IL-24 modulates the high mobility group (HMG) A1/miR222 /AKT signaling in lung cancer cells

Abstract

Interleukin (IL)-24, a novel tumor suppressor/cytokine exhibits antitumor activity against a broad-spectrum of human cancer cells. In a recent study, we showed that IL-24 inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive.The high mobility group (HMG) A1 a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunit B (PPP2R2A) via the oncogenic micro (mi) RNA-222. Based on this report we hypothesized that IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis.To test our hypothesis, in the present study we used a H1299 lung cancer cell line that expressed exogenous human IL-24 when induced with doxycycline (DOX). Induction of IL-24 expression in the tumor cells markedly reduced HMGA1 mRNA and protein levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKTT308/S473 expression. SiRNA-mediated knockdown of HMGA1 in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression.Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. Thus, a therapy combining IL-24 with HMGA1 siRNA or miR-222-3p inhibitor should present effective treatment of lung cancer.

Tumor-targeted Nanoparticle Delivery of HuR siRNA Inhibits Lung Tumor Growth In Vitro and In Vivo By Disrupting the Oncogenic Activity of the RNA-binding Protein HuR

“…Total cell lysates prepared from cultured cells or tumor tissues receiving various treatments were prepared using RIPA buffer and were subjected to western blot analysis as previously described [24, 29, 30]. Primary antibodies against human HuR, Bcl2, Cyclin D1, Cyclin E, and p27 (Santa Cruz Biotechnology, Dallas, TX); caspase 9 and PARP (Cell Signaling, Cambridge, MA), and beta actin (Sigma Chemicals) were all purchased and used as recommended by the manufacturers.…”

“…Primary antibodies against human HuR, Bcl2, Cyclin D1, Cyclin E, and p27 (Santa Cruz Biotechnology, Dallas, TX); caspase 9 and PARP (Cell Signaling, Cambridge, MA), and beta actin (Sigma Chemicals) were all purchased and used as recommended by the manufacturers. Proteins were detected using the appropriate horseradish peroxidase- (HRP)-tagged secondary antibodies and differences in protein expression determined as previously described [24, 29, 30]. …”

“…The cDNA was subsequently used to perform qRT-PCR (Bio-Rad CFX96 Touch Real-Time PCR Detection System) with SYBR chemistry using iQTM SYBR Green super mix (Bio-Rad). The relative gene expression values were quantified by the 2 −ΔΔCT method [30, 31]. …”

“…The cell migration and invasion assay was carried out as previously described [12, 24, 30]. Briefly, C-TfNP and HuR-TfNP-treated A549 cell were seeded in the upper chambers of the migration and invasion inserts.…”

“…The lower chamber was filled with 20% FBS-containing culture medium. After 24 h and 48 h of incubation, the number of migrated and invaded cells were counted and the results represented as previously described [24, 30]. …”