Novel insights into the expression of CGB1 & 2 genes by epithelial cancer cell lines secreting ectopic free hCGβ

BACKGROUND Ectopic secretion of human chorionic gonadotrophin free beta (hCGβ) by epithelial cancer is associated with aggressive tumors which more readily metastasize, possibly by acting as an autocrine anti-apoptotic agent. hCGβ is encoded by six homologous CGB genes, with poorly-understood variable transcriptionally active expression profiles; CGB1 and CGB2 have always been considered pseudogenes. However, transcripts from CGB1 and -2 can be detected in placental, testicular and pituitary tissues. The expression and function of these genes in cancer is less well-known. MATERIALS AND METHODS Expression profiles of CGB genes in epithelial cancer cells by quantitative polymerase chain reaction (qPCR) were explored, along with the consequence of specific siRNA silencing of CGB1 and 2. Immunohistochemical and immunoassay techniques were used to detect the translation and secretion of hCGβ in these cells. RESULTS CGB1 and -2 gene transcripts were only detected in cells which secreted hCGβ. siRNA-mediated silencing of CGB1 and -2 transcripts significantly reduced secreted protein in concordance with a reduction in cell survival to a greater degree than that of other CGB genes. CONCLUSION CGB genes 1 and 2, previously considered as pseudogenes, are notably expressed by epithelial cancer cell lines. The transcription of these genes, but not other CGB genes, correlates with a functionally expressed protein and propensity for cancer growth

Modelling and validating three-dimensional human breast and cancerous human breast tissues in vitro

In this study three dimensional (3-D) in vitro models of normal breast and breast cancer tissues were developed to mimic closely the in vivo tissue microenvironment and therefore providing reliable models for in vitro studies as well as testing of novel cancer therapies. Normal and cancerous human breast cell lines were used to construct 3-D artificial tissues, where de-epidermalised dermis (DED) was used as a scaffold for both models. Morphological analyses were conducted using haematoxylin and eosin staining. Biomarkers including keratin 5 and 19 as well as α smooth muscle actin and mucin 1 were used to confirm and validate the reliability of the proposed models using immunohistochemical techniques. Findings suggest that the 3-D in vitro models described in this work can serve as functional models of both human normal and cancerous breast tissues. Multiple structures similar to ducts and lobules of human breast in vivo were observed in 3-D in vitro models by the use of H&E, some breast cancer colonies seen in the cancerous 3-D model were similar to the ducto-lobular structures observed in normal 3-D model of the breast but the former cells were more loosely connected, irregular and largely disorganized. The established 3-D in vitro model of normal breast showed the development of ducto-lobular structures composed of an inner cell layer which was stained positive with α mucin 1 antibody, a biomarker that is characteristic for luminal cells; and also an outer basal layer of cells that was stained positive for α smooth muscle actin, a biomarker of myoepithelial cells.. Keratin staining in 3-D in vitro models also resembled the pattern observed in vivo where keratin 5 was detected in both luminal and myoepithelial cells of normal breast model (NTERT cells), whereas keratin 19 was present in breast cancer model (C2321 cells). These 3-D models successfully recapitulate both normal and pathological tissue architecture of breast tissue and has the potential for various applications in the evaluation of breast cancer progression and treatment

Regulation of human chorionic gonadotropin beta subunit expression in ovarian cancer

Expression of human chorionic gonadotropin beta subunit by cancers is extensively documented, yet regulation of the multiple genes that can code for this protein is poorly understood. The aim of the study was to examine the mechanisms regulating CGB gene expression in ovarian cancer. Expression of CGB genes and SP1, SP3, TFAP2A transcription factor genes was evaluated by RT-qPCR. The methylation status of CGB genes promoter regions was examined by methylation-specific PCR. mRNA arising from multiple CGB genes was detected in both ovarian control and malignant tissues. However, expression of CGB3-9 genes was shown to be significantly higher in malignant than healthy ovarian tissues. CGB1 and CGB2 transcripts were shown to be present in 20% of ovarian cancers, but were not detected in any of the control samples. Malignant tissues were characterized by DNA demethylation of CGB promoter regions. In ovarian cancer CGB expression positively correlated with TFAP2A transcripts level and expression of TFAP2A transcription factor was significantly higher in cancer than in control tissues. In contrast SP3 expression level was significantly lower in ovarian tumours than in control ovarian tissue. In ovarian cancers increased expression of human chorionic gonadotropin beta subunit is associated with demethylation of CGB promoter regions. CGB3-9 expression level strongly correlates with expression of the TFAP2A transcription factor. Presence of mRNA arising from CGB1 and CGB2 genes appears to be a unique feature of a subset of ovarian cancers

Reduction of human chorionic gonadotropin beta subunit expression by modified U1 snRNA caused apoptosis in cervical cancer cells

BACKGROUND: Secretion of human chorionic gonadotropin, especially its beta subunit by malignant trophoblastic tumors and varieties of tumors of different origin is now well documented; however the role of hCG in tumorogenesis is still unknown. RESULTS: This study documents the molecular presence of human chorionic gonadotropin beta subunit in uterine cervix cancer tissues and investigates a novel technique to reduce hCGbeta levels based on expression of a modified U1 snRNA as a method to study the hormone's role in biology of human cervical cancer cells cultured in vitro. The property of U1 snRNA to block the accumulation of specific RNA transcript when it binds to its donor sequence within the 3' terminal exon was used. The first 10 nucleotides of the human U1 snRNA gene, which normally binds to the 5'ss in pre-mRNA were replaced by a sequence complementary to a 10-nt segment in the terminal exon of the hCGbeta mRNA. Three different 5' end-mutated U1 snRNA expression plasmids were tested, each targeting a different sequence in the hCGbeta mRNA, and we found each one blocked the expression of hCGbeta in HeLa cells, a cervix carcinoma cell line, as shown by immunohistochemistry and qRT-PCR. Reduction of hCGbeta levels resulted in a significantly increased apoptosis rate with almost 90% of cells transfected with modified anti-hCGbeta U1 snRNAs showing morphological changes characteristic of the apoptotic process. CONCLUSION: These data suggest that human chorionic gonadotropin beta subunit may act as a tumor growth-stimulating factor

Geographical and socioeconomic inequalities in female breast cancer incidence and mortality in Iran: A Bayesian spatial analysis of registry data

Background In Iran, trends in breast cancer incidence and mortality have generally been monitored at national level. The purpose of this study is to examine province-level disparities in age-standardised breast cancer incidence versus mortality from 2000 to 2010 and their association with socioeconomic status. Methods In this study, data from Iran’s national cancer and death registry systems, and covariates from census and household expenditure surveys were used. We estimated the age-standardised incidence and mortality rates in women aged more than 30 years for all 31 provinces in the consecutive time intervals 2000–2003, 2004–2007 and 2008–2010 using a Bayesian spatial model. Results Mean age-standardised breast cancer incidence across provinces increased over time from 15.0 per 100,000 people (95% credible interval 12.0,18.3) in 2000–2003 to 39.6 (34.5,45.1) in 2008–2010. The mean breast cancer mortality rate declined from 10.9 (8.3,13.8) to 9.9 (7.5,12.5) deaths per 100,000 people in the same period. When grouped by wealth index quintiles, provinces in the highest quintile had higher levels of incidence and mortality. In the wealthiest quintile, reductions in mortality over time were larger than those observed among provinces in the poorest quintile. Relative breast cancer mortality decreased by 16.7% in the highest quintile compared to 10.8% in the lowest quintile. Conclusions Breast cancer incidence has increased over time, with lower incidence in the poorest provinces likely driven by underdiagnoses or late-stage diagnosis. Although the reported mortality rate is still higher in wealthier provinces, the larger decline over time in these provinces indicates a possible future reversal, with the most deprived provinces having higher mortality rates. Ongoing analysis of incidence and mortality at sub-national level is crucial in addressing inequalities in healthcare systems and public health both in Iran and elsewhere

Monitoring occurrence of SARS-CoV-2 in school populations: A wastewater-based approach

Clinical testing of children in schools is challenging, with economic implications limiting its frequent use as a monitoring tool of the risks assumed by children and staff during the COVID-19 pandemic. Here, a wastewater-based epidemiology approach has been used to monitor 16 schools (10 primary, 5 secondary and 1 post-16 and further education) in England. A total of 296 samples over 9 weeks have been analysed for N1 and E genes using qPCR methods. Of the samples returned, 47.3% were positive for one or both genes with a detection frequency in line with the respective local community. WBE offers a low cost, non-invasive approach for supplementing clinical testing and can provide longitudinal insights that are impractical with traditional clinical testing

Wastewater monitoring for detection of public health markers during the COVID-19 pandemic: Near-source monitoring of schools in England over an academic year

Background Schools are high-risk settings for infectious disease transmission. Wastewater monitoring for infectious diseases has been used to identify and mitigate outbreaks in many near-source settings during the COVID-19 pandemic, including universities and hospitals but less is known about the technology when applied for school health protection. This study aimed to implement a wastewater surveillance system to detect SARS-CoV-2 and other public health markers from wastewater in schools in England. Methods A total of 855 wastewater samples were collected from 16 schools (10 primary, 5 secondary and 1 post-16 and further education) over 10 months of school term time. Wastewater was analysed for SARS-CoV-2 genomic copies of N1 and E genes by RT-qPCR. A subset of wastewater samples was sent for genomic sequencing, enabling determination of the presence of SARS-CoV-2 and emergence of variant(s) contributing to COVID-19 infections within schools. In total, >280 microbial pathogens and >1200 AMR genes were screened using RT-qPCR and metagenomics to consider the utility of these additional targets to further inform on health threats within the schools. Results We report on wastewater-based surveillance for COVID-19 within English primary, secondary and further education schools over a full academic year (October 2020 to July 2021). The highest positivity rate (80.4%) was observed in the week commencing 30th November 2020 during the emergence of the Alpha variant, indicating most schools contained people who were shedding the virus. There was high SARS-CoV-2 amplicon concentration (up to 9.2x106 GC/L) detected over the summer term (8th June - 6th July 2021) during Delta variant prevalence. The summer increase of SARS-CoV-2 in school wastewater was reflected in age-specific clinical COVID-19 cases. Alpha variant and Delta variant were identified in the wastewater by sequencing of samples collected from December to March and June to July, respectively. Lead/lag analysis between SARS-CoV-2 concentrations in school and WWTP data sets show a maximum correlation between the two-time series when school data are lagged by two weeks. Furthermore, wastewater sample enrichment coupled with metagenomic sequencing and rapid informatics enabled the detection of other clinically relevant viral and bacterial pathogens and AMR. Conclusions Passive wastewater monitoring surveillance in schools can identify cases of COVID-19. Samples can be sequenced to monitor for emerging and current variants of concern at the resolution of school catchments. Wastewater based monitoring for SARS-CoV-2 is a useful tool for SARS-CoV-2 passive surveillance and could be applied for case identification and containment, and mitigation in schools and other congregate settings with high risks of transmission. Wastewater monitoring enables public health authorities to develop targeted prevention and education programmes for hygiene measures within undertested communities across a broad range of use cases

Purification and method optimisation for identification of carbohydrate moieties on human chorionic gonadotropin glycoforms using MALDI IT–TOF MS

The expression of human chorionic gonadotropin (hCG) is com-monly associated with a variety of cancers including testicular,bladder and gestational trophoblast diseases. All trophoblastictumours secrete very high amounts of hCG however hyperglyco-sylated form of hCG was found to be highly elevated in chorio-carcinoma patients. Current analytical methods, measuringbranched N-linked glycans in hCG, involve use of antibodieswhich may cause uncertainty due to issues of antibody specificity.In this project, highly sensitive and robust method to identify N-linked glycans, based on their specific chemical composition andmass tolerance, was developed. It was applied to variety of hCGforms, including hCG from pregnancy, choriocarcinoma andrecombinant hCG. hCG and its varied forms, with freebhCG,were isolated using IgG-coupled magnetic beads. N-linked gly-cans were then isolated from hCG by denaturation, reduction,alkylation (100°C, 10mM DTT, 20mM IAA) followed by degly-cosylation (PNGase, 37°C, 20h) and purification using porousgraphite carbon tips (Hypercarb). MALDI IT-TOF MS was usedto separate resulting glycans, which were then characterised usingProtein Scape software. Glycan peaks were reported, m/z 900–5,000 (Da), and carbohydrate library with precise glycan searchparameters (GlycoQuest, Bruker) was designed. A series of gly-cans were successfully identified from selected hCG sources, andsignificant differences in repertoire of glycans composition weredetected. Alteration in cellular glycosylation of proteins con-tribute to cancer metastasis, therefore strategies to understandtumour glycome and glycoproteome provide diagnostic potential,and targets for therapeutic intervention. Rapid and efficientdetection method of specific hCG glycoforms, from crudepatients urine samples, could help to speed and improve diagno-sis of hCG associated cancers including choriocarcinoma andnon-trophoblastic malignancies