IL-33 Enhances the Lipopolysaccharide-Induced Secretion of Inflammatory Cytokines and Ameliorates Lipopolysaccharide Desensitization in Macrophages

Background: Lipopolysaccharide (LPS) desensitization, which is characterized by hyporesponsiveness and a form of immunosuppression, is important in the negative regulation of responses to LPS and inflammatory disease such as sepsis. However, effect of IL-33 in the desensitization to LPS remains unclear. Methods: We used RNA-sequencing technology to analyze changes in mRNA in bone-marrow-derived macrophages (BMDMs) stimulated with LPS. Changes in expression and secretion of inflammatory cytokines were detected by qPCR and ELISA, respectively. Mechanisms were further studied through p65 phosphorylation detection. Results: IL-33 expression was significantly increased in LPS-treated macrophages, indicating its involvement in LPS-induced inflammation. Exogenous IL-33 increased the inflammatory response and ameliorated LPS desensitization by increasing the secretion of proinflammatory cytokines. It also activated p65 phosphorylation in resistant cells. Conclusion: IL-33 can enhance the inflammatory response induced by LPS and ameliorate LPS desensitization possibly by activating the NF-κB pathway in mouse macrophages.

MicroRNA-92a-3p Enhances Cisplatin Resistance by Regulating Krüppel-Like Factor 4-Mediated Cell Apoptosis and Epithelial-to-Mesenchymal Transition in Cervical Cancer

Recent studies have confirmed the existence and key roles of microRNA (miRNAs) in cancer drug resistance, including cervical cancer (CC). The present study aims to establish a novel role for miR-92a-3p and its associated gene networks in cisplatin (DDP) resistance of CC. First, the disparities in miRNA expression between CC tissues and adjacent normal tissues were screened based on GSE19611 microarray data that retrieved from Gene Expression Omnibus (GEO), and we identified several miRs that were significantly downregulated or upregulated in CC tissues including miR-92a-3p. Moreover, miR-92a-3p was significantly up-regulated in DDP-resistant cells and was the most differently expressed miRNA. Functionally, knockdown of miR-92a-3p increased the sensitivity of DDP-resistant cells to DDP via inhibiting cell proliferation, migration and invasion, and promoting apoptosis. Conversely, overexpression of miR-92a-3p significantly induced DDP resistance in CC parental cells including HeLa and SiHa cells. Moreover, Krüppel-like factor 4 (KLF4) was identified as a direct target of miR-92a-3p, and an obvious inverse correlation was observed between the expression of miR-92a-3p and KLF4 in 40 pairs of cancer tissues. Furthermore, KLF4 knockdown reversed the promoting effect of miR-92a-3p inhibition on DDP sensitivity in DDP-resistant CC cells. Besides, high expression of miR-92a-3p was associated with DDP resistance, as well as a short overall survival in clinic. Taken together, these findings provide important evidence that miR-92a-3p targets KLF4 and is significant in DDP resistance in CC, indicating that miR-92a-3p may be an attractive target to increase DDP sensitivity in clinical CC treatment.

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Single-cell multi-omics analyses reveal EZH2 as a main driver of retinoic acid resistance in PLZF-RARA leukemia.

Cancer relapse is caused by a subset of malignant cells that are resistant to treatment. To characterize resistant cells and their vulnerabilities, we studied the retinoic acid (RA)-resistant PLZF-RARA acute promyelocytic leukemia (APL) using single-cell multi-omics. We uncovered transcriptional and chromatin heterogeneity in leukemia cells and identified a subset of cells resistant to RA that depend on a fine-tuned transcriptional network targeting the epigenetic regulator Enhancer of Zeste Homolog 2 (EZH2). Epigenomic and functional analyses validated EZH2 selective dependency of PLZF-RARA leukemia and its driver role in RA resistance. Targeting pan-EZH2 activities (canonical/non-canonical) was necessary to eliminate leukemia relapse initiating cells, which underlies a dependency of resistant cells on an EZH2 non-canonical activity and the necessity to degrade EZH2 to overcome resistance. Our study provides critical insights into the mechanisms of RA resistance that allow us to eliminate treatment-resistant leukemia cells by targeting EZH2, thus highlighting a potential targeted therapy approach.

Alpelisib Exhibits a Synergistic Effect with Pyrotinib and Reverses Acquired Pyrotinib Resistance in HER2+ Breast Cancer

Background: Human epidermal growth factor receptor 2 (HER2) plays a vital role in breast cancer progression in patients who overexpress HER2, thus promoting the rapid progress of targeted drugs development and therapy strategies advancement targeting this gene. Pyrotinib, approved in clinical by the Chinese State Drug Administration, is a novel pan-ErbB kinase inhibitor and exhibits better efficacy than lapatinib. Alpelisib is a selective PI3K p110α inhibitor approved for application in HR+, HER2-, PIK3CA mutated breast cancers. We assumed that combining pyrotinib with alpelisib worked better than single-drug treatment.Methods: We performed the drug combination assay to evaluate the combination index (CI) of pyrotinib and alpelisib in HER2+ breast cancer cell lines. Cell functional assays, RT-qPCR (Real Time-Quantitative Polymerase Chain Reaction) and western blotting were performed to elucidate the combined efficacy of two drugs and explore the underlying mechanism. Then we established the acquired pyrotinib resistant HER2+ breast cancer cell lines and evaluate the combined efficacy of two drugs in pyrotinib resistant cells and explore the potential mechanisms.Results: Our data exhibited that a combination of alpelisib and pyrotinib showed a synergistic effect in HER2+ breast cancer by enhancing cell proliferation and migration decrease, G0-G1 cell cycle arrest, and apoptosis rate increase. Additionally, alpelisib combined with pyrotinib showed a tremendous synergistic effect in acquired pyrotinib resistant cells.Conclusions: Our results provided the preclinical evidence that a combination of pyrotinib and alpelisib as an effective therapeutic strategy in treating HER2+ breast cancer, whether patients were sensitive or resistant to pyrotinib treatment.

SORT1/LAMP2-mediated Extracellular Vesicle Secretion and Cell Adhesion Are Linked to Lenalidomide Resistance in Multiple Myeloma

Multiple myeloma (MM) is a hematopoietic malignancy whose prognosis has improved with the development of new agents such as lenalidomide over the last decade. However, long-term exposure to drugs induces the acquisition of resistance by MM cells and leads to treatment failure and poor prognosis. Here, we show the molecular and cellular mechanisms of lenalidomide resistance in MM. In a comparison between lenalidomide-resistant cell lines and the parental cell lines, the EV (Extracellular versicles) secretion and adherence abilities were significantly elevated in the resistant cells. Whole-transcriptome analysis revealed that the SORT1 and LAMP2 genes were key regulators of EV secretion. Silencing of these genes caused decreased EV secretion and loss of cell adhesion in the resistant cells, resulting in increased sensitivity to lenalidomide. Analysis of publicly available transcriptome data confirmed the relationship between genes related to EV secretion and cell adhesion and patient prognosis. Together, our findings reveal a novel mechanism of lenalidomide resistance in MM mediated by EV secretion and cell adhesion via SORT1 and LAMP2.

Elevated N-Glycosylation Contributes to the Cisplatin Resistance of Non-Small Cell Lung Cancer Cells Revealed by Membrane Proteomic and Glycoproteomic Analysis

Chemoresistance is the major restriction on the clinical use of cisplatin. Aberrant changes in protein glycosylation are closely associated with drug resistance. Comprehensive study on the role of protein glycosylation in the development of cisplatin resistance would contribute to precise elucidation of the complicated mechanism of resistance. However, comprehensive characterization of glycosylated proteins remains a big challenge. In this work, we integrated proteomic and N-glycoproteomic workflow to comprehensively characterize the cisplatin resistance-related membrane proteins. Using this method, we found that proteins implicated in cell adhesion, migration, response to drug, and signal transduction were significantly altered in both protein abundance and glycosylation level during the development of cisplatin resistance in the non-small cell lung cancer cell line. Accordingly, the ability of cell migration and invasion was markedly increased in cisplatin-resistant cells, hence intensifying their malignancy. In contrast, the intracellular cisplatin accumulation was significantly reduced in the resistant cells concomitant with the down-regulation of drug uptake channel protein, LRRC8A, and over-expression of drug efflux pump proteins, MRP1 and MRP4. Moreover, the global glycosylation was elevated in the cisplatin-resistant cells. Consequently, inhibition of N-glycosylation reduced cell resistance to cisplatin, whereas promoting the high-mannose or sialylated type of glycosylation enhanced the resistance, suggesting that critical glycosylation type contributes to cisplatin resistance. These results demonstrate the high efficiency of the integrated proteomic and N-glycoproteomic workflow in discovering drug resistance-related targets, and provide new insights into the mechanism of cisplatin resistance.

Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis

BackgroundDrug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin’s lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression.MethodsImmunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH.ResultsWe demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues.ConclusionsOur findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.

Molecular Alterations Associated with Acquired Drug Resistance during Combined Treatment with Encorafenib and Binimetinib in Melanoma Cell Lines

Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of “drug holiday”, but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.

Targeting CDA-Directed Metabolism With 5-Formyl-2'-Deoxycytidine For ALK Inhibitor–Resistant Lung Cancer Therapy

Background: Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion–positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). While drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. Methods: We used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. Molecular biology was used to characterize the role of CDA in drug resistance. Integrated analysis of scRNA-seq and scATAC-seq identified gene regulatory networks in resistant cells. Clinical relevance of CDA was evaluated using TCGA datasets, patient-derived cells, and tumor biopsies. Results: CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the na¨ıve cell population by scRNA-seq, and their abundance increased in the acquired-resistance population. Knockdown of CDA had antiproliferative e↵ects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2’-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Conclusions: Targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK-inhibitor resistance in NSCLC.