Abstract IK depletion leads to an aberrant mitotic entry because of chromosomal misalignment through the enhancement of Aurora B activity at the interphase. Here, we demonstrate that IK, a spliceosomal component, plays a crucial role in the proper splicing of the ATM pre-mRNA among other genes related with the DNA Damage Response (DDR). Intron 1 in the ATM pre-mRNA, having lengths <200 bp, was not spliced in the IK-depleted cells and led to a deficiency of the ATM protein. Subsequently, the IK depletion-induced ATM protein deficiency impaired the ability to repair the damaged DNA. Because the absence of SMU1 results in IK degradation, the mechanism underlying IK degradation was exploited. IK was ubiquitinated in the absence of SMU1 and then subjected to proteolysis through the 26S proteasome. To prevent the proteolytic degradation of IK, a deubiquitinating enzyme, USP47, directly interacted with IK and stabilized it through deubiquitination. Collectively, our results suggest that IK is required for proper splicing of the ATM pre-mRNA and USP47 contributes toward the stabilization of IK.
Abstract Background Melanoma-associated antigen C2 (MAGEC2) is an oncogene associated with various cancer types. However, the biological function of MAGEC2 in circulating tumor cells is unclear. In this study, we investigated the role of MAGEC2 using adapted suspension cells (ASCs), which were previously developed to study circulating tumor cells (CTCs). Methods Differential gene expression between adherent cells (ADs) and ASCs was examined using RNA-seq analysis. MAGEC2 expression was assessed using RT-qPCR, immunoblotting, and ChIP-seq analysis. Depletion of MAGEC2 expression was carried out using siRNA. MAGEC2-depleted ADs and ASCs were used to investigate the change in proliferation rate and cell cycle. Then, the protein levels of STAT3, phosphorylated STAT3, and downstream of STAT3 were measured using control and MAGEC2-depleted ADs and ASCs. The direct effect of active STAT3 inhibition with Stattic in ASCs was also assessed in terms of proliferation and apoptosis. Finally, an Annexin V/7-AAD assay was performed to determine the percentage of apoptotic cells in Stattic-treated cells. Results MAGEC2 was highly expressed in ASCs compared to ADs. Depletion of MAGEC2 reduced the proliferation rate and viability of ASCs. To elucidate the underlying mechanism, the level of STAT3 was examined because of its oncogenic properties. Tyrosine-phosphorylated active STAT3 was highly expressed in ASCs and decreased in MAGEC2-depleted ASCs. In addition, when ASCs were treated with Stattic, an active STAT3 inhibitor, they were more sensitive to intrinsic pathway-mediated apoptosis. Conclusions High expression of MAGEC2 may play an important role in the survival of ASCs by maintaining the expression of activated STAT3 to prevent apoptotic cell death.
Melanoma-associated antigen C2 (MAGEC2) is an oncogene associated with various types of cancers. However, the biological function of MAGEC2 in circulating tumor cells remains unclear. In this study, we investigated the role of MAGEC2 using adapted suspension cells (ASCs), which were previously developed to study circulating tumor cells (CTCs).Differential gene expression in adherent cells (ADs) and ASCs was examined using RNA-seq analysis. MAGEC2 expression was assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and ChIP-seq analysis. Depletion of MAGEC2 expression was performed using siRNA. MAGEC2-depleted ADs and ASCs were used to investigate changes in the proliferation rate and cell cycle. Then, the protein levels of signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3, and downstream of STAT3 were measured using control and MAGEC2-depleted ADs and ASCs. In ASCs, the direct effect of active STAT3 inhibition with Stattic, a STAT3 inhibitor, was assessed in terms of proliferation and apoptosis. Finally, an Annexin V/7-AAD assay was performed to determine the percentage of apoptotic cells in the Stattic-treated cells.MAGEC2 was highly expressed in ASCs when compared with ADs. Depletion of MAGEC2 reduced the proliferation rate and viability of ASCs. To elucidate the underlying mechanism, the level of STAT3 was examined owing to its oncogenic properties. Tyrosine-phosphorylated active STAT3 was highly expressed in ASCs and decreased in MAGEC2-depleted ASCs. Furthermore, on treating ASCs with Stattic, an active STAT3 inhibitor, the cells were markedly sensitive to intrinsic pathway-mediated apoptosis.High MAGEC2 expression may play an important role in the survival of ASCs by maintaining the expression of activated STAT3 to prevent apoptotic cell death.
Cancer metastasis is the primary cause of cancer-related death and metastatic cancer has circulating-tumor cells (CTCs), which circulate in the bloodstream before invading other organs. Thus, understanding the precise role of CTCs may provide new insights into the metastasis process and reduce cancer mortality. However, the molecular characteristics of CTCs are not well understood due to a lack of number of CTCs. Therefore, suspension cells were generated from MDA-MB-468 cells to mimic CTCs, and we investigate the microRNA (miRNA)-dependent molecular networks and their role in suspension cells. Here, we present an integrated analysis of mRNA and miRNA sequencing data for suspension cell lines, through comparison with adherent cells. Among the differentially regulated miRNA–mRNAs axes, we focus on the miR-146a-Neuropilin2 (NRP2) axis, which is known to influence tumor aggressiveness. We show that miR-146a directly regulates NRP2 expression and inhibits Semaphorin3C (SEMA3C) signaling. Functional studies reveal that miR-146a represses SEMA3C-induced invasion and proliferation by targeting NRP2. Finally, high-NRP2 is shown to be associated with poor outcomes in breast cancer patients. This study identifies the key role of the miR-146a–NRP2 signaling axis that is critical for the regulation of migration and invasion in CTC-mimicking cells.
Abstract Background Lymphocyte antigen 6 complex locus K (LY6K), a glycosylphosphatidylinositol‐anchored protein, plays a dynamic role in cancer metastasis. In the current study, we deciphered the effects of LY6K on transforming growth factor‐β (TGF‐β) and epidermal growth factor (EGF) signaling through clathrin‐ and caveolin‐1 (CAV‐1)‐mediated endocytosis. Methods Analysis of the TCGA and GTEx dataset were performed to explore the expression and survival of LY6K in cancer patients. Short interfering RNA (siRNA) was used to knockdown the expression of LY6K in human cervical cancer patients. The effect of lack of LY6K on cell proliferation, migration, and invasion was performed, and RT‐qPCR and immunoblotting were performed to identify LY6K‐affected TGF‐β and EGF signaling pathways. Additionally, Immunofluorescence (IF) and transmission electron microscope (TEM) were performed to identify the role of LY6K in CAV‐1‐ and Clathrin‐mediated endocytosis. Results Lymphocyte antigen 6 complex locus K expression level is elevated in higher grade cervical cancer patients correlating with poor overall survival, progression‐free survival, and disease‐free survival. LY6K‐depletion in HeLa and SiHa cancer cells suppressed EGF‐induced proliferation and TGF‐β‐enhanced migration and invasion. Both TGF‐β receptor‐I (TβRI) and EGF receptor (EGFR) localized at the plasma membrane regardless of LY6K expression, and LY6K bound TβRI irrespective of the presence of TGF‐β; however, LY6K did not bind EGFR. LY6K‐depleted cells showed impaired Smad2 phosphorylation upon TGF‐β treatment and lower proliferation rates following long‐term treatment with EGF. We revealed the atypical movement of TβRI and EGFR from plasma membrane upon ligand stimulation in LY6K‐depleted cells and an impaired movement of the endocytic proteins clathrin and CAV‐1. Conclusions Our study demonstrates the key role of LY6K in both clathrin‐ and CAV‐1‐mediated endocytic pathways regulated by TGF‐β and EGF, and it suggests a correlation between LY6K overexpression in cervical cancer cells and poor overall survival.
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