epigenome to maximize the benefit of HDAC inhibitor-based radio-sensitization.

Establishing a correlation between RIN and A260/280 along with the multivariate evaluation of factors affecting the quality of RNA in cryopreserved cancer bio-specimen.

Biomarkers are biological characteristic to measure and evaluate normal and pathological states. To define expression-based molecular biomarkers, high-quality tissue samples are a prerequisite for the preparation of standard RNA. It is already known that RIN number defines the RNA quality, however its relation with A260/280 ratio and Ct value is not defined clearly; therefore, understanding an association will provide a reliable method for describing RNA quality. Multiple cryopreserved human tumor tissue types from ACTREC Biorepository and TMH-INTTR were analyzed for the effect of storage time on RNA quality. The RNA from tumor samples were isolated and analyzed by RIN, A260/280 ratio, and Ct value to establish inter-relationships. Around 50% samples had a RIN of ≥ 6.9 and A260/280 ≤ 2.04; 27% had a RIN ≥ 5 and A260/280 ≤ 2.08, and remaining 23% displayed RIN < 5 and A260/280 > 2.08. However, the RNA quality has no association with the storage period of tissue samples. Moreover, all samples which had A260/280 ≤ 2.08 showed acceptable Ct values of 17-24. The data clearly suggests that the A260/280 ratio is able to predict RNA quality. To the best of our knowledge, this is the first Indian report analyzing the labile nucleic acid-RNA quality from different cancer tissue types cryopreserved for diverse time periods. In conclusion, RIN and A260/280 ratio can help in predicting the quality of RNA independently; however, both together provide better assurance for further downstream processing.