LncRNA GAS6-AS1 contributes to 5-fluorouracil resistance in colorectal cancer by facilitating the binding of PCBP1 with MCM3.

5-Fluorouracil (5-FU) resistance has always been a formidable obstacle in the adjuvant treatment of advanced colorectal cancer (CRC). In recent years, long non-coding RNAs have emerged as key regulators in various pathophysiological processes including 5-FU resistance. TRG is a postoperative pathological score of the chemotherapy effectiveness for CRC, of which TRG 0-1 is classified as chemotherapy sensitivity and TRG 3 as chemotherapy resistance. Here, RNA-seq combined with weighted gene correlation network analysis confirmed the close association of GAS6-AS1 with TRG. GAS6-AS1 expression was positively correlated with advanced clinicopathological features and poor prognosis in CRC. GAS6-AS1 increased the 50% inhibiting concentration of 5-FU, enhanced cell proliferation and accelerated G1/S transition, both with and without 5-FU, both in vitro and in vivo. Mechanistically, GAS6-AS1 enhanced the stability of MCM3 mRNA by recruiting PCBP1, consequently increasing MCM3 expression. Furthermore, PCBP1 and MCM3 counteracted the effects of GAS6-AS1 on 5-FU resistance. Notably, the PDX model indicated that combining chemotherapeutic drugs with GAS6-AS1 knockdown yielded superior outcomes in vivo. Together, our findings elucidate that GAS6-AS1 directly binds to PCBP1, enhancing MCM3 expression and thereby promoting 5-FU resistance. GAS6-AS1 may serve as a robust biomarker and potential therapeutic target for combination therapy in CRC.

Cuproptosis-related molecular patterns and gene (ATP7A) in hepatocellular carcinoma and their relationships with tumor immune microenvironment and clinical features.

BACKGROUND: Cuproptosis has been studied in various aspects as a new form of cell death. AIMS: We hope to explore the molecular patterns and genes related to cuproptosis in evaluating and predicting the prognosis of hepatocellular carcinoma (HCC), as well as the impact of tumor immune microenvironment. METHODS AND RESULTS: Sixteen cuproptosis related gene (CRGs) and cuproptosis related molecular and gene characteristics were comprehensively analyzed from 492 HCC samples. Cuproptosis related molecular patterns were generated by consensus clustering algorithm, including cuproptosis clusters, cuproptosis gene clusters (CGC) and cuproptosis score (CS). The characteristics of tumor microenvironment (TME) and tumor immune cells were described by the ssGSEA and ESTIMATE algorithms. Cuproptosis score was established to assess the clinical characteristics, prognostic and immunotherapy. The role and mechanism of CRG (ATP7A) in HCC, as well as its relationship with TME and immune checkpoints, have been further explored. The results of somatic mutation, copy number variations (CNV), and CRGs expression in HCC suggested the CRGs might participate in the HCC oncogenesis. The cuproptosis clusters were closely related to the clinical pathological characteristics, biological processes, and prognosis of HCC. The three CGC was revealed to be consistent with the three immune infiltration characterizations, including immune-high, immune-mid, and immune-low subtypes. Higher CS was characterized by decreased TMB, activated immunity, higher immune cell proportion score (IPS) and better overall survival (OS), which indicated higher CS was immune-high type and with better treatment effect and prognosis. The ATP7A had the highest hazard ratio (HR = 1.465, p < .001), was high expression in HCC tissues and with a shorter 5-year OS. Knocking down ATP7A could enhance intracellular copper concentration, cause a decrease in DLAT expression, and induce cuproptosis and inhibit cell proliferation and migration. ATP7A was also positively correlated with most cancer immune cells and immune checkpoints. CONCLUSION: Taken together, this research revealed the cuproptosis related molecular patterns and genes associated with the clinical pathological characteristics, TME phenotype and prognosis of HCC. The CS will further deepen our understanding of the TME characteristics of HCC, and the involvement of ATP7A in cuproptosis will provide new ideas for predicting HCC prognosis and immunotherapy.

Prediction of prognosis, immune infiltration, and personalized treatment of hepatocellular carcinoma by analysis of cuproptosis-related long noncoding RNAs and verification in vitro.

Background: The correlations between cuproptosis and long noncoding RNAs (lncRNAs) with the tumor microenvironment (TME), immunotherapy, and some other characteristics of hepatocellular carcinoma (HCC) remain unclear. Methods: Sixteen cuproptosis regulators and 356 cuproptosis-related lncRNAs (CRLnc) were identified from 374 HCC profiles in The Cancer Genome Atlas (TCGA) database. Six differentially expressed CRLnc were selected, and a prognostic risk model based on the CRLnc signature (CRLncSig) was constructed. The prognostic power of the model was verified. Moreover, a cuproptosis-related gene cluster (CRGC) was generated based on six lncRNAs and differentially expressed genes. The relationship between immune cell infiltration in the TME, immunotherapy, CRLncSig, and CRGC was demonstrated through various algorithms, Tumor Immune Dysfunction and Exclusion (TIDE), tumor mutational burden (TMB), etc. Potential drugs and sensitivity to those agents were evaluated for the risk model. LncRNA AL158166.1 was selected and verified in HCC tissues and cell lines, the impact of its knockdown and overexpression in HCC cells was examined, and the copper (Cu) concentration and the cuproptosis-related gene expression were detected. Results: A CRLncSig prognostic risk model with good predictive ability was constructed. The low-risk group had a longer overall survival (OS), lower tumor purity, more extensive immune cell infiltration, higher immune score, enrichment in immune-activated pathways, and more positive response to immunotherapy versus the high-risk group. CRGC-B exhibited the best OS and the lowest tumor stage; the immune cell infiltration analysis was similar to the low-risk group in CRLncSig. CRGC-B belonged to the "immune-high" group of the TME. The low-risk group had a higher TIDE score and susceptibility to antitumor drugs. The lncRNA AL158166.1 had the highest hazard ratio. The levels of AL158166.1 were higher in HCC tissues versus healthy tissues. Knockdown of AL158166.1 could lead to an increase in intracellular Cu concentration, induce DLAT low expression, and inhibit the proliferation and migration of HCC cells, whereas overexpression of AL158166.1 exerted the reverse effect. Conclusion: Overall, a new CRLncSig prognostic risk model and a cuproptosis-related molecular signature were constructed and evaluated. The model and signature were associated with the prognosis, immune infiltration, and immunotherapy of HCC. Inhibiting the lncRNA AL158166.1 may induce cuproptosis and showed potential for the inhibition of tumors. Evaluation of the CRLnc, CRLncSig, and CRGC may enhance our understanding of the TME, determine the effectiveness of immunotherapy, and act as a marker for the prognosis of HCC.

Cuproptosis-related miRNAs signature and immune infiltration characteristics in colorectal cancer.

BACKGROUND: A novel form of cell death termed cuproptosis was proposed recently. miRNAs play important roles in colorectal cancer (CRC). However, their relationships have not been reported. METHODS: miRNAs that negatively regulate 16 cuproptosis regulators were predicted using Targetscan database. The univariate Cox, LASSO, and multivariate Cox regression analyses were performed to select cuproptosis-related miRNAs. GSEA and ssGSEA analysis was carried out for functional enrichment analysis. The immune cell proportion score (IPS) and the efficiencies of multiple chemotherapy drugs were compared between different risk groups. The CCK8, cell colony, edu, and flow cytometry assays were performed to validate the roles of miRNA. Luciferase reporter assay confirmed the regulatory mechanism of miRNA on cuproptosis. RESULTS: Six cuproptosis-related miRNAs (hsa-miR-653, hsa-miR-216a, hsa-miR-3684, hsa-miR-4437, hsa-miR-641, and hsa-miR-552) were screened out for model construction. The risk score could act as an independent prognostic indicator in CRC (p < 0.001, 95% HR = 1.243 (1.129-1.369)). The nomogram could efficiently predict the overall survival rate (AUC = 0.836). Then, the level of immunosuppressive pathways, immunosuppressive cells, stromal-activated genes, and stromal score was higher in the high-risk group. The IPS analysis showed a better response to immunotherapy in the low-risk group. Also, the risk score was closely correlated with efficiencies of multiple chemotherapy drugs. Furthermore, miR-653 was highly expressed in CRC tissues (p < 0.001), closely correlated with T stage (p < 0.001), metastasis (p < 0.001), and tumor stage (p < 0.001). High expression of miR-653 predicted a shorter overall survival (p = 0.0282) and disease-free survival (p = 0.0056). In addition, miR-653 promoted cell proliferation, inhibited apoptosis, and negatively regulated the expression of DLD through directly binding to the 3'-UTR of DLD mRNA. CONCLUSION: We constructed a cuproptosis-related miRNA signature for the prediction of CRC patient survival and immunotherapy sensitivity. miR-653 was highly expressed in CRC tissues, promoted cell proliferation, and inhibited apoptosis by negatively regulating the expression of DLD.

Comprehensive analysis of cuproptosis-related lncRNAs to predict prognosis and immune infiltration characteristics in colorectal cancer.

Background: Cuproptosis is a novel form of cell death discovered in recent. A great quantity of researches has confirmed the close relationships and crucial roles between long non-coding RNAs (lncRNAs) with the progression of colorectal cancer (CRC). However, the relationship between cuproptosis and lncRNAs remains unclear in CRC. Methods: 1,111 co-expressed lncRNAs with 16 cuproptosis regulators were retrieved from CRC samples of The Cancer Genome Atlas (TCGA) database. Through univariate Cox and least absolute shrinkage and selection operator regression analysis, a prognosis model was constructed with 15 lncRNAs. The Kaplan-Meier, receiver operating characteristic curve, C-index and principal component analysis identified the prognostic power. Furthermore, a cuproptosis-related cluster was generated based on the 15 lncRNAs by unsupervised methods. The correlations between the cuproptosis-related signatures with immune cell infiltration and anti-tumor therapy were explored by multiple algorithms. Results: A risk score and nomogram with great prediction ability were constructed for CRC prognosis evaluation. The immune activate pathways, immune infiltration cells, immune functions, immune score and immune activation genes were remarkably enriched in the high risk group. The cuproptosis-related cluster was generated, of which the cluster 2 showed longer overall survival. The immune cell infiltration analysis indicated the similar results of cluster 2 with the high risk group, implying a significant marker for "hot tumor." The cluster 2 also presented high expression of immune checkpoint molecules, MSI-H status and higher susceptibility to multiple immunotherapy drugs. Conclusion: We appraised a novel cuproptosis-related prognosis model and molecular signature associated with prognosis, immune infiltration and immunotherapy. The identification of cuproptosis-related lncRNAs improved our understanding of immune infiltration and provided a significant marker for prognosis and immunotherapy in CRC.

Transcriptomic analysis of graft liver provides insight into the immune response of rat liver transplantation.

Background: As an "immune-privileged organ", the liver has higher rates of both spontaneous tolerance and operational tolerance after being transplanted compared with other solid organs. Also, a large number of patients still need to take long-term immunosuppression regimens. Liver transplantation (LT) rejection involves varieties of pathophysiological processes and cell types, and a deeper understanding of LT immune response is urgently needed. Methods: Homogenic and allogeneic rat LT models were established, and recipient tissue was collected on postoperative day 7. The degree of LT rejection was evaluated by liver pathological changes and liver function. Differentially expressed genes (DEGs) were detected by transcriptome sequencing and confirmed by reverse transcription-polymerase chain reaction. The functional properties of DEGs were characterized by the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. The cells infiltrating the graft and recipient spleen and peripheral blood were evaluated by immunofluorescence and flow cytometry. Result: A total of 1,465 DEGs were screened, including 1,177 up-regulated genes and 288 down-regulated genes. GO enrichment and KEGG pathway analysis indicated that DEGs were involved in several immunobiological processes, including T cell activation, Th1, Th2 and Th17 cell differentiation, cytokine-cytokine receptor interaction and other immune processes. Reactome results showed that PD-1 signaling was enriched. Further research confirmed that mRNA expression of multiple immune cell markers increased and markers of T cell exhaustion significantly changed. Flow cytometry showed that the proportion of Treg decreased, and that of PD-1+CD4+ T cells and PD-1+CD8+ T cells increased in the allogeneic group. Conclusion: Using an omic approach, we revealed that the development of LT rejection involved multiple immune cells, activation of various immune pathways, and specific alterations of immune checkpoints, which would benefit risk assessment in the clinic and understanding of pathogenesis regarding LT tolerance. Further clinical validations are warranted for our findings.

A novel cuproptosis-related molecular pattern and its tumor microenvironment characterization in colorectal cancer.

Cuproptosis, or copper-induced cell death, has been reported as a novel noncanonical form of cell death in recent times. However, the potential roles of cuproptosis in the alteration of tumor clinicopathological features and the formation of a tumor microenvironment (TME) remain unclear. In this study, we comprehensively analyzed the cuproptosis-related molecular patterns of 1,274 colorectal cancer samples based on 16 cuproptosis regulators. The consensus clustering algorithm was conducted to identify cuproptosis-related molecular patterns and gene signatures. The ssGSEA and ESTIMATE algorithms were used to evaluate the enrichment levels of the infiltrated immune cells and tumor immune scores, respectively. The cuproptosis score was established to assess the cuproptosis patterns of individuals with principal component analysis algorithms based on the expression of cuproptosis-related genes. Three distinct cuproptosis patterns were confirmed and demonstrated to be associated with distinguishable biological processes and clinical prognosis. Interestingly, the three cuproptosis patterns were revealed to be consistent with three immune infiltration characterizations: immune-desert, immune-inflamed, and immune-excluded. Enhanced survival, activation of immune cells, and high tumor purity were presented in patients with low cuproptosisScore, implicating the immune-inflamed phenotype. In addition, low scores were linked to high tumor mutation burden, MSI-H and high CTLA4 expression, showing a higher immune cell proportion score (IPS). Taken together, our study revealed a novel cuproptosis-related molecular pattern associated with the TME phenotype. The formation of cuproptosisScore will further strengthen our understanding of the TME feature and instruct a more personalized immunotherapy schedule in colorectal cancer.

Exploring immunotherapy in colorectal cancer.

Chemotherapy combined with or without targeted therapy is the fundamental treatment for metastatic colorectal cancer (mCRC). Due to the adverse effects of chemotherapeutic drugs and the biological characteristics of the tumor cells, it is difficult to make breakthroughs in traditional strategies. The immune checkpoint blockades (ICB) therapy has made significant progress in the treatment of advanced malignant tumors, and patients who benefit from this therapy may obtain a long-lasting response. Unfortunately, immunotherapy is only effective in a limited number of patients with microsatellite instability-high (MSI-H), and segment initial responders can subsequently develop acquired resistance. From September 4, 2014, the first anti-PD-1/PD-L1 drug Pembrolizumab was approved by the FDA for the second-line treatment of advanced malignant melanoma. Subsequently, it was approved for mCRC second-line treatment in 2017. Immunotherapy has rapidly developed in the past 7 years. The in-depth research of the ICB treatment indicated that the mechanism of colorectal cancer immune-resistance has become gradually clear, and new predictive biomarkers are constantly emerging. Clinical trials examining the effect of immune checkpoints are actively carried out, in order to produce long-lasting effects for mCRC patients. This review summarizes the treatment strategies for mCRC patients, discusses the mechanism and application of ICB in mCRC treatment, outlines the potential markers of the ICB efficacy, lists the key results of the clinical trials, and collects the recent basic research results, in order to provide a theoretical basis and practical direction for immunotherapy strategies.

Activation of the Aryl Hydrocarbon Receptor Ameliorates Acute Rejection of Rat Liver Transplantation by Regulating Treg Proliferation and PD-1 Expression.

BACKGROUND: Aryl hydrocarbon receptor (AhR) plays important roles in modulating immune responses. However, the role of AhR in rat liver transplantation (LT) has not been explored. METHODS: Safety and side effects of N -(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) and 2-methyl-2H-pyrazole-3-carboxylic acid amide (CH223191) were evaluated. We used optimal doses of 2 drugs, 3,4-DAA, a drug used for mediating AhR activation, and CH223191, antagonist of AhR (3,4-DAA, CH223191, and 3,4-DAA + CH223191), intraperitoneally administered to recipients daily to investigate the role of AhR in the rat LT model. The recipient livers were used to observe the pathological changes, the cells infiltrating the graft, and changes of AhR and programmed death-1 (PD-1) by Western blot, real-time polymerase chain reaction, and immunofluorescence assays. The contents of Foxp3 + and PD-1 + T cells in the recipient spleen and peripheral blood mononuclear cells were evaluated by flow cytometry. In vitro, after isolating CD4 + T cells, they were treated with different AhR ligands to observe the differentiation direction and PD-1 expression level. RESULTS: The activation of AhR by 3,4-DAA prolonged survival time and ameliorated graft rejection, which were associated with increased expression of AhR and PD-1 in the livers and increased Foxp3 + T cells and PD-1 + T cells in recipient spleens, livers, and peripheral blood mononuclear cells. In vitro, primary T cells incubated with 3,4-DAA mediated increased proportion of Treg and PD-1 + T cells. However, the suppression of AhR with CH223191 reverses these effects, both in the LT model and in vitro. CONCLUSIONS: Our results indicated that AhR activation might reduce the occurrence of rat acute rejection by increasing the proportion of Treg and the expression of PD-1.

TDO Promotes Hepatocellular Carcinoma Progression.

PURPOSE: Tryptophan 2,3-dioxygenase (TDO), encoded by the gene TDO2, is an enzyme that catalyses the first and rate-limiting step of tryptophan (Try) degradation in the kynurenine (Kyn) pathway in the liver. Recently, TDO has been demonstrated to be expressed in various human tumours, especially hepatocellular carcinoma (HCC). However, the role of TDO in HCC is still not very clear. Here, we studied the role of TDO in HCC. METHODS: We demonstrated that TDO is overexpressed in human HCC tissues and is significantly correlated with malignant phenotype characteristics, including tumour size, tumour differentiation, vascular invasion, etc. Kaplan-Meier analysis showed a poor overall survival rate in patients with TDO-overexpressing tumours. In addition, the effects of TDO on HCC tumour growth and metastasis were detected both in vivo and in vitro. TDO overexpression facilitated HCC cell growth, invasion and migration. CONCLUSION: Our results suggest that TDO positively regulates HCC proliferation and invasion and acts as a new prognostic biomarker of HCC.

Circular RNA circZNF566 promotes hepatocellular carcinoma progression by sponging miR-4738-3p and regulating TDO2 expression.

As a recently discovered noncoding RNA, circular RNAs (circRNAs) have been identified to play key roles in cancer biology; however, the detailed functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain largely unclarified. RNA-seq analysis was used to screen the expression profiles of circRNAs in HCC. CircZNF566 expression in HCC tissues and cell lines was detected by qRT-PCR. In vitro CCK-8, colony formation, wound healing, transwell migration, and invasion assays and in vivo tumorigenesis and metastasis assays were conducted to determine the functions of circZNF566. Luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed to confirm the relationship between circZNF566 and miR-4738-3p. Bioinformatics analysis and luciferase reporter assays were employed to determine whether miR-4738-3p regulates tryptophan 2,3-dioxygenase (TDO2) expression. Finally, immunohistochemistry (IHC) was used to detect the level of TDO2 and determine its prognostic value. CircZNF566 was significantly upregulated in HCC tissues and cell lines. High circZNF566 expression in HCC tissues was positively correlated with clinicopathological features and poor prognosis. Functionally, in vitro experiments showed that circZNF566 promoted HCC cell migration, invasion, and proliferation, whereas in vivo experiments showed that circZNF566 promoted tumorigenesis and metastasis. Mechanistically, circZNF566 acted as a miR-4738-3p sponge to relieve the repressive effect of miR-4738-3p on its target TDO2. In addition, miR-4738-3p suppressed HCC cell migration, invasion, and proliferation, while TDO2 was positively correlated with pathological features and poor prognosis and promoted cell migration, invasion, and proliferation in HCC. CircZNF566 is a novel tumor promoter in HCC and functions through the circZNF566/ miR-4738-3p /TDO2 axis; in addition, circZNF566 may serve as a novel diagnostic marker, prognostic indicator, and target for the treatment of HCC.

PCDHGA9 represses epithelial-mesenchymal transition and metastatic potential in gastric cancer cells by reducing ß-catenin transcriptional activity.

Gastric cancer (GC) has a high mortality rate, and metastasis is the main reason for treatment failure. It is important to study the mechanism of tumour invasion and metastasis based on the regulation of key genes. In a previous study comparing the expression differences between GES-1 and SGC-7901 cells, PCDHGA9 was selected for further research. In vitro and in vivo experiments showed that PCDHGA9 inhibited invasion and metastasis. A cluster analysis suggested that PCDHGA9 inhibited epithelial-mesenchymal transition (EMT) through the Wnt/ß-catenin and TGF-ß pathways. Laser confocal techniques and western blotting revealed that PCDHGA9 inhibited the nuclear translocation of ß-catenin, regulated T cell factor (TCF)/ /lymphoid enhancer factor (LEF) transcriptional activity, directly impacted the signal transmission of the TGF-ß/Smad2/3 pathway, strengthened the adhesion complex, weakened the effects of TGF-ß, and blocked the activation of the Wnt pathway. In addition, PCDHGA9 expression was regulated by methylation, which was closely related to poor clinical prognosis. The aim of this study was to elucidate the molecular mechanism by which PCDHGA9 inhibits EMT and metastasis in GC to provide a new theoretical basis for identifying GC metastasis and a new target for improving the outcome of metastatic GC.

TDO2 Promotes the EMT of Hepatocellular Carcinoma Through Kyn-AhR Pathway.

Tryptophan 2,3-dioxygenase (TDO2), an enzyme involved in tryptophan (Trp) metabolism has been linked with some malignant traits of various cancers. Kyn, the main product of Trp metabolism pathway catalyzed by TDO2 and indoleamine 2,3-dioxygenase (IDO) in tumor cells, was also demonstrated to activate aryl hydrocarbon receptor (AhR), which may regulate cancer growth and invasion in some malignancies. However, whether TDO2 participates in the metastasis and invasion of HCC has not been explored before. The underlying mechanism played by TDO2 in this process still requires further investigation. Here, we demonstrated that overexpression of TDO2 correlates with advanced stage or malignant traits in HCC patients. Knockdown or inhibition of TDO2 suppressed the migration and invasion of HCC cells in vitro and in vivo. Epithelial to mesenchymal transition (EMT) is an essential program happened in the initial phase of cancer metastasis. We found that in HCC cells, TDO2 promoted the EMT process evidenced by altered levels of biomarkers for EMT. Mechanically, TDO2 regulated the Kyn production in HCC cell via activated aryl hydrocarbon receptor (AhR). Together, these results indicate that TDO2 promotes the EMT of hepatocellular carcinoma through activating Kyn-AhR pathway, thereby participating in the metastasis and invasion of HCC.

RBBP6, a RING finger-domain E3 ubiquitin ligase, induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer.

RBBP6 has been implicated in tumorigenesis but its role in tumor metastasis and progression has not been evaluated. Interestingly, here we show that RBBP6 is upregulated in colorectal cancer (CRC) where its expression level is positively correlated with distant metastasis. In this study, we identified RBBP6, a RING Finger-domain E3 ubiquitin ligase, served as an independent prognostic factor and predicted poor outcome for CRC patients. RBBP6 promoted cell proliferation, migration, and invasion in CRC cells and promoted tumor growth, lung metastasis, and liver metastasis in mouse models. Mechanistically, we revealed that RBBP6 bound and ubiquitylated IκBα, an inhibitor of the NF-κB-signaling pathway. RBBP6-mediated ubiquitination and degradation of IκBα significantly enhanced p65 nuclear translocation, which triggered the activation of NF-κB pathway and then induced the epithelial-mesenchymal transition (EMT) process and cell metastasis. Furthermore, by DNA methylation results and ChIP analysis, we demonstrated that the promoter of RBBP6 was hypomethylated, and was activated by multi-oncogenic transcription factors. In conclusion, our findings suggest that RBBP6 may be a potential prognostic biomarker and therapeutic target for CRC invasion and metastasis.

Elevated HOXA13 expression promotes the proliferation and metastasis of gastric cancer partly via activating Erk1/2.

PURPOSE: HOXA13 is a transcription factor of the Homeobox (HOX) gene family, which is highly evolutionarily conserved. HOXA13 is upregulated and associated with oncogenic properties in some cancers. Here, we studied the potential mechanism of HOXA13-mediated proliferation and metastasis in gastric cancer (GC). METHODS: Quantitative real-time PCR, Western blot, and immunohistochemistry were used to detect HOXA13 expression levels in GC. In vitro and in vivo assays were performed to investigate the function of HOXA13 in GC cell proliferation, migration, and invasion. RNA-Seq transcriptome analysis was performed to study the underlying mechanism of HOXA13-mediated aggressiveness in GC. RESULTS: HOXA13 mRNA and protein expression levels were upregulated in GC tissues. According to Cell Counting Kit-8 and colony formation assays, we found that HOXA13 over-expression promoted proliferation. Flow cytometry analysis showed that HOXA13 overexpression or knockdown led to G1-S phase transition or G1 phase arrest, respectively. Western blot analysis results showed that HOXA13 overexpression increased cyclin D1 expression, while knockdown decreased its expression. Wound healing and transwell assay results demonstrated that HOXA13 overexpression promoted the migration and invasion of GC cells. Western blot analysis results also showed that HOXA13 overexpression upregulated N-cadherin and vimentin and downregulated E-cadherin, while HOXA13 knockdown led to the opposite results, indicating that HOXA13 might participate in epithelial to mesenchymal transition. These results were verified in vivo by tumor xenograft and metastasis assays. Mechanistically, using RNA-Seq transcriptome analysis, we found that Erk1/2 activation played an important role in HOXA13-induced GC progression. CONCLUSION: Our results show that HOXA13 plays an important role in GC development. HOXA13 overexpression promotes proliferation and metastasis partly via activation of Erk1/2 in GC. Thus, HOXA13, together with Erk1/2, may be promising targets for novel anticancer strategies.

Cartilage oligomeric matrix protein is a prognostic factor and biomarker of colon cancer and promotes cell proliferation by activating the Akt pathway.

PURPOSE: Recent studies have determined that cartilage oligomeric matrix protein (COMP) plays a vital role in carcinogenesis. We sought to clarify the role of COMP in colon cancer. METHODS: We investigated gene expression data from The Cancer Genome Atlas (TCGA) dataset. Tissue microarrays (TMA) containing paired samples from 253 patients with colon cancer were subjected to immunostaining. COMP levels in serum of colon cancer patients and healthy donors were measured with ELISA. We established COMP-knockout cells using the CRISPR/Cas9 system and COMP-overexpressing cells using lentiviral vectors to detect the effects of COMP on colon cancer cells using Cell Counting Kit-8 (CCK8), colony formation, apoptosis detection kit, and tumorigenesis assays in nude mice. RESULTS: The analysis of TCGA dataset and the results of the TMA suggested that COMP expression levels were significantly higher in cancer tissues than in adjacent normal tissues. Moreover, high COMP expression was correlated with the poor outcome of colon cancer patients. COMP levels in the sera of preoperative patients with colon cancer were much higher than those in healthy donors and were significantly reduced after colectomy. Colon cancer cells without COMP were defective with respect to the ability to proliferate, colony formation, the ability to resist 5-Fluorouracil-induced apoptosis and the growth of xenograft tumors in mice. Contrasting results were observed in COMP overexpressed cells. COMP promoted colon cancer cell proliferation partially through the activation of PI3K/ Akt/ mTOR/ p70S6K pathway. CONCLUSIONS: COMP may be a novel prognostic indicator and biomarker and also a potential therapeutic target for colon cancer.

PCDHGA9 acts as a tumor suppressor to induce tumor cell apoptosis and autophagy and inhibit the EMT process in human gastric cancer.

The results of a cDNA  array revealed that protocadherin gamma subfamily A, 9 (PCDHGA9) was significantly decreased in SGC-7901 gastric cancer (GC) cells compared with GES-1 normal gastric cells and was strongly associated with the Wnt/ß-catenin and transforming growth factor-ß (TGF-ß)/Smad2/3 signaling pathway. As a member of the cadherin family, PCDHGA9 functions in both cell-cell adhesion and nuclear signaling. However, its role in tumorigenicity or metastasis has not been reported. In the present study, we found that PCDHGA9 was decreased in GC tissues compared with corresponding normal mucosae and its expression was correlated with the GC TNM stage, the UICC stage, differentiation, relapse, and metastasis (p < 0.01). Multivariate Cox analysis revealed that PCDHGA9 was an independent prognostic indicator for overall survival (OS) and disease-free survival (DFS) (p < 0.01). The effects of PCDHGA9 on GC tumor growth and metastasis were examined both in vivo and in vitro. PCDHGA9 knockdown promoted GC cell proliferation, migration, and invasion, whereas PCDHGA9 overexpression inhibited GC tumor growth and metastasis but induced apoptosis, autophagy, and G1 cell cycle arrest. Furthermore, PCDHGA9 suppressed epithelial-mesenchymal transition (EMT) induced by TGF-ß, decreased the phosphorylation of Smad2/3, and inhibited the nuclear translocation of pSmad2/3. Our results suggest that PCDHGA9 might interact with ß-catenin to prevent ß-catenin from dissociating in the cytoplasm and translocating to the nucleus. Moreover, PCDHGA9 overexpression restrained cell proliferation and reduced the nuclear ß-catenin, an indicator of Wnt/ß-catenin pathway activation, suggesting that PCDHGA9 negatively regulates Wnt signaling. Together, these data indicate that PCDHGA9 acts as a tumor suppressor with anti-proliferative activity and anti-invasive ability, and the reduction of PCDHGA9 could serve as an independent prognostic biomarker in GC.

ER-positive breast cancer patients with more than three positive nodes or grade 3 tumors are at high risk of late recurrence after 5-year adjuvant endocrine therapy.

PURPOSE: Currently, although several clinical trials available give strong suggestions that extension of endocrine therapy has benefits, the risk level at which patients may benefit from extended endocrine therapy remains uncertain. This study aimed to identify the proportion of patients at a substantial risk of late recurrence after 5-year adjuvant endocrine therapy. PATIENTS AND METHODS: We reviewed 1,056 female patients with primary breast cancer who underwent curative resection between January 2006 and December 2011. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model to identify prognostic factors. RESULTS: A total of 327 eligible patients were eventually enrolled in this study. Among them, 42 (12.8%) patients suffered from distant metastasis and 34 (10.4%) patients experienced locoregional recurrence after 5-year adjuvant endocrine therapy. In multivariate analysis, patients with more than three positive nodes (hazard ratio [HR] =2.176, 95% CI=1.071-4.421; P=0.032) and histologic grade 3 disease (HR=2.098, 95% CI=1.300-3.385; P=0.002) were significantly associated with high risk of late recurrence. However, only histologic grade 3 (HR=2.212, 95% CI=1.166-4.194; P=0.015) was significantly associated with high risk of distant metastasis. CONCLUSION: Late relapse after completion of 5-year adjuvant endocrine therapy was still common, and grade 3 and more than three positive nodes were the risk factors of late recurrence, while grade 3 was the only risk factor of late distant metastasis. These patients might benefit from extended endocrine therapy.