Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1

Abstract

The incidence of many cancer types is significantly reduced in individuals with Down's syndrome, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down's syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway. Here we show that DSCR1 is increased in Down's syndrome tissues and in a mouse model of Down's syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down's syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.

Figure 1. Dscr1 expression is upregulated in Down syndrome tissues and tumor angiogenesis is suppressed in Down syndrome models

(a) Increased DSCR1 expression in human fetal Down syndrome (DS) tissues versus age-matched control (ctrl) tissues relative to β-actin. (b) Tumor growth is suppressed in the Ts65Dn Down syndrome mouse model. Values are mean ± s.e.m. n=10–12, *p<0.03; **p <0.01. (c) Microvessel density (MVD) per high-powered field (hpf) of tumors is quantified by anti-CD31 immunofluorescence. Bar, 20 µM. Values are mean ± s.e.m. *p <0.02; **p<0.01. (d) Angiogenesis in tumors from induced pluripotent stem cells (iPS) isolated from Down syndrome or cytogenetically normal cells was quantified by human specific anti-CD31 immunofluorescence. Arrows, hCD31-positive vessels. Values are mean ± s.e.m. n=3–6, **p<0.01.

Figure 2. Targeted Dscr1 transgenic mice with three copies of Dscr1 show inhibition of tumor growth

(a) NF-ATc1 nuclear import after VEGF treatment is suppressed in endothelial cells from Dscr1 transgenic mice. Bar, 5 µM. (b) VEGF-induced proliferation of endothelial cells from Dscr1 transgenic mice is significantly inhibited. *p<0.02; **p<0.03. (c) Tumor growth is suppressed in Dscr1 transgenic mice. n=8–12 mice per group, *p<0.05; **p<0.04. (d) Microvessel density (MVD) per high-powered field (hpf) of tumors is quantified by anti-CD31 immunofluorescence. Bar, 20 µM. Values are mean ± sem. *p<0.01. (e) Tumors from Dscr1 transgenic and wild-type mice are co-immunostained with anti-CD31 to detect endothelial cells and anti-Myc to detect the Dscr1 transgene. Bar, 20 µM.

Figure 3. Trisomic expression of Dscr1 is necessary for significant suppression of tumor growth in the Ts65Dn Down syndrome mouse model

(a) Possible genotypes arising from mating Ts65Dn and Dscr1^+/− mice. (b) Suppression of B16F10 tumor growth in Ts65Dn Down syndrome mice is relieved upon loss of the third copy of Dscr1 (Ts65Dn-Dscr1^+/−). Values are mean± sem, n=4–8 per group, *p<0.01. (c) Microvessel density (MVD) per high-powered field (hpf) is quantified by anti-CD31 immunofluorescence of tumors harvested from the indicated mice at comparable volumes (200–400 mm³). Bar, 20 µM. Values are mean ± sem.

Figure 4. Calcineurin suppression by DSCR1 and DYRK1A attenuates endothelial cell activation

(a, b) Cyclooxygenase-2 (COX-2) mRNA (a) and protein expression (b) was quantified by qPCR and Western blot analysis in endothelial cells isolated from Dscr1 wild-type (WT) and transgenic (Tg) mice. (c) Western blot analysis of endothelial cells isolated from Dscr transgenic mice and probed for DYRK1A after either mock infection (mock) or retroviral infection with Dyrk1A (DYRK1A). (d) Endothelial cells isolated from Dscr1 transgenic (Tg) mice with DYRK1A over-expression (+DYRK1A) demonstrate an even greater suppression of VEGF-mediated proliferation as compared to wild-type (WT) and DSCR1 over-expression alone (mock). Values are mean ± sem, *p<0.01.