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. 2019 Mar 19;116(12):5687-5692.
doi: 10.1073/pnas.1819869116. Epub 2019 Mar 6.

MDA-7/IL-24 regulates the miRNA processing enzyme DICER through downregulation of MITF

Anjan K Pradhan  1 , Praveen Bhoopathi  1 , Sarmistha Talukdar  1 , Danielle Scheunemann  1 , Devanand Sarkar  1   2   3 , Webster K Cavenee  4 , Swadesh K Das  1   2   3 , Luni Emdad  1   2   3 , Paul B Fisher  5   2   3
Affiliations

MDA-7/IL-24 regulates the miRNA processing enzyme DICER through downregulation of MITF

Anjan K Pradhan et al. Proc Natl Acad Sci U S A. .

Abstract

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a multifunctional cytokine displaying broad-spectrum anticancer activity in vitro or in vivo in preclinical animal cancer models and in a phase 1/2 clinical trial in patients with advanced cancers. mda-7/IL-24 _targets specific miRNAs, including miR-221 and miR-320, for down-regulation in a cancer-selective manner. We demonstrate that mda-7/IL-24, administered through a replication incompetent type 5 adenovirus (Ad.mda-7) or with His-MDA-7/IL-24 protein, down-regulates DICER, a critical regulator in miRNA processing. This effect is specific for mature miR-221, as it does not affect Pri-miR-221 expression, and the DICER protein, as no changes occur in other miRNA processing cofactors, including DROSHA, PASHA, or Argonaute. DICER is unchanged by Ad.mda-7/IL-24 in normal immortal prostate cells, whereas Ad.mda-7 down-regulates DICER in multiple cancer cells including glioblastoma multiforme and prostate, breast, lung, and liver carcinoma cells. MDA-7/IL-24 protein down-regulates DICER expression through canonical IL-20/IL-22 receptors. Gain- and loss-of-function studies confirm that overexpression of DICER rescues deregulation of miRNAs by mda-7/IL-24, partially rescuing cancer cells from mda-7/IL-24-mediated cell death. Stable overexpression of DICER in cancer cells impedes Ad.mda-7 or His-MDA-7/IL-24 inhibition of cell growth, colony formation, PARP cleavage, and apoptosis. In addition, stable overexpression of DICER renders cancer cells more resistant to Ad.mda-7 inhibition of primary and secondary tumor growth. MDA-7/IL-24-mediated regulation of DICER is reactive oxygen species-dependent and mediated by melanogenesis-associated transcription factor. Our research uncovers a distinct role of mda-7/IL-24 in the regulation of miRNA biogenesis through alteration of the MITF-DICER pathway.

Keywords: DICER; MITF; ROS; mda-7/IL-24; miRNA.

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Conflict of interest statement

Conflict of interest statement: W.K.C. and P.B.F. are cofounders of InterLeukin Combinatorial Therapies, Inc. (ILCT). W.K.C., P.B.F., and Virginia Commonwealth University own stock in ILCT. L.E. is the principal investigator of a sponsored research agreement provided by ILCT to Virginia Commonwealth University. The other authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
mda-7/IL-24 down-regulates mature miR-221, but not pri-miR-221. Real-time quantitative PCR of cancer cells [DU-145 (A), MDA-MB-231 (B), A549 (C), and GBM6 (D)] infected with Ad.mda-7 (2,000 vp/cell for 72 h) indicates significantly less miR-221. miR-221 expression does not change in RWPE-1 cells (A) after infection with Ad.mda-7. No significant change in primary-miR-221 occurs after Ad.mda-7 infection.
Fig. 2.
Fig. 2.
mda-7/IL-24 down-regulates DICER protein. Effect of mda-7/IL-24 on DICER, DROSHA, DGCR8 (PASHA), Ago2, Beclin-1, and GRP78 protein levels determined by Western blotting. RWPE-1 and DU-145 (A), MDA-MB-231 and A549 (B) cells were infected with Ad.null or Ad.mda-7 (2,000 vp/cell) for 72 h. Cells were lysed, and Western blotting was done. EF1α was used as a loading control.
Fig. 3.
Fig. 3.
DICER overexpression rescues miRs regulated by mda-7/IL-24. DU-145 (A) and MDA-MB-231 (B) cells were transfected with DICER overexpressing plasmid and infected with Ad.mda-7. miR-221, miR-320, and miR-451 levels were monitored by real-time quantitative PCR. Treatment with Ad.mda-7 significantly down-regulated miR-221 and miR-320, which were rescued in DICER-transfected cells. The level of miR-451, which is a DICER-independent miRNA, does not change with either Ad.mda-7 infection or DICER overexpression.
Fig. 4.
Fig. 4.
ROS-dependent regulation of DICER by mda-7/IL-24. RWPE-1 and DU-145 (A), MDA-MB-231, and A549 (B) cells were pretreated with 10 mM NAC and infected with either Ad.null or Ad.mda-7 (2,000 vp/cell for 72 h). Western blotting was done with DICER antibody. EF1α was used as a loading control. (C) Bar graph representation of quantitative ROS levels.
Fig. 5.
Fig. 5.
DICER overexpression rescues cancer cells from mda-7/IL-24-mediated cell death. DU-145 (A) and MDA-MB-231 (B) cells were transfected with pCDNA3.1 (control) or DICER and then infected with Ad.null or Ad.mda-7. MTT assays showed that treatment with mda-7/IL-24 resulted in an inhibition in proliferation of DU-145 and MDA-MB-231 cells, which is less in DICER-overexpressing cells. DICER overexpression also rescues inhibition in colony formation in DU-145 (A) and MDA-MB-231 (B) cells treated with Ad.mda-7. Data presented as mean ± SD. DU-145 (C) and MDA-MB-231 (D) cells were transfected with pCDNA3.1 (control) or DICER and then infected with Ad.null or Ad.mda-7 (2,000 vp/cell). After 72 h, cells were lysed and Western blotting was done with PARP, DICER, and MDA-7/IL-24 antibody. Actin was used as a loading control. DU-145 (E) and MDA-MB-231 (F) cells were transfected with pCDNA3.1 (control) or DICER and then treated with Ad.null or Ad.mda-7. After 72 h, cells were stained with Annexin-V/PI and then analyzed by flow cytometry.
Fig. 6.
Fig. 6.
In vivo xenograft study showing DICER-mediated cell death by mda-7/IL-24. DU-145 cells, stably expressing a control pCDNA3.1 vector or DICER, were s.c. implanted in both flanks of athymic male nude mice. Left-sided tumors were injected with eight intratumoral injections of Ad.null or Ad.mda-7 (1 × 108 vp). Once the control tumors reached maximum allowable limits, animals were killed and tumors were isolated. (A) Graphical representation of the tumor weight on both flanks. (B) Tumor volumes on both flanks were measured, and results are presented in a graphical manner. (C) Immunohistochemical analysis of MDA-7/IL-24 and DICER in tumor sections (Left, injected/treated tumors; Right, uninjected/untreated tumors).
Fig. 7.
Fig. 7.
mda-7/IL-24-mediated DICER regulation is controlled by the transcription factor MITF. mda-7/IL-24 down-regulates MITF in different cancer cell lines (A and B), but not in normal RWPE-1 cells (A). DU-145 (C) and MDA-MB-231 cells (D) were transfected with vector or MITF, and then treated with Ad.null or Ad.mda-7. RNA was isolated 72 h postinfection, and real-time quantitative PCR was done to check the level of DICER. DU-145 (E) and MDA-MB-231 cells (F) were treated as described in C and D, total protein was isolated, and Western blotting was done with DICER and MITF antibodies. Actin was used as a loading control. (G) Schematic representation of regulation of the miRNA processing enzyme DICER by mda-7/IL-24. MDA-7/IL-24 down-regulates the transcription factor MITF in a ROS-dependent manner, which in turn down-regulates DICER.
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