الفهرس 
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Abstract
Colorectal cancer is a major cause of morbidity and mortality throughout the world. In Egypt, CRC is the 6th most common cancer, representing 4% of the total cancers and 53% of gastrointestinal cancers. Moreover, Egypt has unusually high proportion of early onset CRC.
Despite advances in curative surgery, chemotherapy and radiotherapy, as well as extensive CRC-focused research over the past 20 years, the clinical outcome of CRC is still very poor. Increasing evidence suggests that the biologically unique population of CSCs, defined by their ability of self-renewal and differentiation may be responsible for tumor progression, metastasis and relapse. Current therapeutic strategies against cancer are not sufficiently selective against CSCs, so frequently lead to treatment failure. Consequently, CSCs are increasingly becoming priority targets for the development of new antitumor therapeutics. A thorough understanding of the specific biomarkers and the signaling pathways governing the functions of CSCs is crucial towards identifying valuable prognostic markers as well as therapeutic targets for CRC.
Several molecular markers have been employed to identify CRCSCs, of which LGR5 was proposed as the most selective and promising marker. Although, recent studies suggested that LGR5 may be involved in colorectal carcinogenesis, further studies are required to endorse the association of LGR5 expression in CRC tissue with the pro¬gression and prognosis of CRC patients.
Molecular pathways that regulate normal development and homeostasis are frequently dysregulated in cancers and represent potential targets for new therapeutics. Notch signaling is one of such evolutionarily conserved pathways which regulates critical cellular pathways and maintains CSCs, hence plays a crucial role in human malignant diseases.
Notch signaling controls the fate of intestinal progenitors by differentially regulating two opposing bHLH transcription factors, HES1 and ATOH1. Several studies have revealed how Notch signaling pathway might regulate HES1 and ATOH1 expressions in normal and cancerous tissues, nevertheless, further studies are needed to fill our gaps of knowledge particularly in clinical applications.
In spite of remarkable and non-stop advances in studying CSCs in CRC, better molecular characterization is crucial especially in Egypt, where molecular aspects of CSCs has not been fully explored among Egyptian CRC patients.
The aim of this study was to delineate the gene expression profile of three CSCs related genes among young CRC Egyptian patients. To this aim, the expression of LGR5, as a putative marker of CRCSCs, was characterized. Additionally, the activity of Notch pathway, as one of CSCs self-renewal pathways, was studied by evaluating the expression of two Notch target genes (HES1, ATOH1). As a further step, aiming at investigating the possible link between CSCs related genes and clinical outcome, correlations between elucidated patterns of gene expression and the clinicopathological criteria of the studied tumors, including response to neoadjuvant CRT, were conducted.
To fulfill these objectives, the study was conducted on 30 young Egyptian RC patients, who were recommended to undergo neoadjuvant therapy. Paired tumor and non-tumor adjacent mucosal tissues were obtained from patients by routine biopsy techniques. Total RNA was extracted followed by reverse transcription. Quantitative real time PCR was then performed using Taqman Hydrolysis probes and SYBR Green DNA binding dye. Expression levels of LGR5, HES1 and ATOH1 in tumor relative to adjacent non-tumor tissues were calculated using the comparative Cq method (2–ΔΔCq) after normalization for the expression of ACTB as reference gene. Patients were followed up for assessment of response to neoadjuvant CRT based on revised RECIST guidelines 1.1. Relative expression levels of different studied genes were then compared with clinicopathological parameters of the tumors and primary response to neoadjuvant CRT.
Overexpression of LGR5 in human RC tissues compared to non- tumor tissues was detected in the current study, which adds evidence to the enrichment of CSCs in RC tissues and endorses the important role of LGR5 in tumorigenesis. Furthermore, correlation analysis showed that higher expression of LGR5 was correlated with depth of tumor invasion, LN metastasis, cTNM stage and MRF involvement, but it was not correlated with gender, age, tumor site nor pathological features. These results suggest that LGR5 may not only play an important role in the progression of RC but also serve as a potential unfavorable prognostic biomarker for RC. In addition, LGR5 expression level was an excellent predictor for response to CRT.
Moreover, our results further validated the role of Notch signaling pathway in RC tumorigenesis, via elucidating HES1 overexpression and ATOH1 downregulation in cancerous tissues. Additionally, the molecular characterization of rectal adenocarcinomas with mucinous components revealed high ATOH1 expression levels, adding supplementary evidence to its critical effect on cellular differentiation. On the contrary, no significant differences were observed in HES1 nor ATOH1 expression levels as regards MRF involvement, pathological grade of differentiation, depth of tumor invasion, LN metastasis and cTNM staging. Likewise, no statistically significant correlation was detected between HES1 nor ATOH1 and response to CRT.
The current study also demonstrated that well optimized SYBR Green qPCR delivers comparable results to TaqMan assay in relative gene expression measurement of LGR5, HES1 and ATOH1 with the added benefit of lower cost.
It is noteworthy that the current study is, to the best of our knowledge, the first Egyptian study to address the molecular features of young RC patients by studying CSCs related genes. Our findings may allow better understanding of the roles of LGR5, HES1 and ATOH1 in cancer. Interpretation of their functions and the mechanism of its regulation may ultimately lead to the development of novel CSCs-based therapeutic strategies.