Histone Deacetylase Inhibitors Inhibit Metastasis By Restoring a Tumor Suppressive microRNA, Mir-150 in Advanced Cutaneous T-Cell Lymphoma

Background: Various types of histone deacetylase inhibitor (HDACi) are currently under development or being used for non-Hodgkin's lymphomas including cutaneous T-cell lymphoma (CTCL). However, there is little molecular-based evidence to support the proposed efficacy of HDACi against advanced CTCL or information on the possible pivotal target genes of HDACi. We have shown that a tumor suppressive microRNA (miRNA), miR-150 inhibits metastasis via combining "seed sequence" of messenger RNA of a chemokine receptor CCR6 in advanced CTCL (Blood 2014). Because HDACi yielded excellent outcome for treating advanced CTCL, HDACi might have a potential to reduce metastasis capability of CTCL via targeting miR-150 and CCR6. Indeed we previously showed that a tumor suppressive miRNA, miR-16 was epigenetically downregulated and HDACi restored its expression leading to induce cellular senescence or apoptosis in various T-cell lymphomas (Oncogene 2015). This led us to consider that HDACis might upregulate variety of tumor suppressive miRNAs and there might be deep association of restoration for some critical gene(s) or/and miRNA(s) expression with HDACis in CTCL oncogenesis. In this study, we focused on to investigate relationship between miRNAs and HDACis, especially focused on to detect CCR6 associated miRNA(s).

Methods: My-La, HH, and HUT78 (CTCL cell lines) were used for functional analysis because their xenografts in NOD/Shi-scid IL-2γnul mice (CTCL mice) shortened survival, owing to multiple metastases. To investigate changes of miRNA expression by HDACis, we conducted miRNA microarray analysis. To examine the expression of miR-150 in primary CTCL, we conducted a qRT-PCR analysis of samples from early (patch and/or plaque phase, n = 26) and advanced cases (tumor phase, n = 14). The control samples were obtained from patients with atopic dermatitis (AD, n = 18).

Results: We demonstrated that pan-HDACis (vorinostat and panobinostat) inhibited migration of CTCL cells with downregulating CCR6. When we examined miRNA array against CTCL cells treated with and without respective HDACi, 161 miRNAs were commonly upregulated by pan-HDACis in My-La, HH and HUT78. Among these 161 miRNAs, 35 miRNAs that possesses seed sequence of CCR6 showed upregulation by HDACis treatment (e.g., miR-150, miR-96, miR-183, miR-185, miR-194, miR-320a, miR-371a, miR-3135b). To examine the miRNAs that has a functional role in CTCL metastasis, we conducted northern blot analysis of these candidate miRNAs in the CTCL cell lines and primary tissue samples. We found that the miR-150 was strongly expressed in normal CD4+ cells with downregulation of advanced CTCL samples, but the others showed very low or no expressions. In addition, the transduction of candidate miRNAs against the CTCL cells revealed that miR-150 downregulated CCR6 and inhibited their migration most efficiently. These data strongly suggest that the most likely target miRNA of the pan-HDACis in metastasis inhibition was the miR-150 in the metastatic CTCL. To examine whether there was a change in miR-150 expression during disease progression of primary CTCL, we conducted a qRT-PCR analysis of samples from early and advanced cases. The qRT-PCR demonstrated that the expression of miR-150 was significantly lower in the advanced specimens than it was in samples from patients with AD. The miR-150 levels did not significantly differ between the early and the AD specimens, but a significant difference was detected between the early and advanced specimens. These results suggest that miR-150 expression declines with disease progression in CTCL. Finally, we conducted in vivo experiment to examine whether miR-150 indeed inhibit tumor metastasis. We demonstrated that administration of miR-150 against CTCL model mice led to prolong their survivals.

Conclusion: miR-150 and CCR6 are essential targets of pan-HDACi in advanced CTCL. Our results provide rational reasons for using pan-HDACi against metastatic CTCL. Furthermore, these results suggest that miR-150 could be not only powerful biomarker for molecular diagnosis, and predictive of metastasis, but also novel therapeutic molecule in advanced CTCL.