Characterisation of cancer stem cells and the renin-angiotensin system in colon adenocarcinoma

Abstract

Colorectal cancer (CRC) is the third most common cancer and the second highest cause of cancer deaths globally. More than 70% of CRC-related deaths are due to metastasis to the liver. The cancer stem cell (CSC) concept hypothesises that CSCs drive tumour growth, chemoresistance, recurrence and metastasis. Markers such as CD133, LGR5 and EpCAM, have been used to identify and isolate CSCs in CRC. However, these markers are often expressed by cells with no stem cell properties and are not expressed by all tumour-initiating cells. An improved range of markers to define CSCs is needed. In 2007, adult mouse and human fibroblasts were reprogrammed into a stem cell state and defined as induced pluripotent stem cells (iPSCs) using transcription factors OCT4, SOX2, NANOG, KLF4 and c-MYC. These genes have well-documented roles in embryonic development and the maintenance of pluripotency, and their expression has been investigated in a range of cancers. <br><br>The renin-angiotensin system (RAS) physiologically maintains blood pressure and volume and is also acknowledged to play a role in cancer. Over-expression of (pro)renin receptor (PRR), angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R), and angiotensin-converting enzyme (ACE) have been reported in cancer. Epidemiological studies investigating the effect of RAS inhibitors on cancer outcomes have shown contradictory results.<br><br>This thesis investigates the expression of iPSC markers and RAS components in colon adenocarcinoma (CA) with three specific aims: (1) to compare CA-derived primary cell lines to their original CA tissues; (2) to investigate the expression profiles of iPSC markers in CA; and (3) to investigate expression of RAS components by CA CSCs and to determine whether CSCs can be targeted by RAS modulators. <br><br>DNA sequencing was carried out to compare the mutational profiles of formalin-fixed paraffin-embedded (FFPE) CA tissues and CA-derived cell lines to confirm whether the cell lines were a suitable in vitro model for the parent tumours.<br><br><div>Proteomics was performed to determine proteomic differences between CA tissues and patient-matched normal colon (NC) tissues, CA-derived cell lines and NC-derived cells, and between low grade CA (LGCA) tissues and cell lines and high grade CA (HGCA) tissues and cell lines. Biological processes which may link the RAS and CA were investigated, revealing enrichment of various signalling pathways that may play roles in CA onset and progression directly or via the RAS.</div><div><br></div>Western blotting and immunohistochemical staining showed elevated protein levels of OCT4, SOX2, NANOG, c-MYC, AT2R, PRR and cathepsin D in CA tissues relative to their patient-matched NC tissues, with SOX2, ACE and cathepsin B at similar levels and KLF4 less abundant in CA compared with NC tissues. Co-expression analysis by immunofluorescence staining showed a small number of epithelial cells co-expressed NANOG, SOX2, KLF4, c-MYC and CD133, as well as PRR, ACE2 and AT2R, while a small number of stromal cells co-expressed OCT4 and AT2R. This indicates the presence of at least one CSC subpopulation in CA, which expresses RAS components. HGCA tissue-derived cell lines expressed higher levels of OCT4 and SOX2 than LGCA-derived cell lines. The primary cell lines were sorted based on EpCAM expression. These EpCAM High and EpCAM Low cell subpopulations could undergo directed differentiation down the three embryonic lineages. A small number of CA-derived cells, particularly within the HGCA-derived cells, formed tumourspheres. Treatment of HGCA-derived cell lines with RAS modulators revealed that β-blockers and AT2R antagonists consistently reduced their metabolism, tumoursphere formation and iPSC marker expression. <br><br>The findings of this thesis suggest that CA-derived cell lines expressing iPSC markers have stem cell function and express RAS components. Furthermore, RAS modulators may directly influence CSCs in CA by reducing iPSC marker gene expression. This indicates a potential role for RAS modulators in regulating CSCs, which merits further investigation.

Colon adenocarcinoma-derived cells possessing stem cell function can be modulated using renin-angiotensin system inhibitors

“…However, there are currently no specific antibodies against AT 1 R. One laboratory has tested six commonly-cited anti-AT 1 R antibodies using various knock-out models and cells known to be negative for AT 1 R, and found that these antibodies all detected bands at around 43 kDa (the predicted size of AT 1 R), and produced a variety of staining intensities and localizations by IHC staining not specific for AT 1 R [48]. AT 1 R was not detected in our CA tissues or primary cell lines when analyzed by mass spectrometry, possibly because integral membrane proteins can be difficult to isolate during sample preparation [38]. Therefore, despite being a potential target for RAS modulation in the treatment of cancer, determination of AT 1 R protein expression and localization was not performed in this study.…”

“…The downstream effects of AT 2 R and Mas receptor agonism counteract those of AT 1 R. Adapted with permission from Munro et al Integr Cancer Sci Therap. [10] and from [38]…”

“…ACE (T; 195 kDa) and ACE2 (U; 110 kDa) protein expression by cell lines could not be detected by WB; positive controls = mouse lung and human kidney, respectively. Adapted from[38] under a CC BY license, with permission from Munro MJ et al 2021. https://doi.org/10.1371/journal.pone.0256280.g001…”

“…PRR protein expression by cell lines was detected by WB (K; 45 kDa); positive control = HepG2 cells. Adapted from[38]. under a CC BY license, with permission from Munro MJ et al 2021. https://doi.org/10.1371/journal.pone.0256280.g002…”