Lysine butyrylation of HSP90 regulated by KAT8 and HDAC11 confers chemoresistance.

Posttranslational modification dramatically enhances protein complexity, but the function and precise mechanism of novel lysine acylation modifications remain unknown. Chemoresistance remains a daunting challenge to successful treatment. We found that lysine butyrylation (Kbu) is specifically upregulated in chemoresistant tumor cells and tissues. By integrating butyrylome profiling and gain/loss-of-function experiments, lysine 754 in HSP90 (HSP90 K754) was identified as a substrate for Kbu. Kbu modification leads to overexpression of HSP90 in esophageal squamous cell carcinoma (ESCC) and its further increase in relapse samples. Upregulation of HSP90 contributes to 5-FU resistance and can predict poor prognosis in cancer patients. Mechanistically, HSP90 K754 is regulated by the cooperation of KAT8 and HDAC11 as the writer and eraser, respectively; SDCBP increases the Kbu level and stability of HSP90 by binding competitively to HDAC11. Furthermore, SDCBP blockade with the lead compound V020-9974 can target HSP90 K754 to overcome 5-FU resistance, constituting a potential therapeutic strategy.

Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis.

BACKGROUND: Metastasis is one of the most lethal hallmarks of esophageal squamous cell carcinoma (ESCC), yet the mechanisms remain unclear due to a lack of reliable experimental models and systematic identification of key drivers. There is urgent need to develop useful therapies for this lethal disease. METHODS: A genome-wide CRISPR/Cas9 screening, in combination with gene profiling of highly invasive and metastatic ESCC sublines, as well as PDX models, was performed to identify key regulators of cancer metastasis. The Gain- and loss-of-function experiments were taken to examine gene function. Protein interactome, RNA-seq, and whole genome methylation sequencing were used to investigate gene regulation and molecular mechanisms. Clinical significance was analyzed in tumor tissue microarray and TCGA databases. Homology modeling, modified ELISA, surface plasmon resonance and functional assays were performed to identify lead compound which targets MEST to suppress cancer metastasis. FINDINGS: High MEST expression was associated with poor patient survival and promoted cancer invasion and metastasis in ESCC. Mechanistically, MEST activates SRCIN1/RASAL1-ERK-snail signaling by interacting with PURA. miR-449a was identified as a direct regulator of MEST, and hypermethylation of its promoter led to MEST upregulation, whereas systemically delivered miR-449a mimic could suppress tumor metastasis without overt toxicity. Furthermore, molecular docking and computational screening in a small-molecule library of 1,500,000 compounds and functional assays showed that G699-0288 targets the MEST-PURA interaction and significantly inhibits cancer metastasis. INTERPRETATION: We identified the MEST-PURA-SRCIN1/RASAL1-ERK-snail signaling cascade as an important mechanism underlying cancer metastasis. Blockade of MEST-PURA interaction has therapeutic potential in management of cancer metastasis. FUNDING: This work was supported by National Key Research and Development Program of China (2021YFC2501000, 2021YFC2501900, 2017YFA0505100); National Natural Science Foundation of China (31961160727, 82073196, 81973339, 81803551); NSFC/RGC Joint Research Scheme (N_HKU727/19); Natural Science Foundation of Guangdong Province (2021A1515011158, 2021A0505030035); Key Laboratory of Guangdong Higher Education Institutes of China (2021KSYS009).

Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner.

Posttranslational modifications add tremendous complexity to proteomes; however, gaps remain in knowledge regarding the function and regulatory mechanism of newly discovered lysine acylation modifications. Here, we compared a panel of non-histone lysine acylation patterns in metastasis models and clinical samples, and focused on 2-hydroxyisobutyrylation (Khib) due to its significant upregulation in cancer metastases. By the integration of systemic Khib proteome profiling in 20 paired primary esophageal tumor and metastatic tumor tissues with CRISPR/Cas9 functional screening, we identified N-acetyltransferase 10 (NAT10) as a substrate for Khib modification. We further showed that Khib modification at lysine 823 in NAT10 functionally contribute to metastasis. Mechanistically, NAT10 Khib modification enhances its interaction with deubiquitinase USP39, resulting in increased NAT10 protein stability. NAT10 in turn promotes metastasis by increasing NOTCH3 mRNA stability in an N4-acetylcytidine-dependent manner. Furthermore, we discovered a lead compound #7586-3507 that inhibited NAT10 Khib modification and showed efficacy in tumor models in vivo at a low concentration. Together, our findings bridge newly identified lysine acylation modifications with RNA modifications, thus providing novel insights into epigenetic regulation in human cancer. We propose that pharmacological inhibition of NAT10 K823 Khib modification constitutes a potential anti-metastasis strategy.

N4-acetylcytidine modification of lncRNA CTC-490G23.2 promotes cancer metastasis through interacting with PTBP1 to increase CD44 alternative splicing.

Although N4-acetylcytidine (ac4C) modification affects the stability and translation of mRNA, it is unknown whether it exists in noncoding RNAs, and its biological function is unclear. Here, nucleotide-resolution method for profiling CTC-490G23.2 ac4C sites and gain- and loss-of-function experiments revealed that N-acetyltransferase 10 (NAT10) is responsible for ac4C modification of long noncoding RNAs (lncRNAs). NAT10-mediated ac4C modification leads to the stabilization and overexpression of lncRNA CTC-490G23.2 in primary esophageal squamous cell carcinoma (ESCC) and its further upregulation in metastatic tissues. CTC-490G23.2 significantly promotes cancer invasion and metastasis in vitro and in vivo. Mechanistically, CTC-490G23.2 acts as a scaffold to increase the binding of CD44 pre-mRNA to polypyrimidine tract-binding protein 1 (PTBP1), resulting in a oncogenic splicing switch from the standard isoform CD44s to the variant isoform CD44v(8-10). CD44v(8-10), but not CD44s, binds to and increases the protein stability of vimentin. Expression levels of CTC-490G23.2 and CD44v(8-10) can predict poor prognosis in cancer patients. Furthermore, the antisense oligonucleotide (ASO)/SV40-LAH4-L1 peptide self-assembled nanocomplexes targeting CTC490G23.2 exerts a significantly suppressive effect on cancer metastasis. The outcome of this study will provide new mechanistic insight into the ac4C modification of lncRNAs and useful clues for the development of novel systemic therapies and prognostic biomarkers.

Blockade of Nuclear ß-Catenin Signaling via Direct Targeting of RanBP3 with NU2058 Induces Cell Senescence to Suppress Colorectal Tumorigenesis.

Colorectal cancer (CRC) is one of the most common malignant tumors in the world, with high prevalence and low 5-year survival. Most of the CRC patients show excessive activation of the Wnt/ß-catenin pathway which is a vital target for CRC treatment. Based on multiple CRC cell lines with different nuclear expression of ß-catenin, NU2058 is identified from a small molecule library consisting of 280 bioactive compounds and found to selectively inhibit the proliferation of CRC cells with nuclear ß-catenin activation in vitro and in vivo. The translational significance of NU2058 alone or in combination with chemotherapeutic drugs oxaliplatin and irinotecan (SN38) in CRC is demonstrated in orthotopic tumor model and patient-derived xenograft models. By integrating limited proteolysis-small molecule mapping (LiP-SMap) and mass spectrometry (MS), Ran-binding protein 3 (RanBP3) is identified as the direct target of NU2058. The results show that RanBP3 is a tumor suppressor in CRC and is associated with patient survival. Mechanistically, NU2058 increases the interaction of RanBP3 and ß-catenin to promote nuclear export of ß-catenin, which further inhibits transcription of c-Myc and cyclin D1 to induce cell senescence. Collectively, NU2058 may serve as a promising therapeutic agent for CRC patients with selective disruption of pathologic Wnt/ß-catenin signaling.

Anti-HIV Drug Elvitegravir Suppresses Cancer Metastasis via Increased Proteasomal Degradation of m6A Methyltransferase METTL3.

N6-methyladenosine (m6A) methylation is an abundant modification in eukaryotic mRNAs. Accumulating evidence suggests a role for RNA m6A methylation in various aspects of cancer biology. In this study, we aimed to explore the biological role of RNA m6A modification in tumor metastasis and to identify novel therapeutic strategies for esophageal squamous cell carcinoma (ESCC). Integration of genome-wide CRISPR/Cas9 functional screening with highly invasive and metastatic ESCC subline models led to the identification of METTL3, the catalytic subunit of the N6-adenosine-methyltransferase complex, as a promoter of cancer metastasis. METTL3 expression was upregulated in ESCC tumors and metastatic tissues. In vitro and in vivo experiments indicated that METTL3 increased m6A in EGR1 mRNA and enhanced its stability in a YTHDF3-dependent manner, activating EGR1/Snail signaling. Investigation into the regulation of METTL3 expression found that KAT2A increased H3K27 acetylation levels in the METTL3 promoter region and activated transcription of METTL3, whereas SIRT2 exerted the opposite effects. Molecular docking and computational screening in a Food and Drug Administration-approved compound library consisting of 1,443 small molecules identified compounds targeting METTL3 to suppress cancer metastasis. Elvitegravir, originally developed to treat human immunodeficiency virus (HIV) infection, suppressed metastasis by directly targeting METTL3 and enhancing its STUB1-mediated proteasomal degradation. Overall, RNA m6A modifications are important in cancer metastasis, and targeting METTL3 with elvitegravir has therapeutic potential for treating ESCC. SIGNIFICANCE: This study finds that METTL3 promotes cancer metastasis by activating EGR1/Snail signaling in an m6A-dependent manner, revealing vulnerability to METTL3 blockade in esophageal squamous cell carcinoma.

Molecular mechanisms of chemo- and radiotherapy resistance and the potential implications for cancer treatment.

Cancer is a leading cause of death worldwide. Surgery is the primary treatment approach for cancer, but the survival rate is very low due to the rapid progression of the disease and presence of local and distant metastasis at diagnosis. Adjuvant chemotherapy and radiotherapy are important components of the multidisciplinary approaches for cancer treatment. However, resistance to radiotherapy and chemotherapy may result in treatment failure or even cancer recurrence. Radioresistance in cancer is often caused by the repair response to radiation-induced DNA damage, cell cycle dysregulation, cancer stem cells (CSCs) resilience, and epithelial-mesenchymal transition (EMT). Understanding the molecular alterations that lead to radioresistance may provide new diagnostic markers and therapeutic targets to improve radiotherapy efficacy. Patients who develop resistance to chemotherapy drugs cannot benefit from the cytotoxicity induced by the prescribed drug and will likely have a poor outcome with these treatments. Chemotherapy often shows a low response rate due to various drug resistance mechanisms. This review focuses on the molecular mechanisms of radioresistance and chemoresistance in cancer and discusses recent developments in therapeutic strategies targeting chemoradiotherapy resistance to improve treatment outcomes.

Lomerizine 2HCl inhibits cell proliferation and induces protective autophagy in colorectal cancer via the PI3K/Akt/mTOR signaling pathway.

Colorectal cancer (CRC) is one of the most common malignancies currently. Despite advances in drug development, the survival and response rates in CRC patients are still poor. In our previous study, a library comprised of 1056 bioactive compounds was used for screening of drugs that could suppress CRC. Lomerizine 2HCl, which is an approved prophylactic drug for migraines, was selected for our studies. The results of in vitro and in vivo assays suggested that lomerizine 2HCl suppresses cell growth and promotes apoptosis in CRC cells. Moreover, lomerizine 2HCl inhibits cell migration and invasion of CRC. RNA sequencing analysis and Western blotting confirmed that lomerizine 2HCl can inhibit cell growth, migration, and invasion through PI3K/AKT/mTOR signaling pathway and induces protective autophagy in CRC. Meanwhile, autophagy inhibition by 3-methyladenine (3-MA) increases lomerizine 2HCl-induced cell apoptosis. Taken together, these results imply that lomerizine 2HCl is a potential anticancer agent, and the combination of lomerizine 2HCl and autophagy inhibitors may serve as a novel strategy to increase the antitumor efficacy of agents in the treatment of CRC.

Direct Targeting of CREB1 with Imperatorin Inhibits TGFß2-ERK Signaling to Suppress Esophageal Cancer Metastasis.

Metastasis accounts for 90% of cancer death worldwide, and effective therapeutic strategies are lacking. The aim of this work is to identify the key drivers in tumor metastasis and screen therapeutics for treatment of esophageal squamous cell carcinoma (ESCC). Gene Ontology analysis of The Cancer Genome Atlas (TCGA) gene expression datasets of ESCC patients with or without lympy metastasis identifies that TGFß2 is highly enriched in the pathways essential for tumor metastasis and upregulates in the metastatic ESCC tumors. High TGFß2 expression in ESCC correlates with metastasis and patient survival, and functionally contributes to tumor metastasis via activating extracellular signal-regulated kinases (ERK) signaling. By screening of a library consisting of 429 bioactive compounds, imperatorin is verified as a novel TGFß2 inhibitor, with robustly suppressive effect on tumor metastasis in multiple mice models. Mechanistically, direct binding of imperatorin and CREB1 inhibits phosphorylation, nuclear translocation of CREB1, and its interaction with TGFß2 promoter, represses TGFß2 expression and fibroblasts-secreted CCL2, and then inactivates ERK signaling to block cancer invasion and abrogates the paracrine effects of fibroblasts on tumor angiogenesis and metastasis. Overall, the findings suggest the use of TGFß2 as a diagnostic and prognostic biomarker and therapeutic target in ESCC, and supports the potential of imperatorin as a novel therapeutic strategy for cancer metastasis.

Benzethonium chloride suppresses lung cancer tumorigenesis through inducing p38-mediated cyclin D1 degradation.

Lung cancer is the leading cause of cancer-related deaths worldwide, but effective therapeutics is limited. This study aims to identify novel anticancer strategy from a Food and Drug Administration (FDA)-approved drug library consisting of 528 compounds. Benzethonium Chloride (BZN), a FDA-approved drug for anti-infective, was found to markedly induce apoptosis and inhibit proliferation and colony formation ability of lung cancer cells in dose- and time-dependent manners. BZN also enhanced the sensitivity of lung cancer cells to gefitinib, the first-line treatment strategy for selected lung cancer patients. Furthermore, BZN significantly delayed the growth of tumor xenografts in nude mice by increasing apoptosis and decreasing Ki-67 proliferation index, without obvious toxic effects to the vital organs of animals. Mechanistically, quantitative proteomics coupled with bioinformatics analyses and a series of functional assays demonstrated that BZN induced cell cycle arrest at G1 phase, and this was associated with an increase in p38-mediated phosphorylation at threonine 286 (T286) and accelerated degradation of cyclin D1. Our findings provide the first evidence that BZN could be a promising therapeutic agent in lung cancer treatment.

Identification of miR-29c and its Target FBXO31 as a Key Regulatory Mechanism in Esophageal Cancer Chemoresistance: Functional Validation and Clinical Significance.

Rationale: Dysregulated microRNA (miRNA) expressions in cancer can contribute to chemoresistance. This study aims to identify miRNAs that are associated with fluorouracil (5-FU) chemoresistance in esophageal squamous cell carcinoma (ESCC). The potential of miR-29c as a novel diagnostic, prognostic and treatment-predictive marker in ESCC, and its mechanisms and therapeutic implication in overcoming 5-FU chemoresistance were explored. Methods: The miRNA profiles of an ESCC cell model with acquired chemoresistance to 5-FU were analyzed using a Taqman miRNA microarray to identify novel miRNAs associated with 5-FU chemoresistance. Quantitative real-time PCR was used to determine miR-29c expression in tissue and serum samples of patients. Bioinformatics, gain- and loss-of-function experiments, and luciferase reporter assay were performed to validate F-box only protein 31 (FBXO31) as a direct target of miR-29c, and to identify potential transcription factor binding events that control miR-29c expression. The potential of systemic miR-29c oligonucleotide-based therapy in overcoming 5-FU chemoresistance was evaluated in tumor xenograft model. Results: MiR-29c, under the regulatory control of STAT5A, was frequently downregulated in tumor and serum samples of patients with ESCC, and the expression level was correlated with overall survival. Functional studies showed that miR-29c could override 5-FU chemoresistance in vitro and in vivo by directly interacting with the 3'UTR of FBXO31, leading to repression of FBXO31 expression and downstream activation of p38 MAPK. Systemically administered miR-29c dramatically improved response of 5-FU chemoresistant ESCC xenografts in vivo. Conclusions: MiR-29c modulates chemoresistance by interacting with FBXO31, and is a promising non-invasive biomarker and therapeutic target in ESCC.

Significance of integrin-linked kinase (ILK) in tumorigenesis and its potential implication as a biomarker and therapeutic target for human cancer.

Integrin-linked kinase (ILK), which is an ankyrin repeat-containing serine/threonine protein kinase, interacts with integrin ß1 and the ß3 cytoplasmic domain and phosphorylates integrin ß1. ILK has multiple functions in cells, such as cell-extracellular matrix interactions, cell cycle, apoptosis, cell proliferation and cell motility, which are associated with the interacting partners of ILK and downstream signaling pathways. Upregulation of ILK is frequently observed in cancer tissues compared to corresponding normal tissues. Emerging evidence has demonstrated that ILK plays an important role in biological processes associated with tumorigenesis, including cancer cell proliferation, angiogenesis, metastasis, and drug resistance. Furthermore, inhibition of ILK expression and activity using siRNA or chemical inhibitors has shown a significant suppressive effect on cancer development and progression, implicating the potential of ILK as a target for cancer treatment. In this review, we summarized the functional role of ILK in tumorigenesis, with the expectation that targeting ILK could provide more evidence for cancer therapy.

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.

Synephrine Hydrochloride Suppresses Esophageal Cancer Tumor Growth and Metastatic Potential through Inhibition of Galectin-3-AKT/ERK Signaling.

A library consisting of 429 food-source compounds was used to screen the natural products with anticancer properties in esophageal squamous cell carcinoma (ESCC). We demonstrated for the first time that synephrine, an active compound isolated from leaves of citrus trees, markedly suppressed cell proliferation (inhibition rate with 20 µM synephrine at day 5:71.1 ± 5.8% and 75.7 ± 6.2% for KYSE30 and KYSE270, respectively) and colony formation (inhibition rate with 10 µM synephrine: 86.5 ± 5.9% and 82.3 ± 4.5% for KYSE30 and KYSE270, respectively), as well as migration (inhibition rate with 10 µM synephrine: 76.9 ± 4.4% and 62.2 ± 5.8% for KYSE30 and KYSE270, respectively) and invasion abilities (inhibition rate with 10 µM synephrine: 73.3 ± 7.5% and 75.3 ± 3.4% for KYSE30 and KYSE270, respectively) of ESCC cells in a dose-dependent manner, without significant toxic effect on normal esophageal epithelial cells. Mechanistically, quantitative proteomics and bioinformatics analyses were performed to explore the synephrine-regulated proteins. Western blot and qRT-PCR data indicated that synephrine may downregulate Galectin-3 to inactivate AKT and ERK pathways. In addition, we found that the sensitivity of ESCC to fluorouracil (5-FU) could be enhanced by synephrine. Furthermore, in vivo experiments showed that synephrine had significant antitumor effect on ESCC tumor xenografts in nude mice (inhibition rate with 20 mg/kg synephrine is 61.3 ± 20.5%) without observed side effects on the animals. Taken together, synephrine, a food-source natural product, may be a potential therapeutic strategy in ESCC.

Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA Signaling as Prognostic Biomarker and Therapeutic Target for Colon Cancer Invasion.

Metastasis is one of the major causes of treatment failure in the patients with colon cancer. The aim of our study is to find key proteins and pathways that drive invasion and metastasis in colon cancer. Eight rounds of selection of cancer cells invading through matrigel-coated chamber were performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture technology was used to identify the differently expressed proteins, and the proteomics data were analyzed by ingenuity pathway analysis. PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes. HCT116-I8 and RKO-I8 cells showed significantly stronger invasive potential as well as decreased E-cadherin and increased vimentin expressions compared with parental cells. The differently expressed proteins in I8 cells compared with parental cells were identified. Bioinformatics analysis of proteomics data suggested that Cdc42BPA protein and Cdc42 signaling pathway are important for colon cancer invasion, which was confirmed by experimental data showing upregulation of Cdc42BPA and higher expression of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon cancer cell invasion and reversed epithelial mesenchymal transition process. Furthermore, compared with adjacent normal tissues, Cdc42BPA expression was significantly higher in colon cancer tissues and further upregulated in metastatic tumors in lymph nodes. More importantly, Cdc42BPA expression was correlated with metastasis and poor survival of the patients with colon cancer. This study provides the first evidence that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion, and Cdc42BPA has potential implications for colon cancer prognosis and treatment.

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.