Cancer/testis antigen CAGE mediates osimertinib resistance in non-small cell lung cancer cells and predicts poor prognosis in patients with pulmonary adenocarcinoma.

CAGE, a cancer/testis antigen, was originally isolated from the sera of patients with gastric cancers. Previously, we have shown the role of CAGE in resistance to chemotherapy and target therapy. The aim of this study was to investigate the role of CAGE in osimertinib resistance and determine the prognostic value of CAGE in patients with pulmonary adenocarcinomas. The clinicopathological correlation with CAGE and autophagy flux in patients was examined using immunohistochemistry and in situ hybridization. The possible role of autophagy in osimertinib resistance was analyzed using immune blot, immune fluorescence staining and immunohistochemistry. This study found that immunohistochemical staining (IHC) showed CAGE expression in more than 50% of patients with pulmonary adenocarcinomas (pADCs). CAGE expression was increased in pADCs after the acquisition of EGFR-TKIs resistance. High expression of CAGE was correlated with shorter overall survival and progression free survival in patients with pADCs. Thus, CAGE mediates osimertinib resistance and predicts poor prognosis in patients with pADCs. Osimertinib-resistant non-small cell lung cancer cells (PC-9/OSI) were established and mechanistic studies of CAGE-mediated osimertinib resistance were performed. PC-9/OSI cells showed increased autophagic flux and CAGE expression compared with parental sensitive PC-9 cells. PC-9/OSI cells showed higher tumorigenic, metastatic, and angiogenic potential compared with parental PC-9 cells. CAGE CRISPR-Cas9 cell lines showed decreased autophagic flux, invasion, migration potential, and tumorigenic potential compared with PC-9/OSI cells in vitro and in vivo. CAGE plays a crucial role in the cancer progression by modulating autophagy and can predict the poor prognosis of patients with pulmonary adenocarcinomas. Our findings propose CAGE as a potential therapeutic target for developing anticancer drugs that can overcome osimertinib resistance.

Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression.

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

The CAGE-MiR-181b-5p-S1PR1 Axis Regulates Anticancer Drug Resistance and Autophagy in Gastric Cancer Cells.

Cancer-associated gene (CAGE), a cancer/testis antigen, has been known to promote anticancer drug resistance. Since the underlying mechanisms of CAGE-promoted anticancer drug resistance are poorly understood, we established Anticancer drug-resistant gastric cancer cells (AGS R ) to better elucidate possible mechanisms. AGS R showed an increased expression level of CAGE and autophagic flux compared with anticancer drug-sensitive parental gastric cancer cells (AGS cells). AGS R cells showed higher invasion potential, growth rate, tumor spheroid formation, and angiogenic potential than AGS cells. CAGE exerted effects on the response to anticancer drugs and autophagic flux. CAGE was shown to bind to Beclin1, a mediator of autophagy. Overexpression of CAGE increased autophagic flux and invasion potential but inhibited the cleavage of PARP in response to anticancer drugs in CAGE CRISPR-Cas9 cell lines. TargetScan analysis was utilized to predict the binding of miR-302b-5p to the promoter sequences of CAGE, and the results show that miR-302b-5p directly regulated CAGE expression as illustrated by luciferase activity. MiR-302b-5p regulated autophagic flux and the response to anticancer drugs. CAGE was shown to bind the promoter sequences of miR-302b-5p. The culture medium of AGS R cells increased CAGE expression and autophagic flux in AGS cells. ImmunoEM showed CAGE was present in the exosomes of AGS R cells; exosomes of AGS R cells and human recombinant CAGE protein increased CAGE expression, autophagic flux, and resistance to anticancer drugs in AGS cells. MicroRNA array revealed miR-181b-5p as a potential negative regulator of CAGE. MiR-181b-5p inhibitor increased the expression of CAGE and autophagic flux in addition to preventing anticancer drugs from cleaving poly(ADP-ribose) polymerase (PARP) in AGS cells. TargetScan analysis predicted sphingosine 1-phosphate receptor 1 (SIPR1) as a potential target for miR-181b-5p. CAGE showed binding to the promoter sequences of S1PR1. The downregulation or inhibition of S1PR1 led to decreased autophagic flux but enhanced the sensitivity to anticancer drugs in AGS R cells. This study presents a novel role of the CAGE-miR-181b-5p-S1PR1 axis in anticancer drug resistance and autophagy.

Histone Deacetylase Inhibitors to Overcome Resistance to Targeted and Immuno Therapy in Metastatic Melanoma.

Therapies that target oncogenes and immune checkpoint molecules constitute a major group of treatments for metastatic melanoma. A mutation in BRAF (BRAF V600E) affects various signaling pathways, including mitogen activated protein kinase (MAPK) and PI3K/AKT/mammalian target of rapamycin (mTOR) in melanoma. Target-specific agents, such as MAPK inhibitors improve progression-free survival. However, BRAFV600E mutant melanomas treated with BRAF kinase inhibitors develop resistance. Immune checkpoint molecules, such as programmed death-1 (PD-1) and programmed death ligand-1(PD-L1), induce immune evasion of cancer cells. MAPK inhibitor resistance results from the increased expression of PD-L1. Immune checkpoint inhibitors, such as anti-PD-L1 or anti-PD-1, are main players in immune therapies designed to target metastatic melanoma. However, melanoma patients show low response rate and resistance to these inhibitors develops within 6-8 months of treatment. Epigenetic reprogramming, such as DNA methylaion and histone modification, regulates the expression of genes involved in cellular proliferation, immune checkpoints and the response to anti-cancer drugs. Histone deacetylases (HDACs) remove acetyl groups from histone and non-histone proteins and act as transcriptional repressors. HDACs are often dysregulated in melanomas, and regulate MAPK signaling, cancer progression, and responses to various anti-cancer drugs. HDACs have been shown to regulate the expression of PD-1/PD-L1 and genes involved in immune evasion. These reports make HDACs ideal targets for the development of anti-melanoma therapeutics. We review the mechanisms of resistance to anti-melanoma therapies, including MAPK inhibitors and immune checkpoint inhibitors. We address the effects of HDAC inhibitors on the response to MAPK inhibitors and immune checkpoint inhibitors in melanoma. In addition, we discuss current progress in anti-melanoma therapies involving a combination of HDAC inhibitors, immune checkpoint inhibitors, and MAPK inhibitors.

CAGE-miR-140-5p-Wnt1 Axis Regulates Autophagic Flux, Tumorigenic Potential of Mouse Colon Cancer Cells and Cellular Interactions Mediated by Exosomes.

Although the cancer/testis antigen CAGE has been implicated in tumorigenesis, the molecular mechanisms of CAGE-promoted tumorigenesis remain largely unknown. CT26Flag-CAGE cells, CT26 (mouse colon cancer cells) cells stably expressing CAGE, were established to investigate CAGE-promoted tumorigenesis. Down-regulation of CAGE led to decreased autophagic flux in CT26Flag-CAGE cells. CAGE interacted with Beclin1, a mediator of autophagy. The CT26Flag-CAGE cells showed enhanced autophagosome formation and displayed greater tumor spheroid-forming potential than CT26 cells. MicroRNA array analysis revealed that CAGE decreased the expression of various microRNAs, including miR-140-5p, in CT26 cells. CAGE was shown to bind to the promoter sequences of miR-140-5p. MiR-140-5p inhibition increased the tumorigenic potential of and autophagic flux in CT26 cells. A miR-140-5p mimic exerted negative effects on the tumorigenic potential of CT26Flag-CAGE cells and autophagic flux in CT26Flag-CAGE cells. MiR-140-5p was predicted to bind to the 3'-UTR of Wnt1. CT26Flag-CAGE cells showed higher expression of Wnt1 than CT26 cells. Down-regulation of Wnt1 decreased autophagic flux. Luciferase activity assays showed the direct regulation of wnt1 by miR-140-5p. Tumor tissue derived from the CT26Flag-CAGE cells revealed higher expressions of factors associated with activated mast cells and tumor-associated macrophages than tumor tissue derived from CT26 cells. Culture medium from the CT26Flag-CAGE cells increased autophagic flux in CT26 cells, mast cells and macrophages. Culture medium from the CT26Flag-CAGE cells increased CD163 and autophagic flux in CT26 cells, mast cells, and macrophages in a Wnt1-dependent manner. Exosomes from CT26Flag-CAGE cells increased autophagc flux in CT26 cells, mast cells, and macrophages. Exosomes from CT26Flag-CAGE cells increased the tumorigenic potential of CT26 cells. Wnt1 was shown to be present within the exosomes. Recombinant Wnt1 protein increased autophagic flux in CT26, mast cells, and macrophages. Recombinant wnt1 protein mediated interactions between the CT26 cells, mast cells, and macrophages. Our results showed novel roles for the CAGE-miR-140-5p-Wnt1 axis in autophagic flux and cellular interactions mediated by exosomes.

CAGE Binds to Beclin1, Regulates Autophagic Flux and CAGE-Derived Peptide Confers Sensitivity to Anti-cancer Drugs in Non-small Cell Lung Cancer Cells.

The objective of this study was to determine the role of CAGE, a cancer/testis antigen, in resistance of non-small cell lung cancers to anti-cancer drugs. Erlotinib-resistant PC-9 cells (PC-9/ER) with EGFR mutations (ex 19 del + T790M of EGFR), showed higher level of autophagic flux than parental sensitive PC-9 cells. Erlotinib and osimertinib increased autophagic flux and induced the binding of CAGE to Beclin1 in PC-9 cells. The inhibition or induction of autophagy regulated the binding of CAGE to Beclin1 and the responses to anti-cancer drugs. CAGE showed binding to HER2 while HER2 was necessary for binding of CAGE to Beclin1. CAGE was responsible for high level of autophagic flux and resistance to anti-cancer drugs in PC-9/ER cells. A peptide corresponding to the DEAD box domain of CAGE, 266AQTGTGKT273, enhanced the sensitivity of PC-9/ER cells to erlotinib and osimertinib, inhibited the binding of CAGE to Beclin1 and regulated autophagic flux in PC-9/ER cells. Mutant CAGE-derived peptide 266AQTGTGAT273 or 266AQTGTGKA273 did not affect autophagic flux or the binding of CAGE to Beclin1. AQTGTGKT peptide showed binding to CAGE, but not to Beclin1. FITC-AQTGTGKT peptide showed co-localization with CAGE. AQTGTGKT peptide decreased tumorigenic potentials of PC-9/ER and H1975 cells, non-small cell lung cancer (NSCLC) cells with EGFR mutation (L885R/T790M), by inhibiting autophagic fluxand inhibiting the binding of CAGE to Beclin1. AQTGTGKT peptide also enhanced the sensitivity of H1975 cells to anti-cancer drugs. AQTGTGKT peptide showed tumor homing potential based on ex vivo homing assays of xenograft of H1975 cells. AQTGTGKT peptide restored expression levels of miR-143-3p and miR-373-5p, decreased autophagic flux and conferred sensitivity to anti-cancer drugs. These results present evidence that combination of anti-cancer drug with CAGE-derived peptide could overcome resistance of non-small cell lung cancers to anti-cancer drugs.

DDX53 Regulates Cancer Stem Cell-Like Properties by Binding to SOX-2.

This study investigated the role of cancer/testis antigen DDX53 in regulating cancer stem cell-like properties. DDX53 shows co-expression with CD133, a marker for cancer stem cells. DDX53 directly regulates the SOX-2 expression in anticancer drug-resistant Malme3MR cells. DDX53 and miR-200b were found to be involved in the regulation of tumor spheroid forming potential of Malme3M and Malme3MR cells. Furthermore, the self-renewal activity and the tumorigenic potential of Malme3MR-CD133 (+) cells were also regulated by DDX53. A miR-200b inhibitor induced the direct regulation of SOX-2 by DDX53 We therefore, conclude that DDX53 may serve as an immunotherapeutic target for regulating cancer stem-like properties of melanomas.