Shaping immune landscape of colorectal cancer by cholesterol metabolites.

Cancer immunotherapies have achieved unprecedented success in clinic, but they remain largely ineffective in some major types of cancer, such as colorectal cancer with microsatellite stability (MSS CRC). It is therefore important to study tumor microenvironment of resistant cancers for developing new intervention strategies. In this study, we identify a metabolic cue that determines the unique immune landscape of MSS CRC. Through secretion of distal cholesterol precursors, which directly activate RORγt, MSS CRC cells can polarize T cells toward Th17 cells that have well-characterized pro-tumor functions in colorectal cancer. Analysis of large human cancer cohorts revealed an asynchronous pattern of the cholesterol biosynthesis in MSS CRC, which is responsible for the abnormal accumulation of distal cholesterol precursors. Inhibiting the cholesterol biosynthesis enzyme Cyp51, by pharmacological or genetic interventions, reduced the levels of intratumoral distal cholesterol precursors and suppressed tumor progression through a Th17-modulation mechanism in preclinical MSS CRC models. Our study therefore reveals a novel mechanism of cancer-immune interaction and an intervention strategy for the difficult-to-treat MSS CRC.

Comprehensive Analyses of Immune Subtypes of Stomach Adenocarcinoma for mRNA Vaccination.

Background: Although messenger RNA (mRNA) vaccines have unique advantages against multiple tumors, mRNA vaccine targets in stomach adenocarcinoma (STAD) remain unknown. The potential effectiveness of mRNA vaccines is closely associated with the tumor immune infiltration microenvironment. The present study aimed to identify tumor antigens of STAD as mRNA vaccine targets and systematically determine immune subtypes (ISs) of STAD that might be suitable for immunotherapy. Methods: Gene expression profiles and clinical data of patients with gastric cancer were downloaded from The Cancer Genome Atlas (TCGA; n = 409) and the Gene Expression Omnibus (GEO; n = 433), and genomic data were extracted from cBioPortal. Differential gene expression was analyzed using the limma package, genetic alterations were visualized using maftools, and prognosis was analyzed using ToPP. Correlations between gene expression and immune infiltration were calculated using TIMER software, and potential ISs were identified using ConsensusClusterPlus. Functional enrichment was analyzed in clusterProfiler, and r co-expression networks were analyzed using the weighted gene co-expression network analysis (WGCNA) package in R. Results: Overexpression of the prognostic and highly mutated antigens ADAMTS18, COL10A1, PPEF1, and STRA6 was associated with infiltration by antigen-presenting cells in STAD. Five ISs (IS1-IS5) in STAD with distinct prognoses were developed and validated in TCGA and GEO databases. The tumor mutational burden and molecular and clinical characteristics significantly differed among IS1-IS5. Both IS1 and IS2 were associated with a high mutational burden, massive infiltration by immune cells, especially antigen-presenting cells, and better survival compared with the other subtypes. Both IS4 and IS5 were associated with cold immune infiltration and correlated with advanced pathological stages. We analyzed the immune microenvironments of five subtypes of immune modulators and biomarkers to select suitable populations for mRNA vaccination and established four co-expressed key modules to validate the characteristics of the ISs. Finally, the correlation of these four mRNA vaccine targets with the transcription factors of DC cells, including BATF3, IRF4, IRF8, ZEB2, ID2, KLF4, E2-2, and IKZF1, were explored to reveal the underlying mechanisms. Conclusions: ADAMTS18, COL10A1, PPEF1, and STRA6 are potential mRNA vaccine candidates for STAD. Patients with IS1 and IS2 are suitable populations for mRNA vaccination immunotherapy.

SQLE, A Key Enzyme in Cholesterol Metabolism, Correlates With Tumor Immune Infiltration and Immunotherapy Outcome of Pancreatic Adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a treatment-refractory cancer with poor prognosis. Accumulating evidence suggests that squalene epoxidase (SQLE) plays a pivotal role in the development and progression of several cancer types in humans. However, the function and underlying mechanism of SQLE in PAAD remain unclear. Methods: SQLE expression data were downloaded from The Cancer Genome Atlas and the Genotype-Tissue Expression database. SQLE alterations were demonstrated based on the cBioPortal database. The upstream miRNAs regulating SQLE expression were predicted using starBase. The function of miRNA was validated by Western blotting and cell proliferation assay. The relationship between SQLE expression and biomarkers of the tumor immune microenvironment (TME) was analyzed using the TIMER and TISIDB databases. The correlation between SQLE and immunotherapy outcomes was assessed using Tumor Immune Dysfunction and Exclusion. The log-rank test was performed to compare prognosis between the high and low SQLE groups. Results: We demonstrated a potential oncogenic role of SQLE. SQLE expression was upregulated in PAAD, and it predicted poor disease-free survival (DFS) and overall survival (OS) in patients with PAAD. "Amplification" was the dominant type of SQLE alteration. In addition, this alteration was closely associated with the OS, disease-specific survival, DFS, and progression-free survival of patients with PAAD. Subsequently, hsa-miR-363-3p was recognized as a critical microRNA regulating SQLE expression and thereby influencing PAAD patient outcome. In vitro experiments suggested that miR-363-3p could knock down the expression of SQLE and inhibit the proliferation of PANC-1. SQLE was significantly associated with tumor immune cell infiltration, immune checkpoints (including PD-1 and CTLA-4), and biomarkers of the TME. KEGG and GO analyses indicated that cholesterol metabolism-associated RNA functions are implicated in the mechanisms of SQLE. SQLE was inversely associated with cytotoxic lymphocytes and predicted immunotherapy outcomes. Conclusions: Collectively, our results indicate that cholesterol metabolism-related overexpression of SQLE is strongly correlated with tumor immune infiltration and immunotherapy outcomes in patients with PAAD.

Construction and Validation of Convenient Clinicopathologic Signatures for Predicting the Prognosis of Stage I-III Gastric Cancer.

Background: Patients with stage I-III gastric cancer (GC) undergoing R0 radical resection display extremely different prognoses. How to discriminate high-risk patients with poor survival conveniently is a clinical conundrum to be solved urgently. Methods: Patients with stage I-III GC from 2010 to 2016 were included in our study. The associations of clinicopathological features with disease-free survival (DFS) and overall survival (OS) were examined via Cox proportional hazard model. Nomograms were developed which systematically integrated prognosis-related features. Kaplan-Meier survival analysis was performed to compare DFS and OS among groups. The results were then externally validated by The Sixth Affiliated Hospital, Sun Yat-sen University. Results: A total of 585 and 410 patients were included in the discovery cohort and the validation cohort, respectively. T stage, N stage, lymphatic/vascular/nerve infiltration, preoperative CEA, and CA19-9 were independent prognostic factors (P < 0.05). Two prognostic signatures with a concordance index (C-index) of 0.7502 for DFS and 0.7341 for OS were developed based on the nomograms. The 3-year and 5-year calibration curves showed a perfect correlation between predicted and observed outcomes. Patients were divided into three risk groups (low, intermediate, high), and distinct differences were noticed (p < 0.001). Similar results were achieved in the validation cohort. Notably, a free website was constructed based on our signatures to predict the recurrence risk and survival time of patients with stage I-III GC. Conclusions: The signatures demonstrate the powerful ability to conveniently identify distinct subpopulations, which may provide significant suggestions for individual follow-up and adjuvant therapy.

Clinicopathological Features Combined With Immune Infiltration Could Well Distinguish Outcomes in Stage II and Stage III Colorectal Cancer: A Retrospective Study.

BACKGROUND: The Immunoscore predicts prognosis in patients with colorectal cancer (CRC). However, a few studies have incorporated the Immunoscore into the construction of comprehensive prognostic models in CRC, especially stage II CRC. We aimed to construct and validate multidimensional models integrating clinicopathological characteristics and the Immunoscore to predict the prognosis of patients with stage II-III CRC. METHODS: Patients (n = 254) diagnosed with stage II-III CRC from 2009 to 2016 were used to generate Cox models for predicting disease-free survival (DFS) and overall survival (OS). The variables included basic clinical indicators, blood inflammatory markers, preoperative tumor biomarkers, mismatch repair status, and the Immunoscore (CD3+ and CD8+ T-cell densities). Univariate and multivariate Cox proportional regressions were used to construct the prognostic models for DFS and OS. We validated the predictive accuracy and ability of the prognostic models in our cohort of 254 patients. RESULTS: We constructed two predictive prognostic models with C-index values of 0.6941 for DFS and 0.7138 for OS in patients with stage II-III CRC. The Immunoscore was the most informative predictor of DFS (11.92%), followed by pN stage, carcinoembryonic antigen (CEA), and vascular infiltration. For OS, the Immunoscore was the most informative predictor (8.59%), followed by pN stage, age, CA125, and CEA. Based on the prognostic models, nomograms were developed to predict the 3- and 5-year DFS and OS rates. Patients were divided into three risk groups (low, intermediate, and high) according to the risk scores obtained from the nomogram, and significant differences were observed in the recurrence and survival of the different risk groups (p < 0.0001). Calibration curve and time-dependent receiver operating characteristic (ROC) analysis showed good accuracy of our models. Furthermore, the decision curve analysis indicated that our nomograms had better net benefit than pathological TNM (pTNM) stage within a wide threshold probability. Especially, we developed a website based on our prognostic models to predict the risks of recurrence and death of patients with stage II-III CRC. CONCLUSIONS: Multidimensional models including the clinicopathological characteristics and the Immunoscore were constructed and validated, with good accuracy and convenience, to evaluate the risks of recurrence and death of stage II-III CRC patients.

Clinical Significances of Positive Postoperative Serum CEA and Post-preoperative CEA Increment in Stage II and III Colorectal Cancer: A Multicenter Retrospective Study.

Background: Carcinoembryonic antigen (CEA) is the most common serum tumor marker in colorectal cancer (CRC). Nevertheless, few previous studies demonstrated the impacts of postoperative CEA and post-preoperative CEA increment on prognosis of CRC. Methods: Patients with stage II and III CRC were included from January 2009 to December 2015. All clinical and follow-up data were collected. Patients were divided into four different groups according to the levels of postoperative serum CEA and post-preoperative CEA trends. Chi-square test was used to analyze the relationship between clinical variables and categorized postoperative CEA and CEA increment. Cox proportional hazard regression was used for univariate and multivariable analyses. The log-rank test was performed to compare PFS and OS among groups. Results: Patients, 1,008, who underwent radical surgery, were enrolled. Our results showed that positive postoperative CEA and CEA increment were related to clinical stage, T stage, N stage, tumor differentiation, and lymphatic invasion (p < 0.05). Univariate and multivariable analysis results suggested that positive postoperative CEA and CEA increment were independent prognostic factors for PFS (HR = 3.149, 95% CI, 2.426-4.088, p = 0.000 for postoperative CEA; HR = 2.708, 95% CI, 2.106-3.482, p = 0.000 for CEA increment) and OS (HR = 3.414, 95% CI, 2.549-4.574, p = 0.000 for postoperative CEA; HR = 2.373, 95% CI, 1.783-3.157, p = 0.000 for CEA increment). The survival analyses revealed positive postoperative CEA, and CEA increment predicted worse prognosis. Furthermore, our results indicated that the 3- and 5-year PFS rates were 86.6 and 78.4% in group A, but decreased to 25.3 and 7.2% in group D (p < 0.001). Similarly, the 3- and 5-year OS rates for group A were 92.5 and 83.9%, much higher than group D (p < 0.001). In other words, patients with both postoperative CEA elevation and CEA increment had the worst prognosis. Conclusions: Positive postoperative CEA and CEA increment were independent prognostic factors for stage II and III CRC. Additionally, postoperative CEA and CEA increment had significant impacts on PFS and OS of CRC.

The difference in prognosis of stage II and III colorectal cancer based on preoperative serum tumor markers.

Background: Preoperative serum tumor markers have been widely used to predict prognosis in stage II and III colorectal cancer (CRC). However, few previous studies addressed the effect of increased preoperative numbers of tumor markers. Methods: Patients with stage II and III CRC who underwent curative resection were included from January 2009 to October 2015. The relationship between serum tumor markers and clinicopathological parameters was analyzed. DFS and OS were compared in stage II and III CRC. Results: The median follow-up was 45 months. In this study, 735 enrolled patients were assessed based on the numbers of increased tumor markers. We found that these increased tumor markers were closely associated with clinical stage, T stage, N stage, tumor location, pathology type, differentiation, lymphatic invasion and vascular invasion (all p values < 0.05). Furthermore, the number of increased tumor markers directly affected the survival of patients with CRC after curative surgery. The 3-year DFS and OS of patients with a score of 0 were 84.0% and 91.0%, respectively, which are much higher than those of patients with a score of 4 (42.9% and 37.8%, respectively) (p < 0.05). The 5-year DFS and OS of patients with a score of 0 were 75.9% and 77.9%, respectively, which are much higher than those of patients with a score of 4 (31.7% and 23.6%, respectively). Interestingly, our results suggested that stage III CRC patients with a score of 0 had longer DFS and OS times than stage II patients with scores of 3 and 4. Further analysis revealed statistically significant differences in OS (p < 0.05) but not in DFS. Conclusions: The number of increased tumor markers could significantly predict prognosis in stage II and III CRC. In addition, these increased tumor markers had direct impacts on metastasis as well as the recurrence status and survival time of stage II and III CRC patients.

A CREB1/miR-433 reciprocal feedback loop modulates proliferation and metastasis in colorectal cancer.

Increasing evidence has indicated the prognostic value of miR-433 across a series of malignancy types. However, the underlying mechanisms involved in cancer progression haven't been sufficiently elucidated. In the present work, we found that miR-433 was downregulated in CRC tissues and cell lines. Ectopic expression of miR-433 obviously suppressed the proliferation, invasion and metastasis activity of CRC cells in vitro and in vivo. CREB1, CCAR1 and JNK1 were highly expressed and negatively correlated with miR-433 expression in CRC. CRC patients with higher expression of CREB1, CCAR1 or JNK1 presented a worse outcome relative to those with lower expression. CREB1 transactivated the expression of miR-433, and CREB1, CCAR1 and JNK1 simultaneously served as its targets, which in turn composed a feedback loop between CREB1 and miR-433. miR-433 blocked cell cycle progression and abolished EMT. Collectively, our study demonstrated the CREB1/miR-433 reciprocal feedback loop restrained the propagation, invasion and metastasis activities of CRC cells through abrogation of cell cycle progression and constraint of EMT.

TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common neoplasms worldwide. However, the mechanisms underlying its development are still poorly understood. Thyroid hormone Receptor Interactor 13 (TRIP13) is a key mitosis regulator, and recent evidence has shown that it is an oncogene. Here, we report that TRIP13, which is overexpressed in CRC, is correlated with the CEA (carcino-embryonic antigen), CA19-9 (carbohydrate antigen 19-9) and pTNM (pathologic primary tumor, lymph nodes, distant metastasis) classification. Multivariate analyses showed that TRIP13 might serve as an independent prognostic marker of CRC. We also found that TRIP13 promoted CRC cell proliferation, invasion and migration in vitro and subcutaneous tumor formation in vivo. Furthermore, the potential mechanism underlying these effects involves the interaction of TRIP13 with a 14-3-3 protein, YWHAZ, which mediates G2-M transition and epithelial-mesenchymal transition (EMT). Together, these findings suggest that TRIP13 may be a potential biomarker and therapeutic target for CRC.

Knockdown of SLC39A7 inhibits cell growth and induces apoptosis in human colorectal cancer cells.

SLC39A7 (zip7) is a zinc transporter that plays a key role in intestinal epithelial self-renewal. However, little is known about SLC39A7 in colorectal cancer. To assess the biological function of SLC39A7 in colorectal cancer, the expression of SLC39A7 in human colorectal tumors and five colorectal cancer cell lines were evaluated by Oncomine Cancer Microarray Database and western blot analysis. In addition, short hairpin RNAs specifically targeting SLC39A7 were transfected into HCT116 and SW1116 cells to knockdown SLC39A7 expression. Then, the effects of SLC39A7 knockdown on colorectal cancer cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, colony-forming assay and flow cytometry. Our results showed that colorectal tumors have higher expression levels of SLC39A7 than normal colon tissues. Knockdown of SLC39A7 exhibited a significant decrease in cell viability and proliferation of colorectal cancer cells. It was also shown that knockdown of SLC39A7 interfered with cell cycle progression and induced G2/M cell cycle arrest, as well as boosted early and late apoptosis in colorectal cancer cells. Furthermore, downregulation of SLC39A7 promoted the cleavage of PARP and enhanced the expression of Bad, Caspase-9, and cleaved-Caspase-3, as well as suppressed Bcl-2 expression. In conclusion, our results suggest that SLC39A7 plays a crucial role in the proliferation and survival of colorectal cancer cells, which associates with colorectal tumorigenesis.

MiR-145 inhibits human colorectal cancer cell migration and invasion via PAK4-dependent pathway.

MicroRNA-145 (miR-145), as a tumor-suppressive miRNA, has been demonstrated down-regulated in colorectal cancer (CRC) cells, and could inhibit CRC cells growth. However, the molecular pathway in which miR-145 modulates CRC malignant transformation has not been fully revealed. Here, we reported an intense correlation between the expressions of PAK4 and miR-145 in human CRC cell lines. Transwell assay verified overexpression of miR-145, as well as knockdown of PAK4, significantly suppressed cell migration and invasion ability. The impaired migration and invasion ability of SW1116 cells was affected through the down-regulation of phosphorylation level of LIMK1 and cofilin in a PAK4-dependent manner. Collectively, we have demonstrated that miR-145 suppressed CRC migration and invasion through PAK4 pathway, which provides an attractive microRNA-based therapeutic target for CRC.

Targeted treatment of liver metastasis from gastric cancer using specific binding peptide.

Gastric cancer ranks the first in China among all gastrointestinal cancers in terms of incidence, and liver metastasis is the leading cause of death for patients with advanced gastric cancer. Tumor necrosis factor (TNF) is a cytokine commonly chosen as the target for gene therapy against cancers. The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human TNF to obtain the pd20-TNF gene using DNA recombinant technique. The expression of the fusion protein was induced and the protein was purified. In vitro activity test showed that the fusion protein greatly improved the membrane permeability of liver cells in nude mice with liver metastasis from gastric cancer. The tumor implantation experiment in nude mice showed that the fusion protein effectively mitigated the cancer lesions. The results provide important clues for developing the drugs for targeted treatment of liver metastasis from gastric cancer.

Downregulation of myosin VI reduced cell growth and increased apoptosis in human colorectal cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with the mortality increasing steadily over the last decade. Myosin VI (MYO6) expression is found to be elevated in some types of human carcinoma cell types, suggesting that it may be a sensitive biomarker for the diagnosis and follow-up. In this study, we first used the Oncomine database to explore the expression of MYO6 in CRC tissues, and then constructed a plasmid of RNA interference targeting MYO6 gene. After transfection of lentivirus targeting MYO6 into SW1116 cells, cell viability and proliferation were measured with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Cell cycle distribution was assayed by flow cytometry and apoptosis was evaluated by Annexin V. MYO6 expression was detected by quantitative real-time polymerase chain reaction and western blot analysis. It was found that MYO6 mRNA was upregulated in CRC tissues using data mining of public Oncomine microarray datasets. Depletion of MYO6 significantly inhibited cell proliferation and colony formation. In addition, knockdown of MYO6 slightly arrested cell cycle in G0/G1 phase, but remarkably increased the proportion of the sub-G1 phase of cell with the increase of apoptotic cells. These results suggest that MYO6 may promote cell growth and may be used as a potential target for anticancer therapy of CRC.