Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin.

Proteins containing a CAAX motif at the C-terminus undergo prenylation for localization and activity and include a series of key regulatory proteins, such as RAS superfamily members, heterotrimeric G proteins, nuclear lamina protein, and several protein kinases and phosphatases. However, studies of prenylated proteins in esophageal cancer are limited. Here, through research on large-scale proteomic data of esophageal cancer in our laboratory, we found that paralemmin-2 (PALM2), a potential prenylated protein, was upregulated and associated with poor prognosis in patients. Low-throughput verification showed that the expression of PALM2 in esophageal cancer tissues was higher than that in their paired normal esophageal epithelial tissues, and it was generally expressed in the membrane and cytoplasm of esophageal cancer cells. PALM2 interacted with the two subunits of farnesyl transferase (FTase), FNTA and FNTB. Either the addition of an FTase inhibitor or mutation in the CAAX motif of PALM2 (PALM2C408S) impaired its membranous localization and reduced the membrane location of PALM2, indicating PALM2 was prenylated by FTase. Overexpression of PALM2 enhanced the migration of esophageal squamous cell carcinoma cells, whereas PALM2C408S lost this ability. Mechanistically, PALM2 interacted with the N-terminal FERM domain of ezrin of the ezrin/radixin/moesin (ERM) family. Mutagenesis indicated that lysine residues K253/K254/K262/K263 in ezrin's FERM domain and C408 in PALM2's CAAX motif were important for PALM2/ezrin interaction and ezrin activation. Knockout of ezrin prevented enhanced cancer cell migration by PALM2 overexpression. PALM2, depending on its prenylation, increased both ezrin membrane localization and phosphorylation of ezrin at Y146. In summary, prenylated PALM2 enhances the migration of cancer cells by activating ezrin.

Sulconazole Induces PANoptosis by Triggering Oxidative Stress and Inhibiting Glycolysis to Increase Radiosensitivity in Esophageal Cancer.

Esophageal cancer is the seventh most common cancer in the world. Although traditional treatment methods such as radiotherapy and chemotherapy have good effects, their side effects and drug resistance remain problematic. The repositioning of drug function provides new ideas for the research and development of anticancer drugs. We previously showed that the Food and Drug Administration-approved drug sulconazole can effectively inhibit the growth of esophageal cancer cells, but its molecular mechanism is not clear. Here, our study demonstrated that sulconazole had a broad spectrum of anticancer effects. It can not only inhibit the proliferation but also inhibit the migration of esophageal cancer cells. Both transcriptomic sequencing and proteomic sequencing showed that sulconazole could promote various types of programmed cell death and inhibit glycolysis and its related pathways. Experimentally, we found that sulconazole induced apoptosis, pyroptosis, necroptosis, and ferroptosis. Mechanistically, sulconazole triggered mitochondrial oxidative stress and inhibited glycolysis. Finally, we showed that low-dose sulconazole can increase radiosensitivity of esophageal cancer cells. Taken together, these new findings provide strong laboratory evidence for the clinical application of sulconazole in esophageal cancer.

Co-Expression of Chromatin Assembly Factor 1 Subunit A and Proliferating Cell Nuclear Antigen Is a Prognostic Biomarker of Esophageal Cancer.

(1) Background: Esophageal cancer (EC) is an important global health challenge. Due to the lack of necessary biomarkers and therapeutic targets, the survival of EC patients is poor. The EC proteomic data of 124 patients recently published by our group provides a database for research in this field. (2) Methods: Bioinformatics analysis was used to identify DNA replication and repair-related proteins in EC. Proximity ligation assay, colony formation assay, DNA fiber assay, and flow cytometry were used to study the effects of related proteins on EC cells. Kaplan-Meier survival analysis was used to evaluate the relationship between gene expression and the survival time of EC patients. (3) Results: Chromatin assembly factor 1 subunit A (CHAF1A) was highly correlated with proliferating cell nuclear antigen (PCNA) expression in EC. CHAF1A and PCNA colocalized in the nucleus of EC cells. Compared with the knockdown of CHAF1A or PCNA alone, the double knockdown of CHAF1A and PCNA could significantly inhibit EC cell proliferation. Mechanistically, CHAF1A and PCNA synergistically accelerated DNA replication and promoted S-phase progression. EC patients with high expression of both CHAF1A and PCNA had a worse survival rate. (4) Conclusion: we identify CHAF1A and PCNA as key cell cycle-related proteins leading to the malignant progression of EC, and these proteins could serve as important prognostic biomarkers and targets for EC.

Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma.

Anillin (ANLN) is a mitosis-related protein that promotes contractile ring formation and cytokinesis, but its cell cycle-dependent degradation mechanisms in cancer cells remain unclear. Here, we show that high expression of ANLN promotes cytokinesis and proliferation in esophageal squamous cell carcinoma (ESCC) cells and is associated with poor prognosis in ESCC patients. Furthermore, the findings of the study showed that the deubiquitinating enzyme USP10 interacts with ANLN and positively regulates ANLN protein levels. USP10 removes the K11- and K63-linked ubiquitin chains of ANLN through its deubiquitinase activity and prevents ANLN ubiquitin-mediated degradation. Importantly, USP10 promotes contractile ring assembly at the cytokinetic furrow as well as cytokinesis by stabilizing ANLN. Interestingly, USP10 and the E3 ubiquitin ligase APC/C co-activator Cdh1 formed a functional complex with ANLN in a non-competitive manner to balance ANLN protein levels. In addition, the macrolide compound FW-04-806 (F806), a natural compound with potential for treating ESCC, inhibited the mitosis of ESCC cells by targeting USP10 and promoting ANLN degradation. F806 selectively targeted USP10 and inhibited its catalytic activity but did not affect the binding of Cdh1 to ANLN and alters the balance of the USP10-Cdh1-ANLN complex. Additionally, USP10 expression was positively correlated with ANLN level and poor prognosis of ESCC patients. Overall, targeting the USP10-ANLN axis can effectively inhibit ESCC cell-cycle progression.

AKT serine/threonine kinase 2-mediated phosphorylation of fascin threonine 403 regulates esophageal cancer progression.

Fascin is the main actin-bundling protein in filopodia and is highly expressed in metastatic tumor cells. The overexpression of Fascin has been associated with poor clinical prognosis and metastatic progression. Post-translational modifications of Fascin, such as phosphorylation, can affect the proliferation and invasion of tumor cells by regulating the actin-bundling activity of Fascin. However, the phosphorylation sites of Fascin and their corresponding kinases require further exploration. In the current study, we identified novel phosphorylation of Fascin Threonine 403 (Fascin-T403) mediated by AKT serine/threonine kinase 2 (AKT2), which was studied using mass spectrometry data from esophageal cancer tissues (iProX database: IPX0002501000). A molecular dynamics simulation revealed that Fascin-Threonine 403 phosphorylation (Fascin-T403D) had a distinct spatial structure and correlation of amino acid residues, which was different from that of the wild type (Fascin-WT). Low-speed centrifugation assay results showed that Fascin-T403D affected actin cross-linking. To investigate whether Fascin-T403D affected the function of esophageal cancer cells, either Fascin-WT or Fascin-T403D were rescued in Fascin-knockout or siRNA cell lines. We observed that Fascin-T403D could suppress the biological behavior of esophageal cancer cells, including filopodia formation, cell proliferation, and migration. Co-immunoprecipitation (Co-IP) and Duolink in situ proximity ligation assay (PLA) were performed to measure the interaction between Fascin and AKT2. Using in vitro and in vivo kinase assays, we confirmed that AKT2, but not AKT1 or AKT3, is an upstream kinase of Fascin Threonine 403. Taken together, the AKT2-catalyzed phosphorylation of Fascin Threonine 403 suppressed esophageal cancer cell behavior, actin-bundling activity, and filopodia formation.

Cisplatin-induced pyroptosis is mediated via the CAPN1/CAPN2-BAK/BAX-caspase-9-caspase-3-GSDME axis in esophageal cancer.

Esophageal cancer is the seventh most common cancer globally. Chemotherapy resistance remains a significant challenge in the treatment of esophageal cancer patients. Cisplatin can damage tumor cells by inducing pyroptosis. However, the underlying molecular mechanisms remain unclear. In this work, we aim to investigate pyroptosis-dependent molecular mechanisms underlying cisplatin sensitivity and find potential biomarkers to predict response to cisplatin-based chemotherapy for esophageal cancer patients. Pyroptosis-associated proteins were screened via proteomics for esophageal cancer (n = 124) and bioinformatics analysis. We observed that high calpain-1 (CAPN1) and calpain-2 (CAPN2) expression were associated with favorable clinical outcomes and prolonged survival in esophageal cancer patients. We employed immunohistochemistry to evaluate the expression of CAPN1 and CAPN2 in pretreatment tumor biopsies from 108 patients with esophageal cancer who received concurrent chemoradiotherapy (CCRT). These results suggested that esophageal cancer patients with high expression of both CAPN1 and CAPN2 are likely to experience a complete response to CCRT and have significantly better survival. Western blotting, LDH release, calpain activity and cell viability assays indicated that cisplatin could activate calpain activity, while calpain inhibition or knockout suppressed cisplatin-induced pyroptosis. Mechanistically, we uncovered a novel mechanism whereby cisplatin induced pyroptosis via activation of a CAPN1/CAPN2-BAK/BAX-caspase-9-caspase-3-GSDME signaling axis in esophageal cancer cells. Collectively, this study is the first to explore the effects of calpain on cisplatin-induced pyroptosis in esophageal cancer cells. Further, our findings also imply that the combination of CAPN1 and CAPN2 could be considered as a promising biomarker of cisplatin sensitivity and prognosis in patients with esophageal cancer, providing a possibility to guide individualized treatment.

Large-scale and high-resolution mass spectrometry-based proteomics profiling defines molecular subtypes of esophageal cancer for therapeutic targeting.

Esophageal cancer (EC) is a type of aggressive cancer without clinically relevant molecular subtypes, hindering the development of effective strategies for treatment. To define molecular subtypes of EC, we perform mass spectrometry-based proteomic and phosphoproteomics profiling of EC tumors and adjacent non-tumor tissues, revealing a catalog of proteins and phosphosites that are dysregulated in ECs. The EC cohort is stratified into two molecular subtypes-S1 and S2-based on proteomic analysis, with the S2 subtype characterized by the upregulation of spliceosomal and ribosomal proteins, and being more aggressive. Moreover, we identify a subtype signature composed of ELOA and SCAF4, and construct a subtype diagnostic and prognostic model. Potential drugs are predicted for treating patients of S2 subtype, and three candidate drugs are validated to inhibit EC. Taken together, our proteomic analysis define molecular subtypes of EC, thus providing a potential therapeutic outlook for improving disease outcomes in patients with EC.