Chemoresistance In Colorectal Cancer Research Articles

Ubiquitin-Specific Peptidase 22 Contributes to Colorectal Cancer Stemness and Chemoresistance via Wnt/β-Catenin Pathway

Background/Aims: Two major barriers to the successful treatment of colorectal cancer (CRC) are the development of stem cell-like characteristics (stemness) and chemoresistance. Ubiquitin-specific peptidase 22 (USP22) is a deubiquitinating enzyme and putative CRC marker that has emerged as a potential cause of both phenomena in CRC. There is evidence that USP22 acts through the Wnt/β-catenin pathway and that downregulation of the latter may reduce chemoresistance. Methods: In this study, we used CRC tissue specimens from human patients as well as human CRC cell lines to evaluate the role of USP22 in CRC stemness and chemoresistance in vitro and in vivo. RT-PCR and western blot were used for gene expression analyses. Immunohistochemistry was performed for USP22 expression in clinical samples. CD133 levels were analyzed by flow cytometry. Sphere formation and MTT assays were used for self-renewal and proliferation analysis. Chemoresistance was evaluated by cell viability and sphere formation assays. Results: We found a significant increase of USP22 in recurrent CRC and chemoresistant CRC cells as compared to primary CRC and non-chemoresistant CRC cells, respectively. We then demonstrated that USP22 mediates CRC cell chemoresistance through the Wnt/β-catenin pathway and that reducing USP22 in CRC cells diminishes chemoresistance. Conclusions: Having established the crucial role of USP22 in CRC stemness and chemoresistance, this study suggests that USP22 may be an ideal genetic target in the treatment of chemoresistant CRC.

Knockdown of Long Non-Coding RNA XIST Inhibited Doxorubicin Resistance in Colorectal Cancer by Upregulation of miR-124 and Downregulation of SGK1

Doxorubicin (DOX) is a widely used chemotherapeutic agent for colorectal cancer (CRC). However, the acquirement of DOX resistance limits its clinical application for cancer therapy. Mounting evidence has suggested that aberrantly expressed lncRNAs contribute to drug resistance of various tumors. Our study aimed to explore the role and molecular mechanisms of lncRNA X-inactive specific transcript (XIST) in chemoresistance of CRC to DOX. The expressions of XIST, miR-124, serum and glucocorticoid-inducible kinase 1 (SGK1) mRNA in DOX-resistant CRC tissues and cells were detected by qRT-PCR or western blot analysis. DOX sensitivity was assessed by detecting IC50 value of DOX, the protein levels of P-glycoprotein (P-gp) and glutathione S-transferase-π (GST-π) and apoptosis. The interactions between XIST, miR-124 and SGK1 were confirmed by luciferase reporter assay, qRT-PCR and western blot. Xenograft tumor assay was used to verify the role of XIST in DOX resistance in CRC in vivo. XIST expression was upregulated and miR-124 expression was downregulated in DOX-resistant CRC tissues and cells. Knockdown of XIST inhibited DOX resistance of CRC cells, as evidenced by the reduced IC50 value of DOX, decreased P-gp and GST-π levels and enhanced apoptosis in XIST-silenced DOX-resistant CRC cells. Additionally, XIST positively regulated SGK1 expression by interacting with miR-124 in DOX-resistant CRC cells. miR-124 suppression strikingly reversed XIST-knockdown-mediated repression on DOX resistance in DOX-resistant CRC cells. Moreover, SGK1-depletion-elicited decrease of DOX resistance was greatly restored by XIST overexpression or miR-124 inhibition in DOX-resistant CRC cells. Furthermore, XIST knockdown enhanced the anti-tumor effect of DOX in CRC in vivo. XIST exerted regulatory function in resistance of DOX possibly through miR-124/SGK1 axis, shedding new light on developing promising therapeutic strategy to overcome chemoresistance in CRC patients.

Vitexin induces apoptosis by suppressing autophagy in multi-drug resistant colorectal cancer cells.

Cancer treatment is limited due to the diverse multidrug resistance acquired by cancer cells and the collateral damage caused to adjacent normal cells by chemotherapy. The flavonoid compound vitexin exhibits anti-oxidative, anti-inflammatory and anti-tumor activity. This study elucidated the antitumor effects of vitexin and its underlying mechanisms in a multi-drug resistant human colon cancer cell line (HCT-116DR), which exhibits higher levels of multidrug-resistant protein 1 (MDR1) expression as compared with its parental cell line (HCT-116). Here, we observed that vitexin suppressed MDR-1 expression and activity in HCT-116DR cells and showed cytotoxic effect in HCT-116DR cells by inhibiting autophagy and inducing apoptosis in a concentration-dependent manner. Additionally, vitexin treatment caused cleavage of caspase-9 and caspase-3, and upregulated the expression of the pro-apoptotic proteins, BID and Bax. Moreover, the expression of autophagy-related proteins, such as ATG5, Beclin-1 and LC3-II, was markedly reduced by vitexin treatment. Furthermore, in vivo experiments showed that vitexin induced apoptosis and suppressed tumor growth in HCT-116DR xenograft model. These results revealed that vitexin induced apoptosis through suppression of autophagy in vitro and in vivo and provide insight into the therapeutic potential of vitexin for the treatment of chemo-resistant colorectal cancer.

The Immune-microenvironment Confers Chemoresistance of Colorectal Cancer through Macrophage-Derived IL6.

Purpose: Tumor-associated macrophages (TAMs) are frequently associated with poor prognosis in human cancers. However, the effects of TAMs in colorectal cancer are contradictory. We therefore investigated the functions, mechanisms, and clinical significance of TAMs in colorectal cancer.Experimental Design: We measured the macrophage infiltration (CD68), P-gp, and Bcl2 expression in colorectal cancer tissues using IHC staining. Coculture of TAMs and colorectal cancer cells both in vitro and in vivo models was used to evaluate the effects of TAMs on colorectal cancer chemoresistance. Cytokine antibody arrays, ELISA, neutralizing antibody, and luciferase reporter assay were performed to uncover the underlying mechanism.Results: TAM infiltration was associated with chemoresistance in patients with colorectal cancer. Colorectal cancer-conditioned macrophages increased colorectal cancer chemoresistance and reduced drug-induced apoptosis by secreting IL6, which could be blocked by a neutralizing anti-IL6 antibody. Macrophage-derived IL6 activated the IL6R/STAT3 pathway in colorectal cancer cells, and activated STAT3 transcriptionally inhibited the tumor suppressor miR-204-5p. Rescue experiment confirmed that miR-204-5p is a functional target mediating the TAM-induced colorectal cancer chemoresistance. miR-155-5p, a key miRNA regulating C/EBPβ, was frequently downregulated in TAMs, resulting in increased C/EBPβ expression. C/EBPβ transcriptionally activated IL6 in TAMs, and TAM-secreted IL6 then induced chemoresistance by activating the IL6R/STAT3/miR-204-5p pathway in colorectal cancer cells.Conclusions: Our data indicate that the maladjusted miR-155-5p/C/EBPβ/IL6 signaling in TAMs could induce chemoresistance in colorectal cancer cells by regulating the IL6R/STAT3/miR-204-5p axis, revealing a new cross-talk between immune cells and tumor cells in colorectal cancer microenvironment. Clin Cancer Res; 23(23); 7375-87. ©2017 AACR.

Downregulation of miR-874-3p promotes chemotherapeutic resistance in colorectal cancer via inactivation of the Hippo signaling pathway.

Overcoming resistance to chemotherapy is an arduous challenge in the treatment of colorectal cancer (CRC), particularly since the underlying molecular mechanisms remain obscure. In the present study, we reported that miR-874-3p was markedly downregulated in CRC tissues compared with that in adjacent normal colorectal epithelial tissues. Upregulation of miR-874-3p attenuated the chemoresistance of CRC cells to 5-fluorouracil (5-FU) in vitro and in vivo. Conversely, inhibition of miR-874-3p yielded an opposite effect. Furthermore, our results demonstrated that miR-874-3p directly inhibited the expression of transcriptional co-activators YAP and TAZ of the Hippo signaling pathway, resulting in the inactivation of the TEAD transcription. Thus, our findings clarify a novel mechanism by which miR-874-3p restores chemotherapeutic sensitivity of CRC to 5-FU, indicating that offering miR-874-3p mimics in combination with 5-FU may serve as a new therapeutic strategy to circumvent the chemoresistance in CRC.

MiR-92a promotes stem cell-like properties by activating Wnt/β-catenin signaling in colorectal cancer.

We previously reported the oncogenic function of miR-92a in colorectal cancer. This study identified that miR-92a was upregulated in chemoresistant colorectal cancer cells and tissues. Ectopic expression of miR-92a conferred resistance to 5-fluorouracil-induced apoptosis in vitro, while antagomiR-92a significantly enhanced chemosensitivity in vivo. Moreover, Overexpression of miR-92a promoted the tumor sphere formation and the expression of stem cell markers. MiR-92a overexpression also displayed higher tumourigenesis in vivo. Furthermore, we demonstrated that miR-92a upregulates the Wnt/β-catenin signaling activity via directly targeting KLF4, GSK3β and DKK3, which are multiple level negative regulators of the Wnt/β-catenin signaling cascade. In addition, our results indicate IL-6/STAT3 pathway increases miR-92a expression by directly targeting its promoter, resulting in Wnt/β-catenin signaling activation and consequent promotion of stem-like phenotypes of colorectal cancer cells. Our present results suggest the essential role of IL-6/STAT3/miR-92a/Wnt/β-catenin pathway in regulating the stem cell-like traits of colorectal cancer cells and provide a potential target for colorectal cancer therapy.

Deregulated expression of microRNA-200b/c and SUZ12, a Polycomb repressive complex 2 subunit, in chemoresistant colorectal cancer cells.

In colorectal cancer, chemotherapy and/or radiotherapy can lead to the formation of resistant cells that become metastatic through Epithelial-Mesenchymal Transition (EMT). Invasive and metastatic characteristics of carcinoma cells in primary tumors are mediated by EMT. During EMT, the primary tumor cells lose cell-cell adhesion, have increased intercellular separation, and gain an elongated shape with pseudopodia. There is also dysregulation of Polycomb group proteins (such as BMI1, SUZ12, and EZH2), and changes in the expression of microRNA-200 (miR-200) family. In this study, we developed a chemoresistant colorectal cancer cell line (DLD-1-OxR) by exposing DLD-1 colorectal cancer cells to increasing concentrations of oxaliplatin (a chemotherapy drug used for colorectal cancer), and tested for EMT characteristics. We found that DLD-1-OxR exhibited EMT characteristics by morphologic, biochemical and molecular markers. SUZ12, a Polycomb repressive complex 2 subunit, was upregulated in DLD-1-OxR. The miRNA-200 family members that target SUZ12 were downregulated. Drug resistance is an impediment to chemotherapy and understanding the molecular mechanisms of chemoresistance can lead to its reversal and improvement of chemotherapy outcomes.

Participation of CCL1 in Snail-Positive Fibroblasts in Colorectal Cancer Contribute to 5-Fluorouracil/Paclitaxel Chemoresistance.

PurposeCancer-associated fibroblasts (CAFs) activated by cancer cells has a central role in development and malignant biological behavior in colorectal cancer (CRC). Adult fibroblasts do not express Snail, but Snail-positive fibroblasts are discovered in the stroma of malignant CRC and reported to be the key role to chemoresistance. However, the reciprocal effect of CAFs expressed Snail to chemoresistance on CRC cells and the underlying molecular mechanisms are not fully characterized.Materials and MethodsSnail-overexpressed 3T3 stable cell lines were generated by lipidosome and CT26 mixed with 3T3-Snail subcutaneous transplanted CRC models were established by subcutaneous injection. Cell Counting Kit-8, flow cytometry and western blotting assays were performed, and immunohistochemistry staining was studied. The cytokines participated in chemoresistance was validated with reverse transcriptase-polymerase chain reaction and heatmap.ResultsSnail-expression fibroblasts are discovered in human and mouse spontaneous CRCs. Overexpression of Snail induces 3T3 fibroblasts transdifferentiation to CAFs. CT26 co-cultured with 3T3-Snail resisted the impairment from 5-fluorouracil and paclitaxel in vitro. The subcutaneous transplanted tumor models included 3T3-Snail cells develop without restrictions even after treating with 5-fluorouracil or paclitaxel. Moreover, these chemoresistant processes may be mediated by CCL1 secreted by Snail-expression fibroblasts via transforming growth factor β/nuclear factor-κB signaling pathways.ConclusionTaken together, Snail-expressing 3T3 fibroblasts display CAFs properties that support 5-fluorouracil and paclitaxel chemoresistance in CRC via participation of CCL1 and suggest that inhibition of the Snail-expression fibroblasts in tumor may be a useful strategy to limit chemoresistance.

Upregulation of microRNA-135b and microRNA-182 promotes chemoresistance of colorectal cancer by targeting ST6GALNAC2 via PI3K/AKT pathway.

MicroRNAs (miRNAs) are increasingly involved in the development of drug resistance, including 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC). Aberrant sialylation is correlated with human CRC. The study was to explore whether miR-135b and miR-182 modulated 5-FU chemoresistance of CRC by targeting ST6GALNAC2 via PI3K/AKT pathway. MiR-135b and miR-182 were found to be up-regulated in CRC tissues and 5-FU resistant CRC cell lines. Forced miR-135b and miR-182 expression also affected ST6GALNAC2 levels. Using reporter-gene assay, ST6GALNAC2 was identified as direct target of miR-135b and miR-182, while ST6GALNAC2 expression exhibited patterns opposite to that of miR-135b and miR-182 in CRC samples and cell lines. Interestingly, up-regulation of miR-135b or miR-182 increased drug resistance and proliferation, but decreased apoptosis in 5-FU resistant CRC cell lines. Suppression of these miRNAs implicated an inverse function, while altered expression of ST6GALNAC2 mediated CRC progression upon transfection with miR-135b/-182 mimic or inhibitor. Furthermore, miR-135b and miR-182 were clarified to regulate the activity of phosphoinositide-3 kinase (PI3K)/AKT pathway. Inhibition of the PI3K/AKT pathway enhanced the chemosensitivity to 5-FU in HCT-8/5-FU and LoVo/5-FU. Taken together, miR-135b and miR-182 may reverse the resistance to 5-FU in CRC cells by targeting ST6GALNAC2 via PI3K/AKT pathway, which render potential chemotherapy targets for the treatment of CRC.

Abstract 2048: Saturated fatty acids in cell membrane phospholipids play an important role in chemoresistance in colorectal cancer

Abstract [Background and Aim] Resistance to chemotherapy represents the major obstacle to survival in patients with chemoresistant colorectal cancer. Various factors, such as drug efflux, autocrine survival signaling, and alterations in DNA damage repair mechanisms, can contribute to chemoresistance; however, the actual underlying mechanism is yet to be elucidated. The aim of this study was to elucidate the mechanism behind anticancer drug resistance based on lipidomics. [Materials and Methods] 1) Spheroids were derived from three colorectal cancer patient-derived xenograft models (A, B, and C) that were incubated with chemotherapeutic agents, such as 5-fluorouracil (5-FU). We analyzed the response rate of spheroids under 5-FU treatment. Global lipid analysis was performed on 5-FU resistance spheroids (A) and sensitive spheroids (C) using a Q Exactive LC-MS/MS mass spectrometer, and lipid identification was performed using the Lipid Search software. 2) We tried to identify genes that were responsible for changes in phospholipid profiles in the cell membrane. 3) To determine phospholipid profile changes in the cell membrane that occur in colon cancer cell lines (HCT116, SW480) during anticancer drug resistance, the genes responsible for such resistance were knocked down using siRNA. [Results and discussion] 1) Response to 5-FU was different in individual spheroids. Global lipid analysis and chemosensitivity assay revealed that phospholipids of 5-FU resistant spheroids were rich in saturated fatty acids (SFAs), while those of the 5-FU sensitive spheroids were rich in monounsaturated fatty acids (MUFAs). 2) The enzyme stearoyl-CoA desaturase (SCD) converts SFAs to MUFAs in the phospholipids. Based on these findings we hypothesized that decreased SCD expression is possibly associated with chemoresistance. 3) Cell lines with SCD knock down demonstrated increased SFA containing phospholipids and chemoresistance towards 5-FU. Viable cells after the administration of the anti-cancer agent confirmed that the expression of CD44 was upregulated. Therefore, changes in the phospholipid composition of cell membranes regulate 5-FU resistance in colon cancer. [Conclusions] Identification and analysis of the role of membrane lipids has become possible using high-sensitivity and high-resolution mass spectrometers and the relevant software, such as Lipid Search. Changes of phospholipid composition regulates chemoresistance. In future, investigation of the pathophysiology of membrane lipids in cancer may help to reveal novel strategies for cancer therapy. Citation Format: Takanori Hiraide, Takanori Sakaguchi, Satoru Furuhashi, Tomohiro Matsumoto, Yusuke Ozaki, Ryota Kiuchi, Makoto Takeda, Yasushi Shibasaki, Yoshifumi Morita, Hirotoshi Kikuchi, Megumi Baba, Mitsutoshi Setou, Hiroyuki Konno. Saturated fatty acids in cell membrane phospholipids play an important role in chemoresistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2048. doi:10.1158/1538-7445.AM2017-2048

Drugs, Bugs, and Cancer: Fusobacterium nucleatum Promotes Chemoresistance in Colorectal Cancer

The tumor microenvironment has recently been shown to play decisive roles in chemotherapeutic response. In this issue of Cell, Yu etal. add to these findings by identifying the bacterium Fusobacterium nucleatum as a previously unrecognized chemoresistance mediator in colorectal cancer, thereby establishing the microbiota as a potential therapeutic target.

Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy

Gut microbiota are linked to chronic inflammation and carcinogenesis. Chemotherapy failure is the major cause of recurrence and poor prognosis in colorectal cancer patients. Here, we investigated the contribution of gut microbiota to chemoresistance in patients with colorectal cancer. We found that Fusobacterium (F.) nucleatum was abundant in colorectal cancer tissues in patients with recurrence post chemotherapy, and was associated with patient clinicopathological characterisitcs. Furthermore, our bioinformatic and functional studies demonstrated that F.nucleatum promoted colorectal cancer resistance to chemotherapy. Mechanistically, F.nucleatum targeted TLR4 and MYD88 innate immune signaling and specific microRNAs to activate the autophagy pathway and alter colorectal cancer chemotherapeutic response. Thus, F.nucleatum orchestrates a molecular network of the Toll-like receptor, microRNAs, and autophagy to clinically, biologically, and mechanistically control colorectal cancer chemoresistance. Measuring and targeting F.nucleatum and its associated pathway will yield valuable insight into clinical management and may ameliorate colorectal cancer patient outcomes.

Epithelial-mesenchymal transition and cancer stem cell-like phenotype induced by Twist1 contribute to acquired resistance to irinotecan in colon cancer.

Inherent and acquired chemoresistance reduce the effectiveness of irinotecan in the treatment of metastatic colorectal cancer (CRC). However, the molecular mechanisms underlying this resistance process are still unclear. Twist1 is one of the master transcription factors of epithelial-mesenchymal transition (EMT). Our previous study indicated that Twist1 is overexpressed in colon cancer tissues, and demonstrated that Twist1 plays a crucial role in the chemoresistance of CRC. In the present study, we further investigated how Twist1 contribute to acquired resistance to irinotecan in colon cancer. The irinotecan-resistant cells were established by gradual adaptation of increasing irinotecan concentrations in LoVo cells, named LoVo/CPT-11R cells. Results showed that cell viabilities to different anticancer drugs were markedly increased in LoVo/CPT-11R cells compared to LoVo cells. Moreover, LoVo/CPT-11R cells displayed EMT, CSC-like cellular morphology and relative biomarkers were also significantly increased. In addition, overexpressed Twist1 LoVo cells were established by lentivirus transfection assay, named LoVo/Twist1 cells. Results showed that the LoVo/Twist1 cells perform a distinctly decreased sensitivity to irinotecan, downregulated expression of E-cadherin, upregulated expression of cluster of differentiation44 (CD44), and a significant enhancement of invasion and migration potential by regulation of MMP2 compared with control cells. In contrast, the inhibition of Twist1 transfected with siRNA could enhance the irinotecan sensitivity in LoVo/CPT-11R cells and downregulate the expression of vimentin and CD44. Our data provide evidence that EMT and CSC-like phenotype induced by Twist1 contribute to acquire resistance to irinotecan and enhanced migration and invasion in colon cancer.

DKK4 is important in Snail1-induced chemoresistance to fluorouracilin colorectal cancer

Background: The overexpression of Snail1 is correlated with the epithelial-mesenchymal transition (EMT) in many cancers. However, the correlation between Snail1 and chemoresistance in colorectal cancer (CRC) as well as the underlying mechanism remain unknown. In this study, we evaluated the relationship between Snail1 and chemoresistance in CRC and determined whether DKK4 was involved in Snail1-induced chemoresistance. Methods: IHC was performed to determine the correlation between Snail1 and DKK4 and their relationship to chemoresistance in CRC. Additionally, a cellular functional assay was performed to study the DKK4 expression regulated by Snail1 in CRC cell lines. Results: In our cohort, a high level of Snail1 indicated chemoresistance and poor prognosis. Furthermore, a strong correlation between DKK4 and Snail1 was observed in our CRC samples. Knockdown of Snail1 in LoVo cells resulted in the downregulation of DKK4, whereas the overexpression of Snail1 in CRC cell lines resulted in resistance to 5-fluorourcil (5 FU) treatment, with an up-regulation of the DKK4 level. Knockdown of DKK4 by siRNA in Snail1-overexpressing CRC cell lines reversed the resistance to 5 FU treatment induced by Snail1 overexpression. Conclusions: Taken together, our results indicated that DKK4 could be a downstream gene that is regulated by Snail1 and could play an important role in Snail1-induced CRC chemoresistance to 5 FU. Targeting downstream Snail1 genes may provide a new therapeutic strategy for the treatment of advanced CRC.

Scutellaria barbata D. Don inhibits 5-fluorouracil resistance in colorectal cancer by regulating PI3K/AKT pathway.

5-Fluorouracil (5-FU) resistance or multidrug resistance (MDR) has become a major obstacle in clinical treatment of cancers including colorectal cancer (CRC). Aberrant activation of phosphatidylinositol 3 kinase (PI3K)/protein kinaseB (AKT) pathway may lead to unlimited growth and chemoresistance in CRC cells, which thus could be a promising therapeutic target. As a long-term used traditional Chinese folk-medicine, Scutellaria barbata D. Don (SB) processes specific anticancer activity, but its activity against cancer chemoresistance is less known. Therefore, using a 5-FU-resistant CRC cell line HCT-8/5-FU, in this study we evaluated the therapeutic efficacy of the ethanol extracts of SB (EESB) against 5-FU resistance and explored the possible molecular mechanisms. We found that EESB significantly suppressed proliferation and promoted apoptosis in HCT-8/5-FU cells. Additionally, EESB displayed remarkable effect enhancing the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine‑123 (Rh‑123) in HCT-8/5-FU cells. Furthermore, EESB obviously downregulated the expression of cyclinD1, Bcl-2 and ABCG2, while upregulated p21 and Bax expression. Moreover, EESB showed a prominent suppressive effect on the activation of PI3K/AKT pathway. The findings suggested that Scutellaria barbata D.Don was able to inhibit chemoresistance in colorectal cancer by suppression of the PI3K/AKT pathway.

MALAT1 Is Associated with Poor Response to Oxaliplatin-Based Chemotherapy in Colorectal Cancer Patients and Promotes Chemoresistance through EZH2.

A major reason for oxaliplatin chemoresistance in colorectal cancer is the acquisition of epithelial-mesenchymal transition (EMT) in cancer cells. The long noncoding RNA (lncRNA), MALAT1, is a highly conserved nuclear ncRNA and a key regulator of metastasis development in several cancers. However, its role in oxaliplatin-induced metastasis and chemoresistance is not well known. In this study, we aim to investigate the prognostic and therapeutic role of lncRNA MALAT1 in colorectal cancer patients receiving oxaliplatin-based therapy and further explore the potential transcriptional regulation through interaction with EZH2 based on the established HT29 oxaliplatin-resistant cells. Our results showed that high MALAT1 expression was associated with reduced patient survival and poor response to oxaliplatin-based chemotherapy in advanced colorectal cancer patients. Oxaliplatin-resistant colorectal cancer cells exhibited high MALAT1 expression and EMT. LncRNA MALAT1 knockdown enhances E-cadherin expression and inhibits oxaliplatin-induced EMT in colorectal cancer cells. EZH2 is highly expressed and associated with the 3' end region of lncRNA MALAT1 in colorectal cancer, and this association suppressed the expression of E-cadherin. Furthermore, targeted inhibition of MALAT1 or EZH2 reversed EMT and chemoresistance induced by oxaliplatin. Finally, the interaction between lncRNA MALAT1 and miR-218 was observed, which further indicated its prognostic value in patients who received standard FOLFOX (oxaliplatin combine with 5-fluorouracil and leucovorin) treatment. In conclusion, this study illuminates the prognostic role of lncRNA MALAT1 in colorectal cancer patients receiving oxaliplatin-based treatment and further demonstrates how lncRNA MALAT1 confers a chemoresistant function in colorectal cancer. Thus, lncRNA MALAT1 may serve as a promising prognostic and therapeutic target for colorectal cancer patients. Mol Cancer Ther; 16(4); 739-51. ©2017 AACR.

2-Deoxy-D-glucose suppresses the migration and reverses the drug resistance of colon cancer cells through ADAM expression regulation.

Cancer cell resistance to chemotherapy is associated with a poor prognosis. The compound 2-deoxy-D-glucose (2-DG) enhances the effect of chemotherapy against cancer cells lines in vitro and in vivo. However, its effect on the epithelial to mesenchymal transition (EMT) in drug-resistant cancer cells has not been fully elucidated. In this study, we investigated whether treatment of 5-fluorouracil or oxaliplatin-resistant colorectal cancer (CRC) cells with 2-DG suppressed their migratory activity and enhanced their susceptibility to chemotherapy. Chemoresistant CRC cells stably expressed drug resistance-related proteins (MDR1, MRP1, MRP2, and MRP3) and showed mesenchymal characteristics and a migratory phenotype. 2-DG treatment attenuated glycolysis-related enzyme expression, invasion activity, and EMT-related cytokine secretion in drug-resistant CRC cells. In addition, 2-DG inhibited the activation of a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17. Gene silencing of ADAM10 and ADAM17 with small interfering RNA downregulated mesenchymal properties, reduced EMT-associated cytokine secretion, and rendered chemoresistant cells susceptible to anticancer drug treatment. Collectively, these findings suggest that increased glycolytic metabolism in drug-resistant cells has an effect on both migratory activity and cell viability through the activation of ADAM10 and ADAM17.

The extracellular domain of E cadherin linked to invasiveness in colorectal cancer: a new resistance and relapses monitoring serum-bio marker?

Multiple studies have attempted to demonstrate the interest of the cell adhesion marker, E cadherin, as a diagnostic and prognosis marker in colorectal cancer (CRC). However, it was considered non specific. Studies were carried out with CRC cell lines and patients' cohort operated for CRC. The expression of E cadherin was studied after 5 fluorouracil (5FU) treatment and correlated to CRC relapse, chemo-resistance and survival. In CRC cell lines derived from high tumor stages, extracellular domain of E cadherin expression decreased after 5FU treatment whereas it increased in supernatants. Interestingly, only specific cleaved forms at 55kDa of E cadherin were detected in supernatants. In CRC surgical patients, more importantly concerning extracellular E cadherin domain, a decreased expression was observed in tissues in function of CRC stages whereas an increased expression was found in sera. Moreover, there is an increasing trend of survival with weak serum E cadherin secretion, reinforcing the implication of this protein in CRC evolution. The extracellular domain can be defined as a 5FU resistance marker and allow CRC monitoring.

MiR-506 enhances the sensitivity of human colorectal cancer cells to oxaliplatin by suppressing MDR1/P-gp expression.

Chemoresistance development represents a major obstacle to the successful treatment of colorectal cancer (CRC). The aim of this study was to elucidate the mechanism by which miR-506 reverses oxaliplatin chemoresistance in CRC. In this study, miR-506 levels were measured in 74 patients with colon cancer via quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). We subsequently analysed the relationship between miR-506 expression and CRC patient survival via the Kaplan-Meier method. MTT assay demonstrated the fractional survival rates and cell viability of HCT116-OxR, HCT116-OxR-miR-Ctrl and HCT116-OxR-miR-506 cells treated with oxaliplatin at different concentrations. Cell proliferation and apoptosis were assessed via flow cytometry (FCM) analysis and apoptosis assay. MDR1 mRNA expression and P-gp protein expression were assessed via qRT-PCR and Western blotting (WB) respectively. Immunofluorescence (IF) staining demonstrated P-gp expression in HCT116-OxR and HCT116-OxR-miR-506 cells. qRT-PCR and WB were used to detect Wnt/β-catenin pathway activity after miR-506 overexpression. In the present study, in ISH and qRT-PCR results demonstrated that miR-506 is weakly expressed in chemoresistant CRC tissues. The low miR-506 expression group exhibited lower 5-year OS and lower 5-year RFS than the high miR-506 expression group. miR-506 overexpression inhibited cell growth and increased oxaliplatin-induced cell apoptosis in HCT116-OxR cells, as shown via FCM and apoptosis assay. We subsequently noted low MDR1/P-gp expression in HCT116-OxR-miR-506 cells via qRT-PCR, WB and IF. Lastly, we demonstrated low MDR1/P-gp expression in HCT116-OxR-miR-506 cells via inhibition of the Wnt/β-catenin by WB, MTT and FCM analysis. Taken together, the findings of our study demonstrate that miR-506 overexpression in HCT116-OxR cells enhances oxaliplatin sensitivity by inhibiting MDR1/P-gp expression via down-regulation of the Wnt/β-catenin pathway and thus provide a rationale for the development of miRNA-based strategies to reverse oxaliplatin resistance in CRC cells.

Base excision repair imbalance in colorectal cancer has prognostic value and modulates response to chemotherapy.

Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients’ clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG, OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG, but not XRCC1, sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.