Microrna-16 Mediates the Regulation of a Senescence-Apoptosis Switch in Cutaneous T-Cell and Other Non-Hodgkin Lymphomas
Authors: Kitadate Akihiro , Ikeda Sho , Teshima Kazuaki , Ito Mitsugu , Takahashi Naoto , Miyagaki Tomomitsu , Sugaya Makoto , Tagawa Hiroyuki 
Published: October 2019

Multiple sequential genetic and epigenetic alterations, including alterations in microRNAs (miRNAs), underlie the development and progression of cancers. For example, overcoming cellular senescence is an early step in cancer pathogenesis. We show here that a tumor-suppressive miRNA, microRNA-16/miR-16 has a potential to induce cellular senescence in cutaneous T-cell lymphoma (CTCL). Firstly, to examine the role of miR-16 in the progression of primary CTCL, we performed q-PCR analysis against early (n=26) and advanced (n=14) Mycosis Fungoides samples. As for control (CD4+ T-cells), we used samples from patients with atopic dermatitis (AD; n=18). We found that expression of miR-16 in both early and advanced stages were significantly lower than AD, with the expression of advanced showing significantly more reduced than early stage. These results suggest that miR-16 is associated with lymphomagenesis from early to advanced stages of CTCL. To examine in vivo tumor-suppressive effects of miR-16, we used NOD/Shi-scid IL2Rgnull (NOG) mouse xenograft model, which would die around day 30-40 after CTCL cells (My-La, MJ, HH or HUT78) injection by invasion and metastasis (Ito et al, Blood 2014). By use of the xenograft model, we found that miR-16 transduced CTCL cells transplanted NOG mice showed significant prolonged survivals than control xenograft mice. We previously demonstrated that miR-16 did not inhibit migration capability of CTCL cells. When we considered that miR-16 successfully contribute to extend survival of mice models despite of lacking migration-inhibition capability, the result suggested that other tumor suppressive mechanisms might exist. Therefore we conducted functional analysis of miR-16, and found that miR-16 could induce cellular senescence against CTCL cells. In CTCL cells that expressed wild-type p53 (My-La and MJ), forced expression of miR-16 enhanced p21, which is a key mediator of senescence, in a p53 dependent manner. Because miR-16 family members are also able to directly suppress the polycomb group protein Bmi1, which is known to negatively regulate p21, we assessed Bmi1 expression in miR-16-transduced CTCL cells and found it to be diminished in all four CTCL cell lines tested. When we knocked down BMI1, expression of p21 was elevated in cells expressing wild-type p53 and the incidence of senescence was increased. We further demonstrated that Bmi1 could directly interact with the promoter lesion of CDKN1A/p21 by cross-linking/chromatin immunoprecipitation assays. From these results, miR-16 has potential to induce cellular senescence via targeting Bmi1-p21 pathway in p53-wild type CTCL cells. Although miR-16 could not induce senescence in p53 non-functional CTCL cells (HH and HUT78), in that case, we found that miR-16 can compensatory induce apoptosis by negative regulation of Bmi1-Survivin pathway. To elucidate this senescence-apoptosis switch mechanism, we examined co-transfection of miR-16 and siCDKN1A/p21 against p53 wild-type CTCL cells. We found that co-transfection of miR-16 and siCDKN1A significantly decreased senescent cells and increased apoptotic cells, with downregulation of Bmi1 and Survivin. In addition, miR-16 negatively regulated cyclinD1-Rb pathway regardless of p53 status, leading to cell growth inhibition against CTCL cells. Together, these results strongly support our idea that presence or absence of functional p53-p21 is an essential factor in miR-16-mediated senescence-to-apoptosis switch in CTCL. Finally, because a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA, vorinostat) has been recently applied and marks good responses in advanced CTCL, we tried to examine whether SAHA might share essential targets with miR-16 in CTCL lymphomagenesis. We found that SAHA restored miR-16 and its essential targets (p21, Bmi1, Survivin), and induced senescence in CTCL cells with wild-type p53 and apoptosis in cells with non-functional p53. Moreover, we found that T or NK/T-cell lymphoma cells (n=8) derived from various subtypes also showed similar tumor-suppressive effects by miR-16 transduction or SAHA treatment. In conclusion, through detailed study of miR-16 function, we have shed light on the mechanism by which lymphoma cells avoid senescence. Furthermore, identification of essential SAHA targets may provide supporting evidence of its clinical application for various non-Hodgkin T/NK-cell lymphomas.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.