Evaluation of the role of HMGA1 protein in colon cancer stem cells

• Autor(a) principal: Monteiro, Inês Filipa Soares
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/40519
• topic: Colorectal cancer; Cancer stem cells; HMGA1; SORE6; Viral transduction; Stemness

Colorectal cancer, with over 1.9 million cases in 2020 and ranking second in cancer-related deaths, is increasing in highly developed countries. Cancer stem cells (CSCs), related to tumour initiation and chemotherapy resistance, are a significant focus in cancer research. Transcription factors (TFs) like SOX2, CMYC, and OCT4 are used to identify these cells. The lentiviral SORE6 reporter system, developed by Tang et al. (2015), allows the detection and characterization of CSCs by using a green fluorescent protein (GFP) under the control of a promoter with binding sites for SOX2 and OCT4. HMGA1 protein, overexpressed in several types of cancer, influences gene expression through chromatin changes, and is linked to stemness TFs, such as SOX2, OCT4, KLF4, and NANOG. In colorectal cancer, HMGA1 is crucial for maintaining a stem cell phenotype. The aim of this study was to investigate and establish the functional relevance of this protein in colon CSCs. We started by evaluating if HMGA1 levels, assessed by immunohistochemistry in 219 stage II CRC patient samples, were statistically correlated with clinicopathological and molecular characteristics of the tumours. We found that tumours with high HMGA1 expression were significantly associated with MMR-proficiency and SOX2 expression. Nevertheless, no prognostic or predictive value for HMGA1 expression was found. Aiming to use the SORE6 reporter to detect CSCs, a transduction of two CRC cell lines, HCT116 and SW480, with the SORE6 reporter vector was performed, and later two cell subpopulations, SORE6⁺ (GFP⁺) and SORE6ˉ (GFPˉ) were established through Fluorescence Activated Cell Sorting (FACS). Even though SOX2 expression was higher in SORE6⁺ cells (CSC-like), as expected, HCT116 cells showed less HMGA1 expression, when comparing to SORE6ˉ cells. Only SW480 had more expression of these two proteins in SORE6⁺ cells. Then, HMGA1 overexpression was performed by transducing both SORE6ˉ and SORE6⁺ sub-populations of HCT116 and SW480 cell lines with a DOX-inducible lentiviral vector, to understand if this protein has a role in the acquisition of stemness characteristics by colon cells and to evaluate phenotypic changes related to the establishment of colon CSCs. Unfortunately, the infection of SW480 revealed unsuccessful. The HCT116 HMGA1 transduced cells had a GFP gain was observed, through flow cytometry, but only in SORE6⁺ HMGA1 transduced cells. Regarding SOX2 and C-MYC expression in these cells, while SOX2 appeared to be inhibited by HMGA1, C-MYC seemed up-regulated by this protein. OCT4 had very low levels of expression in both cells and was not further evaluated. Through functional assays, no significant differences in stemness were observed after transducing SORE6 cells with HMGA1, either by using clonogenic or 5-FU resistance assays.