PTPLAD1 Regulates PHB-Raf Interaction to Orchestrate Epithelial-Mesenchymal and Mitofusion-Fission Transitions in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The poor prognosis of this malignancy is attributed mainly to the persistent activation of cancer signaling for metastasis. Here, we showed that protein tyrosine phosphatase-like A domain containing 1 (PTPLAD1) is down-regulated in highly metastatic CRC cells and negatively associated with poor survival of CRC patients. Systematic analysis reveals that epithelial-to-mesenchymal transition (EMT) and mitochondrial fusion-to-fission (MFT) transition are two critical features for CRC patients with low expression of PTPLAD1. PTPLAD1 overexpression suppresses the metastasis of CRC in vivo and in vitro by inhibiting the Raf/ERK signaling-mediated EMT and mitofission. Mechanically, PTPLAD1 binds with PHB via its middle fragment (141-178 amino acids) and induces dephosphorylation of PHB-Y259 to disrupt the interaction of PHB-Raf, resulting in the inactivation of Raf/ERK signaling. Our results unveil a novel mechanism in which Raf/ERK signaling activated in metastatic CRC induces EMT and mitochondrial fission simultaneously, which can be suppressed by PTPLAD1. This finding may provide a new paradigm for developing more effective treatment strategies for CRC.

Targeting ARF1-IQGAP1 interaction to suppress colorectal cancer metastasis and vemurafenib resistance.

INTRODUCTION: Acquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. OBJECTIVES: We identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. METHODS: DIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co­immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. RESULTS: We found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently. CONCLUSION: This study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

MEST promotes lung cancer invasion and metastasis by interacting with VCP to activate NF-κB signaling.

BACKGROUND: Cell invasion is a hallmark of metastatic cancer, leading to unfavorable clinical outcomes. In this study, we established two highly invasive lung cancer cell models (A549-i8 and H1299-i8) and identified mesoderm-specific transcript (MEST) as a novel invasive regulator of lung cancer. We aim to characterize its biological function and clinical significance in lung cancer metastasis. METHODS: Transwell invasion assay was performed to establish high-invasive lung cancer cell model. Immunohistochemistry (IHC) was used to detect MEST expression in tumor tissues. Mass spectrometry and bioinformatic analyses were used to identify MEST-regulated proteins and binding partners. Co-immunoprecipitation assay was performed to detect the interaction of MEST and VCP. The biological functions of MEST were investigated in vitro and in vivo. Immunofluorescence staining was conducted to explore the colocalization of MEST and VCP. RESULTS: MEST overexpression promoted metastasis of lung cancer cells in vivo and in vitro by activating NF-κB signaling. MEST increased the interaction between VCP and IκBα, which accelerated IκBα degradation and NF-κB activation. Such acceleration was abrogated by VCP silencing, indicating that MEST is an upstream activator of the VCP/IκBα/NF-κB signaling pathway. Furthermore, high expressions of MEST and VCP were associated with poor survival of lung cancer patients. CONCLUSION: Collectively, these results demonstrate that MEST plays an important role in driving invasion and metastasis of lung cancer by interacting with VCP to coordinate the IκBα/NF-κB pathway. Targeting the MEST/VCP/IκBα/NF-κB signaling pathway may be a promising strategy to treat lung cancer.

Anti-allergic drug azelastine suppresses colon tumorigenesis by directly targeting ARF1 to inhibit IQGAP1-ERK-Drp1-mediated mitochondrial fission.

This study aimed to screen novel anticancer strategies from FDA-approved non-cancer drugs and identify potential biomarkers and therapeutic targets for colorectal cancer (CRC). Methods: A library consisting of 1056 FDA-approved drugs was screened for anticancer agents. WST-1, colony-formation, flow cytometry, and tumor xenograft assays were used to determine the anticancer effect of azelastine. Quantitative proteomics, confocal imaging, Western blotting and JC-1 assays were performed to examine the effects on mitochondrial pathways. The target protein of azelastine was analyzed and confirmed by DARTS, WST-1, Biacore and tumor xenograft assays. Immunohistochemistry, gain- and loss-of-function experiments, WST-1, colony-formation, immunoprecipitation, and tumor xenograft assays were used to examine the functional and clinical significance of ARF1 in colon tumorigenesis. Results: Azelastine, a current anti-allergic drug, was found to exert a significant inhibitory effect on CRC cell proliferation in vitro and in vivo, but not on ARF1-deficient or ARF1-T48S mutant cells. ARF1 was identified as a direct target of azelastine. High ARF1 expression was associated with advanced stages and poor survival of CRC. ARF1 promoted colon tumorigenesis through its interaction with IQGAP1 and subsequent activation of ERK signaling and mitochondrial fission by enhancing the interaction of IQGAP1 with MEK and ERK. Mechanistically, azelastine bound to Thr-48 in ARF1 and repressed its activity, decreasing Drp1 phosphorylation. This, in turn, inhibited mitochondrial fission and suppressed colon tumorigenesis by blocking IQGAP1-ERK signaling. Conclusions: This study provides the first evidence that azelastine may be novel therapeutics for CRC treatment. ARF1 promotes colon tumorigenesis, representing a promising biomarker and therapeutic target in CRC.

Identification of miR-515-3p and its targets, vimentin and MMP3, as a key regulatory mechanism in esophageal cancer metastasis: functional and clinical significance.

Metastasis is the main factor of treatment failure in cancer patients, but the underlying mechanism remains to be elucidated and effective new treatment strategies are urgently needed. This study aims to explore novel key metastasis-related microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). By comparing miRNA profiles of the highly metastatic ESCC cell sublines, we established through serial in vivo selection with the parental cells, we found that the expression level of miR-515-3p was lower in ESCC tumor tissues than adjacent normal tissues, further decreased in metastatic tumors, and moreover, markedly associated with advanced stage, metastasis and patient survival. The in vitro and in vivo assays suggested that miR-515-3p could increase the expression of the epithelial markers as well as decrease the expression of the mesenchymal markers, and more importantly, suppress invasion and metastasis of ESCC cells. Mechanistically, we revealed that miR-515-3p directly regulated vimentin and matrix metalloproteinase-3 (MMP3) expression by binding to the coding sequence and 3'untranslated region, respectively. In addition, the data from whole-genome methylation sequencing and methylation-specific PCR indicated that the CpG island within miR-515-3p promoter was markedly hypermethylated in ESCC cell lines and ESCC tumor tissues, which may lead to deregulation of miR-515-3p expression in ESCC. Furthermore, our preclinical experiment provides solid evidence that systemic delivery of miR-515-3p oligonucleotide obviously suppressed the metastasis of ESCC cells in nude mice. Taken together, this study demonstrates that miR-515-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic strategy in ESCC.

Liensinine perchlorate inhibits colorectal cancer tumorigenesis by inducing mitochondrial dysfunction and apoptosis.

SCOPE: Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options, and there is an urgent need to develop novel therapeutic agents with good treatment efficiency and low toxicity. This study aims to examine the anticancer bioactivity of liensinine, a constituent of Nelumbo nucifera Gaertn, in CRC and investigate the action mechanisms involved. METHODS AND RESULTS: Liensinine was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any observed cytotoxicity on normal colorectal epithelial cells. Mechanistically, our data from quantitative proteomics, western blot analysis and flow cytometry analyses demonstrated that exposure of CRC cells to liensinine caused cell cycle arrest, mitochondrial dysfunction and apoptosis, accompanied by the activation of the JNK signaling pathway. Furthermore, animal experiments showed that liensinine markedly suppressed the growth of CRC tumor xenografts in nude mice by reducing the Ki-67 proliferation index, but did not damage the vital organs of the animals. CONCLUSION: This study demonstrated for the first time that liensinine, a food-source natural product, could be a novel therapeutic strategy for treating CRC without obvious side effects.

Propafenone suppresses esophageal cancer proliferation through inducing mitochondrial dysfunction.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with poor survival and limited therapeutic options. The aim of this study is to identify novel anticancer strategies from existing Food and Drug Administration (FDA)-approved drugs that have been used to clinically treat other diseases. Here, propafenone, an antiarrhythmic medication, was found to induce apoptosis and exert a significantly inhibitory effect on the proliferation and colony-forming ability of ESCC cells in a dose-dependent manner without observed cytotoxicity on normal esophageal epithelial cells. Furthermore, propafenone markedly suppressed growth of tumor xenografts in nude mice by reducing the Ki-67 proliferation index and angiogenesis but did not damage the vital organs of the animals. Mechanistically, our data from the proteomics, Western blot and flow cytometry analyses demonstrated that propafenone caused mitochondrial dysfunction as indicated by a decreased mitochondrial membrane potential and reduced expression of Bcl-xL and Bcl-2. In summary, this study provides the first evidence that propafenone, an FDA-approved drug to treat arrhythmias, could be a novel therapeutic strategy for treating ESCC without obvious side effects.

Dynamic quantitative proteomics characterization of TNF-α-induced necroptosis.

Emerging evidence suggested that necroptosis has essential functions in many human inflammatory diseases, but the molecular mechanisms of necroptosis remain unclear. Here, we employed SILAC quantitatively dynamic proteomics to compare the protein changes during TNF-α-induced necroptosis at different time points in murine fibrosarcoma L929 cells with caspase-8 deficiency, and then performed the systematical analysis on the signaling networks involved in the progress using bioinformatics methods. Our results showed that a total of 329, 421 and 378 differentially expressed proteins were detected at three stages of necroptosis, respectively. Gene ontology and ingenuity pathway analysis (IPA) revealed that the proteins regulated at early stages of necroptosis (2, 6 h) were mainly involved in mitochondria dysfunction, oxidative phosphorylation and Nrf-2 signaling, while the expression levels of the proteins related to ubiquitin, Nrf-2, and NF-κB pathways were found to have changes at last stages of necroptosis (6, 18 h). Taken together, we demonstrated for the first time that dysfunction of mitochondria and ubiquitin-proteasome signaling contributed to the initiation and execution of necroptosis. These findings may provide clues for the identification of important regulators in necroptosis and the development of novel therapeutic strategies for the related diseases.