Epigenetic regulation of embryonic stem cell marker miR302C in human chondrosarcoma as determinant of antiproliferative activity of proline-rich polypeptide 1

Abstract

Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. We described recently that tumor growth inhibiting cytostatic proline-rich polypeptide 1, (PRP-1) significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocytes culture. In this study we hypothesized the existence and regulation of a functional marker in cancer stem cells, correlated to peptides antiproliferative activity. Experimental results indicated that among significantly downregulated miRNA after PRP-1treatment was miRNAs 302c*. This miRNA is a part of the cluster miR302-367, which is stemness regulator in human embryonic stem cells and in certain tumors, but is not expressed in adult hMSCs and normal tissues. PRP-1 had strong inhibitory effect on viability of chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type). Unlike chondrosarcoma, in glioblastoma, PRP-1 does not have any inhibitory activity on cell proliferation, because in glioblastoma miR-302-367 cluster plays an opposite role, its expression is sufficient to suppress the stemness inducing properties. The observed correlation between the antiproliferative activity of PRP-1 and its action on downregulation of miR302c explains the peptides opposite effects on the upregulation of proliferation of adult mesenchymal stem cells, and the inhibition of the proliferation of human bone giant-cell tumor stromal cells, reported earlier. PRP-1 substantially downregulated the miR302c targets, the stemness markers Nanog, c-Myc and polycomb protein Bmi-1. miR302c expression is induced by JMJD2-mediated H3K9me2 demethylase activity in its promoter region. JMJD2 was reported to be a positive regulator for Nanog. Our experimental results proved that PRP-1 strongly inhibited H3K9 activity comprised of a pool of JMJD1 and JMJD2. We conclude that inhibition of H3K9 activity by PRP-1 leads to downregulation of miR302c and its targets, defining the PRP-1 antiproliferative role.

Effect of cytostatic proline rich polypeptide-1 on tumor suppressors of inflammation pathway signaling in chondrosarcoma

“…It is known that H3K9 methylation is eliminated from the promoters of inflammation-associated genes (44), and, therefore the mechanisms underpinning the inhibition of H3K9 demethylases merit further attention. The ability of PRP-1 to inhibit H3K9 demethylase activity was previously reported by our group (8), and a possible mechanism of the peptide's function in the upregulation of tumor suppressors and in the downregulation of oncoproteins was proposed. In the present study, the PRP-1-mediated inhibition of H3K9 demethylase was associated with the process of inflammation, and the IC 50 value has been demonstrated.…”

“…The upregulation of SOCS3, TET1 and TET2 expression in a dose-dependent manner by PRP-1 further demonstrates the ability of this cytokine peptide to upregulate tumor suppressor genes in general (8)(9)(10). Even though PRP-1 did not exert any effect on tumor suppressors or oncoproteins of the Hippo or Hedgehog signaling pathways, there was an important conclusion to consider: The inhibition of tumor suppressors by PRP-1 depends on the pathway these tumor suppressors represent or are involved in.…”

“…Understanding the differences in their expression patterns between the normal and malignant states, and the signaling pathways that lead to these differences, require further investigation as important targets for future therapeutic interventions. Metastatic chondrosarcoma does not respond to conventional therapies and the search for new therapeutic approaches is urgent (2)(3)(4)(5)(6)(7)(8)(9)(10)36,46). The ability of PRP-1 as an antiproliferative agent to restore the expression of anti-inflammatory cytokines with tumor suppressor function proves the importance of this neuropeptide for future clinical consideration.…”

“…The effect of the mammalian target of rapamycin complex 1 (mTORC1) inhibitor and anti-proliferative immunomodulator, proline-rich polypeptide 1 (PRP-1) (3-7), on the expression of cytokines, tumor suppressors SOCS3, TET1/2 and oncoproteins of inflammatory oncogenic pathways, including the Hedgehog and Hippo pathways, was also investigated. PRP-1 is a powerful upregulator of tumor suppressor microRNAs (miRNAs) and proteins, and a downregulator of oncoproteins, as reported in previous publications from our laboratory (8)(9)(10). Ten eleven translocation (TET) enzymes, a family of α-ketoglutarate (α-KG)-dependent dioxygenases, catalyse the oxidative reactions of 5-methylcytosine (5mC) to promote the DNA demethylation process.…”

Epigenetic control of cancer by neuropeptides

“…miR302C, a component of the miR302 367 cluster, was significantly downregulated by PRP-1. Notably, this cluster is not expressed in adult mesenchymal stem cells and normal tissue (28), although it is particularly characteristic, with a significant role in tumors and human embryonic stem cells, where it regulates renewal of stem cells and the process of differentiation (29). This may explain the strong tumor growth inhibitory role of PRP-1 in certain tumors, particularly in chondrosarcomas and multipotent adult stem cells (marrow-isolated adult multilineage inducible cells) of embryonic primitive type (28,30).…”