Browsing by Author "Parker, Mohamed"

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    Open Access
    Analysis of driver gene mutations in oesophageal squamous cell carcinoma
    (2025) Shipanga, Hendrina Nelao Mwiiwete; Parker, Mohamed; Hendricks, Denver
    Oesophageal cancer (OC) is the eleventh most diagnosed cancer and the seventh most common cause of cancer-related deaths worldwide. The two main subtypes are oesophageal adenocarcinoma (OAC) and oesophageal squamous cell carcinoma (OSCC). OAC is more common in North America and Europe, while OSCC predominantly occurs in Eastern Asia, Sub-Saharan Africa and Latin America. Over 80% of the OSCC cases and deaths worldwide occur in less developed regions, including Sub-Saharan Africa. The asymptomatic development of OSCC, results in late diagnosis of the disease with a poor prognosis, typically ranging from 5-10% at 5-year post-diagnosis in Africa. This study investigated the genomic landscape of OSCC in the South African population by whole-genome sequencing (WGS) and whole-exome sequencing (WES). Normal and tumour DNA and RNA was isolated from OSCC patient biopsies prior to the commencement of any form of chemotherapy or radiotherapy. WGS was performed on 31 samples, while WES was conducted on 67 samples. The mRNA levels of selected genes in OSCC were quantitated by RT-qPCR. KYSE30 cells were used for siRNA-mediated knockdown experiments targeting p14ARF and p16INK4a in OSCC. In silico structural analysis of missense mutations in p14ARF and p16INK4a was conducted using UCSF Chimera tool. WGS analysis identified 35 frequently mutated genes in OSCC, among these, TP53, CDKN2A.p16INK4a, CDKN2A.p14ARF, and KMT2D were identified as OSCC driver genes. Based on the mutation spectrum analysis, samples clustered into two distinct groups, cluster 1 and cluster 2b, characterized by TP53 alterations and mutation rates per megabase (Mb). WES expanded findings across 67 samples, identifying TP53, NFE2L2, CDKN2A.p16INK4a, ZNF750, and NOTCH1 as OSCC driver genes. Samples clustered into three groups: cluster 1, cluster 2a, and cluster 2b, expanding upon the two clusters identified in our WGS analysis. In both WGS and WES analyses, cluster 1 exhibited TP53 mutations and relatively high somatic mutation rates per Mb, while cluster 2 lacked TP53 mutations. Cluster 2 is further subdivided into clusters 2a and 2b in WES. Cluster 2a samples display a high mutation rate per Mb, while cluster 2b samples display fewer genomic alterations. By quantifying the contribution of the mutational signatures to the mutation spectrum, we found a relatively high contribution of mutation signature SBS1, SBS2, and SBS13, implicating aging and AID/APOBEC (activation induced cytidine deaminase/apolipoprotein B mRNA editing enzyme catalytic subunit) activation in OSCC tumourigenesis. WGS analysis revealed three novel mutational signatures that had not been previously identified. Interestingly, these signatures were not observed in the samples analysed by WES, even though the WES cohort included a larger sample size. The significance of these novel mutational signatures remains unclear. Evaluation of selected differentially expressed genes in OSCC involved in cell cycle control, the KEAP1-NFE2L2 (NRF2) pathway, and DNA damage response pathways showed variable expression of these genes in OSCC suggests potential dysregulation of the genes in OSCC. Furthermore, p16INK4a and p14ARF mRNA levels were significantly lower in 61% and 48% of OSCC tumour samples, respectively, while elevated levels were observed in 16% and 25% of tumours, respectively. Knockdown of p14ARF and p16INK4a in KYSE30 cells resulted in dysregulation of key regulators involved in multiple cancer signalling pathways, including cell cycle, apoptosis, and KEAP1-NFE2L2 pathways. This dysregulation could promote cell survival, growth of apoptosis-resistant cells, and resistance to stress, which are critical events in tumorigenesis. In-silico mutation analysis revealed damaging mutations in p16INK4a, such as p.A68V, p.D84N, p.D108H, p.D108N, p.D108Y, and p.L130P. These mutations cause significant structural alterations that disrupt interactions crucial for p16INK4a stability and function, possibly affecting cell cycle regulation and potentially promoting tumorigenesis in OSCC. Our findings highlights novel molecular features of OSCC and provides comprehensive insights into the genomic and molecular mechanisms driving OSCC within the South African population
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    Open Access
    The analysis of genetic aberrations in South African oesophageal squamous cell carcinoma patients
    (2023) Patten, Victoria Alexandra; Parker, Mohamed; Hendricks Denver
    Estimates for 2017 indicate that 20% of cancers globally are gastrointestinal tract (GIT) cancers, with oesophageal cancer being the 8th most common cancer. Oesophageal squamous cell carcinoma (OSCC) occurs in the upper to mid oesophagus and is present at high incidence in developing countries including South Africa. There are no early symptoms, resulting in late diagnosis and poor prognosis. In this study, tumour and blood DNA was obtained from 35 OSCC patients and subjected to whole genome sequencing (WGS). Bioinformatics analysis pipelines were designed to identify the possibility of novel viral insertions, investigating Human Endogenous Retroviruses (HERV's) insertions alongside the presence of somatic mutations in patient samples. The aims being to identify integration of any foreign DNA, to investigate if there is any linkage between HERV insertion and somatic mutations, and to identify any somatic mutations of potential interest in the OSCC cohort. The novel virus investigations however, proved to be inconclusive and there appeared to be no link between HERV insertions and somatic mutations present in the patients. Very significantly, it was determined that numerous somatic mutations were present in the MUC3A gene of the patient cohort, an interesting observation as no such previous association with OSCC has been recorded. MUC3A is a membrane-bound glycoprotein component of mucous gels, and its aberrant expression has been correlated with invasion and metastasis in a variety of other cancers. However, due to the complexity of the particular gene sequence and the known inconsistencies of variant calling performed on complex data sets, these mutations should be viewed with extreme caution as they are likely to be false positives. Analysis of RNA-seq data showed a 4.6 log2 fold increase in MUC3A expression in the tumour samples of these OSCC patients, with a P-adjusted value of 7.05e-06, suggesting highly significant differential gene expression. Functional enrichment analysis further showed that MUC3A was significantly associated with one of the top 5 gene ontologies (extracellular matrix structural constituent) for molecular function ontology class together with a number of collagen (COL) and MMP genes known to play a role in oncogenic progression and membrane stiffness. GSEA and KEGG analysis indicated predominantly chemokine/cytokine pro-inflammatory enriched pathways. Immunohistochemistry staining showed 10 out of 13 of the samples had no detectable levels of MUC3A protein, suggesting that the production of a non-functional truncated protein may lead to the upregulation of MUC3A expression that could possibly play a role in downstream pro-oncogenic signalling.
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    The role of BCLFA1 in cellular transformation
    (2018) Shipanga, Hendrina; Parker, Mohamed
    The malignant transformation of normal cells into cancer cells result in the loss of control of cellular regulatory mechanisms such as loss of function of tumour suppressors and gain of function of oncogenes. Genetic mutations may be inherited or acquired during the process of malignant transformation, such that the normal mechanisms responsible for control cellular proliferation become dysfunctional. Aberrations of the BCLAF1 gene located on chromosome 6q23 has previously been detected by whole genome sequence analysis of DNA from oesophageal cancer biopsies. Although the role of BCLAF1 is not well defined, some studies have shown BCLAF1 to have functional connections linked to some of the known hallmarks of cancer such as cell proliferation, apoptosis and genome stability, thus linking BCLAF1 to cellular transformation. The objective of the study was to examine the effects of BCLAF1 knockdown/BCLAF1 knockout studies in cellular gene expression and tumorigenesis. BCLAF1 expression was significantly reduced in the immortalized keratinocyte cell line (HaCaT), a lung transformed fibroblasts cell line (CT1), cervical cancer cell line (HeLa), breast cancer cell line (MDA-231) and two oesophageal cancer cell lines (KYSE30 and WHCO1), with a highly significant reduction in the breast cancer cell line (MDA-231). siRNA mediated knockdown of BCLAF1 resulted in altered expression of several downstream genes including downregulation the proapoptotic genes Caspase-3 and BAX and the DNA damage repair genes EXO1, ATRIP and BACH1. BCLAF1 deficiency also attenuated P53 expression and slightly increased P21 expression resulting in a P21-dependent G1 phase cell cycle arrest. To our knowledge, this is the first study to identify H2AX as a downstream gene of BCLAF1 that is downregulated by BCLAF1 knockdown. Abrogation of BCLAF1 in CT1 cells resulted in P53-dependent uncontrolled growth of cells, survival and accumulation of apoptosis-resistant cells, and genomic instability; all events that are crucial in tumourigenesis.