Oxaliplatin-resistant Colorectal Cancer Cells Research Articles

Upregulated lncRNA PRNT promotes progression and oxaliplatin resistance of colorectal cancer cells by regulating HIPK2 transcription.

Colorectal cancer (CRC) is the third most common cancer and a significant cause of cancer-related mortality globally. Resistance to chemotherapy, especially during CRC treatment, leads to reduced effectiveness of drugs and poor patient outcomes. Long noncoding RNAs (lncRNAs) have been implicated in various pathophysiological processes of tumor cells, including chemotherapy resistance, yet the roles of many lncRNAs in CRC remain unclear. To identify and analyze the lncRNAs involved in oxaliplatin resistance in CRC and to understand the underlying molecular mechanisms influencing this resistance. Gene Expression Omnibus datasets GSE42387 and GSE30011 were reanalyzed to identify lncRNAs and mRNAs associated with oxaliplatin resistance. Various bioinformatics tools were employed to elucidate molecular mechanisms. The expression levels of lncRNAs and mRNAs were assessed via quantitative reverse transcription-polymerase chain reaction. Functional assays, including MTT, wound healing, and Transwell, were conducted to investigate the functional implications of lncRNA alterations. Interactions between lncRNAs and transcription factors were examined using RIP and luciferase reporter assays, while Western blotting was used to confirm downstream pathways. Additionally, a xenograft mouse model was utilized to study the in vivo effects of lncRNAs on chemotherapy resistance. LncRNA prion protein testis specific (PRNT) was found to be upregulated in oxaliplatin-resistant CRC cell lines and negatively correlated with homeodomain interacting protein kinase 2 (HIPK2) expression. PRNT was demonstrated to sponge transcription factor zinc finger protein 184 (ZNF184), which in turn could regulate HIPK2 expression. Altered expression of PRNT influenced CRC cell sensitivity to oxaliplatin, with overexpression leading to decreased sensitivity and decreased expression reducing resistance. Both RIP and luciferase reporter assays indicated that ZNF184 and HIPK2 are targets of PRNT. The PRNT/ZNF184/HIPK2 axis was implicated in promoting CRC progression and oxaliplatin resistance both in vitro and in vivo. The study concludes that PRNT is upregulated in oxaliplatin-resistant CRC cells and modulates the expression of HIPK2 by sponging ZNF184. This regulatory mechanism enhances CRC progression and resistance to oxaliplatin, positioning PRNT as a promising therapeutic target for CRC patients undergoing oxaliplatin-based chemotherapy.

Wnt/β-catenin signalling activates IMPDH2-mediated purine metabolism to facilitate oxaliplatin resistance by inhibiting caspase-dependent apoptosis in colorectal cancer

BackgroundOxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC.MethodsAn oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5ʹ-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro.ResultsMetabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/β-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/β-catenin and IMPDH2. Blocking the Wnt/β-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP.ConclusionsIMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC.

LINC00982-encoded protein PRDM16-DT regulates CHEK2 splicing to suppress colorectal cancer metastasis and chemoresistance.

Metastasis is one of the key factors of treatment failure in late-stage colorectal cancer (CRC). Metastatic CRC frequently develops resistance to chemotherapeutic agents. This study aimed to identify the novel regulators from "hidden" proteins encoded by long noncoding RNAs (lncRNAs) involved in tumor metastasis and chemoresistance. Methods: CRISPR/Cas9 library functional screening was employed to identify the critical suppressor of cancer metastasis in highly invasive CRC models. Western blotting, immunofluorescence staining, invasion, migration, wound healing, WST-1, colony formation, gain- and loss-of-function experiments, in vivo experimental metastasis models, multiplex immunohistochemical staining, immunohistochemistry, qRT-PCR, and RT-PCR were used to assess the functional and clinical significance of FOXP3, PRDM16-DT, HNRNPA2B1, and L-CHEK2. RNA-sequencing, co-immunoprecipitation, qRT-PCR, RT-PCR, RNA affinity purification, RNA immunoprecipitation, MeRIP-quantitative PCR, fluorescence in situ hybridization, chromatin immunoprecipitation and luciferase reporter assay were performed to gain mechanistic insights into the role of PRDM16-DT in cancer metastasis and chemoresistance. An oxaliplatin-resistant CRC cell line was established by in vivo selection. WST-1, colony formation, invasion, migration, Biacore technology, gain- and loss-of-function experiments and an in vivo experimental metastasis model were used to determine the function and mechanism of cimicifugoside H-1 in CRC. Results: The novel protein PRDM16-DT, encoded by LINC00982, was identified as a cancer metastasis and chemoresistance suppressor. The down-regulated level of PRDM16-DT was positively associated with malignant phenotypes and poor prognosis of CRC patients. Transcriptionally regulated by FOXP3, PRDM16-DT directly interacted with HNRNPA2B1 and competitively decreased HNRNPA2B1 binding to exon 9 of CHEK2, resulting in the formation of long CHEK2 (L-CHEK2), subsequently promoting E-cadherin secretion. PRDM16-DT-induced E-cadherin secretion inhibited fibroblast activation, which in turn suppressed CRC metastasis by decreasing MMP9 secretion. Cimicifugoside H-1, a natural compound, can bind to LEU89, HIS91, and LEU92 of FOXP3 and significantly upregulated PRDM16-DT expression to repress CRC metastasis and reverse oxaliplatin resistance. Conclusions: lncRNA LINC00982 can express a new protein PRDM16-DT to function as a novel regulator in cancer metastasis and drug resistance of CRC. Cimicifugoside H-1 can act on the upstream of the PRDM16-DT signaling pathway to alleviate cancer chemoresistance.

The role of p62 in cell death and survival of 5-fluorouracil and oxaliplatin-resistant colorectal cancer cells.

The protein sequestosome 1 (p62/SQSTM1) is primarily known as a selective autophagy cargo receptor, but due to its multidomain structure, it also has roles in the ubiquitin-proteasome system, metabolism, cell death and survival signalling. The increase in p62 levels is detected in some types of cancers, including colorectal cancer (CRC). Chemoresistance is the main cause of high mortality rates of CRC patients. Since p62 can regulate both cell survival and death, it is a potential modulator of chemoresistance. The impact of p62 on molecular causes of chemoresistance in CRC cells is insufficiently analysed. Therefore, we aimed to determine the impact of p62 on apoptosis, RIPK1-pRIPK3 axis, and IL-8 levels in chemoresistant CRC cells. Our data revealed that p62 levels are higher in the 5-fluorouracil (5-FU)-resistant HCT116/FU subline compared to the parental cell line. 5-FUand oxaliplatin (OxaPt) treatment decreased p62 protein levels and it correlated with chemoresistance of HCT116 and DLD1 cell lines. The silencing of p62 increased CRC cell sensitivity to 5-FU and OxaPt, hence p62 is one of the factors supporting chemoresistance. The downregulation of p62 reduced the activation of caspase-3 and the levels of RIPK1 and pRIPK3. Furthermore, p62 silencing decreased the BAX/BCL2 ratio in the HCT116/FU subline and did not change the levels of apoptosis. Instead, p62 silencing reduced the amount of IL-8 protein. Our results show that p62 impacts chemoresistance by stimulating prosurvival signalling.

Inhibition of the RBMS1/PRNP axis improves ferroptosis resistance-mediated oxaliplatin chemoresistance in colorectal cancer.

The majority of patients with advanced colorectal cancer have chemoresistance to oxaliplatin, and studies on oxaliplatin resistance are limited. Our research showed that RNA-binding motif single-stranded interacting protein 1 (RBMS1) caused ferroptosis resistance in tumor cells, leading to oxaliplatin resistance. We employed bioinformatics to evaluate publically accessible data sets and discovered that RBMS1 was significantly upregulated in oxaliplatin-resistant colorectal cancer cells, in tandem with ferroptosis suppression. In vivo and in vitro studies revealed that inhibiting RBMS1 expression caused ferroptosis in colorectal cancer cells, restoring tumor cell sensitivity to oxaliplatin. Mechanistically, this is due to RBMS1 inducing prion protein translation, resulting in ferroptosis resistance in tumor cells. Validation of clinical specimens revealed that RBMS1 is similarly linked to tumor development and a poor prognosis. Overall, RBMS1 is a potential therapeutic target with clinical translational potential, particularly for oxaliplatin chemoresistance in colorectal cancer.

Oxaliplatin Enhances the Apoptotic Effect of Mesenchymal Stem Cells, Delivering Soluble TRAIL in Chemoresistant Colorectal Cancer.

A key problem in colorectal cancer (CRC) is the development of resistance to current therapies due to the presence of cancer stem cells (CSC), which leads to poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a protein that activates apoptosis in cancer cells through union with TRAIL death receptors. Cell therapies as delivery systems can produce soluble TRAIL (sTRAIL) and full-length TRAIL (flTRAIL), showing a high capacity to produce apoptosis in vitro and in vivo assays. However, the apoptotic activity of TRAIL as monotherapy had limitations, so it is important to explore other ways to enhance susceptibility to TRAIL. This study evaluated the cytotoxic and proapoptotic activity of soluble TRAIL overexpressed by mesenchymal stem cells (MSC) in an oxaliplatin-resistant CRC cell line. Bone marrow-MSC were lentiviral transduced for soluble TRAIL expression. DR5 death receptor expression was determined in Caco-2 and CMT-93 CRC cell lines. Sensitivity to first-line chemotherapies and recombinant TRAIL was evaluated by half-maximal inhibitory concentrations. Cytotoxic and proapoptotic activity of soluble TRAIL-MSC alone and combined with chemotherapy pre-treatment was evaluated using co-cultures. Caco-2 and CMT-93 cell lines expressed 59.08 ± 5.071 and 51.65 ± 11.99 of DR5 receptor and had IC50 of 534.15 ng/mL and 581.34 ng/mL for recombinant murine TRAIL (rmTRAIL), respectively. This finding was classified as moderate resistance to TRAIL. The Caco-2 cell line showed resistance to oxaliplatin and irinotecan. MSC successfully overexpressed soluble TRAIL and induced cancer cell death at a 1:6 ratio in co-culture. Oxaliplatin pre-treatment in the Caco-2 cell line increased the cell death percentage (50%) and apoptosis by sTRAIL. This finding was statistically different from the negative control (p < 0.05), and activity was even higher with the oxaliplatin-flTRAIL combination. Thus, oxaliplatin increases apoptotic activity induced by soluble TRAIL in a chemoresistant CRC cell line.

TGM2, HMGA2, FXYD3, and LGALS4 genes as biomarkers in acquired oxaliplatin resistance of human colorectal cancer: A systems biology approach.

Acquired resistance to oxaliplatin is considered as the primary reason for failure in colorectal cancer (CRC) therapy. Identifying the underlying resistance mechanisms may improve CRC treatment. The present study aims to identify the key genes involved in acquired oxaliplatin-resistant in CRC by confirming the oxaliplatin resistance index (OX-RI). To this aim, two public microarray datasets regarding oxaliplatin-resistant CRC cells with different OX-RI, GSE42387, and GSE76092 were downloaded from GEO database to identify differentially expressed genes (DEGs). The results indicated that the OX-RI affects the gene expression pattern significantly. Then, 54 common DEGs in both datasets including 18 up- and 36 down-regulated genes were identified. Protein-protein interaction (PPI) analysis revealed 13 up- (MAGEA6, TGM2, MAGEA4, SCHIP1, ECI2, CD33, AKAP12, MAGEA12, CALD1, WFDC2, VSNL1, HMGA2, and MAGEA2B) and 12 down-regulated (PDZK1IP1, FXYD3, ALDH2, CEACAM6, QPRT, GRB10, TM4SF4, LGALS4, ALDH3A1, USH1C, KCNE3, and CA12) hub genes. In the next step, two novel up-regulated hub genes including ECI2 and SCHIP1 were identified to be related to oxaliplatin resistance. Functional enrichment and pathway analysis indicated that metabolic pathways, proliferation, and epithelial-mesenchymal transition may play dominant roles in CRC progression and oxaliplatin resistance. In the next procedure, two in vitro oxaliplatin-resistant sub-lines including HCT116/OX-R4.3 and HCT116/OX-R10 cells with OX-IR 3.93 and 10.06 were established, respectively. The results indicated the up-regulation of TGM2 and HMGA2 in HCT116/OX-R10 cells with high OX-RI and down-regulation of FXYD3, LGALS4, and ECI2 in both cell types. Based on the results, TGM2, HMGA2, FXYD3, and LGALS4 genes are related to oxaliplatin-resistant CRC and may serve as novel therapeutic targets.

CYP4F3 is associated with poor prognosis and resistance to oxaliplatin based chemotherapy in col orectal cancer

Purpose: To screen the expression of different genes related to oxaliplatin resistance in colorectal cancer (CRC) therapy.&#x0D; Methods: Limma and principal component analysis (PCA) techniques were used to determine genes with significantly different expression levels in the Gene Expression Omnibus (GEO) dataset. A lasso regression model and Venn diagram were used to analyze the intersecting genes. Gene Expression Profiling Interactive Analysis (GEPIA) and the University of Alabama at Birmingham Cancer data analysis Portal (UALCAN) online platform were used to analyze the expression of CYP4F3. The relationship between CYP4F3 expression and survival rate in colorectal cancer was analyzed by Kaplan–Meier curve, while the related pathways involving CYP4F3 were determined by Metascape and gene Ontology-Kyoto Encyclopedia of Genes and Genomes (GO-KEGG) analysis. Furthermore, the correlation between CYP4F3 and TME-related cells was analyzed by Pearson score. In addition, analysis of clinically tested and FDA-approved drugs significantly associated with CYP4F3 was carried out using CellMiner database.&#x0D; Results: PCA and volcano plot analysis indicated there are four upregulated genes and 11 down-regulated genes in oxaliplatin-resistant CRC cells. The intersection gene was CYP4F3 in the lasso regression model and differentially expressed genes (DEG). Moreover, CYP4F3 was upregulated and associated with poor survival in CRC. Gene set enrichment analysis (GSEA), KEGG enrichment, and PPI analysis showed that CYP4F3 and GNG3 are the most significant genes in oxaliplatin-resistant CRC. Furthermore, CYP4F3 expression negatively correlated with the enrichment of T cells, while the expression of CYP4F3 was not associated with drug sensitivity in CRC cells.&#x0D; Conclusion: The findings of this study suggest that CYP4F3 may be a target for the treatment of oxaliplatin-resistant CRC. However, the underlying mechanism of CYP4F3 in the regulation of sensitivity to oxaliplatin needs further investigation.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells

BackgroundTumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy.MethodsImmunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The “one-two punch” sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models.ResultsWe found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3β/Wnt/β-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo.ConclusionOur study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a “one-two punch” approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

Abstract 420: Dicer-dependent of ERK activation promotes the acquired oxaliplatin resistance in colorectal cancer

Abstract Introduction: Chemoresistance is one of the major challenges for cancer therapy. Oxaliplatin is the first-line therapy for most cancers, including colorectal cancer (CRC). However, the underlying mechanisms of oxaliplatin resistance remain elusive. Previous reports indicated that microRNAs might regulate the development of CRC. Dicer, an endonuclease involved in miRNA biogenesis, may play a critical role in oxaliplatin resistance. Here, we investigated the mechanism of Dicer-mediated oxaliplatin resistance in CRC. Materials and Methods: Oxaliplatin-resistant CRC cells were established. Colony formation, migration, invasion, and sphere formation assays were performed to characterize the properties of oxaliplatin-resistant CRC cells. cDNA microarray and NGS were used to identify the altered expression of mRNAs and miRNAs in oxaliplatin-resistant cells. RT-qPCR determined gene expression. A connectivity map (CMAP) database was used to find the potential drugs which can reduce cell viability in oxaliplatin-resistant cells. CCK-8 assay was used to measure the anti-tumor effect of those potential drugs in oxaliplatin-resistant CRC cells. PANTHER was used to find the potential pathways that may involve drug resistance in CRC. Both reporter assay and western blot analyzed the activation of the Wnt/β-catenin and MAPK signaling pathways. Results: Dicer is overexpressed in oxaliplatin-resistant CRC cells and CRC recurrent patients. Overexpressed Dicer attenuated the cytotoxicity of oxaliplatin and promoted the cell migration, invasion, and stem cell properties of the oxaliplatin-resistant cells. Knocked-down of Dicer restored the chemosensitivity and decreased all of those abilities in oxaliplatin-resistant CRC cells. The expression of several potential mRNAs and microRNAs was changed in drug-resistant cells and chemo-resistant CRC patients' plasma. MAPK and Wnt/β-catenin pathways are hyper-activated in oxaliplatin-resistant CRC cells. PRI-724, a Wnt/β-catenin pathway inhibitor, can increase the cell apoptosis of oxaliplatin-resistant CRC cells. Trametinib, the MEK inhibitor, can induce cell apoptosis in oxaliplatin-resistant CRC cells and present an anti-tumor effect in oxaliplatin-resistant CRC cell-derived xenograft tumors. Knocked-down expression of Dicer did not affect the Wnt/β-catenin pathway but attenuated the ERK activation. A combination of oxaliplatin and trametinib can synergistically inhibit tumor growth and reverses oxaliplatin resistance in vitro and in vivo. Conclusion: Dicer might be an acquired factor contributing to cancer malignancies in oxaliplatin-resistant CRC cells via activating the ERK pathway. The activation of the MAPK signaling pathway is Dicer-dependent in oxaliplatin-resistant CRC cells. Wnt/β-catenin inhibitors and ERK inhibitors have an anti-tumor effect in oxaliplatin-resistant cells. Citation Format: Yu-Heng Kuan, Ming-Ern Wong, Liang-Yi Hung. Dicer-dependent of ERK activation promotes the acquired oxaliplatin resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 420.

Shikonin potentiates therapeutic efficacy of oxaliplatin through reactive oxygen species-mediated intrinsic apoptosis and endoplasmic reticulum stress in oxaliplatin-resistant colorectal cancer cells.

Oxaliplatin (OXA) has been recognized as a third-generation platinum-based chemotherapeutic agent with stellar therapeutic efficacy in managing colorectal cancer (CRC). Nevertheless, resistance to OXA in CRC patients hinders its effectiveness. Shikonin (SHI), a natural naphthoquinone derived from Arnebia euchroma (Royle) Johnst., features a broad pharmacological profile and minimal toxicities. To assess the synergism of SHI andOXAtowards OXA-resistant CRC cells (HCT116R ), we employed in vitro and in vivo pharmacological assays. Our experiments provided evidence that SHI, either alone or in combination with OXA, considerably reduced cell proliferation, triggered apoptosis, and induced the generation of reactive oxygen species (ROS) in HCT116R cells. Furthermore, the combination of SHI and OXA dramatically curbed the extent of HCT116R -initiated xenograft growth in mouse models. Bioinformatics, western blot, and ROS assays highlighted that the mechanisms of SHI against OXA-resistant CRC cells may involve the induction of cellular responses to chemical stress, intrinsic apoptosis, as well as endoplasmic reticulum stress pathways mediated by ROS. Notably, the synergism of SHI+OXA was partially abrogated by an ROS inhibitor N-acetyl cysteine. Our findings imply the potential of SHI to boost the sensitivity of OXA to CRC, offering promising benefits for clinical strategies to combat OXA resistance.

LncRNA-NEF suppressed oxaliplatin resistance and epithelial-mesenchymal transition in colorectal cancer through epigenetically inactivating MEK/ERK signaling.

A major cause of oxaliplatin chemoresistance in colorectal cancer (CRC) is acquired epithelial-mesenchymal transition (EMT) in cancer cells, making the cancer cells easy to metastasis and recurrence. LncRNA Neighboring Enhancer of FOXA2 (lncRNA-NEF) has been characterized as a tumor suppressor to mediate cancer metastasis in multiple cancer types. However, whether it mediated the drug resistance remains unknown. In the present study, an oxaliplatin-resistant CRC cell line (SW620R) was established and lncRNA-NEF was obviously down-regulated in this resistant cell line. The further loss and gain-of-function studies demonstrated that this lncRNA suppressed oxaliplatin resistance as well as EMT programme in vitro and inhibited metastasis in vivo. Mechanistically, lncRNA-NEF epigenetically promoted the expression of DOK1 (Downstream of Tyrosine kinase 1), a negative regulator of MEK/ERK signaling, by disrupting DNA methyltransferases (DNMTs)-mediated DNA methylation. DOK1, in turn, induced the inactivation of MEK/ERK signaling, forming the lncRNA-NEF/DOK1/MEK/ERK regulatory axis to mediate oxaliplatin resistance in CRC. Collectively, our work reveals the critical function of lncRNA-NEF in mediating the oxaliplatin chemotherapy resistance in CRC, and provides a promising therapeutic strategy for CRC patients with oxaliplatin resistance.

A Novel Protein Encoded by Exosomal CircATG4B Induces Oxaliplatin Resistance in Colorectal Cancer by Promoting Autophagy.

Oxaliplatin is commonly used in chemotherapeutic regimens for colorectal cancer (CRC) after surgical resection. However, acquired chemoresistance seriously affects the curative effect in CRC patients, and the mechanism is still unclear. Here, a circular RNA, circATG4B is identified, which plays an important role in oxaliplatin resistance in CRC. circATG4B expression is found to be increased in exosomes secreted by oxaliplatin-resistant CRC cells. In addition, the results suggest that circATG4B induces oxaliplatin resistance by promoting autophagy. Further in vivo and in vitro studies indicate that the effect of circATG4B is attributed to its potential to encode a novel protein, circATG4B-222aa. Next, circATG4B-222aa is found to function as a decoy to competitively interact with TMED10 and prevent TMED10 from binding to ATG4B, which leads to increased autophagy followed by induction of chemoresistance. Therefore, this study reveals that exosomal circATG4B participates in the decreased chemosensitivity of CRC cells, providing a new rationale for a potential therapeutic target for oxaliplatin resistance in CRC.

Role of AMPK in Regulation of Oxaliplatin-Resistant Human Colorectal Cancer.

Oxaliplatin is a platinum analog that can interfere with DNA replication and transcription. Continuous exposure to oxaliplatin results in chemoresistance; however, this mechanism is not well known. In this study, oxaliplatin-resistant (OR) colorectal cancer (CRC) cells of HCT116, HT29, SW480 and SW620 were established by gradually increasing the drug concentration to 2.5 μM. The inhibitory concentrations of cell growth by 50% (IC50) of oxaliplatin were 4.40-12.7-fold significantly higher in OR CRC cells as compared to their respective parental (PT) CRC cells. Phospho-Akt and phospho-mammalian target of rapamycin (mTOR) decreased in PT CRC cells but was overexpressed in OR CRC cells in response to oxaliplatin. In addition, an oxaliplatin-mediated decrease in phospho-AMP-activated protein kinase (AMPK) in PT CRC cells induced autophagy. Contrastingly, an increased phospho-AMPK in OR CRC cells was accompanied by a decrease in LC3B, further inducing the activity of glycolytic enzymes, such as glucose transporter 1 (GLUT1), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), to mediate cell survival. Inhibition of AMPK in OR CRC cells induced autophagy through inactivation of Akt/mTOR pathway and a decrease in GLUT1, PFKFB3, and PFK1. Collectively, targeting AMPK may provide solutions to overcome chemoresistance in OR CRC cells and restore chemosensitivity to anticancer drugs.

Fermented Ginger Extract in Natural Deep Eutectic Solvent Enhances Cytotoxicity by Inhibiting NF-κB Mediated CXC Chemokine Receptor 4 Expression in Oxaliplatin-Resistant Human Colorectal Cancer Cells.

Ginger extracts have been shown to have health-promoting pharmacological activity and beneficial effects, including antioxidant and anticancer properties. The extraction of ginger by natural deep eutectic solvents (NaDES) has been shown to enhance bioactivity, but the cytotoxicity of NaDES extracts needs to be further determined. Signaling through the CXC chemokine receptor 4 (CXCR4) expressed on colorectal cancer (CRC) cells has a pivotal role in tumor cell chemosensitivity. Oxaliplatin is a third-generation platinum compound used as an effective chemotherapeutic drug for CRC treatment. However, whether ginger extract and oxaliplatin could induce a synergistic cytotoxic effect in oxaliplatin-resistant CRC cells through modulating CXCR4 expression is not known. In this study, oxaliplatin-resistant HCT-116 (HCT-116/R) cells were generated first. Ginger was extracted using the NaDES mixture betaine/lactate/water (1:2:2.5). Lactobacillus reuteri fermentation of NaDES-ginger extract increased the total polyphenol content (12.42 mg gallic acid/g in non-fermented NaDES-ginger extract and 23.66 mg gallic acid/g in fermented NaDES-ginger extract). It also increased the antioxidant activity by about 20–30% compared to non-fermented NaDES-ginger extract. In addition, it achieved low cytotoxicity to normal colonic mucosal cells and enhanced the anticancer effect on HCT-116/R cells. On the other hand, the inhibition of NF-κB activation by fermented NaDES-ginger extract significantly decreased the CXCR4 expression (p < 0.05) in HCT-116/R cells. The inactivation of NF-κB by pharmacological inhibitor pyrrolidine dithiocarbamate further enhanced the fermented NaDES-ginger extract-reduced CXCR4 expression levels (p < 0.05). Moreover, fermented NaDES-ginger extract could synergistically increase the cytotoxicity of oxaliplatin by inhibiting CXCR4 expression and inactivating NF-κB, resulting in HCT-116/R cell death. These findings demonstrate that fermented NaDES-ginger extract reduces the NF-kB-mediated activation of CXCR4 and enhances oxaliplatin-induced cytotoxicity in oxaliplatin-resistant CRC cells.

YTHDF3 Facilitates eIF2AK2 and eIF3A Recruitment on mRNAs to Regulate Translational Processes in Oxaliplatin-Resistant Colorectal Cancer.

Oxaliplatin, as a first-line drug, frequently causes chemo-resistance in colorectal cancer (CRC). The role of N6-methyladenosine (m6A) modification in multiple biological functions has been well studied. However, the molecular mechanisms underlying m6A methylation in modulating anti-cancer drug resistance in CRC remain obscure. In the present study, we found that YTH m6A RNA-binding protein 3 (YTHDF3) was highly expressed in oxaliplatin-resistant (OXAR) CRC tissues and cells. Moreover, we observed that YTHDF3 could recognize the 5' untranslated region of significantly m6A-methylated RNAs, which were associated with tumor resistance and recruit eukaryotic translation initiation factor 3 subunit A (eIF3A) to facilitate the translation of these target genes. Furthermore, we determined that eukaryotic translation initiation factor 2 alpha kinase 2 (eIF2AK2) bridged YTHDF3 and eIF3A, enhancing the stability of the YTHDF3/eIF3A complex in OXAR CRC cells. Taken together, our data identified YTHDF3 as a novel hallmark and revealed the molecular mechanism of YTHDF3 on gene translation via coordination with eIF2AK2 in OXAR CRC cells.

WD repeat domain 62 (WDR62) promotes resistance of colorectal cancer to oxaliplatin through modulating mitogen-activated protein kinase (MAPK) signaling

ABSTRACT WD repeat domain 62 (WDR62) is involved in embryonic brain growth through regulation of glial and neural cell populations. WDR62 is also implicated in the carcinogenesis of various cancers. The role of WDR62 in progression and chemoresistance of colorectal cancer (CRC) was investigated. Firstly, oxaliplatin-resistant CRC cells (HCT116/R and HT29/R) were sequentially exposed to an increasing concentration of oxaliplatin. The results showed that WDR62 was elevated in CRC tissues, and oxaliplatin resistance conferred up-regulation of WDR62 in CRC cells. Knockdown of WDR62 reduced cell proliferation and promoted the apoptosis of oxaliplatin-resistant CRC cells. Moreover, silencing of WDR62 increased fluorescence intensity of γH2AX, and decreased protein expression of p-DNA-PK and Rad51 in the oxaliplatin-resistant CRC cells. The protein expression of p-ERK, p-JNK, and p-p38 in oxaliplatin-resistant CRC cells were down-regulated by knockdown of WDR62. In conclusion, silencing of WDR62 suppressed oxaliplatin resistance and DNA damage repair of CRC cells through inactivation of MAPK signaling.

LIM-Domain-Binding Protein 1 Mediates Cell Proliferation and Drug Resistance in Colorectal Cancer.

Objective: It has been shown that LIM-domain-binding protein 1 (LDB1) is involved in the tumorigenesis of several cancers, but its function in colorectal cancer (CRC) has not been fully explained. This study is aimed to investigate whether LDB1 is involved in regulating the cell growth and drug sensitivity of CRC.Methods: To analyze the protein expression of LDB1 in CRC tissues, western blot was used. KM plotter and UALCAN databases were used to predict the prognosis of CRC patients with low or high LDB1 expression. To do the correlation analysis in CRC tissues, GEPIA database was used. CCK-8 assay and xenograft models were used to evaluate the effects of LDB1 in CRC cell growth. An oxaliplatin-resistant cell line was constructed to evaluate the effect of LDB1 in drug sensitivity of CRC cells.Results: Our current research confirmed that LDB1 was upregulated in CRC tumor tissues, and its elevation predicted a poor prognosis for CRC patients. LDB1 was also found positively correlated with CCNA1, BCL2 and BCLW, but negatively correlated with the pro-apoptotic signals (BID, BAX and BAK). Silence of LDB1 significantly inhibited CRC cell growth in vitro, and CRC cells with low expression of LDB1 had a lower tumorigenesis rate in tumor-bearing nude mice. Our experiments also showed that LDB1 silence enhanced the anti-tumor activity of oxaliplatin in CRC cells. The expression of LDB1 was also found increased in oxaliplatin-resistant CRC cell lines, and silence of LDB1 partly restored the antitumor effect of oxaliplatin in an oxaliplatin-resistant CRC cell line.Conclusion: Our current results revealed the roles of LDB1 in the growth and drug resistance of CRC cells, and may provide the new theoretical support for LDB1 as a potential target for the treatment of CRC in the future.

EVs delivery of miR-1915-3p improves the chemotherapeutic efficacy of oxaliplatin in colorectal cancer.

Oxaliplatin is a crucial component of the combinatorial chemotherapeutic standard of care for advanced colorectal cancer (CRC). Unfortunately, a serious barrier to effective oxaliplatin treatment is drug resistance due to epithelial-mesenchymal transitioning (EMT). Interestingly, stable oxaliplatin-resistant CRC cell lines show differential expression of miR-1915-3p; thus, this microRNA may represent a potential modifier of oxaliplatin resistance in CRC cells. miR-1915-3p was over-expressed in oxaliplatin-resistant CRC cells and a non-tumorigenic intestinal cell line (FHC) via lentiviral transduction. Extracellular vesicles (EVs) were purified from transduced FHC cells and co-incubated with CRC cells. Expression levels of miR-1915-3p and other RNA species were assessed by RT-qPCR, while protein expression levels were assessed by Western blotting. The effects of miR-1915-3p on CRC viability were evaluated by proliferation, apoptosis assays, and Transwell assays. Effects of miR-1915-3p over-expression on in vivo oxaliplatin sensitivity was tested via murine xenograft models. miRNA-1915-3p decreased EMT marker expression in oxaliplatin-resistant CRC cell lines and in vivo. FHC cells were able to produce and secrete miR-1915-3p-containingEVs, which we employed to mediate miR-1915-3p delivery to oxaliplatin-resistant CRC cells and increase their oxaliplatin sensitivity in vivo and in vitro. Mechanistically, miR-1915-3p overexpression downregulated the EMT-promoting oncogenes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and ubiquitin carboxyl-terminal hydrolase 2 (USP2) as well as upregulated E-cadherin (a cell adhesionmediator). miR-1915-3p's effects on chemosensitivity and EMT were mediated by its regulation of PFKFB3 and USP2. Exosomal delivery of miR-1915-3p can improve the chemotherapeutic efficacy of oxaliplatin in CRC cells by suppressing the EMT-promoting oncogenes PFKFB3 and USP2.

KIF18b-dependent hypomethylation of PARPBP gene promoter enhances oxaliplatin resistance in colorectal cancer

As the new platinum drug oxaliplatin has been widely used in clinical treatment of colorectal cancer (CRC), oxaliplatin resistance has become a burning problem. In this study, higher expression of PARP-1 binding protein (PARPBP) was detected in oxaliplatin-resistant CRC (OR-CRC) cells than in non-resistant cells. Further research showed that kinesin family member 18 b (KIF18b) induced the overexpression of PARPBP, sustaining oxaliplatin resistance in OR-CRC cells. Through exploring the PARPBP gene promoter, we found that SP1-recruited DNMT3b methylated PARPBP promoter to suppress transcription in CRC cells, and increased KIF18b attenuated the recruitment of DNMT3b to PARPBP promoter by directly interacting with SP1 in OR-CRC cells. Clinical analysis suggested a positive relationship between KIF18b and PARPBP in CRC tissues and indicated poor prognosis in CRC patients with high level of KIF18b or PARPBP. In summary, KIF18b-induced PARPBP contributes to the resistant phenotype of OR-CRC.