Chemoresistance In Colorectal Cancer Research Articles

CX43 down-regulation promotes cell aggressiveness and 5-fluorouracil-resistance by attenuating cell stiffness in colorectal carcinoma

ABSTRACT Chemotherapy is one of the most commonly treatments of advanced colorectal cancer (CRC). However, the drug resistant following chemotherapeutic treatment is a significant challenge in the clinical management of CRC. Therefore, understanding the resistance mechanisms and developing new strategies for enhancing the sensitivity are urgently needed to improve CRC outcome. Connexins contribute to the formation of gap junctions among neighboring cells and then advance gap junctional intercellular communication (GJIC) for transportation of ions and small molecules. Although the drug resistance resulted from GJIC dysfunctional by aberrant expression of connexins is relatively well understood, the underlying mechanisms of mechanical stiffness mediated by connexin responsible for chemoresistance are largely unknown in CRC. Here, we demonstrated that connexin 43 (CX43) expression was downregulated in CRC and that loss of CX43 expression was positively correlated with metastasis and poor prognosis of CRC patients. The CX43 overexpressing suppressed CRC progression and increased the sensitivity to 5-fluorouracil (5-FU) via enhanced GJIC in vitro and in vivo. Moreover, we also highlight that the downregulation of CX43 in CRC increases the stemness of cells via reducing the cell stiffness, thus promoting the drug resistance. Our results further suggest that both effects, that is changes in the mechanical stiffness of the cell and GJIC mediated by CX43 deregulated, are closely related to drug resistance in CRC, which indicating CX43 as a target against cancer growth and chemoresistance in CRC.

Restoration of miR-650 leads to down-regulation of KISS1, a possible route involved in overcoming 5-FU resistance and induction of apoptosis in CRC cells in-vitro.

Colorectal cancer (CRC) is one of the most common cancers and the fourth leading cause of cancer-related deaths worldwide. We aimed to determine the role of miR-650 in CRC pathogenesis. In this study, we examined the expression of miR-650 and KISS1 in 80 CRC patients who either received or did not receive chemo agents. For this aim, we assessed the miR-650 and KISS1 expression levels in 80 CRC tissues, 30 of which had no history of chemotherapy. The effect of miR-650 and 5-FU on KISS1 expression was measured using qPCR and Western blotting. Also, the 5- FU effect on miR-650 expression in the CRC cell lines was measured by qRT-PCR. Next, MTT assay and Flowcytometry assays were conducted to determine the role of miR-650 in cell viability and apoptosis. The results showed that miR-650 was down-regulated in CRC tissues. However, patients who received 5-FU before surgery showed increased expression of miR-650. The results for KISS1 were insignificant while administering 5-FU to patients preoperatively increased its expression. In-vitro studies showed that 5-FU led to the up-regulation of miR-650 in the SW480 CRC cell line. Furthermore, the administration of miR-650 and 5-FU downregulated KISS1, especially when combined. Moreover, miR-650 with 5-FU significantly reduced cell viability in CRC cell lines by inducing apoptosis. These results indicate that miR-650 has a tumor suppressive function, overcoming 5-FU chemoresistance in CRC, and induces apoptosis probably by alleviating KISS1. These results suggest that miR-650 is a potential contributor to CRC pathogenesis.

PCTAIRE Protein Kinase 1 (PCTK1) Suppresses Proliferation, Stemness, and Chemoresistance in Colorectal Cancer through the BMPR1B-Smad1/5/8 Signaling Pathway.

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality worldwide. Even with advances in therapy, CRC mortality remains high. Therefore, there is an urgent need to develop effective therapeutics for CRC. PCTAIRE protein kinase 1 (PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family, and the function of PCTK1 in CRC is poorly understood. In this study, we found that patients with elevated PCTK1 levels had a better overall survival rate in CRC based on the TCGA dataset. Functional analysis also showed that PCTK1 suppressed cancer stemness and cell proliferation by using PCTK1 knockdown (PCTK1-KD) or knockout (PCTK1-KO) and PCTK1 overexpression (PCTK1-over) CRC cell lines. Furthermore, overexpression of PCTK1 decreased xenograft tumor growth and knockout of PCTK1 significantly increased in vivo tumor growth. Moreover, knockout of PCTK1 was observed to increase the resistance of CRC cells to both irinotecan (CPT-11) alone and in combination with 5-fluorouracil (5-FU). Additionally, the fold change of the anti-apoptotic molecules (Bcl-2 and Bcl-xL) and the proapoptotic molecules (Bax, c-PARP, p53, and c-caspase3) was reflected in the chemoresistance of PCTK1-KO CRC cells. PCTK1 signaling in the regulation of cancer progression and chemoresponse was analyzed using RNA sequencing and gene set enrichment analysis (GSEA). Furthermore, PCTK1 and Bone Morphogenetic Protein Receptor Type 1B (BMPR1B) in CRC tumors were negatively correlated in CRC patients from the Timer2.0 and cBioPortal database. We also found that BMPR1B was negatively correlated with PCTK1 in CRC cells, and BMPR1B expression was upregulated in PCTK1-KO cells and xenograft tumor tissues. Finally, BMPR1B-KD partially reversed cell proliferation, cancer stemness, and chemoresistance in PCTK1-KO cells. Moreover, the nuclear translocation of Smad1/5/8, a downstream molecule of BMPR1B, was increased in PCTK1-KO cells. Pharmacological inhibition of Smad1/5/8 also suppressed the malignant progression of CRC. Taken together, our results indicated that PCTK1 suppresses proliferation and cancer stemness and increases the chemoresponse of CRC through the BMPR1B-Smad1/5/8 signaling pathway.

The ubiquitin E3 ligase MDM2 induces chemoresistance in colorectal cancer by degradation of ING3.

Chemoresistance is an obstacle for colorectal cancer (CRC) treatment. This study investigates the role of the ubiquitin E3 ligase MDM2 in affecting cell growth and chemosensitivity in CRC cells by modifying the transcription factor inhibitor of growth protein 3 (ING3). The expression of MDM2 and ING3 in CRC tissues was predicted by bioinformatics analysis, followed by expression validation and their interaction in CRC HCT116 and LS180 cells. Ectopic overexpression or knockdown of MDM2/ING3 was performed to test their effect on proliferation and apotptosis as well as chemosensitivity of CRC cells. Finally, the effect of MDM2/ING3 expression on the in vivo tumorigenesis of CRC cells was examined through subcutaneous tumor xenograft experiment in nude mice. MDM2 promoted ubiquitin-proteasome pathway degradation of ING3 through ubiquitination and diminished its protein stability. Overexpression of MDM2 downregulated ING3 expression, which promoted CRC cell proliferation and inhibited the apoptosis. The enhancing role of MDM2 in tumorigenesis and resistance to chemotherapeutic drugs was also confirmed in vivo. Our findings highlight that MDM2 modifies the transcription factor ING3 by ubiquitination-proteasome pathway degradation, thus reducing ING3 protein stability, which finally promotes CRC cell growth and chemoresistance.

SNHG15 promotes chemoresistance and glycolysis in colorectal cancer.

Long noncoding RNAs (lncRNAs) play an important role in tumor progression. Small nucleolar RNA host gene 15 (SNHG15) is a lncRNA that has been confirmed to play an oncogenic role in multiple cancer types. However, its role in glycolysis and chemoresistance in colorectal cancer (CRC) is unclear. The expression of SNHG15 in CRC was analyzed using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases by bioinformatics methods. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to evaluate cell viability. Cell sensitivity to 5-fluorouracil (5-FU) was detected by CCK-8. Glucose absorption and lactate production were used to evaluate the impact of SNHG15 on glycolysis. RNA-seq, real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) and Western blotting (WB) were used to reveal the potential molecular mechanism of SNHG15 in CRC. SNHG15 was upregulated in CRC tissues compared with paired noncancerous tissues. Ectopic SNHG15 expression increased proliferation, 5-FU chemoresistance, and glycolysis in CRC cells. In contrast, SNHG15 knockdown inhibited CRC proliferation, 5-FU chemoresistance and glycolysis. Multiple pathways, including apoptosis and glycolysis, were potentially regulated by SNHG15 based on RNA-seq and pathway enrichment analyses. RT-qPCR and WB experiments confirmed that SNHG15 promoted the expression of TYMS, BCL2, GLUT1 and PKM2 in CRC cells. In conclusion, SNHG15 promotes 5-FU chemoresistance and glycolysis in CRC by potentially regulating the expression of TYMS, BCL2, GLUT1 and PKM2 and appears to be a new target for cancer therapy.

Proteomic profiling of FOLFOX-resistant colorectal cancer cells.

e15588 Background: Chemoresistance is a major cause of colorectal cancer (CRC) mortality. Although multidrug regimen has been established as the backbone of CRC chemotherapy for better treatment efficacy, chemoresistance is common among late-stage CRC. Proteomic profiling of both the chemoresistant and wild-type CRC cells may provide hints for the mechanisms of chemoresistance and suggest targets for improving chemotherapy efficacy. Methods: Human CRC cell lines (DLD-1 and HCT-116) were treated with progressively increased dosage of FOLFOX (5-flurouracil (5-FU), folinic acid (LEU) and oxaliplatin (OXA)) to develop chemoresistance as DLD-1-R and HCT-116-R respectively. After confirmation of the successful development of chemoresistance by Cell Counting Kit-8 test (CCK-8), the comparison of cell samples between chemoresistant and wild-type cell lines were conducted by proteomic profiling using quantitative mass spectrometry (MS)-based techniques. MS and MS/ MS peptides spectra were analysed by Progenesis QI for proteomics software compared with Swiss-Prot dataset. Differentially expressed proteins (DEP) are defined as -1 > log2FC > 1, p < 0.05. Gene-list enrichment by Enrichr (https://maayanlab.cloud/Enrichr/) was employed for identifying the hallmark gene sets of the significantly enriched molecular processes and pathways of each group. All experiments were performed with at least 3 replications. Results: The development of FOLFOX-resistance took one year. The highest drug concentrations achieved were 0.5mM 5-FU, 50µM LEU, and 50µM OXA for DLD-1-R, and 5µM 5-FU, 0.5µM LEU, and 0.5µM OXA for HCT-116-R. The chemoresistance was found to be 14.73 (DLD-1-R) and 1.61 (HCT-116-R) times of the wild-type DLD-1 and HCT-116 respectively from CCK-8 tests. A total of 345 DEP with 154 up-regulated and 191 down-regulated proteins were found in DLD-1-R. While a total of 95 DEP with 66 up-regulated and 29 down-regulated proteins were found in HCT-116-R. Gene ontology molecular process (2021) analyses found that cadherin binding were significantly enriched in both DLD-1-R and HCT-116-R (Adjusted p-value < 0.05). KEGG pathway (2021) analysis found that oxidative phosphorylation, regulation of actin cytoskeleton and HIF-1 signalling pathway are among the significantly enriched pathways in DLD-1-R (Adjusted p value < 0.05). There was no significantly enriched pathway found in HCT-116-R in the same analysis. Conclusions: Our study has successfully established CRC cells with chemoresistance to the FOLFOX regimen. Proteomic profiling has found the cadherin binding as the significantly altered molecular process along with several important signalling pathways in chemoresistant CRC cells. These findings could suggest new directions in the development of anti-chemoresistance strategies of CRC.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

AimChemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches.MethodsGene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses.ResultsWe identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells.ConclusionsIGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

FOXD1 promotes chemotherapy resistance by enhancing cell stemness in colorectal cancer through β‑catenin nuclear localization.

Forkhead box D1 (FOXD1) serves a critical role in colorectal cancer (CRC). FOXD1 expression is an independent prognostic factor in patients with CRC; however, the molecular mechanism and signaling pathway of FOXD1 that regulates cell stemness and chemoresistance has not been fully characterized. The aim of the present study was to further validate the effect of FOXD1 on the proliferation and migration of CRC cells, and to delve into the possible potential of FOXD1 in the clinical treatment of CRC. The effect of FOXD1 on cell proliferation was assessed using Cell Counting Kit 8 (CCK‑8) and colony formation assays. The effect of FOXD1 on cell migration was assessed by wound‑healing and Transwell assays. The effect of FOXD1 on cell stemness was assessed by spheroid formation in vitro and limiting dilution assays invivo. The expression of stemness associated proteins, leucine rich repeat containing Gprotein‑coupled receptor5 (LGR5), OCT4, Sox2 and Nanog, and epithelial‑mesenchymal transition associated proteins, E‑cadherin, N‑cadherin and vimentin, were detected by western blotting. Proteins interrelationships were assessed by a co‑immunoprecipitation assay. Oxaliplatin resistance was assessed using CCK‑8 and apoptosis assays invitro, and using a tumor xenograft model invivo. By constructing FOXD1 overexpression and knockdown stably transfected strains of colon cancer cells, it was revealed that the overexpression of FOXD1 increased CRC cell stemness and chemoresistance. By contrast, knockdown of FOXD1 produced the opposite effects. These phenomena were caused by the direct interaction between FOXD1 and β‑catenin, thus promoting its nuclear translocation and the activation of downstream target genes, such as LGR5 and Sox2. Notably, inhibition of this pathway with a specific β‑catenin inhibitor (XAV‑939) could impair the effects induced by the overexpression of FOXD1. In summary, these results indicated that FOXD1 may promote cell stemness and the chemoresistance of CRC by binding directly to β‑catenin and enhancing β‑catenin nuclear localization; therefore, it may be considered a potential clinical target.

KIAA1549 promotes the development and chemoresistance of colorectal cancer by upregulating ERCC2.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Chemotherapy is the mainstay of treatment for patients with CRC in II-IV stages. Resistance to chemotherapy occurs commonly, which results in treatment failure. Therefore, the identification of novel functional biomarkers is essential for recognizing high-risk patients, predicting recurrence, and developing new therapeutic strategies. Herein, we assessed the roles of KIAA1549 in promoting tumor development and chemoresistance in colorectal cancer. As a result, we found that KIAA1549 expression is up-regulation in CRC. Public databases revealed a progressive up-regulation of KIAA1549 expression from adenomas to carcinomas. Functional characterization uncovered that KIAA1549 promotes tumor malignant phenotypes and boosts the chemoresistance of CRC cells in an ERCC2-dependent manner. Inhibition of KIAA1549 and ERCC2 effectively enhanced the sensitivity to chemotherapeutic drugs oxaliplatin and 5-fluorouracil. Our findings suggest that endogenous KIAA1549 might function as a tumor development-promoting role and trigger chemoresistance in colorectal cancer partly by upregulating DNA repair protein ERCC2. Hence, KIAA1549 could be an effective therapeutic target for CRC and inhibition of KIAA1549 combined with chemotherapy might be a potential therapeutic strategy in the future.

Delivery of Engineered Primary Tumor-Derived Exosomes Effectively Suppressed the Colorectal Cancer Chemoresistance and Liver Metastasis.

Liver metastasis is one of the major causes of colorectal cancer (CRC)-related morbidity and mortality. Delivering small interfering RNAs (siRNAs) or noncoding RNAs has been reported as a promising method to target liver metastasis and chemoresistance in CRC. Here, we report a noncoding RNA delivery system using exosomes derived from primary patient cells. Coiled-coil domain-containing protein 80 (CCDC80) was strongly associated with CRC liver metastasis and chemoresistance, a finding validated by bioinformatic analysis and clinical specimens. Silencing CCDC80 significantly increased sensitivity to chemotherapy agents in OXA-resistant cell lines and a mouse model. The primary cell-derived exosome delivery system was designed to simultaneously deliver siRNAs targeting CCDC80 and increase chemotherapy sensitivity in the distant CRC liver metastasis mouse models and patient-derived xenograft mouse models. We further validated the antitumor effect in an ex vivo model of chemoresistant CRC organoids and a patient-derived organoid xenograft model. Tumor-bearing mice treated with the siRNA-delivering exosomes and hepatectomy showed ideal overall survival. Our results provide a therapeutic target and represent a possible therapeutic alternative for patients with CRC and distant metastasis and in cases of chemoresistance.

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

Abstract 5900: Tolerance to colibactin correlates with response to chemotherapeutic agents in colorectal cancer

Abstract Introduction: The bacterial genotoxin colibactin is enriched in colorectal cancer (CRC) and promotes the accumulation of mutations that drive tumorigenesis. However, systematic assessment of its impact on DNA damage response is lacking and the effect of colibactin exposure on response to other genotoxic agents (such as chemotherapy) is missing. Materials and Methods: We implemented an in vitro bacteria-coculture system to assess the effect of colibactin on a representative subset of 40 molecularly and pharmacologically annotated CRC cell lines and in a panel of isogenic DDR KO cell lines we generated. We further validated our results in patient-derived organoids. Finally, we recapitulated prolonged exposure to colibactin occurring during tumorigenesis by chronically infecting sensitive cells until the emergence of a tolerant phenotype.} Results: We found that different cell lines display specific sensitivity to colibactin’s genotoxic stress: while colibactin-tolerant cells are capable to quickly and efficiently repair colibactin-induced DNA damage, sensitive cells lack this ability. Moreover, we found that homologous recombination (HR) proficiency discriminates colibactin-tolerant cells, which display higher levels of RAD51 foci (as marker of activation of HR) compared to sensitive cells upon infection with colibactin. Screening of isogenic DDR KO cell lines revealed that genetic inactivation of the intertwined pathways of HR (through KO of ATM) and replication stress (RS) response (through KO of ATRIP) significantly sensitized cells to colibactin. In addition, we found that restoration of HR activity was sufficient to induce a colibactin-tolerant phenotype in initially sensitive cell lines. Notably, thanks to a previous effort of pharmacological characterization of CRC cell lines in our lab, we found a significant correlation between sensitivity to colibactin and irinotecan active metabolite SN38, but not oxaliplatin. We validated the same correlation in patient-derived organoids annotated for response to SN38. While colibactin, SN38 and oxaliplatin all induced RS in treated cells, we found that colibactin and SN38 showed a similar DNA damage response which involved activation of ATM. Finally, chronic re-infection of sensitive, HR-deficient CRC cells with colibactin selected a tolerant phenotype characterized by restoration of HR activity. Of translational relevance, colibactin-tolerant derivative cells acquired cross-resistance to SN38 and PARP inhibitor olaparib but not to oxaliplatin. Conclusion: Our results shed novel insight into colibactin’s genotoxic mechanism and support a model in which colibactin both promotes tumorigenesis and acts as an evolutionary bottleneck which selects HR proficient CRC cells. Furthermore, our study provides preclinical evidence on colibactin’s role in promoting chemoresistance in colorectal cancer. Citation Format: Alberto Sogari, Emanuele Rovera, Nicole Megan Reilly, Simona Lamba, Mariangela Russo, Annalisa Lorenzato, Erika Durinikova, Livio Trusolino, Sabrina Arena, Manuela Donalisio, Federica Di Nicolantonio, David Lembo, Alberto Bardelli. Tolerance to colibactin correlates with response to chemotherapeutic agents 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 5900.

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.

Aberrant HMGA2 Expression Sustains Genome Instability That Promotes Metastasis and Therapeutic Resistance in Colorectal Cancer

Colorectal cancer (CRC) is one of the most lethal cancers worldwide, accounting for nearly ~10% of all cancer diagnoses and deaths. Current therapeutic approaches have considerably increased survival for patients diagnosed at early stages; however, ~20% of CRC patients are diagnosed with late-stage, metastatic CRC, where 5-year survival rates drop to 6-13% and treatment options are limited. Genome instability is an enabling hallmark of cancer that confers increased acquisition of genetic alterations, mutations, copy number variations and chromosomal rearrangements. In that regard, research has shown a clear association between genome instability and CRC, as the accumulation of aberrations in cancer-related genes provides subpopulations of cells with several advantages, such as increased proliferation rates, metastatic potential and therapeutic resistance. Although numerous genes have been associated with CRC, few have been validated as predictive biomarkers of metastasis or therapeutic resistance. A growing body of evidence suggests a member of the High-Mobility Group A (HMGA) gene family, HMGA2, is a potential biomarker of metastatic spread and therapeutic resistance. HMGA2 is expressed in embryonic tissues and is frequently upregulated in aggressively growing cancers, including CRC. As an architectural, non-histone chromatin binding factor, it initiates chromatin decompaction to facilitate transcriptional regulation. HMGA2 maintains the capacity for stem cell renewal in embryonic and cancer tissues and is a known promoter of epithelial-to-mesenchymal transition in tumor cells. This review will focus on the known molecular mechanisms by which HMGA2 exerts genome protective functions that contribute to cancer cell survival and chemoresistance in CRC.

Research progress of traditional Chinese medicine as sensitizer in reversing chemoresistance of colorectal cancer.

In recent years, the incidences and mortalities from colorectal cancer (CRC) have been increasing; therefore, there is an urgent need to discover newer drugs that enhance drug sensitivity and reverse drug tolerance in CRC treatment. With this view, the current study focuses on understanding the mechanism of CRC chemoresistance to the drug as well as exploring the potential of different traditional Chinese medicine (TCM) in restoring the sensitivity of CRC to chemotherapeutic drugs. Moreover, the mechanism involved in restoring sensitivity, such as by acting on the target of traditional chemical drugs, assisting drug activation, increasing intracellular accumulation of anticancer drugs, improving tumor microenvironment, relieving immunosuppression, and erasing reversible modification like methylation, have been thoroughly discussed. Furthermore, the effect of TCM along with anticancer drugs in reducing toxicity, increasing efficiency, mediating new ways of cell death, and effectively blocking the drug resistance mechanism has been studied. We aimed to explore the potential of TCM as a sensitizer of anti-CRC drugs for the development of a new natural, less-toxic, and highly effective sensitizer to CRC chemoresistance.

Fully human monoclonal antibody targeting activated ADAM10 on colorectal cancer cells

Metastasis and chemoresistance in colorectal cancer are mediated by certain poorly differentiated cancer cells, known as cancer stem cells, that are maintained by Notch downstream signaling initiated upon Notch cleavage by the metalloprotease ADAM10. It has been shown that ADAM10 overexpression correlates with aberrant signaling from Notch, erbBs, and other receptors, as well as a more aggressive metastatic phenotype, in a range of cancers including colon, gastric, prostate, breast, ovarian, uterine, and leukemia. ADAM10 inhibition, therefore, stands out as an important and new approach to deter the progression of advanced CRC. For targeting the ADAM10 substrate-binding region, which is located outside of the catalytic domain of the protease, we generated a human anti-ADAM10 monoclonal antibody named 1H5. Structural and functional characterization of 1H5 reveals that it binds to the substrate-binding cysteine-rich domain and recognizes an activated ADAM10 conformation present on tumor cells. The mAb inhibits Notch cleavage and proliferation of colon cancer cell lines in vitro and in mouse models. Consistent with its binding to activated ADAM10, the mAb augments the catalytic activity of ADAM10 towards small peptide substrates in vitro. Most importantly, in a mouse model of colon cancer, when administered in combination with the therapeutic agent Irinotecan, 1H5 causes highly effective tumor growth inhibition without any discernible toxicity effects. Our singular approach to target the ADAM10 substrate-binding region with therapeutic antibodies could overcome the shortcomings of previous intervention strategies of targeting the protease active site with small molecule inhibitors that exhibit musculoskeletal toxicity.

STAT3 regulates 5-Fu resistance in human colorectal cancer cells by promoting Mcl-1-dependent cytoprotective autophagy.

Chemoresistance to 5-fluorouracil (5-Fu)-based chemotherapy is one of the primary reasons for the failure of colorectal cancer (CRC) management. STAT3 can mediate tumor drug resistance through a variety of diverse mechanisms. Nonetheless, the underlying mechanisms of STAT3-induced 5-Fu resistance in CRC are still poorly understood. Here, we aimed to investigate the potential mechanism(s) of STAT3-induced 5-Fu resistance in CRC. Quantitative RT-PCR and Western blot were used to test the expression of STAT3 and Mcl-1 in chemosensitive and chemoresistant CRC tissues and cell lines. After overexpression or knockdown of STAT3 or Mcl-1, and/or treatment with or without 5-Fu or chloroquine (CQ), we tested cell viability, inhibitory concentration 50% (IC50 ) value of 5-FU, cell apoptosis, proliferation, migration, and autophagy. STAT3 and Mcl-1 were significantly upregulated in the chemoresistant CRC tissues and cell lines, and STAT3 positively regulated Mcl-1. Functional studies demonstrated that STAT3 promoted 5-Fu resistance in CRC. Mechanistically, STAT3 triggered autophagy via Mcl-1 to induce cancer chemoresistance. Our results show that STAT3 regulates 5-Fu resistance in CRC by promoting Mcl-1-dependent cytoprotective autophagy. Our results provide a novel role of STAT3 and may offer a new approach for managing CRC 5-Fu resistance.

LncRNA CACClnc promotes chemoresistance of colorectal cancer by modulating alternative splicing of RAD51.

Long non-coding RNAs (lncRNAs) play important roles in carcinogenesis. However, the effect of lncRNA on chemoresistance and RNA alternative splicing remains largely unknown. In this study, we identified a novel lncRNA, CACClnc, which was upregulated and associated with chemoresistance and poor prognosis in colorectal cancer (CRC). CACClnc promoted CRC resistance to chemotherapy via promoting DNA repair and enhancing homologous recombination in vitro and in vivo. Mechanistically, CACClnc specifically bound to Y-box binding protein 1 (YB1, a splicing factor) and U2AF65 (a subunit of U2AF splicing factor), promoting the interaction between YB1 and U2AF65, and then modulated alternative splicing (AS) of RAD51 mRNA, and consequently altered CRC cell biology. In addition, expression of exosomal CACClnc in peripheral plasma of CRC patients can effectively predict the chemotherapy effect of patients before treatment. Thus, measuring and targeting CACClnc and its associated pathway could yield valuable insight into clinical management and might ameliorate CRC patients' outcomes.

USP35 promotes cell proliferation and chemotherapeutic resistance through stabilizing FUCA1 in colorectal cancer

Ubiquitin-specific-processing proteases 35 (USP35) is an under-characterized deubiquitinase and its role in colorectal cancer (CRC) remains unclear. Here, we focus on delineating the impact of USP35 on CRC cell proliferation and chemo-resistance, as well as the possible regulatory mechanism. By examining the genomic database and clinical samples, we found that USP35 was overexpressed in CRC. Further functional studies showed that enhanced USP35 expression promoted CRC cell proliferation and resistance to oxaliplatin (OXA) and 5-fluorouracil (5-FU), whereas USP35 depletion impeded cell proliferation and sensitized cells to OXA and 5-FU treatments. Then, to explore the possible mechanism underlying USP35-triggered cellular responses, we performed co-immunoprecipitation (co-IP) followed by mass spectrometry (MS) analysis and identified α-L-fucosidase 1 (FUCA1) as a direct deubiquitiation target of USP35. Importantly, we demonstrated that FUCA1 was an essential mediator for USP35-induced cell proliferation and chemo-resistance in vitro and in vivo. Finally, we observed that nucleotide excision repair (NER) components (e.g., XPC, XPA, ERCC1) were up-regulated by USP35-FUCA1 axis, indicating a potential mechanism for USP35-FUCA1-mediated platinum resistance in CRC. Together, our results for the first time explored the role and important mechanism of USP35 in CRC cell proliferation and chemotherapeutic response, providing a rationale for USP35-FUCA1-targeted therapy in CRC.

Up-regulation of STRA6 predicts poor prognosis and contributes to oxaliplatin resistance in colorectal cancer.

Signaling receptor and transporter of retinol STRA6 (STRA6) plays a primary role in transporting retinol. Several studies have reported that STRA6 is involved in several pathways related to tumorigenesis and progression. However, the exact functions and mechanisms of STRA6 in colorectal cancer (CRC) remain unclear. In our work, STRA6 was highly up-regulated in CRC and promoted the proliferation of CRC cells. Additionally, we discovered that STRA6 suppresses apoptosis partly by controlling BCL2 expression, which in turn causes CRC to become resistant to LOHP treatment. Our study demonstrates that STRA6 is a potential prognostic factor and oncogene in CRC by promoting CRC growth and chemoresistance.