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

10.26686/wgtn.14331389 ◽

2021 ◽

Author(s):

Matthew Munro

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Colon Adenocarcinoma ◽

Renin Angiotensin System ◽

Primary Cell ◽

Low Grade ◽

Angiotensin System ◽

Renin Angiotensin ◽

Primary Cell Lines

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.

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

PLoS ONE ◽

10.1371/journal.pone.0256280 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0256280

Author(s):

Matthew J. Munro ◽

Lifeng Peng ◽

Susrutha K. Wickremesekera ◽

Swee T. Tan

Keyword(s):

Stem Cell ◽

Cell Lines ◽

Colon Adenocarcinoma ◽

Cellular Metabolism ◽

Renin Angiotensin System ◽

Primary Cell ◽

Immunofluorescence Staining ◽

Angiotensin System ◽

Primary Cell Lines ◽

Ras Inhibitors

The cancer stem cell (CSC) concept proposes that cancer recurrence and metastasis are driven by CSCs. In this study, we investigated whether cells from colon adenocarcinoma (CA) with a CSC-like phenotype express renin-angiotensin system (RAS) components, and the effect of RAS inhibitors on CA-derived primary cell lines. Expression of RAS components was interrogated using immunohistochemical and immunofluorescence staining in 6 low-grade CA (LGCA) and 6 high-grade CA (HGCA) tissue samples and patient-matched normal colon samples. Primary cell lines derived from 4 HGCA tissues were treated with RAS inhibitors to investigate their effect on cellular metabolism, tumorsphere formation and transcription of pluripotency genes. Immunohistochemical and immunofluorescence staining showed expression of AT2R, ACE2, PRR, and cathepsins B and D by cells expressing pluripotency markers. β-blockers and AT2R antagonists reduced cellular metabolism, pluripotency marker expression, and tumorsphere-forming capacity of CA-derived primary cell lines. This study suggests that the RAS is active in CSC-like cells in CA, and further investigation is warranted to determine whether RAS inhibition is a viable method of targeting CSCs.

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Cancer Stem Cells in Head and Neck Metastatic Malignant Melanoma Express Components of the Renin-Angiotensin System

Life ◽

10.3390/life10110268 ◽

2020 ◽

Vol 10 (11) ◽

pp. 268

Author(s):

Sam Siljee ◽

Tessa Pilkington ◽

Helen D. Brasch ◽

Nicholas Bockett ◽

Josie Patel ◽

...

Keyword(s):

Stem Cells ◽

Malignant Melanoma ◽

Cancer Stem Cells ◽

Cell Lines ◽

Renin Angiotensin System ◽

Metastatic Malignant Melanoma ◽

Primary Cell ◽

Tissue Samples ◽

Angiotensin System ◽

Primary Cell Lines

Components of the renin-angiotensin system (RAS) are expressed by cancer stem cells (CSCs) in many cancer types. We here investigated expression of the RAS by the CSC subpopulations in human head and neck metastatic malignant melanoma (HNmMM) tissue samples and HNmMM-derived primary cell lines. Immunohistochemical staining demonstrated expression of pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all; renin in one; and ACE2 in none of the 20 HNmMM tissue samples. PRR was localized to cells within the tumor nests (TNs), while AT2R was expressed by cells within the TNs and the peritumoral stroma (PTS). ACE was localized to the endothelium of the tumor microvessels within the PTS. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) detected transcripts for PRR, ACE, ACE2, and AT1R, in all the five HNmMM tissue samples and four HNmMM-derived primary cell lines; renin in one tissue sample and one cell line, and AT2R in none of the five HNmMM tissue samples and cell lines. Western blotting showed variable expression of ACE, PRR, and AT2R, but not ACE2, in six HNmMM tissue samples and two HNmMM-derived primary cell lines. Immunofluorescence staining of two HNmMM tissue samples demonstrated expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the OCT4+ CSCs within the PTS, with ACE localized to the endothelium of the tumor microvessels within the PTS.

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The Role of the Renin–Angiotensin System in the Cancer Stem Cell Niche

Journal of Histochemistry & Cytochemistry ◽

10.1369/00221554211026295 ◽

2021 ◽

pp. 002215542110262

Author(s):

Ethan J. Kilmister ◽

Swee T. Tan

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Signaling Pathways ◽

Renin Angiotensin System ◽

Epidemiological Data ◽

Malignant Cells ◽

Angiotensin System ◽

Renin Angiotensin

Cancer stem cells (CSCs) drive metastasis, treatment resistance, and tumor recurrence. CSCs reside within a niche, an anatomically distinct site within the tumor microenvironment (TME) that consists of malignant and non-malignant cells, including immune cells. The renin–angiotensin system (RAS), a critical regulator of stem cells and key developmental processes, plays a vital role in the TME. Non-malignant cells within the CSC niche and stem cell signaling pathways such as the Wnt, Hedgehog, and Notch pathways influence CSCs. Components of the RAS and cathepsins B and D that constitute bypass loops of the RAS are expressed on CSCs in many cancer types. There is extensive in vitro and in vivo evidence showing that RAS inhibition reduces tumor growth, cell proliferation, invasion, and metastasis. However, there is inconsistent epidemiological data on the effect of RAS inhibitors on cancer incidence and survival outcomes, attributed to different patient characteristics and methodologies used between studies. Further mechanistic studies are warranted to investigate the precise effects of the RAS on CSCs directly and/or the CSC niche. Targeting the RAS, its bypass loops, and convergent signaling pathways participating in the TME and other key stem cell pathways that regulate CSCs may be a novel approach to cancer treatment:

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Cancer stem cells in liver metastasis from colon adenocarcinoma express components of the renin-angiotensin system

Journal of Cancer Metastasis and Treatment ◽

10.20517/2394-4722.2018.77 ◽

2019 ◽

Vol 2019 ◽

Cited By ~ 3

Author(s):

Anantha Narayanan ◽

Susrutha K. Wickremesekera ◽

Bede Van Schaijik ◽

Reginald W. Marsh ◽

Helen D. Brasch ◽

...

Keyword(s):

Stem Cells ◽

Liver Metastasis ◽

Cancer Stem Cells ◽

Colon Adenocarcinoma ◽

Renin Angiotensin System ◽

Angiotensin System ◽

Renin Angiotensin

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Abstract 356: A Role for Renal Proximal Tubule Sodium Bicarbonate Cotransporter SLC4A5 in Salt-Sensitivity of Blood Pressure

Hypertension ◽

10.1161/hyp.60.suppl_1.a356 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Julia M Carlson ◽

John J Gildea ◽

Helen E McGrath ◽

Robin A Felder

Keyword(s):

Sodium Bicarbonate ◽

Proximal Tubule ◽

Mrna Expression ◽

Cell Lines ◽

Renin Angiotensin System ◽

Salt Sensitivity ◽

Renal Proximal Tubule ◽

Angiotensin System ◽

Renin Angiotensin ◽

Homozygous Variant

SLC4A5 is a sodium-bicarbonate co-transporter involved with sodium homeostasis. Based on unpublished data, two SLC4A5 single nucleotide polymorphisms (SNPs rs1017783 and rs7571842) have been highly associated with an individual’s salt-sensitivity status. Since the renal proximal tubule (RPT) regulates a large percentage of renal sodium transport, we investigated whether SLC4A5 was present in this nephron segment. Using confocal immunofluorescence microscopy, we found expression of SLC4A5 in human RPT cell plasma membrane and intracellular membrane vesicles. We then examined the physiologic implications of the SLC4A5 SNPs in human RPT cells. Using immunoblotting and RT-PCR, we found no significant differences in basal SLC4A5 expression in RPT cells between individuals that are homozygous variant at both SNPs and individuals that are wild-type (WT) for both alleles. Stimulation of the dopaminergic system with 1μM fenoldopam, or the renin-angiotensin system with 10 nM angiotensin II or 10 nM angiotensin III (n=18 per treatment) over 3 and 24 hours did not significantly alter SLC4A5 protein or 24 hour mRNA expression. These data indicate that SLC4A5 is not directly regulated by either the renal dopaminergic or renin-angiotensin system. However, 24 hour stimulation with the sodium ionophore monensin (MON, 1μM) significantly increased overall mRNA expression of SLC4A5 by 182±0.098% over vehicle (VEH) (ΔCq VEH=0.283±0.035; n=18, p<0.001). There was also a significant increase in SLC4A5 mRNA in three cell lines homozygous variant for both alleles compared to three WT cell lines following MON treatment at both 3 hours (138±0.10%; ΔCq WT MON = 0.5±0.052; n=9, p<0.05) and 24 hours (161±0.11%; ΔCq WT MON = 0.39±0.066; n=9, p<0.02). Three but not 24 hour stimulation with MON also significantly increased overall expression of SLC4A5 protein (137±0.00041%; RFU VEH=0.0030±0.00022; n=18, p<0.01). MON, by allowing salt to enter a cell, may be activating an enhancer that leads to increased transcription of SLC4A5 mRNA that is more effective in homozygous variant cell lines. These novel observations demonstrate that SNPs located in a non-promoter DNA intron are associated with enhanced promoter activity that is regulated by altered intracellular sodium.

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