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Dysregulated cholesterol metabolism is a hallmark of colorectal cancer (CRC). However, the usage of cholesterol-lowering agents seemed to have no benefit in CRC patients. In this study, we focused on the cholesterol-nuclear receptors (NRs) axis as a strategy. Cholesterol and its derivatives work as ligands for different nuclear receptors, thus promoting cancer progression. The key NR downstream of cholesterol in CRC is unknown. Here, we treated CRC cells with a cholesterol-lowering agent and lipoprotein-depleted conditioned medium, and then detected the change of the putative NRs. The results revealed that RORα/γ (Retinoic acid receptor-related Orphan Receptor α/γ) levels exhibited the most obvious increases in CRC cells subjected them to cholesterol deprivation. RORα/γ agonists significantly inhibited CRC cells proliferation and migration in vitro and in vivo. Also, RORα/γ overexpression repressed CRC cells proliferation and migration in vitro and in vivo and RORα/γ knockdown promoted it. Mechanistically, RORα/γ agonists promoted c-myc degradation by activating the transcription of the ubiquitinase NEDD4. Intriguingly, the combination of RORα/γ agonists and atorvastatin had a synergistic effect on inhibiting CRC cells. These findings demonstrate that the cholesterol- RORα/γ axis is important for maintaining c-myc protein levels. Combination therapy with atorvastatin and RORα/γ agonist is a promising therapeutic strategy for CRC.

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.

Supplementary Table 2

Supplementary Methods

Supplementary Figures

Supplementary Table 1

Genomic alterations of tumor suppressorsoften encompass collateral protein-coding genes that create therapeutic vulnerability to further inhibition of their paralogs. Here, we report that malic enzyme 2 (ME2) is frequently hemizygously codeleted with SMAD4 in gastric cancer. Its isoenzyme ME1 was upregulated to replenish the intracellular reducing equivalent NADPH and to maintain redox homeostasis. Knockdown of ME1 significantly depleted NADPH, induced high levels of reactive oxygen species (ROS), and ultimately cell apoptosis under oxidative stress conditions, such as glucose starvation and anoikis, in ME2-underexpressed cells. Moreover, ME1 promoted tumor growth, lung metastasis, and peritoneal dissemination of gastric cancer in vivo. Intratumoral injection of ME1 siRNA significantly suppressed tumor growth in cell lines and patient-derived xenograft–based models. Mechanistically, ME1 was transcriptionally upregulated by ROS in an ETV4-dependent manner. Overexpression of ME1 was associated with shorter overall and disease-free survival in gastric cancer. Altogether, our results shed light on crucial roles of ME1-mediated production of NADPH in gastric cancer growth and metastasis.Significance: These findings reveal the role of malic enzyme in growth and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/8/1972/F1.large.jpg. Cancer Res; 78(8); 1972–85. ©2018 AACR.

Figure legends for Figure S1-7.

Supplementary Methods provide experimental details on bioinformatics analysis, standard assays using commercial reagents and kits; Supplementary Figures S1-8 describe additional data to support the function of MNX1-AS1, which promotes the tumorigenesis in both CRC cells and tissues; Supplementary tables S1-6 show clinical information of sequenced patients, detailed screening candidates for validation, regression analysis together with mass spectrometry data used in this study.

Supplementary Figure S1. SMAD4 and ME2 copy number are frequently altered in human malignancies. Supplementary Figure S2. ME1 maintains cell survival during glucose deprivation. Supplementary Figure S3. Effects of ME1 on epithelial mesenchymal transition in gastric cancer. Supplementary Figure S4. Simultaneous knockdown of ME1 and ME2 in HGC27 cells significantly suppressed tumor growth in vivo. Supplementary Figure S5. Immunoblots show increased ME1 and decreased ME2 expression in the tumor tissues compared with corresponding normal tissues of PDX models. Supplementary Figure S6. ETV4 regulates ME1 expression in gastric cancer. Supplementary Figure S7. Clinical significance of ETV4/ME1 axis in gastric cancer.

Long noncoding RNAs (lncRNA) are involved in tumorigenesis and drug resistance. However, the roles and underlying mechanisms of lncRNAs in colorectal cancer are still unknown. In this work, through transcriptomic profiling analysis of 21 paired tumor and normal samples, we identified a novel colorectal cancer–related lncRNA, MNX1-AS1. MNX1-AS1 expression was significantly upregulated in colorectal cancer and associated with poor prognosis. In vitro and in vivo gain- and loss-of-function experiments showed that MNX1-AS1 promotes the proliferation of colorectal cancer cells. MNX1-AS1 bound to and activated Y-box-binding protein 1 (YB1), a multifunctional RNA/DNA-binding protein, and prevented its ubiquitination and degradation. A marked overlap between genes that are differentially expressed in MNX1-AS1 knockdown cells and transcriptional targets of YB1 was observed. YB1 knockdown mimicked the loss of viability phenotype observed upon depletion of MNX1-AS1. In addition, MYC bound the promoter of the MNX1-AS1 locus and activated its transcription. In vivo experiments showed that ASO inhibited MNX1-AS1, which suppressed the proliferation of colorectal cancer cells in both cell-based and patient-derived xenograft models. Collectively, these findings suggest that the MYC–MNX1-AS1–YB1 axis might serve as a potential biomarker and therapeutic target in colorectal cancer.Significance:This study highlights the discovery of a novel colorectal cancer biomarker and therapeutic target, MNX1-AS1, a long noncoding RNA that drives proliferation via a MYC/MNX1-AS1/YB1 signaling pathway.

Supplementary Figures 1-5 from miR-125b Is Methylated and Functions as a Tumor Suppressor by Regulating the ETS1 Proto-oncogene in Human Invasive Breast Cancer

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.

Metastasis is the main cause of death for patients suffering gastric cancer. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critical attributes of metastasis, both of which are regulated tightly by DNA methylation and Wnt/β-catenin signaling. Here, we studied the functions of DNA dioxygenase TET1 in regulating Wnt signaling and in gastric cancer metastasis. Knocking-down and overexpressing TET1 in gastric cancer cells promoted and inhibited metastatic spreading to the liver in immune-deficient mice, respectively. TET1 showed inhibitory effects on metastasis-related features -EMT and CSC, which were reversed by interfering with Wnt/β-catenin signaling. RNA-sequencing identified FOXO4 as a direct transactivating target of TET1. FOXO4 directly interacted with β-catenin and recruited it in the cytoplasm, so as to inhibit β-catenin-mediated transcription of Wnt target genes, including CSC marker EpCAM. Moreover, modulation of FOXO4 could reverse the effects of TET1 manipulation on EMT and self-renewal of CSCs. The analysis with clinical samples confirmed the value of FOXO4 as an independent prognostic predictor of patients’ overall survival. Taken together, regulation of Wnt signaling by TET1/FOXO4 is essential for metastasis-associated cellular properties, and targeting TET1/FOXO4/β-catenin pathway may serve as promising therapeutics in the prevention and treatment of gastric cancer metastasis.

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.

Long noncoding RNAs (lncRNA) are involved in tumorigenesis and drug resistance. However, the roles and underlying mechanisms of lncRNAs in colorectal cancer are still unknown. In this work, through transcriptomic profiling analysis of 21 paired tumor and normal samples, we identified a novel colorectal cancer–related lncRNA, MNX1-AS1. MNX1-AS1 expression was significantly upregulated in colorectal cancer and associated with poor prognosis. In vitro and in vivo gain- and loss-of-function experiments showed that MNX1-AS1 promotes the proliferation of colorectal cancer cells. MNX1-AS1 bound to and activated Y-box-binding protein 1 (YB1), a multifunctional RNA/DNA-binding protein, and prevented its ubiquitination and degradation. A marked overlap between genes that are differentially expressed in MNX1-AS1 knockdown cells and transcriptional targets of YB1 was observed. YB1 knockdown mimicked the loss of viability phenotype observed upon depletion of MNX1-AS1. In addition, MYC bound the promoter of the MNX1-AS1 locus and activated its transcription. In vivo experiments showed that ASO inhibited MNX1-AS1, which suppressed the proliferation of colorectal cancer cells in both cell-based and patient-derived xenograft models. Collectively, these findings suggest that the MYC–MNX1-AS1–YB1 axis might serve as a potential biomarker and therapeutic target in colorectal cancer. Significance: This study highlights the discovery of a novel colorectal cancer biomarker and therapeutic target, MNX1-AS1, a long noncoding RNA that drives proliferation via a MYC/MNX1-AS1/YB1 signaling pathway.

Gastrointestinal cancer causes countless deaths every year due to therapeutic resistance. However, whether metabolic alterations contribute to chemoresistance is not well understood. In this study, we report that fatty acid (FA) catabolism was activated in gastrointestinal cancer cells treated with oxaliplatin, which exhibited higher expression of the rate-limiting enzymes carnitine palmitoyltransferase 1B (CPT1B) and CPT2. The clinical analysis also showed that high expression of these enzymes was associated with poor oxaliplatin-based chemotherapy outcomes in patients. Furthermore, genetic or pharmacological inhibition of CPT2 with perhexiline disturbed NADPH and redox homeostasis and increased reactive oxygen species (ROS) generation and cell apoptosis in gastrointestinal cancer cells following oxaliplatin treatment. Specifically, the combination of oxaliplatin and perhexiline significantly suppressed the progression of gastrointestinal cancer in cell-based xenograft and patient-derived xenograft (PDX) models. Mechanistically, CPT2 was transcriptionally upregulated by nuclear factor of activated T cells 3 (NFATc3), which translocated to the nucleus in response to oxaliplatin treatment. In summary, our study suggests that the inhibition of CPT-mediated FA catabolism combined with conventional chemotherapy is a promising therapeutic strategy for patients with gastrointestinal cancers.

Rationale: STING is a critical player in the innate and adaptive immune system, sensing cytosolic DNA to activate the expression of interferon genes and regulate T lymphocytes, which drives immunogenic responses to cancer cells. Tumor-associated macrophages (TAMs), abundantly present in the tumor microenvironment, play a key role in cancer development. Gastric cancer is one of the most common and leading causes in cancer-related death worldwide. However, studies on the function and regulation of STING in TAMs and their roles in gastric cancer progression are still limited. Methods: We analyzed STING and CD68 expression of 200 pairs of gastric cancer and adjacent normal tissues by immunohistochemistry to identify the prognostic values of STING, as well as the correlations between STING and CD68 in gastric cancer. The characteristics of STING-altered macrophages, as well as their effects on cancer cell apoptosis and T cell differentiation were examined by flow cytometry. Cytokines secreted by STING-altered macrophages were identified by the Human Inflammation Array3 kit. Concentrations of soluble IL24 and IFN-β were measured by ELISA. In vivo models, including spontaneous gastric cancer in p53+/- mice and cell line-based xenografts, were established, and clinical benefits of STING-altered macrophages were examined. Results: Our study identifies STING as a prognostic factor for gastric cancer, and for the first time demonstrated that knocking-down STING and STING activation by 2'3'-c-GAMP both promote TAMs polarizing into pro-inflammatory subtype and induce apoptosis of gastric cancer cells, mechanistically through IL6R-JAK-IL24 pathway. Conclusions : This study evaluated effects of targeting STING in TAMs in anti-gastric-cancer therapies. Moreover, we unveil a novel function of STING to activate the IL6R-JAK-IL24 pathway in macrophages.

Esophageal squamous cell carcinoma (ESCC) is one of the most life- and health-threatening malignant diseases worldwide, especially in China. Long noncoding RNAs (lncRNAs) have emerged as important regulators of tumorigenesis and tumor progression. However, the roles and mechanisms of lncRNAs in ESCC require further exploration. Here, in combination with a small interfering RNA (siRNA) library targeting specific lncRNAs, we performed MTS and Transwell assays to screen functional lncRNAs that were overexpressed in ESCC. TMPO-AS1 expression was significantly upregulated in ESCC tumor samples, with higher TMPO-AS1 expression positively correlated with shorter overall survival times. In vitro and in vivo functional experiments revealed that TMPO-AS1 promotes the proliferation and metastasis of ESCC cells. Mechanistically, TMPO-AS1 bound to fused in sarcoma (FUS) and recruited p300 to the TMPO promoter, forming biomolecular condensates in situ to activate TMPO transcription in cis by increasing the acetylation of histone H3 lysine 27 (H3K27ac). Targeting TMPO-AS1 led to impaired ESCC tumor growth in a patient-derived xenograft (PDX) model. We found that TMPO-AS1 is required for cell proliferation and metastasis in ESCC by promoting the expression of TMPO, and both TMPO-AS1 and TMPO might be potential biomarkers and therapeutic targets in ESCC.

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 genetic basis of colorectal cancer (CRC) and its clinical associations remain poorly understood due to limited samples or targeted genes in current studies. Here, we perform ultradeep whole-exome sequencing on 1015 patients with CRC as part of the ChangKang Project. We identify 46 high-confident significantly mutated genes, 8 of which mutate in 14.9% of patients: LYST, DAPK1, CR2, KIF16B, NPIPB15, SYTL2, ZNF91, and KIAA0586. With an unsupervised clustering algorithm, we propose a subtyping strategy that classisfies CRC patients into four genomic subtypes with distinct clinical characteristics, including hypermutated, chromosome instability with high risk, chromosome instability with low risk, and genome stability. Analysis of immunogenicity uncover the association of immunogenicity reduction with genomic subtypes and poor prognosis in CRC. Moreover, we find that mitochondrial DNA copy number is an independent factor for predicting the survival outcome of CRCs. Overall, our results provide CRC-related molecular features for clinical practice and a valuable resource for translational research.

Background Gastric cancer (GC) is one of the most threatening malignant diseases in east Asia with largely unknown mechanisms. Protein kinases and their signaling pathways were closely related to cancer occurrence and progression. We decide to investigate the role of related protein kinases and their signaling pathways in GC. Methods We analyze the protein kinases activity using the phosphorylated proteomic data of gastric cancer and find that the substrates of HIPK family with abnormal phosphorylated levels. HIPK3 with less known function and mechanisms is chosen for further research, and its low expression in GC is confirmed by qPCR and immunohistochemistry analysis in a large GC patient cohort. MTS assay, colony formation assay, transwell migration and invasion assays are performed after ectopic express or knockdown of HIPK3 in vitro. Additionally, subcutaneous xenograft and in situ xenograft models are built in vivo. We detect the HIPK3 mRNA and protein level in cisplatin-resistant GC cells by qPCR and western blotting and measure the tolerance of cisplatin after overexpression of HIPK3. Coimmunopreicipitation and mass spectrometry analysis identify the interaction between HIPK3 and MAP7. Immunofluorescence confirms their co-localization. Downstream mechanisms were examined by regular molecular biological methods. Results We discovered the aberrant phosphorylation of substrates for HIPK family kinases based on the profiled gastric cancer phosphoproteome data and bioinformatical pipeline for kinase activity analysis. Then the low expression of HIPK3 in the gastric tumor was validated and correlated with patient overall survival. Cell line and mouse model experiments for gastric cancer showed that reducing the HIPK3 expression promoted the growth, proliferation, migration, invasion and metastasis, and high expression of HIPK3 could reverse the cisplatin resistance. Further molecular mechanism revealed that HIPK3 might regulate the progression of gastric cancer through phosphorylating MSH6 to regulate DNA mismatch repair and interaction with MAP7 to regulate cell morphology. Conclusions Our study explicit the potential of HIPK3 as a key regulator for progression, an effective biomarker for prognosis and drug resistance, and a potential therapy to target gastric cancer.

Background Improving the prognosis of Colorectal Cancer (CRC) depends on the identification of the mechanisms of recurrence and metastasis, and developing new therapeutic targets. lncRNA has become an attractive potential therapeutic target because it is more precise and less toxic when compared with traditional protein targeted drugs. We aimed to identify novel lincRNAs that significantly affects the development of CRC, and investigate the potential associated therapeutic targets. Methods Based on RNA-seq analysis, we screened out ten candidates and identified highly expressed lncRNAs in the 21 CRC tissue samples. An siRNA library was established to identify lncRNAs that significantly affected CRC cell proliferation and metastasis. RT-PCR and RNAi assays were performed to investigate the functional role of lncRNA MNX1-AS1 and clinical relevance. In vivo, cell-based and patient-derived xenograft (PDX) models were used to further explore roles of MNX1-AS1 in CRC tumorigenesis, metastasis and potential therapeutic target. RNA pull-down, mass spectrometry analyses, western blot and RNA-binding protein immunoprecipitation (RIP), DNA-binding protein immunoprecipitation (Chip), and Double Luciferase Report experiment were performed to identify interaction proteins and related mechanisms. Results MNX1-AS1 was upregulated in CRC tissues from patients with poor overall survival (OS), and MNX1-AS1 inhibition led to the impaired CRC cell line growth. Moreover, knockdown of MNX1-AS1 resulted in a decreased level of Y-box binding protein 1 (YB1), a multifunctional RNA/DNA binding protein. MNX1-AS1 blocked ubiquitination of YB1 and maintained its stability. This process prevented the degradation of YB1 through the MYC pathway. Therefore, knockdown of MNX1-AS1 attenuated the downstream effects of YB1. In addition, the transcription of MNX1-AS1 could be inhibited by MYC in CRC cells. In vivo experiments showed that the inhibition of MNX1-AS1 suppressed the proliferation of tumors in orthotopic models and patient-derived xenograft (PDX) models. Conclusions The newly identified MNX1-AS1, which is regulated by MYC, plays a pivotal role in CRC proliferation by enhancing YB1 stability, thus facilitating the development of CRC. Collectively, our study suggests that MYC- MNX1-AS1-YB1 axis might serve as potential biomarkers and therapeutic targets in CRC treatment.

Background The human ERBB receptor tyrosine kinase family is deregulated in multiple cancer types either through amplification or mutations. The ERBB4 mutation of gastric cancer (GC) is listed as the top 2 alteration frequency, which was around 10%. Methods In order to understand the biological functions of ERBB4 wild-type (WT) and mutants, ERBB4 WT and its several hot spot mutants including R106H, R393W, S774G, and L798R were constructed into adenovirus with red fluorescent protein. We examined the ERBB4 WT and mutants for their roles in cell proliferation and metastasis and validated their sensitivity to tyrosine kinase inhibitors (TKIs) in vitro and in vivo. Results We found that the ERBB4 amplification and mutations played the oncogenic roles in GC, in which the ERBB4 mutations had more significant effects. ERBB4 overexpression and mutants increased the sensitivity of GC cells to TKIs in vitro and in vivo, especially the ERBB4 mutants had stronger effects. The mechanisms of ERBB4 oncogenic activities were dependent or independent on kinase active ERBB2. Conclusions This finding broadened the clinical application of TKIs targeted to ERBB2 or pan-ERBB, suggesting that they may provide therapeutic benefits to GC patients with ERBB4 overexpression or mutations. Moreover, ERBB4 is supposed to be a significant biomarker for GC treatment.

The acidic tumor microenvironment provides an energy source driving malignant tumor progression. Adaptation of cells to an acidic environment leads to the emergence of cancer stem cells. The expression of the vitamin D receptor (VDR) is closely related to the initiation and development of colorectal carcinoma (CRC), but its regulatory mechanism in CRC stem cells is still unclear. Our study revealed that acidosis reduced VDR expression by downregulating peroxisome proliferator-activated receptor delta (PPARD) expression. Overexpression of VDR effectively suppressed the stemness and oxaliplatin resistance of cells in acidosis. The nuclear export signal in VDR was sensitive to acidosis, and VDR was exported from the nucleus. Chromatin immunoprecipitation (ChIP) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses showed that VDR transcriptionally repressed SRY-box 2 (SOX2) by binding to the vitamin D response elements in the promoter of SOX2, impairing tumor growth and drug resistance. We demonstrated that a change in the acidic microenvironment combined with overexpression of VDR substantially restricted the occurrence and development of CRC in vivo. These findings reveal a new mechanism by which acidosis could affect the stemness of CRC cells by regulating the expression of SOX2 and show that abnormal VDR expression leads to ineffective activation of vitamin D signaling, resulting in a lack of efficacy of vitamin D in antineoplastic process.

Background: Programmed death ligand 1 (PD-L1) expression has been shown to predict benefit from anti-PD-1 treatment in several cancers. However, its predictive value in colorectal cancer seems limited. This study was aimed to explore the clinical and biomarker association of programmed death ligand 1 and its spatial heterogeneous expression in colorectal cancer. Methods: Tissue microarrays of 422 primary colorectal cancers from our hospital were used for the interpretation of PD-L1 and programmed death 1 (PD-1) expression, cluster of differentiation 4 (CD4) and CD8 density and microsatellite instability (MSI) status by immunohistochemistry. To assess the spatial heterogeneity of PD-L1 expression, Tissue microarrays of 383 paired intra-primary-tumor tissues, and 105 paired lymph node metastatic tumors and 64 paired distant metastatic tumors were also used. Results: PD-L1 was positive in 188 (44.5%) primary colorectal cancers. PD-L1 expression was associated with less advanced N category (P

Purpose: Tumour cells produce energy through glycolysis and lactate dehydrogenase (LDH) is a key part of glycolysis. Elevation of serum LDH may indicate poor prognosis in primary duodenum adenocarcinoma. We aim to explore the prognostic significance of LDH in this disease. Methods and materials: Two hundred forty-four patients diagnosed with primary duodenum adenocarcinoma who were treated at the Sun Yat-sen Cancer Center from February 1996 to January 2016 were retrospectively analysed. We collected routine clinical data, including baseline LDH. Patients were classified into a normal LDH group (≤ 245U/L) and higher LDH group (>245U/L). Correlations of the LDH level and other clinicopathological characteristics were explored using the Chi-square test. Prognostic factors for overall survival were identified using univariate and multivariate analyses. Results: Two hundred seven patients (84.9%) had normal LDH levels, while 37 patients (15.1%) had abnormally high LDH levels. Higher LDH levels were significantly associated with more distant metastasis, node metastasis, poor differentiation and TNM stage Ⅲ-Ⅳ (P

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.

Purpose: Patients with advanced or metastatic adenocarcinoma of the stomach or esophagogastric junction for whom trastuzumab therapy is being considered, assessment for tumor human epidermal growth factor receptor-2 (HER2) status is necessary. Can the HER2 status and Lauren classification of the biopsy sample truly represent the HER2 status in the gastric cancer? Methods: Formalin-fixed, paraffin-embedded sections of 116 pair surgically resected and biopsy specimens as well as 80 pair primary and metastatic lesions of gastric cancer patients were analyzed. Protein expression was assessed using immunohistochemistry (IHC) and graded by the modified scoring criteria for gastric cancer. Gene amplification was evaluated by fluorescence in situ hybridization (FISH) in IHC 2+ cases. Results: The positive rate of HER2 was 11.2% in both surgical and biopsy samples. The consistent rate of HER2 expression was 91.4% (106/116) between biopsy and surgical samples, P=0.666. The positive rate of HER2 was 20.5% in primary and 15.9% in metastatic samples, P=0.1876. The consistent rate of HER2 expression was 90.9% (40/44) between primary and metastatic samples, P=0.580. The consistent rate of Lauren classification was 64.7% (75/116) between biopsy and surgical sample, and 92.5% (74/80) between primary and metastatic samples. Discussion: For gastric cancer, HER2 expression and Lauren classification were highly homogenous in biopsy and surgical samples, primary and their corresponding metastatic samples. The high concordance observed between these two cohorts indicated that the HER2 examination and Lauren classification of biopsy samples from the primary tumor could well represent the metastatic lesions of the patients.

Objectives Although the genomic landscape of small‐cell carcinoma of the oesophagus (SCCE) has been dissected, its transcriptome‐level aberration and immune microenvironment status are unknown. Methods Using ultra‐deep whole transcriptome sequencing, we analysed the expression profile of nine paired SCCE samples and compared the transcriptome with public transcriptomic data set of normal oesophageal mucosa and other cancer types. Based on the transcriptome data, the immune signatures were investigated. The genomic data of 55 SCCE samples were also applied for immune checkpoint blockade therapy (ICBT) biomarker evaluation including microsatellite instability (MSI) status, tumor mutation burden (TMB) and neoantigen burden (TNB). Also, we evaluated the CD8, CD68 and programmed death‐ligand 1 (PD‐L1) in 62 retrospective SCCE samples with IHC assay. Results Differential expression analysis revealed that the cell cycle, p53, and Wnt pathways are significantly deregulated in SCCE. Immune microenvironment analysis showed that high leucocyte infiltration and adaptive immune resistance did occur in certain individuals, while the majority showed a relatively suppressive immune status. Immune checkpoints such as CD276 and LAG‐3 were upregulated, and higher M2 macrophage infiltration in tumor tissues. Furthermore, normal tissues adjacent to the tumors of SCCE presented a more activated inflammatory status than tumor‐free healthy controls. These observations showed that ICBT might benefit SCCE patients. As the critical biomarker of ICBT, TMB of SCCE was 3.64 with the predictive objective response rate 13.2%, while the PD‐L1‐positive rate was 43%. Conclusions Our study systematically characterized the immune microenvironment in small‐cell carcinoma of the esophagus and provided evidence that several patients with SCCE may benefit from immune checkpoint blockade therapy., In this study, transcriptomic aberrance and immune microenvironment of small‐cell carcinoma of the esophagus (SCCE) was comprehensively characterized with transcriptome and immunohistochemistry analysis. Assessment of immune checkpoint blockade (ICB) treatment biomarkers provides a rationale for use of ICB treatment in patients with SCCE.

Methyltransferase-like 3 (METTL3), a major component of the N6-methyladenosine (m6A) methyltransferase complex, has been suggested to function as an oncogene in several cancers. However, its biological mechanism and the involved pathways in gastric cancer (GC) remain unknown. Here, we reported that frequent upregulation of METTL3 was responsible for the aberrant m6A levels in gastric carcinoma. On the other hand, a high level of METTL3 was significantly associated with several clinicopathological features and poor survival in patients with GC. The knockdown of METTL3 effectively inhibited cell proliferation and migration and invasion capacity. Moreover, overexpression of METTL3 considerably augmented its oncogenic function. Integrated RNA-seq and m6A-seq analysis first indicated that several component molecules (e.g., MCM5, MCM6, etc.) of MYC target genes were mediated by METTL3 via altered m6A modification. Our work uncovers the oncogenic roles of METTL3 in GC and suggests a critical mechanism of GC progression.

In the original version of [this article][1] ([1][2]), the immunostaining of Ki-67 (si#ME1 group of PDX#2 and PDX#3) in Fig. 5G was inadvertently duplicated during data arrangement. The image has been replaced with the intended image for Fig. 5G. The error does not affect the conclusion and has been

Background Colorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and methyltransferase-like 3 (METTL3) participates in tumor progression in several cancer types. However, its role in CRC remains unexplored. Methods Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect METTL3 expression in cell lines and patient tissues. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3. The biological functions of METTL3 were investigated in vitro and in vivo. RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. RNA stability assay was used to detect the half-lives of the downstream genes of METTL3. Results Using TCGA database, higher METTL3 expression was found in CRC metastatic tissues and was associated with a poor prognosis. MeRIP-seq revealed that SRY (sex determining region Y)-box 2 (SOX2) was the downstream gene of METTL3. METTL3 knockdown in CRC cells drastically inhibited cell self-renewal, stem cell frequency and migration in vitro and suppressed CRC tumorigenesis and metastasis in both cell-based models and PDX models. Mechanistically, methylated SOX2 transcripts, specifically the coding sequence (CDS) regions, were subsequently recognized by the specific m6A “reader”, insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), to prevent SOX2 mRNA degradation. Further, SOX2 expression positively correlated with METTL3 and IGF2BP2 in CRC tissues. The combined IHC panel, including “writer”, “reader”, and “target”, exhibited a better prognostic value for CRC patients than any of these components individually. Conclusions Overall, our study revealed that METTL3, acting as an oncogene, maintained SOX2 expression through an m6A-IGF2BP2-dependent mechanism in CRC cells, and indicated a potential biomarker panel for prognostic prediction in CRC.

Background Long non-coding RNAs (lncRNAs) are emerging as key molecules in gastric cancer(GC), yet their potential molecular mechanisms are not well understood. The aim of our study is to identify lncRNAs that are up-regulated in gastric cancer tissues and explore their function in gastric cancer metastasis. Methods RNA sequencing of 48 paired gastric tumor and non-tumor tissues in SYSUCC was conducted. The upregulated lncRNAs were selected and overlapped with those in TCGA database. A siRNA library was established using the top 50 lncRNAs highly expressed in GC and used to identify lncRNAs that significantly affected cell migration based on transwell assays. The expression of our focused lncRNA CRCAL-2(ColoRectal Cancer Associated LncRNA-2) was measured using quantitative reverse transcription PCR assays and lncRNA in situ hybridization. In vivo,cell-based and patient-derived xenograft (PDX) models were used to further explore roles of CRCAL-2 in GC metastasis. Then, RNA pull-down, mass spectrometry analyses, western blot and RNA-binding protein immunoprecipitation (RIP) were performed to identify interaction proteins and related mechanisms of CRCAL-2. Results LncRNA CRCAL-2, significantly overexpressed in the tumor tissues, was identified as the key regulator of GC metastasis. Upregulated CRCAL-2 in GC correlated with poor overall survival. Knockdown of CRCAL-2 significantly reduced GC cells migration, invasion, and metastasis of xenograft tumors in nude mice. Mechanistically, CRCAL-2 promoted GC metastasis by directly interacting with and stabilizing the nuclear transport protein RAN, resulting in increasement of nuclear import of beta-catenin and ultimately, activation of downstream targeted EMT(Epithelial-Mesenchymal Transition) genes. Additionally, the transcription factor YY1 could bind to the promoter of CRCAL-2 to upregulate its expression. Conclusions Our results suggest that YY1/CRCAL2/beta-catenin axis play a crucial role in GC metastasis, and the newly identified lncRNA CRCAL-2 might be developed as a biomarker and potential therapeutic target of GC in patients.

Background Rapid proliferation and glucose metabolism remodeling are hallmarks of cancers. Long non-coding RNAs (lncRNAs) are potentially involved in Warburg effect. We aimed to identify oncogenic lncRNAs that significantly affect the development of esophageal squamous cell carcinoma (ESCC) and investigate the metabolism-related mechanisms. Methods Bioinformatics analysis and siRNA library screening were used to pinpoint lncRNAs that significantly affected cell glycolysis and proliferation. RNAScope(R) in situ hybridization and qRT-PCR assays were performed in clinical samples to investigate expression levels and clinical relevance of the lncRNA. RNA interference and CRISPR-Cas9 were used to explore the functional roles of the lncRNA. In vivo, cell-based and patient-derived xenograft (PDX) models were used. Extracellular acidification rate and 13C-labeled intracellular metabolites were determined. RNA pull-down, MS2-Tagged RNA affinity purification, RNA-binding protein immunoprecipitation and cross-linking immunoprecipitation were performed to identify lncRNA associated proteins and related mechanisms. Results The lncRNA LOC148709 was identified as a metabolism-related lncRNA. Increased expression of LOC148709 was observed in ESCC and was correlated with poor prognosis. LOC148709 knockdown significantly decreased cell proliferation and glycolysis. Mechanistically, LOC148709 was directly associated with PFKFB3 and significantly affected PFKFB3 stability. LOC148709 interacted mainly with the C-terminal fragment of PFKFB3 and the T5 (2031–2321) fragment of LOC148709 mediated the interaction with PFKFB3. Through inhibiting K302 ubiquitination, LOC148709 protected PFKFB3 from proteasomal degradation, which subsequently activated glycolytic flux and promoted cell cycle progression by regulating p27 and CDK1. P53 could bind to the LOC148709 promoter and repress its transcription, p53 loss or mutation triggered striking LOC148709 upregulation. Multiple micro-environmental factors, including hypoxia and oncogenic stress, were also involved in the LOC148709 regulatory network via affecting the status of p53. Notably, our patient-derived xenograft (PDX) model studies demonstrated that LOC148709 knockdown dramatically impaired tumor growth. Conclusions The lncRNA LOC148709 plays an essential role in glycolytic reprogramming by binding to and stabilizing PFKFB3. This study identified a novel metabolism-related lncRNA and revealed a novel mechanism underlying lncRNA-mediated cancer metabolism remodeling. Translational studies further implicated that LOC148709 is a promising biomarker for cancer diagnosis and therapy.