Parental HCT116 Cells Research Articles

Identification of key lncRNAs associated with oxaliplatin resistance in colorectal cancer cells and isolated exosomes: From In-Silico prediction to In-Vitro validation.

One of the critical challenges in managing colorectal cancer (CRC) is the development of oxaliplatin (OXP) resistance. Long non-coding RNAs (lncRNAs) have a crucial role in CRC progression and chemotherapy resistance, with exosomal lncRNAs emerging as potential biomarkers. This study aimed to predict key lncRNAs involved in OXP-resistance using in-silico methods and validate them using RT-qPCR methods in CRC cells and their isolated exosomes. Two public datasets, GSE42387 and GSE119481, were downloaded from the GEO database to identify differentially expressed genes (DEGs) and miRNAs (DEmiRNAs) associated with OXP-resistance in the HCT116 cell line. The analysis of GSE42387 revealed 210 DEGs, and GSE119481 identified 73 DEmiRNAs. A protein-protein interaction (PPI) network analysis of the DEGs identified 133 interconnected genes, from which the top ten genes with the highest degree scores were selected. By intersecting predicted miRNAs targeting these genes with the DEmiRNAs, 38 common miRNAs were found. Subsequently, 224 lncRNAs targeting these common miRNAs were predicted. LncRNA-miRNA-mRNA network were constructed and the top five lncRNAs with the highest degree scores were identified. Analysis using the Kaplan-Meier plotter database revealed that the key lncRNAs NEAT1, OIP5-AS1, and MALAT1 are significantly associated with the overall survival of CRC patients. To validate these lncRNAs, OXP-resistant HCT116 sub-cell line (HCT116/OXR) was developed by exposing parental HCT116 cells to gradually increasing concentrations of OXP. Exosomes derived from both HCT116 and HCT116/OXR cells were isolated and characterized utilizing dynamic light scattering (DLS), transmission electron microscopy (TEM), and Western blotting. RT-qPCR confirmed elevated levels of NEAT1, OIP5-AS1, and MALAT1 in HCT116/OXR cells and their exosomes compared to parental HCT116 cells and their exosomes. This study concludes that NEAT1, OIP5-AS1, and MALAT1 are associated with the OXP-resistance in CRC. The high levels of these lncRNAs in exosomes of resistant cells suggest their involvement in intercellular communication and resistance propagation. This positioning makes them promising biomarkers for OXP-resistance in CRC.

Acquired Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Resistance of Human Colorectal Cancer Cells Is Linked to Histone Acetylation and Is Synergistically Ameliorated by Combination with HDAC Inhibitors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for the treatment of various malignancies; however, its therapeutic potential is limited because of the frequent occurrence of tumor cell resistance. In this study, we determined whether TRAIL resistance acquired by repeated administration could be overcome by HDAC inhibition in human colorectal cancer cells. TRAIL-resistant HCT116 human colorectal cancer cells (HCT116-TR) were generated by repeated treatment with 10 and 25 ng/mL TRAIL twice weekly for 28 days. The resulting TRAIL-resistant cells were noncross-resistant to other chemotherapeutic agents. The levels of histone acetylation-related proteins, such as ac-histone H4 and HDAC1, were altered in HCT116-TR cells compared with the parental HCT116 cell line. The combined treatment with TRAIL and HDAC inhibitors significantly increased apoptosis in HCT116-TR cells and indicated a synergistic effect. The mechanism by which HDAC inhibition sensitizes HCT116-TR cells to TRAIL is dependent on the intrinsic pathway. In addition, we found that HDAC inhibition enhanced the sensitivity of cells to TRAIL through mitogen-activated protein kinases/CCAAT/enhancer-binding protein homologs of protein-dependent upregulation of death receptor 5. These results suggest that histone acetylation is responsible for acquired TRAIL resistance after repeated exposure and acquired resistance to TRAIL may be overcome by combination therapies with HDAC inhibitors.

In-Silico and In-Vitro Investigation of Key Long Non-coding RNAs Involved in 5-Fluorouracil Resistance in Colorectal Cancer Cells: Analyses Highlighting NEAT1 and MALAT1 as Contributors.

Background Acquired resistance to 5-fluorouracil (5-FU) frequently results in chemotherapy failure and disease recurrence in advanced colorectal cancer (CRC) patients. Research has demonstrated that dysregulation of long non-coding RNAs (lncRNAs) mediates the development of chemotherapy resistance in cancerous cells. The present study aims to identify key lncRNAs associated with 5-FU resistance in CRC using bioinformatic and experimental validation approaches. Methods The Gene Expression Omnibus (GEO) dataset GSE119481, which contains miRNA expression profiles of the parental CRC HCT116 cell line (HCT116/P) and its in-vitro established 5-FU-resistant sub-cell line (HCT116/FUR), was downloaded. Firstly, differentially expressed microRNAs (DEmiRNAs) between the parental and 5-FU resistance cells were identified. LncRNAs and mRNAs were then predicted using online databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to uncover relevant biological mechanisms and pathways. Networks integrating lncRNAs, miRNAs, and mRNAs interactions were constructed, and topological analyses were used to identify key lncRNAs associated with 5-FU resistance. An in-vitro model of the HCT116/FUR sub-cell line was developed by exposing the HCT116/P cell line to increasing concentrations of 5-FU. Finally, real-time quantitative PCR (RT-qPCR) was performed on total RNA extracted from the HCT116/P cell line and the HCT116/FUR sub-cell line to validate the in-silico predictions of key lncRNAs. Results A total of 32 DEmiRNAs were identified. Enrichment analysis demonstrated that these DEmiRNAs were mainly enriched in several cancer hallmark pathways that regulate cell growth, cell cycle, cell survival, inflammation, immune response, and apoptosis. The predictive analysis identified 237 unique lncRNAs and 123 mRNAs interacting with these DEmiRNAs. The pathway analysis indicated that most of these predicted genes were enriched in the cellular response to starvation, protein polyubiquitination, chromatin remodeling, and negative regulation of gene expression. Topological analyses of the lncRNA-miRNA-mRNA network highlighted the nuclear enriched abundant transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and Opa interacting protein 5 antisense RNA 1 (OIP5-AS1) as central lncRNAs. Experimental analysis by RT-qPCR confirmed that the expression levels of NEAT1 and MALAT1 were significantly increased in HCT116/FUR cells compared to HCT116/P cells. However, no significant difference was observed in the OIP5-AS1 expression level between the two cells. Conclusion Our findings specifically highlight MALAT1 and NEAT1 as significant contributors to 5-FU resistance in CRC. These lncRNAs are promising biomarkers for diagnosing and predicting outcomes in CRC.

Abstract 1822: Artemisia annua Lpolyphenols augment b-lapachone effects on oxaliplatin-resistant HCT 116 colorectal cancer cells by downregulating TERT, CD44, EGFR and oxaliplatin resistance-related proteins of OXP-R-Hct 116 cells

Abstract We previously demonstrated that β-lapachone (β-Lap) exerts stronger anticancer activity on oxaliplatin-resistant HCT116 colorectal cancer cells (Oxp-R-HCT 116 cells) than parental HCT116 cells (p-HCT 116 cells) by downregulating the oxaliplatin resistance-related upregulated proteins (Oxp-RR-Proteins) proteins in Oxp-R-HCT 116 cells, such as SOD1, MAPK, and nucelophosmin. We also demonstrated polyphenols extracted from Korean Artemisia annua L. (pKAL) exerts anticancer activity on radio-resistant MDA-MB-231 human breast cancer cells by suppressing stem cell phenotype, β-catenin, and MMP-9. In this study, we found that pKAL significantly enhanced the anticancer activity of β-lapachone (β-Lap) on oxaliplatin-resistant (Oxp-R) HCT116 colorectal cancer cells. The aim of this study is to elucidate how pKAL enhance the anticancer effects of β-Lap on Oxp-R-HCT 116 cells. CCK-8 assay, phase-contrast microscopy analysis, and morphological analysis with hematoxylin stain revealed that the anticancer effect of β-Lap is more enhanced by pKAL on Oxp-R-HCT 116 cells over 60 hr. This phenomenon was associated with suppression of oxaliplatin resistance-related upregulated proteins (Oxp-RR-Proteins) (p53, ERK, and β-catenin), and downregulation of cell survival- and stemness-related proteins (TERT, CD44 and EGFR) that were not up-regulated in Oxp-R-HCT 116 cells. Protein-protein interaction network analysis of the 21 proteins that showed large difference in expression between before and after the combined treatment of pKAL and β-Lap in HCT116-OxPt-R cells revealed that the enhanced anticancer effect of β-Lap by pKAL was related to the inhibition of negative regulation of apoptotic process and the induction of DNA damage through TERT, CD44, and EGFR-mediated multiple signaling networks. The detailed western blot analysis for anti-cancer effects of pKAL revealed that pKAL alone also suppressed the expression of TERT, CD44 and EGFR in p-HCT 116 cells as well as Oxp-R-HCT 116 cells while β-Lap alone did not influence the expression of them. In addition, for some of the Oxp-RR-Proteins of Oxp-R-HCT 116 cells (p53, ERK, and β-catenin) were suppressed by pKAL alone, but not others. However, some other Oxp-RR-Proteins of Oxp-R-HCT 116 cells that were not suppressed by pKAL alone were significantly suppressed by the combination treatment of pKAL and β-Lap. These findings suggest that pKAL enhanced the anti-cancer effects β-Lap by suppressing TERT, CD44, and EGFR-mediated multiple signaling networks as well as some of the Oxp-RR-Proteins of Oxp-R-HCT 116 cells. In conclusion, this study suggests that pKAL enhance the anticancer effects of β-Lap on OXP-R-Hct 116 cells by downregulating CD44, EGFR and some of the Oxp-RR-Proteins of Oxp-R-HCT 116 cells. Citation Format: Won Sup Lee, Eun Joo Jung, Hye Jung Kim, Sung Chul Shin, Gon Sup Kim, Jin-Myung Jung, Soon Chan Hong, Choong Won Kim. Artemisia annua Lpolyphenols augment b-lapachone effects on oxaliplatin-resistant HCT 116 colorectal cancer cells by downregulating TERT, CD44, EGFR and oxaliplatin resistance-related proteins of OXP-R-Hct 116 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1822.

Artemisia annua L. Polyphenols Enhance the Anticancer Effect of β-Lapachone in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

Recent studies suggest that the anticancer activity of β-lapachone (β-Lap) could be improved by different types of bioactive phytochemicals. The aim of this study was to elucidate how the anticancer effect of β-Lap is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in parental HCT116 and oxaliplatin-resistant (OxPt-R) HCT116 colorectal cancer cells. Here, we show that the anticancer effect of β-Lap is more enhanced by pKAL in HCT116-OxPt-R cells than in HCT116 cells via a CCK-8 assay, Western blot, and phase-contrast microscopy analysis of hematoxylin-stained cells. This phenomenon was associated with the suppression of OxPt-R-related upregulated proteins including p53 and β-catenin, the downregulation of cell survival proteins including TERT, CD44, and EGFR, and the upregulation of cleaved HSP90, γ-H2AX, and LC3B-I/II. A bioinformatics analysis of 21 proteins regulated by combined treatment of pKAL and β-Lap in HCT116-OxPt-R cells showed that the enhanced anticancer effect of β-Lap by pKAL was related to the inhibition of negative regulation of apoptotic process and the induction of DNA damage through TERT, CD44, and EGFR-mediated multiple signaling networks. Our results suggest that the combination of pKAL and β-Lap could be used as a new therapy with low toxicity to overcome the OxPt-R that occurred in various OxPt-containing cancer treatments.

Butyrate-mediated Resistance to Trichostatin A Accompanied by Elevated Expression of Glucose Transporter 3 (GLUT3) in Human Colorectal Carcinoma HCT116 Cells.

The aim of study was to investigate the correlation of GLUT3 upregulation and butyrate-mediated acquired chemoresistance. A butyrate-resistant CRC cell model was established from parental (PT) HCT116 cells by gradually increasing the concentration of sodium butyrate (NaBu), followed by evaluation of resistance to butyrate and trichostatin A (TSA) by the MTT method. The expression of SLC2A3 gene and GLUT3 protein were assessed by semi-quantitative RT-PCR and western blotting, respectively. The correlation of GLUT3 and butyrate-induced acquired chemoresistance was investigated using SLC2A3 silencing. Butyrate-resistant (BR) HCT116 cells were more tolerant to butyrate-induced cell death and also resist to 750 and 1000 nM TSA when compared with HCT116-PT cells (p <0.05). Long-term exposure to butyrate revealed that upregulation of the SLC2A3 gene was significantly increased by more than 20 fold (p < 0.01), and that of GLUT3 was elevated by approximately 2 fold (p < 0.05) in HCT116-BR cells. Silencing of the SLC2A3 gene increased the sensitivity of HCT116-BR cells to the effects of TSA. Upregulation of GLUT3 is associated with resistance to butyrate and TSA. GLUT3 is a molecular target for the detection of chemoresistant CRC cells and thus a potential target for diagnostic strategies.

Driver gene KRAS aggravates cancer-associated stroke outcomes

The incidence of cancer-associated stroke has increased with the prolonged survival times of cancer patients. Recent genetic studies have led to progress in cancer therapeutics, but relationships between oncogenic mutations and stroke remain elusive. Here, we focused on the driver gene KRAS, which is the predominant RAS isoform mutated in multiple cancer types, in cancer associated stroke study. KRASG13D/− and parental human colorectal carcinoma HCT116 cells were inoculated into mice that were then subjected to a photochemically-induced thrombosis model to establish ischemic stroke. We found that cancer inoculation exacerbated neurological deficits after stroke. Moreover, mice inoculated with KRASG13D/− cells showed worse neurological deficits after stroke compared with mice inoculated with parental cells. Stroke promoted tumor growth, and the KRASG13D/− allele enhanced this growth. Brain RNA sequencing analysis and serum ELISA showed that chemokines and cytokines mediating pro-inflammatory responses were upregulated in mice inoculated with KRASG13D/− cells compared with those inoculated with parental cells. STAT3 phosphorylation was promoted following ischemic stroke in the KRASG13D/− group compared with in the parental group, and STAT3 inhibition significantly ameliorated stroke outcomes by mitigating microglia/macrophage polarization. Finally, we compared the prognosis and mortality of colorectal cancer patients with or without stroke onset between 1 January 2007 and 31 December 2020 using a hospital-based cancer registry and found that colorectal cancer patients with stroke onset within 3 months after cancer diagnosis had a worse prognosis. Our work suggests an interplay between KRAS and ischemic stroke that may offer insight into future treatments for cancer-associated stroke.

The Role of Cyanidin-3-O-glucoside in Modulating Oxaliplatin Resistance by Reversing Mesenchymal Phenotype in Colorectal Cancer.

Epithelial-mesenchymal transition (EMT) plays an important role in the biological and biochemical processes of cells, and it is a critical process in the malignant transformation, and mobility of cancer. Additionally, EMT is one of the main mechanisms contributing to chemoresistance. Resistance to oxaliplatin (OXA) poses a momentous challenge in the chemotherapy of advanced colorectal cancer (CRC) patients, highlighting the need to reverse drug resistance and improve patient survival. In this study, we explored the response of cyanidin-3-O-glucoside (C3G), the most abundant anthocyanin in plants, on the mechanisms of drug resistance in cancer, with the purpose of overcoming acquired OXA resistance in CRC cell lines. We generated an acquired OXA-resistant cell line, named HCT-116-ROx, by gradually exposing parental HCT-116 cells to increasing concentrations of OXA. To characterize the resistance, we performed cytotoxicity assays and shape factor analyses. The apoptotic rate of both resistant and parental cells was determined using Hoechst 33342/Propidium Iodide (PI) fluorescence staining. Migration capacity was evaluated using a wound-healing assay. The mesenchymal phenotype was assessed through qRT-PCR and immunofluorescence staining, employing E-cadherin, N-cadherin, and Vimentin markers. Resistance characterization announced decreased OXA sensitivity in resistant cells compared to parental cells. Moreover, the resistant cells exhibited a spindle cell morphology, indicative of the mesenchymal phenotype. Combined treatment of C3G and OXA resulted in an augmented apoptotic rate in the resistant cells. The migration capacity of resistant cells was higher than parental cells, while treatment with C3G decreased the migration rate of HCT-116-ROx cells. Analysis of EMT markers showed that HCT-116-ROx cells exhibited loss of the epithelial phenotype (E-cadherin) and gain of the mesenchymal phenotype (N-cadherin and Vimentin) compared to HCT-116 cells. However, treatment of resistant cells with C3G reversed the mesenchymal phenotype. The morphological observations of cells acquiring oxaliplatin resistance indicated the loss of the epithelial phenotype and the acquisition of the mesenchymal phenotype. These findings suggest that EMT may contribute to acquired OXA resistance in CRC. Furthermore, C3G decreased the mobility of resistant cells, and reversed the EMT process, indicating its potential to overcome acquired OXA resistance.

Prostaglandin F2α synthase promotes oxaliplatin resistance in colorectal cancer through prostaglandin F2α-dependent and F2α-independent mechanism.

Oxaliplatin (Oxa) is the first-line chemotherapy drug for colorectal cancer (CRC), and Oxa resistance is crucial for treatment failure. Prostaglandin F2α synthase (PGF2α) (PGFS), an enzyme that catalyzes the production of PGF2α, is involved in the proliferation and growth of a variety of tumors. However, the role of PGFS in Oxa resistance in CRC remains unclear. To explore the role and related mechanisms of PGFS in mediating Oxa resistance in CRC. The PGFS expression level was examined in 37 pairs of CRC tissues and paracancerous tissues at both the mRNA and protein levels. Overexpression or knockdown of PGFS was performed in CRC cell lines with acquired Oxa resistance (HCT116-OxR and HCT8-OxR) and their parental cell lines (HCT116 and HCT8) to assess its influence on cell proliferation, chemoresistance, apoptosis, and DNA damage. For determination of the underlying mechanisms, CRC cells were examined for platinum-DNA adducts and reactive oxygen species (ROS) levels in the presence of a PGFS inhibitor or its products. Both the protein and mRNA levels of PGFS were increased in the 37 examined CRC tissues compared to the adjacent normal tissues. Oxa induced PGFS expression in the parental HCT116 and HCT8 cells in a dose-dependent manner. Furthermore, overexpression of PGFS in parental CRC cells significantly attenuated Oxa-induced proliferative suppression, apoptosis, and DNA damage. In contrast, knockdown of PGFS in Oxa-resistant HCT116 and HCT8 cells (HCT116-OxR and HCT8-OxR) accentuated the effect of Oxa treatment in vitro and in vivo. The addition of the PGFS inhibitor indomethacin enhanced the cytotoxicity caused by Oxa. Treatment with the PGFS-catalyzed product PGF2α reversed the effect of PGFS knockdown on Oxa sensitivity. Interestingly, PGFS inhibited the formation of platinum-DNA adducts in a PGF2α-independent manner. PGF2α exerts its protective effect against DNA damage by reducing ROS levels. PGFS promotes resistance to Oxa in CRC via both PGF2α-dependent and PGF2α-independent mechanisms.

Relationship between anticancer sensitivities and cellular respiration properties in 5-fluorouracil-resistant HCT116 human colorectal cancer cells.

5-Fluorouracil (5-FU) is widely used for colorectal cancer (CRC) treatment; however, continuous treatment of CRC cells with 5-FU can result in acquired resistance, and the underlying mechanism of 5-FU resistance remains unclear. We previously established an acquired 5-FU-resistant CRC cell line, HCT116RF10 , and examined its biological features and 5-FU resistance mechanisms. In this study, we evaluated the 5-FU sensitivity and cellular respiration dependency of HCT116RF10 cells and parental HCT116 cells under conditions of high- and low-glucose concentrations. Both HCT116RF10 and parental HCT116 cells were more sensitive to 5-FU under low-glucose conditions compared with high-glucose conditions. Interestingly, HCT116RF10 and parental HCT116 cells exhibited altered cellular respiration dependence for glycolysis and mitochondrial respiration under high- and low-glucose conditions. Additionally, HCT116RF10 cells showed a markedly decreased ATP production rate compared with HCT116 cells under both high- and low-glucose conditions. Importantly, glucose restriction significantly reduced the ATP production rate for both glycolysis and mitochondrial respiration in HCT116RF10 cells compared with HCT116 cells. The ATP production rates in HCT116RF10 and HCT116 cells were reduced by approximately 64% and 23%, respectively, under glucose restriction, suggesting that glucose restriction may be effective at enhancing 5-FU chemotherapy. Overall, these findings shed light on 5-FU resistance mechanisms, which may lead to improvements in anticancer treatment strategies.

MiR-7-5p/KLF4 signaling inhibits stemness and radioresistance in colorectal cancer

Resistance to radiotherapy remains a major unmet clinical obstacle in the treatment of locally advanced rectal cancer. Cancer stem cells (CSCs) are considered to mediate tumor development and radioresistance. However, the role of CSCs in regulating resistance to radiotherapy in colorectal cancer (CRC) remains largely unknown. We established two radioresistant CRC cell lines, HCT116-R and RKO-R, using fractionated irradiation. Analysis using miRNA sequencing and quantitative real-time PCR confirmed lower levels of miR-7-5p in both of the radioresistant cells compared to their parental cells. Subsequently, we validated that miR-7-5p expression was decreased in cancerous tissues from radiotherapy-resistant rectal cancer patients. The Cancer Genome Atlas (TCGA) database analyses revealed that low miR-7-5p expression was significantly correlated with poor prognosis in CRC patients. Overexpression of miR-7-5p led to a rescue of radioresistance and an increase in radiation-induced apoptosis, and attenuated the stem cell-like properties in HCT116-R and RKO-R cells. Conversely, knocking down miR-7-5p in parental HCT116 and RKO cells suppressed the sensitivity to radiation treatment and enhance cancer cell stemness. Stemness-associated transcription factor KLF4 was demonstrated as a target of miR-7-5p. Rescue experiments revealed that miR-7-5p/KLF4 axis could induce radiosensitivity by regulating CSCs in colorectal cancer cells. Furthermore, we used CRC tumor tissues which exhibited resistance to neoadjuvant radiotherapy to establish a patient-derived xenograft (PDX) mouse model. Tail vein injection of magnetic nanoparticles carrying miR-7-5p mimics into the PDX mice significantly inhibited tumor growth with or without irradiation treatment in vivo. Our current studies not only demonstrate an anti-cancer function of miR-7-5p in regulating CSC properties and radiosensitivity in colorectal cancer, but also provide a novel potential strategy for delaying or reverse radiation resistance in preoperative radiotherapy of CRC patients.

Prognostic risk analysis related to radioresistance genes in colorectal cancer.

Radiotherapy (RT) is one of the most important treatments for patients with colorectal cancer (CRC). Radioresistance is the crucial cause of poor therapeutic outcomes in colorectal cancer. However, the underlying mechanism of radioresistance in colorectal cancer is still poorly defined. Herein we established a radioresistant colorectal cancer cell line and performed transcriptomics analyses to search for the underlying genes that contribute to radioresistance and investigate its association with the prognosis of CRC patients. The radioresistant cell line was developed from the parental HCT116 cell by a stepwise increased dose of irradiation. Differential gene analysis was performed using cellular transcriptome data to identify genes associated with radioresistance, from which extracellular matrix (ECM) and cell adhesion-related genes were screened. Survival data from a CRC cohort in the TCGA database were used for further model gene screening and validation. The correlation between the risk score model and tumor microenvironment, clinical phenotype, drug treatment sensitivity, and tumor mutation status were also investigated. A total of 493 different expression genes were identified from the radioresistant and wild-type cell line, of which 94 genes were associated with ECM and cell adhesion-related genes. The five model genes TNFRSF13C, CD36, ANGPTL4, LAMB3, and SERPINA1 were identified for CRC radioresistance via screening using the best model. A ROC curve indicated that the AUC of the resulting prognostic model (based on the 5-gene risk score and other clinical parameters, including age, sex, and tumor stages) was 0.79, 0.77, and 0.78 at 1, 2, and 3 years, respectively. The calibration curve showed high agreement between the risk score prediction and actual survival probability. The immune microenvironment, drug treatment sensitivity, and tumor mutation status significantly differed between the high- and low-risk groups. The risk score model built with five radioresistance genes in this study, including TNFRSF13C, CD36, ANGPTL4, LAMB3, and SERPINA1, showed favorable performance in prognosis prediction after radiotherapy for CRC.

Sulforaphane is Synergistic with CB-5083 and Inhibits Colony Formation of CB-5083-Resistant HCT116 Cells.

Human p97 is a potential drug target in oncology. Mutation-driven drug resistance is an obstacle to the long-term efficacy of targeted therapy. We found that the ATPase activity for one of the CB-5083-resistant p97 mutants was reduced, which also attenuated the degradation of K48 ubiquitinated proteins in cells. To understand how p97 mutant cells with significantly reduced ATPase activity can still grow, we discovered reduced levels of CHOP and NF-κB activation in the p97 mutant cells and these cellular changes can potentially protect HCT116 cells from death due to lowered p97 activity. In addition, the NF-kB inhibitor Sulforaphane reduces proliferation of CB-5083 resistant cells and acts synergistically with CB-5083 to block proliferation of the parental HCT116 cells. The combination of Sulforaphane and CB-5083 may be a useful treatment strategy to combat CB-5083 resistance.

Improvement of resistance to oxaliplatin by vorinostat in human colorectal cancer cells through inhibition of Nrf2 nuclear translocation

Vorinostat (suberoylanilide hydroxamic acid: SAHA), a histone deacetylase inhibitor, has potential benefit of improving the resistance to conventional other anti-cancer drugs. This study was aimed to clarify whether SAHA improves the resistance to oxaliplatin (L-OHP), a platinum-based anticancer drug using L-OHP-resistant HCT116 cells (HCT116/OxR), established from colorectal cancer (CRC) cell line HCT116. HCT116/OxR cells showed cross-resistance to other platinum-based drugs. Pre-treatment with SAHA improved the sensitivity of both L-OHP and its metabolite in HCT116/OxR cells, but not in parental HCT116 cells. However, pre-treatment with SAHA did not affect the sensitivity of other platinum-based drugs. These results indicated that SAHA specifically improved the sensitivity of L-OHP in HCT116/OxR cells. Focusing on NF-E2 p45-related factor 2-Kelch-like ECH-associated protein 1 pathway (Nrf2-Keap1) pathway, which is activated by oxidative stress such as the treatment with anti-cancer drugs, mechanisms behind these observations were elucidated. In HCT116/OxR cells transfected with Nrf2 siRNA, the improving effects on L-OHP resistance by SAHA were abolished, suggesting that Nrf2-Keap1 pathway was involved in L-OHP-resistance. In addition, L-OHP metabolite significantly induced the expression of the nuclear protein Nrf2 and its target gene mRNA expression in HCT116/OxR cells. Pre-treatment with SAHA suppressed these changes observed in HCT116/OxR cells. In conclusion, this study demonstrated that SAHA improved L-OHP resistance by inhibiting Nrf2-Keap1 activation via Nrf2 nuclear translocation by L-OHP metabolite.

Trapping of 5-Fluorodeoxyuridine Monophosphate by Thymidylate Synthase Confers Resistance to 5-Fluorouracil.

The major metabolite of the anticancer agent 5-fluorouracil (5-FU) is 5-fluorodeoxyuridine monophosphate (FdUMP), which is a potent inhibitor of thymidylate synthase (TS). Recently, we hypothesized that 5-FU-resistant colorectal cancer (CRC) cells have increased levels of TS protein relative to 5-FU-sensitive CRC cells and use a fraction of their TS to trap FdUMP, which results in resistance to 5-FU. In this study, we analyzed the difference between the regulation of the balance of the free, active form of TS and the inactive FdUMP-TS form in 5-FU-resistant HCT116 cells and parental HCT116 cells. Silencing of TYMS, the gene that encodes TS, resulted in greater enhancement of the anticancer effect of 5-FU in the 5-FU-resistant HCT116RF10 cells than in the parental HCT116 cells. In addition, the trapping of FdUMP by TS was more effective in the 5-FU-resistant HCT116RF10 cells than in the parental HCT116 cells. Our observations suggest that the regulation of the balance between the storage of the active TS form and the accumulation of FdUMP-TS is responsible for direct resistance to 5-FU. The findings provide a better understanding of 5-FU resistance mechanisms and may enable the development of anticancer strategies that reverse the sensitivity of 5-FU resistance in CRC cells.

Abstract PO-157: Calcium/calmodulin-dependent protein kinase-related peptide (CARP) increases stemness of colorectal cancer cells

Abstract Calcium/calmodulin-dependent protein kinase-related peptide (CARP) is one of the five alternative splicing products of the doublecortin-like kinase 1 (DCLK1) gene, which is a specific marker for cancer stem cells in multiple cancers. Up-regulation of DCLK1 is correlated with progression and poor prognosis of malignant cancers, but the role of individual DCLK1 isoforms during tumorigenesis is unclear. CARP was reported to play an important role in the hippocampus and in the facilitation of apoptosis in granule cells of the rat dentate gyrus. Here we aimed to reveal the effects of CARP on the stemness of cancer cells. In order to achieve our goal, isogenic CARP over-expressing cells were established using HCT116 cells, a colorectal cancer cell line. Spheroid formation assay, proliferation assay, and clonogenic capacity assay were applied to assess the effects of CARP on cancer biology. Our results demonstrated that over-expression of CARP increased the number of spheroids, but decreased the proliferation rate in comparison to parental control HCT116 cells. In conclusion, CARP can increase the self-renewal capability of cancer cells under spheroid formation conditions, but can also inhibit cell proliferation under normal cell culture conditions. CARP may be used to develop a therapeutic target for cancer treatment. Citation Format: Leo Mei, Jinghe Mao. Calcium/calmodulin-dependent protein kinase-related peptide (CARP) increases stemness of colorectal cancer cells [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-157.

MLH1 mediates cytoprotective nucleophagy to resist 5-Fluorouracil-induced cell death in colorectal carcinoma.

Colorectal Cancer (CRC) with Microsatellite instability (MSI) and mutLhomolog-1 (MLH1) gene deficiency are less aggressive than MLH1 proficient cancers. MLH1 is involved in several cellular processes, but its connection with the autophagy-dependent cellular response towards anticancer drugs remains unclear. In this study, we aimed to investigate the interaction between MLH1 and the autophagy marker LC3, which facilitated nucleophagy induction, and its potential role in determining sensitivity to 5-Fluorouracil (5-FU) induced cell death. To examine the role of MLH1 in DNA-damage-induced nucleophagy in CRC cells, we utilized a panel of MLH1 deficient and MLH1 proficient CRC cell lines. We included a parental HCT116 cell line (MLH1-/-) and its isogenic cell line HCT116 MLH1+/- in which a single allele of the MLH1 gene was introduced using CRISPR-Cas9 gene editing. We observed that MLH1 proficient cells were less sensitive to the 5-FU-induced cytotoxic effect. The 5-FU induced DNA damage led to LC3 up-regulation, which was dependent on MLH1 overexpression. Moreover, immunofluorescence and immunoprecipitation data showed LC3 and MLH1 were colocalized in CRC cells. Consequently, MLH1 dependent 5-FU-induced DNA damage contributed to the formation of micronuclei. These micronuclei colocalize with autolysosome, indicating a cytoprotective role of MLH1 dependent nucleophagy. Interestingly, siRNA knockdown of MLH1 in HCT116 MLH1+/- prevented LC3 upregulation and micronuclei formation. These novel data are the first to show an essential role of MLH1 in mediating the chemoresistance and survival of cancer cells by increasing the LC3 expression and inducing nucleophagy in 5-FU treated CRC cells.

Targeted Protein Depletion Using the Auxin-Inducible Degron 2 (AID2) System.

Targeted protein depletion using a conditional degron is a powerful method to probe the role of proteins in living cells because of the speed with which depletion can be induced and its reversibility. The auxin-inducible degron (AID) is one of the most common degron-based technologies used in cell biology. We recently established an improved system, called AID2, which involves expressing a mutant E3 ligase subunit, OsTIR1(F74G), and fusing a protein of interest to the mini-AID (mAID) tag, and that employs a new and more potent ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA). The AID2 system overcomes some of the drawbacks associated with the original AID system, i.e., leaky degradation without auxin and the requirement of high auxin doses. With AID2 it is, therefore, now possible to control a degron-fused protein more precisely, enabling target proteins to be degraded with a half-life of 10 to 45 min via the addition of a low dose of 5-Ph-IAA. Importantly, in AID2, it is not necessary to control the expression of OsTIR1(F74G) for suppressing leaky degradation and a parental cell line constitutively expressing OsTIR1(F74G) can be used for the generation of multiple mAID-tagged proteins. Here, we describe a protocol for the tagging of endogenous proteins with mAID in diploid HCT116 cells. Our protocol can be applied to other mammalian cell lines and will enhance the utility of AID2 for studying protein functions in living cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of a parental HCT116 cell line expressing OsTIR1(F74G) Basic Protocol 2: Construction of CRISPR and donor plasmids for tagging endogenous genes Basic Protocol 3: Generation of cell lines expressing a protein of interest fused with mAID.

Downregulation of miR-423-5p Contributes to the Radioresistance in Colorectal Cancer Cells.

Resistance to radiotherapy is the main reason causing treatment failure in locally advanced rectal cancer. MicroRNAs (miRNAs) have been well demonstrated to regulate cancer development and progression. However, how miRNAs regulate radiotherapy resistance in colorectal cancer remains unknown. Herein, we established two human colorectal cancer cell lines resistant to radiotherapy, named HCT116-R and RKO-R, using the strategy of fractionated irradiation. The radioresistant phenotypical changes of the two cell lines were validated by cell viability assay, colony formation assay and apoptosis assay. The miRNA expression profilings of HCT116-R and RKO-R were determined using RNA-seq analyses, and further confirmed by quantitative real-time PCR. Multiple miRNAs, including miR-423-5p, miR-7-5p, miR-522-3p, miR-3184-3p, and miR-3529-3p, were identified with altered expression in both of the radiotherapy-resistant cells, compared to the parental cells. The downregulation of miR-423-5p was further validated in the rectal cancer tissues from radiotherapy-resistant patients. Silencing of miR-423-5p in parental HCT116 and RKO cells decreased the sensitivity to radiation treatment, and inhibited the radiation-induced apoptosis. In consistence, overexpression of miR-423-5p in HCT116-R and RKO-R cells partially rescued their sensitivity to radiotherapy, and promoted the radiation-induced apoptosis. Bcl-xL (Bcl-2-like protein 1) was predicted to be a potential target gene for miR-423-5p, and miR-423-5p/Bcl-xL axis could be a critical mediator of radiosensitivity in colorectal cancer cells. The current finding not only revealed a novel role of miR-423-5p in regulating the radiosensitivity in colorectal cancer, but also suggested miR-423-5p as a molecular candidate for combination therapy with radiation to treat colorectal cancer.

101P HIF1-α depletion overcomes resistance to oxaliplatin in colorectal cancer via ERK signalling pathway

Chemoresistance is a major reason for treatment failure and results in poor prognosis in patients with advanced colorectal cancer (CRC). Hypoxia-inducible factor-1 (HIF1-α) is a regulator of the transcriptional response to oxygen deprivation in cancer cells and activates the transcription of genes that are involved in angiogenesis, cell survival and invasion. However, the effect of HIF1-α activation on oxaliplatin resistance in CRC is unclear. This study aimed to investigate the effects and potential mechanism of HIF1-α on oxaliplatin resistance in CRC. Oxaliplatin-resistant cells were generated from HCT116 cells. Cell Counting Kit-8, flow cytometry, Transwell assay were used to compare the characteristics of oxaliplatin resistant HCT116 (HCT116_OxR) cells and the corresponding parental HCT116 cells. The expression of HIF1-α, extracellular signal-related kinase (ERK), and transforming growth factor β (TGFβ) were confirmed by RT-PCR and western blotting in HCT116_OxR and HCT116 cells. Next, we evaluated the combination efficacy of inhibitors of HIF1-α (YC-1), ERK (Ravoxertinib), and TGFβ1 (SB431542) with oxaliplatin by in vitro and in vivo experiments. We found that cell viability of HCT116_OxR was higher than that in parental cells in the presence of oxaliplatin. The relative expression of HIF1-α was significantly increased in HCT116_OxR cells compared with the parental HCT116 cells. Downregulation of HIF1-α in HCT116_OxR cells increased oxaliplatin sensitivity, and diminished cell survival and invasion. Significantly, ERK signaling in HCT116_OxR cells is dependent on TGFβ1, promoting oxaliplatin resistance by activating HIF1-α. Inhibition of ERK by Ravoxertinib or TGFβ1 by SB431542 efficiently overcame oxaliplatin resistance in vitro and in vivo. HIF1-α activation is regulated by TGFβ1/ERK axis in HCT116_OxR cells. Hence, HIF1-α, ERK, and TGFβ1 are potential therapeutic targets for overcoming oxaliplatin resistance in patients with CRC.