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Tumor-associated macrophages (TAM) are among the most abundant immune cells in the tumor microenvironment and are important mediators of tumor development and progression. Here, we identified a macrophage-associated long noncoding RNA (lncRNA), MALR, that facilitates progression of esophageal squamous cell carcinoma (ESCC). TAM-mediated secretion of TNFα drove MALR upregulation in ESCC cells. MALR promoted aerobic glycolytic activity and facilitated angiogenesis by activating the HIF1α signaling pathway. Mechanistically, MALR bound the dsRBD1 domain of interleukin enhancer-binding factor 3 (ILF3), promoting ILF3 protein stability and ILF3-mediated liquid–liquid phase separation (LLPS), thereby enhancing HIF1α mRNA stability by preventing PARN-mediated degradation. Loss of MALR suppressed cell line–based and patient-derived xenograft tumor growth. Clinically, high expression of MALR positively correlated with HIF1α target gene expression and indicated poor prognoses for patients with esophageal cancer. Overall, this study uncovers the physiologic roles of MALR/ILF3-mediated LLPS in tumor microenvironment remodeling, highlighting the MALR–ILF3–HIF1α axis as a potential target for cancer therapy.Significance:Secretion of TNFα by tumor-associated macrophages stimulates cancer cells to upregulate lncRNA MALR, which induces ILF3 liquid–liquid phase separation and activation of HIF1α signaling to promote cancer progression.

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ObjectiveMethionine metabolism is involved in a myriad of cellular functions, including methylation reactions and redox maintenance. Nevertheless, it remains unclear whether methionine metabolism, RNA methylation and antitumour immunity are molecularly intertwined.DesignThe antitumour immunity effect of methionine-restricted diet (MRD) feeding was assessed in murine models. The mechanisms of methionine and YTH domain-containing family protein 1 (YTHDF1) in tumour immune escape were determined in vitro and in vivo. The synergistic effects of MRD or YTHDF1 depletion with PD-1 blockade were also investigated.ResultsWe found that dietary methionine restriction reduced tumour growth and enhanced antitumour immunity by increasing the number and cytotoxicity of tumour-infiltrating CD8+T cells in different mouse models. Mechanistically, the S-adenosylmethionine derived from methionine metabolism promoted the N6-methyladenosine (m6A) methylation and translation of immune checkpoints, including PD-L1 and V-domain Ig suppressor of T cell activation (VISTA), in tumour cells. Furthermore, MRD or m6A-specific binding protein YTHDF1 depletion inhibited tumour growth by restoring the infiltration of CD8+T cells, and synergised with PD-1 blockade for better tumour control. Clinically, YTHDF1 expression correlated with poor prognosis and immunotherapy outcomes for cancer patients.ConclusionsMethionine and YTHDF1 play a critical role in anticancer immunity through regulating the functions of T cells. Targeting methionine metabolism or YTHDF1 could be a potential new strategy for cancer immunotherapy.

Tumor-associated macrophages (TAM) are among the most abundant immune cells in the tumor microenvironment and are important mediators of tumor development and progression. Here, we identified a macrophage-associated long noncoding RNA (lncRNA), MALR, that facilitates progression of esophageal squamous cell carcinoma (ESCC). TAM-mediated secretion of TNFα drove MALR upregulation in ESCC cells. MALR promoted aerobic glycolytic activity and facilitated angiogenesis by activating the HIF1α signaling pathway. Mechanistically, MALR bound the dsRBD1 domain of interleukin enhancer-binding factor 3 (ILF3), promoting ILF3 protein stability and ILF3-mediated liquid–liquid phase separation (LLPS), thereby enhancing HIF1α mRNA stability by preventing PARN-mediated degradation. Loss of MALR suppressed cell line–based and patient-derived xenograft tumor growth. Clinically, high expression of MALR positively correlated with HIF1α target gene expression and indicated poor prognoses for patients with esophageal cancer. Overall, this study uncovers the physiologic roles of MALR/ILF3-mediated LLPS in tumor microenvironment remodeling, highlighting the MALR–ILF3–HIF1α axis as a potential target for cancer therapy. Significance: Secretion of TNFα by tumor-associated macrophages stimulates cancer cells to upregulate lncRNA MALR, which induces ILF3 liquid–liquid phase separation and activation of HIF1α signaling to promote cancer progression.

Background Numerous reports on microRNAs have illustrated their role in tumor growth and metastasis. Recently, a new prognostic factor, miR‐125b‐2‐3p, has been identified for predicting chemotherapeutic sensitivity in advanced colorectal cancer (CRC). However, the specific mechanisms and biological functions of miR‐125b‐2‐3p in advanced CRC under chemotherapy have yet to be elucidated. Methods MiR‐125b‐2‐3p expression was detected by real‐time PCR (RT‐PCR) in CRC tissues. The effects of miR‐125b‐2‐3p on the growth, metastasis, and drug sensitivity of CRC cells were tested in vitro and in vivo. Based on multiple databases, the upstream competitive endogenous RNAs (ceRNAs) and the downstream genes for miR‐125b‐2‐3p were predicted by bioinformatic analysis, followed by the experiments including luciferase reporter assays, western blot assays, and so on. Results MiR‐125b‐2‐3p was significantly lowly expressed in the tissues and cell lines of CRC. Higher expression of miR‐125b‐2‐3p was associated with relatively lower proliferation rates and fewer metastases. Moreover, overexpressed miR‐125b‐2‐3p remarkably improved chemotherapeutic sensitivity of CRC in vivo and in vitro. Mechanistically, miR‐125b‐2‐3p was absorbed by long noncoding RNA (lncRNA) XIST regulating WEE1 G2 checkpoint kinase (WEE1) expression. The upregulation of miR‐125b‐2‐3p inhibited the proliferation and epithelial‐mesenchymal transition (EMT) of CRC induced by lncRNA XIST. Conclusions Lower miR‐125b‐2‐3p expression resulted in lower sensitivity of CRC to chemotherapy and was correlated with poorer survival of CRC patients. LncRNA XIST promoted CRC metastasis acting as a ceRNA for miR‐125b‐2‐3p to mediate WEE1 expression. LncRNA XIST‐miR‐125b‐2‐3p‐WEE1 axis not only regulated CRC growth and metastasis but also contributed to chemotherapeutic resistance to CRC., Low expression of miR‐125b‐2‐3p in CRC was linked to lower chemotherapeutic sensitivity and poor survival. The lncRNA XIST promoted CRC invasion and migration by functioning as a ceRNA for miR‐125b‐2‐3p to mediate WEE1 expression. Our findings suggest that the lncRNA XIST‐miR‐125b‐2‐3p‐WEE1 axis not only regulated CRC growth and metastasis but also contributed to chemotherapeutic resistance to CRC, which may shed light on their targeted applications.

BACKGROUND Overcoming oxidative stress is a critical step for tumor progression; however, the underlying mechanisms in colorectal cancer (CRC) remain unclear. METHODS We investigated nicotinamide adenine dinucleotide (phosphate) (NAD(P))-dependent enzyme methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) expression, clinical relevance, redox modification, and molecular mechanisms using the CRC cells and tissues (n = 462 paired samples). The antitumor effects of MTHFD2 inhibitor LY345899 on CRC tumorigenesis and metastasis were evaluated in vitro and in vivo. Data analysis used Kaplan-Meier, Pearson's correlation, and Student t test where appropriate. All statistical tests were two-sided. RESULTS Here, we report that the patients with high expression of MTHFD2 have a shorter overall survival (HR = 1.62, 95% CI = 1.12 to 2.36, P = .01) and disease-free survival (HR = 1.55, 95% CI = 1.07 to 2.27, P = .02) than patients with low MTHFD2 expression. Suppression of MTHFD2 disturbs NADPH and redox homeostasis and accelerates cell death under oxidative stress, such as hypoxia or anchorage independence (P ≤ .01 for all). Also, genetic or pharmacological inhibition of MTHFD2 suppresses CRC cell growth and lung and peritoneal metastasis in cell-based xenografts (n = 5-8 mice per group). Importantly, LY345899 treatment statistically significantly suppresses tumor growth and decreases the tumor weight in CRC patient-derived xenograft models (n = 10 mice per group, mean [SD] tumor weight of the vehicle-treated group was 1.83 [0.19] mg vs 0.74 [0.30] mg for the LY345899-treated group, P < .001). CONCLUSIONS Our study presents evidence that MTHFD2 confers redox homeostasis and promotes CRC cell growth and metastasis. The folate analog LY345899 as MTHFD2 inhibitor displays therapeutic activity against CRC and warrants further clinical investigation for CRC treatment.

The efficacy of susceptible variants derived from genome-wide association studies (GWAs) optimizing discriminatory accuracy of colorectal cancer (CRC) in Chinese remains unclear. In the present validation study, we assessed 75 recently identified variants from GWAs. A risk predictive model combining 19 variants using the least absolute shrinkage and selection operator (LASSO) statistics offered certain clinical advantages. This model demonstrated an area under the receiver operating characteristic (AUC) of 0.61 during training analysis and yielded robust AUCs from 0.59 to 0.61 during validation analysis in three independent centers. The individuals carrying the highest quartile of risk score revealed over 2-fold risks of CRC (ranging from 2.12 to 2.90) compared with those who presented the lowest quartile of risk score. This genetic model offered the possibility of partitioning risk within the average risk population, which might serve as a first step toward developing individualized CRC prevention strategies in China.

Rationale: The antitumor activity of high-dose ascorbate has been re-evaluated recently, but the mechanism underlying cell-specific sensitivity to ascorbate has not yet been clarified. Methods: The effects of high-dose ascorbate on gastric cancer were assessed using cancer cell lines with high and low expression of GLUT1 via flow cytometry and colony formation assays in vitro and patient-derived xenografts in vivo. Results: In this study, we demonstrated that gastric cancer cells with high GLUT1 expression were more sensitive to ascorbate treatment than cells with low GLUT1 expression. GLUT1 knockdown significantly reversed the therapeutic effects of pharmacological ascorbate, while enforced expression of GLUT1 enhanced the sensitivity to ascorbate treatment. The efficacy of pharmacological ascorbate administration in mice bearing cell line-based and patient-derived xenografts was influenced by GLUT1 protein levels. Mechanistically, ascorbate depleted intracellular glutathione, generated oxidative stress and induced DNA damage. The combination of pharmacological ascorbate with genotoxic agents, including oxaliplatin and irinotecan, synergistically inhibited gastric tumor growth in mouse models. Conclusions: The current study showed that GLUT1 expression was inversely correlated with sensitivity of gastric cancer cells to pharmacological ascorbate and suggested that GLUT1 expression in gastric cancer may serve as a marker for sensitivity to pharmacological ascorbate.

Circulating tumor DNA (ctDNA) has emerged as a useful diagnostic and prognostic biomarker in many cancers. Here, we conducted a study to investigate the potential use of ctDNA methylation markers for the diagnosis and prognostication of colorectal cancer (CRC) and used a prospective cohort to validate their effectiveness in screening patients at high risk of CRC. We first identified CRC-specific methylation signatures by comparing CRC tissues to normal blood leukocytes. Then, we applied a machine learning algorithm to develop a predictive diagnostic and a prognostic model using cell-free DNA (cfDNA) samples from a cohort of 801 patients with CRC and 1021 normal controls. The obtained diagnostic prediction model discriminated patients with CRC from normal controls with high accuracy (area under curve = 0.96). The prognostic prediction model also effectively predicted the prognosis and survival of patients with CRC (P < 0.001). In addition, we generated a ctDNA-based molecular classification of CRC using an unsupervised clustering method and obtained two subgroups of patients with CRC with significantly different overall survival (P = 0.011 in validation cohort). Last, we found that a single ctDNA methylation marker, cg10673833, could yield high sensitivity (89.7%) and specificity (86.8%) for detection of CRC and precancerous lesions in a high-risk population of 1493 participants in a prospective cohort study. Together, our findings showed the value of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of CRC.

Background: Accumulating evidence has demonstrated that microRNAs regulate diverse tumorigenic processes and play important roles in tumor metastasis and growth. Recently, miR-125b-2-3p was identified as a meaningful prognostic factor for predicting chemotherapeutic sensitivity in advanced colorectal cancer (CRC). However, the biological functions and molecular mechanisms of miR-125b-2-3p in the chemotherapy of advanced CRC have yet to be elucidated. Methods: MiR-125b-2-3p expression was detected by real-time PCR in CRC tissues. Gain-of-function experiments were performed to assess the effect of miR-125b-2-3p on CRC growth, metastasis, invasion and drug sensitivity in vitro and in vivo. Based on multiple databases, the competitive endogenous RNAs (ceRNAs) and target genes for miR-125b-2-3p were predicted and have been confirmed by bioinformatic analysis, luciferase reporter assays, rescue experiments and western blot assays. Findings: MiR-125b-2-3p expression was significantly downregulated in CRC tissues and cell lines. MiR-125b-2-3p overexpression was correlated with lower proliferation rates, fewer metastases and better chemotherapeutic sensitivity in vitro and in vivo. Mechanistically, miR-125b-2-3p was regulated by lncRNA XIST and influenced the expression of the WEE1 G2 checkpoint kinase (WEE1). Interpretation: These findings suggested that the lncRNA XIST-miR-125b-2-3p-WEE1 axis not only regulated CRC growth and metastasis but also contributed to chemotherapeutic resistance, thus identifying a potential therapeutic target for CRC. Funding: National Natural Science Foundation of China (81572392), Guangdong special support program (2016TQ03R614), National Key RD Natural Science Foundation of Guangdong Province (2014A030312015); Science and Technology Program of Guangdong (2019B020227002); Science and Technology Program of Guangzhou (201904020046, 201803040019, 201704020228). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: This study was approved by the institutional ethics review board of SYSUCC (Guangzhou, China). Our animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

Accurate risk stratification for patients with stage II/III colon cancer is pivotal for postoperative treatment decisions. Here, we aimed to identify and validate a circRNA‐based signature that could improve postoperative prognostic stratification for these patients. In current retrospective analysis, we included 667 patients with R0 resected stage II/III colon cancer. Using RNA‐seq analysis of 20 paired frozen tissues collected postoperation, we profiled differential circRNA expression between patients with and without recurrence, followed by quantitative validation. With clinical information, we generated a four‐circRNA‐based cirScore to classify patients into high‐risk and low‐risk groups in the training cohort. The patients with high cirScores in the training cohort had a shorter disease‐free survival (DFS) and overall survival (OS) than patients with low cirScores. The prognostic capacity of the classifier was validated in the internal and external cohorts. Loss‐of‐function assays indicated that the selected circRNAs played functional roles in colon cancer progression. Overall, our four‐circRNA‐based classifier is a reliable prognostic tool for postoperative disease recurrence in patients with stage II/III colon cancer.

Prognostic and predictive markers are needed to predict the clinical outcomes of patients with advanced colorectal cancer (CRC) who receive standard first-line treatments. We performed a prospective cohort study in advanced CRC patients to identify a miRNA signature that could predict the benefit of receiving first-line chemotherapy for these patients. Twenty-one paired tumours and adjacent normal tissues were collected from advanced CRC patients and analysed by miRNA microarrays. Between tumour and normal tissues, 33 miRNAs were differentially expressed and was confirmed by qRT-PCR from another group of 67 patients from a prospective cohort study. A two-miRNA-based signature was obtained using the LASSO Cox regression model based on the association between the expression of each miRNA and the PFS of individual patients. Internal and external validation cohorts, including 40 and 44 patients with advanced CRC, respectively, were performed to prove the prognostic and predictive value of this signature. A signature was built based on two miRNAs, miR-125b-2-3p and miR-933. CRC patients were classified into low- and high-risk groups for disease progression based on this tool. The patients with low risk scores generally had better PFS than those with high risk scores. In the training set, the median PFS in the low- and high-risk groups were 12.00 and 7.40 months, respectively. In the internal validation set, the median PFS in the low- and high-risk groups were 9.90 and 5.10 months, respectively. In the external validation set, the median PFS in the low- and high-risk groups were 9.90 and 6.40 months, respectively. Furthermore, we detected miR-125b-2-3p associated with CRC cell sensitivity to first-line chemotherapy. Our two-miRNA-based signature was a reliable prognostic and predictive tool for tumour progression in patients with advanced CRC, and might be able to predict the benefit of receiving standard first-line chemotherapy in CRC.

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor for the redox reactions in the metabolic pathways of cancer cells that have elevated aerobic glycolysis (Warburg effect). Cancer cells are reported to rely on NAD recycling and inhibition of the NAD salvage pathway causes metabolic collapse and cell death. However, the underlying regulatory mechanisms and clinical implications for the NAD salvage pathway in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study showed that the expression of Nampt, the rate-limiting enzyme of the NAD salvage pathway, was significantly increased in PDAC cells and PDAC tissues. Additionally, inhibition of Nampt impaired tumor growth in vitro and tumorigenesis in vivo, which was accompanied by a decreased cellular NAD level and glycolytic activity. Mechanistically, the Nampt expression was independent of Kras and p16 status, but it was directly regulated by miR-206, which was inversely correlated with the expression of Nampt in PDAC tissues. Importantly, pharmacological inhibition of Nampt by its inhibitor, FK866, significantly enhanced the antitumor activity of gemcitabine in PDAC cells and in orthotopic xenograft mouse models. In conclusion, the present study revealed a novel regulatory mechanism for Nampt in PDAC and suggested that Nampt inhibition may override gemcitabine resistance by decreasing the NAD level and suppressing glycolytic activity, warranting further clinical investigation for pancreatic cancer treatment.

Constitutive activation and gemcitabine induction of nuclear factor-κB (NF-κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF-κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF-κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF-κB by suppressing IκBα phosphorylation and decreased the expression of NF-κB response genes in MiaPaCa-2, AsPc-1, Panc-28 cells and gemcitabine resistance MiaPaCa-2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor-independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug-treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF-κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF-κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.

Nafamostat mesilate is an anti-inflammatory drug that is usually used to treat pancreatitis. Recent studies show that it can suppress pancreatic cancer via inhibition of the nuclear factor κB (NF-κB) pathway. However, whether it has anti-tumor activity in some other cancer, including colorectal cancer (CRC), has not been investigated and remained unclear. Here, our study showed that nafamostat mesilate abrogated the constitutive NF-κB activation in CRC cells, which is mediated through inhibition of phosphorylation of IκBα and nuclear translocation of p65. Also, we found that nafamostat mesilate inhibited phosphorylation of Erk in CRC cells. Consistently, our study demonstrated that nafamostat mesilate inhibited the CRC cell proliferation, invasion and migration and induced mitochondria-dependent apoptosis. Furthermore, nafamostat mesilate could reverse oxaliplatin induced NF-κB and Erk activation in CRC cells, and enhance the sensitivity of CRC cells to oxaliplatin. Nafamostat mesilate combined with oxaliplatin repressed subcutaneous tumor growth and hepatic metastasis in vivo. Overall, our data suggest that nafamostat mesilate, a relatively non-toxic drug that targets NF-κB and Erk, may, in combination with oxaliplatin, represent a novel therapeutic strategy for CRC treatment.

Colorectal cancer (CRC) is a common neoplastic disease and a frequent cause of death. Drug resistance is a major challenge to CRC treatment and stem-like side-population (SP) cells may play a key role in this resistance. Although it has been recognized that cancer stem cells may be affected by redox status, the underlying mechanisms for this effect and the roles of celllular redox adaptation and antioxidant capacity in CRC remain elusive. Our study shows that CRC SP cells are highly dependent on cellular GSH to maintain ROS levels below those of non-SP cells. Exposing CRC cells to H2O2 produced a significant decrease in the percentage of SP cells, which was rescued by adding N-acetylcysteine. Mechanistically, CD44v interacts with and stabilizes xCT and thereby promotes the uptake of cysteine for GSH synthesis and stimulates SP cell enrichment. Additionally, miR-1297 levels were inversely correlated with the expression of xCT; thus, reduced miR-1297 contributes to SP cell enrichment in CRC tumors, which results in tumor aggressiveness and poor clinical outcomes. Importantly, redox modification by PEITC significantly reduces CRC SP cells in vitro and impairs tumors growth in vivo. The combination of 5FU and PEITC led to synergistic cytotoxic effects against CRC cells in vitro and in vivo. Taken together, our data suggest that a GSH-mediated reduction in cellular ROS levels is an essential regulator of CRC SP cells mediated by the CD44v-xCT axis, and disrupting the redox status may eliminate the chemotherapy-resistant CRC SP cells with potentially significant benefits for cancer treatment.

Oesophageal squamous cell carcinoma (ESCC) is the sixth most common cause of cancer-associated death in the world and novel therapeutic alternatives are urgently warranted. In this study, we investigated the anti-tumour activity and underlying mechanisms of melatonin, an indoleamine compound secreted by the pineal gland as well as naturally occurring plant products, in ESCC cells and revealed that melatonin inhibited proliferation, migration, invasion and induced mitochondria-dependent apoptosis of ESCC cells in vitro and suppressed tumour growth in the subcutaneous mice model in vivo. Furthermore, after treatment with melatonin, the expressions of pMEK, pErk, pGSK3β and pAkt were significantly suppressed. In contrast, treatment of the conventional chemotherapeutic drug fluorouracil (5-Fu) resulted in activation of Erk and Akt, which could be reversed by co-treatment with melatonin. Importantly, melatonin effectively enhanced cytotoxicity of 5-Fu to ESCC in vitro and in vivo. Together, these results suggested that inhibition of Erk and Akt pathway by melatonin have an important role in sensitization of ESCC cells to 5-Fu. Combined 5-Fu and melatonin treatment may be appreciated as a useful approach for ESCC therapy that warrants further investigation.