5-Azacytidine inhibits human rhabdomyosarcoma cell growth by downregulating insulin-like growth factor 2 expression and reactivating the H19 gene product miR-675, which negatively affects insulin-like growth factors and insulin signaling

Abstract

Abstract. insulin-like growth factor 2 (igf2) and 1 (igf1) and insulin (inS) promote proliferation of rhabdomyosarcoma (rMS) cells by interacting with the insulin-like growth factor 1 receptor (igf1r) and the insulin receptor (inSr). loss of imprinting (loi) by Dna hypermethylation at the differentially methylated region (DMr) for the igf2-H19 locus is commonly observed in rMS cells and results in an increase in the expression of proliferation-promoting igf2 and downregulation of proliferation-inhibiting non-coding H19 mirnas. one of these mirnas, mir-675, has been reported in murine cells to be a negative regulator of igf1r expression. to better address the role of igf2 and 1, as well as inS signaling in the pathogenesis of rMS and the involvement of loi at the igf2-H19 locus, we employed the Dna demethylating agent 5-azacytidine (azac). We observed that azac-mediated demethylation of the DMr at the igf2-H19 locus resulted in downregulation of igf2 and an increase in the expression of H19. this epigenetic change resulted in a decrease in rMS proliferation due to downregulation of igf2 and, igf1r expression in an mir-675-dependent manner. interestingly, we observed that mir-675 not only inhibited the expression of igf1r in a similar manner in human and murine cells, but we also observed its negative effect on the expression of the INSR. These results confirm the crucial role of loi at the igf2-H19 DMr in the pathogenesis of rMS and are relevant to the development of new treatment strategies.

Insulin-Like Growth Factor 2 in Physiology, Cancer, and Cancer Treatment

“…Since the maternal ICR in IGF2 gene is not methylated and as a consequence of 5-azacytidine treatment, ICR on the paternal IGF2 becomes hypomethylated and IGF2 expression from the paternal gene would be strongly inhibited [149]. In line with this, 5azacytidine treatment inhibited rhabdomyosarcoma cell growth through repression of IGF2 expression and re-expression of H19 in vitro [150]. Additionally, it has been shown that IGF2 can maintain cancer stem cell populations in breast cancer [151] and HCC [152].…”

Parentally imprinted genes regulate hematopoiesis—new evidence from the Dlk1–Gtl2 locus

“…Because paternally expressed imprinted genes (Igf2 and Rasgrf1) enhance embryonic growth, and maternally expressed genes (H19, p57 KIP2 , and Igf2r) inhibit cell proliferation, the unique genomic imprinting pattern observed in VSELs demonstrates the growth-repressive influence of imprinted genes, including the Igf2-H19 locus, on the proliferation of these cells. One miRNA derived from the H19 ncRNA has been demonstrated to inhibit expression of the signaling receptor for Igf2, the insulin-like growth factor 1 receptor (Igf1r) (8), and in addition we have proposed the same phenomenon for the insulin receptor itself (17). Therefore, VSELs are in a quiescent state because of attenuation of insulin/insulinlike growth factor signaling (18,19).…”

Picropodophyllin (PPP) is a potent rhabdomyosarcoma growth inhibitor both in vitro and in vivo

“…The expression of IGF1R, InsR in RMS cell lines was evaluated by flow cytometry as previously described [ 24 ]. Briefly, the receptor expression was assayed with phycoerythrin (PE)-anti-IGF1R monoclonal antibody Clone 33,255 and anti–human/mouse Insulin R/CD220 conjugated with APC (R&D Systems).…”