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Wickremesekera et al. J Cancer Metastasis Treat 2019;5:62 I http://dx.doi.org/10.20517/2394-4722.2019.09 Page 7 of 9

Figure 4. Relative expression of mRNA transcripts of PRR, ACE, ATIIR1 and ATIIR2 by NanoString mRNA analysis of four samples
of metastatic melanoma to the brain showing high levels of expression of PRR, moderate levels for ACE, low levels for ATIIR1 and
undetectable levels for ATIIR2, relative to the housekeeping gene GAPDH. PRR: pro-renin receptor; ATIIR1: angiotensin II receptor 1;
ATIIR2: angiotensin II receptor 2; ACE: angiotensin converting enzyme

OCT4 and NANOG as surrogate markers, IF IHC staining revealed localization of PRR, ATIIR1 and
+
+
+
+
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ATIIR2 to the OCT4 /OCT4 /SALL4 /SOX2 /NANOG CSC subpopulations while ACE was localized the
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CSC subpopulation on ERG endothelium of the microvessels which we have shown to express pSTAT3
previously. The expression of PRR, ACE, ATIIR1 and ATIIR2 was confirmed by WB. NanoString mRNA
analysis showed high levels of expression of PRR, lower expression levels of ACE and ATIIR1. ATIIR2
was below detectable levels which may be due to mRNA degradation or its transient presence during
transcription within the samples examined.
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We have previously reported expression of PRR, ATIIR1 and ATIIR2 by the SOX2 CSC subpopulation,
and exclusive expression of ACE by the subpopulation on the endothelium of the microvessels, in human
[26]
isocitrate dehydrogenase-wildtype GB and OCSCC affecting different subsites [27-29] . mRNA expression
of renin, ACE, ATIIR1 and ATIIR2 has been demonstrated on primary cultured human keratinocytes,
melanocytes, dermal fibroblasts and dermal capillary endothelial cells, but ATIIR2 is not detected in
melanocytes .
[31]
Cellular proliferation associated with ATI-treated and ATII-treated infantile hemangioma cell culture has
shown to be diminished by ACE inhibitor ramipril and the ATIIR2 antagonist PD123319, and is enhanced
by ATIIR2 agonist CGP42112 , implying a role for the RAS peptides in stem cell proliferation.
[32]

Blockade of the RAS leads to inhibited growth of colorectal cancer liver metastases in the regenerating
[33]
liver and recent reports of increased overall survival of GB patients treated with angiotensin receptor
blockers , support the role of the RAS in cancer. It is exciting to speculate that CSCs in MM to the brain
[34]
maybe a novel therapeutic target by modulating the RAS, although more work including a larger sample
size, control tissue samples and in vitro and in vivo functional work is needed to determine the precise role
of the RAS in this aggressive cancer.

In conclusion, in this study we have demonstrated the expression and localization of components of the
components of the RAS: PRR, ACE, ATIIR1 and ATIIR2 in MM to the brain. PRR, ATIIR1 and ATIIR2 are
localized to the CSC subpopulations within the tumor while ACE is expressed by the CSC subpopulation
on the endothelium of the microvessels. Targeting the CSCs using modulators of the RAS may be a novel
therapeutic approach for MM to the brain.