Intercellular adhesion molecule 2 as a novel prospective tumor suppressor induced by ERG promotes ubiquitination-mediated radixin degradation to inhibit gastric cancer tumorigenicity and metastasis.

BACKGROUND: Gastric cancer (GC) is a fatal cancer with unclear pathogenesis. In this study, we explored the function and potential mechanisms of intercellular adhesion molecule 2 (ICAM2) in the development and advancement of GC. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to quantify ICAM2 expression in harvested GC tissues and cultured cell lines. Immunohistochemical analyses were conducted on a GC tissue microarray to quantify ICAM2 expression and explore its implication on the prognosis of GC patients. In vitro experiments were carried out to reveal the biological functions of ICAM2 in GC cell lines. Further, in vivo experiments were conducted using xenograft models to assess the impact of ICAM2 on GC development and metastasis. Western blot, immunofluorescence, immunoprecipitation, luciferase assay, chromatin immunoprecipitation, and ubiquitination analysis were employed to investigate the underlying mechanisms. RESULTS: ICAM2 expression was downregulated in GC, positively correlating with advanced T stage, distant metastasis, advanced clinical stage, vessel invasion, and shorter patient survival time. ICAM2 overexpression suppressed the proliferation, migration, invasion, metastasis of GC cells as well as their ability to form tumors, whereas ICAM2 knockdown yielded opposite results. Erythroblast transformation-specific-related gene (ERG) as a transcription factor promoted the transcription of ICAM2 by binding to the crucial response element localized within its promoter region. Further analysis revealed that ICAM2 reduced radixin (RDX) protein stability and expression. In these cells, ICAM2 bound to the RDX protein to promote the ubiquitination and degradation of RDX via NEDD4 Like E3 Ubiquitin Protein Ligase (NEDD4L), and this post-translational modification resulted in the inhibition of GC. CONCLUSIONS: In summary, this study demonstrates that ICAM2, which is induced by ERG, suppresses GC progression by enhancing the ubiquitination and degradation of RDX in a NEDD4L-dependent manner. Therefore, these results suggest that ICAM2 is a potential prognostic marker and a therapeutic target for GC.

Epigenetic silencing and tumor suppressor gene of HAND2 by targeting ERK signaling in colorectal cancer.

BACKGROUND: The screening biomarkers for early detection of colorectal cancer (CRC) is lacking. The aim is to identify epigenetic silenced genes and clarify its roles and underlying mechanism in CRC. We conducted integrative analyses of epigenome-wide Human Methylation 450 K arrays and transcriptome to screen out candidate epigenetic driver genes with transcription silencing. Methylated silencing HAND2 were identified and verified in large CRC cohort. The mechanism of HAND2 expression by promoter inhibition were clarified both in vitro and vivo assays. Cell biofunctional roles of HAND2 methylation was investigated in CRC cells. HAND2 reconstitution were constructed by lentivirus plasmid and tumor xenograft model of HAND2 were built subcutaneously. Genomic mRNA analysis by RNA-sequencing and subsequent GSEA analysis were performed to identify potential target of HAND2 and qPCR/WB was conducted to identify the results. RESULTS: We firstly reported high frequency of HAND2 methylation in promoter in CRC and hypermethylation was negatively correlated with expression silencing and leaded to poor survival in several CRC cohort patients. 5-Aza treatment to demethylated HAND2 could revert its expression in CRC cells. Functionally, HAND2 reconstitution can inhibit cell proliferation, invasion and migration in vitro. In tumor xenograft, HAND2 reconstruction significantly repressed tumor growth when compared to control vector. Thousands of aberrant expressed genes were observed in the heatmap of RNA-sequencing data. HAND2 reconstitution could bind to ERK and reduce its phosphorylation by CoIP assay. These above results showed HAND2 reconstitution perturbed the activation of MAPK/ERK signaling by reduction of ERK phosphorylation. CONCLUSIONS: HAND2 is one tumor suppressor by targeting ERK signaling and one potential epigenetic driver gene in CRC. Video Abstract.

ADAMTS19 Suppresses Cell Migration and Invasion by Targeting S100A16 via the NF-κB Pathway in Human Gastric Cancer.

A Disintegrin and Metalloproteinase with Thrombospondin motifs 19 (ADAMTS19) has been reported to participate in the pathogenesis of solid cancers. However, its role in gastric cancer (GC) remains undocumented. Using immunohistochemistry (IHC) staining and quantitative real-time polymerase chain reaction (qRT-PCR) on GC tissues and adjacent normal tissues, we found that ADAMTS19 was downregulated in GC tissues (IHC: p < 0.001; qRT-PCR: p = 0.017). Further investigation revealed that ADAMTS19 correlated with distant metastasis (p = 0.008) and perineural invasion (p = 0.018) and that patients with low ADAMTS19 had worse overall survival (p = 0.021). Gain- and loss-of-function assays showed that ADAMTS19 suppressed cell migration and invasion in vitro. Using bioinformatics analysis and co-immunoprecipitation, immunofluorescence, and dual-luciferase reporter gene assays, we confirmed that ADAMTS19 binds with cytoplasm P65, decreasing the nucleus phosphorylation of P65, a crucial transcription factor in the nuclear factor kappa-B (NF-κB) pathway, thereby downregulating S100 calcium-binding protein A16 (S100A16) expression. S100A16 acted as the downstream of ADAMTS19, reversing the suppression of cell migration and invasion by ADAMTS19 in vitro. A combination of ADAMTS19 and S100A16 expression provided the optimal prognostic indicator for GC. Patients with ADAMTS19high-S100A16low had better overall survival than ADAMTS19low-S100A16high patients (p = 0.006). These results suggest that ADAMTS19 suppresses cell migration and invasion by targeting S100A16 via the NF-κB pathway and that ADAMTS19 and S100A16 are potential metastasis and survival biomarkers for GC.

Impact of Cold Ischemic Time and Freeze-Thaw Cycles on RNA, DNA and Protein Quality in Colorectal Cancer Tissues Biobanking.

Tissue-derived RNA, DNA and protein samples become more and more crucial for molecular detection in clinical research, personalized and targeted cancer therapy. This study evaluated how to biobanking colorectal tissues through examining the influences of cold ischemic time and freeze-thaw cycles on RNA, DNA and protein integrity. Here, 144 pairs of tumor and normal colorectal tissues were used to investigate the impact of cold ischemic times (0-48h) on RNA, DNA and protein integrity at on ice or room temperature conditions. Additionally, 45 pairs of tissues experienced 0-9 freeze-thaw cycles, and then the RNA, DNA and protein quality were analyzed. On ice, RNA, DNA and protein from colorectal tumor and normal tissues were all stable up to 48h after surgery. At room temperature, RNA in colorectal tumor and normal tissues began to degrade at 8h and 24h, respectively. Meanwhile, the tumor tissues DNA degradation occurred at 24h after surgery at room temperature. Similarly, the protein expression level of tumor and normal tissues began to change at 24h after the surgery at room temperature. Interestingly, tissue RNA and DNA remained stable even after 9 freeze-thaw cycles, whereas the proteins levels were remarkably changed after 7 freeze-thaw cycles. This study provided a useful evidence on how to store human colorectal tissues for biobanking. Preserving the surgical colorectal tissue on ice was an effective way to prevent RNA, DNA and protein degradation. Importantly, more than 7 repeated freeze-thaw cycles were not recommended for colorectal tissues.

eIF6 Promotes Colorectal Cancer Proliferation and Invasion by Regulating AKT-Related Signaling Pathways.

Although abnormal expression of eukaryotic initiation factor 6 (eIF6) has been found in several human solid tumors, the functions and underlying mechanisms of eIF6 in the progression of colorectal cancer (CRC) still needs further elucidation. In the present study, large-scale gene analysis based on Oncomine and The Cancer Genome Atlas (TCGA) database revealed significantly higher baseline expression of eIF6 in colorectal cancer than in normal tissues. Furthermore, our Chinese cohort study revealed that high expression of eIF6 was correlated with aggressive characteristics and poor survival in CRC patients. Functional studies using magnetic nanoparticle extraction indicated that eIF6 was an oncogene in CRC cells. Regarding its mechanism, through Gene ontology (GO) and KEGG pathway analysis based on TCGA RNAseq database, we found that eIF6 can activate multiple AKT-related cancer signaling pathways, such as p-AKT\MMP1\cyclinD1\Bcl2-related signaling, to regulate cell proliferation, invasion, cell cycle and apoptosis in CRC. Collectively, these findings suggested that eIF6 can positively regulate AKT-related cancer signaling and enhance tumorigenicity in CRC, and may serve as a potential prognostic indicator and therapeutic target in CRC.

The novel long noncoding RNA u50535 promotes colorectal cancer growth and metastasis by regulating CCL20.

Long noncoding RNAs (lncRNAs) have been emerging as master regulators of tumor growth and metastasis, but the functions and underlying mechanisms of lncRNAs in colorectal cancer (CRC) still need to be clarified. Here, we found a novel lncRNA u50535, which was greatly overexpressed in CRC tissues and was associated with poor prognosis in CRC patients. Function studies showed that u50535 was an oncogene in CRC both in vitro and in vivo. In mechanism, through RNA sequencing and rescue assay, we found that u50535 activates CCL20 signaling to promote cell proliferation and migration in CRC. Taken together, these findings suggest that u50535 can promote CRC growth and metastasis and may serve as a potential biomarker in CRC.

Erectile and urinary function in men with rectal cancer treated by neoadjuvant chemoradiotherapy and neoadjuvant chemotherapy alone: a randomized trial report.

OBJECTIVE: The aim was to evaluate erectile and urinary function of male patients with rectal cancer treated by neoadjuvant (NA) chemoradiotherapy (CRT) or NA chemotherapy only. METHODS: In this prospective randomized trial (ClinicalTrials.gov NCT01211210; "FOWARC"), we included 102 men who received NA therapy for stage II-III rectal cancer between January 2011 and June 2013. Before surgery, patients received either NA mFOLFOX6 chemotherapy with radiation (RCS group, n = 54) or NA mFOLFOX6 chemotherapy alone (CS group, n = 48). Erectile and urinary dysfunctions were assessed with the five-item International Index of Erectile Function (IIEF-5) scale and the International Prostatic Symptom Score (IPSS), respectively. Questionnaires were completed at baseline and at 3, 6, and 12 months (t0-t3, respectively) after surgery. RESULTS: At t3, mean IIEF-5 score was significant higher in the CS group (15.3 ± 5.5) than in the RCS group (12.6 ± 5.7; P < 0.05). And score difference was statistically higher in RCS group compared with CS group at t2 and t3, especially t3 (9.3 ± 5.5 vs. 6.1 ± 5.1, P < 0.01). Univariate analysis of the RCS group's IIEF-5 scores associated age, stoma, location, and tumor size to erectile dysfunction. However, mean IPSS scores did not differ between the two groups at any point. CONCLUSIONS: Compared with the CS group, erectile and urinary functions were significantly affected by NA CRT. Age, stoma, tumor location, and tumor size were also correlated with erectile dysfunction in the RCS group.

Colostomy is a simple and effective procedure for severe chronic radiation proctitis.

AIM: To assess the efficacy and safety of diverting colostomy in treating severe hemorrhagic chronic radiation proctitis (CRP). METHODS: Patients with severe hemorrhagic CRP who were admitted from 2008 to 2014 were enrolled into this study. All CRP patients were diagnosed by a combination of pelvic radiation history, clinical rectal bleeding, and endoscopic findings. Inclusion criteria were CRP patients with refractory bleeding with moderate to severe anemia with a hemoglobin level < 90 g/L. The study group included patients who were treated by diverting colostomy, while the control group included patients who received conservative treatment. The remission of bleeding was defined as complete cessation or only occasional bleeding that needed no further treatment. The primary outcome was bleeding remission at 6 mo after treatment. Quality of life before treatment and at follow-up was evaluated according to EORTC QLQ C30. Severe CRP complications were recorded during follow-up. RESULTS: Forty-seven consecutive patients were enrolled, including 22 in the colostomy group and 27 in the conservative treatment group. When compared to conservative treatment, colostomy obtained a higher rate of bleeding remission (94% vs 12%), especially in control of transfusion-dependent bleeding (100% vs 0%), and offered a better control of refractory perianal pain (100% vs 0%), and a lower score of bleeding (P < 0.001) at 6 mo after treatment. At 1 year after treatment, colostomy achieved better remission of both moderate bleeding (100% vs 21.5%, P = 0.002) and severe bleeding (100% vs 0%, P < 0.001), obtained a lower score of bleeding (0.8 vs 2.0, P < 0.001), and achieved obvious elevated hemoglobin levels (P = 0.003), when compared to the conservative treatment group. The quality of life dramatically improved after colostomy, which included global health, function, and symptoms, but it was not improved in the control group. Pathological evaluation after colostomy found diffused chronic inflammation cells, and massive fibrosis collagen depositions under the rectal wall, which revealed potential fibrosis formation. CONCLUSION: Diverting colostomy is a simple, effective and safe procedure for severe hemorrhagic CRP. Colostomy can improve quality of life and reduce serious complications secondary to radiotherapy.

Overexpression of long non-coding RNA-CTD903 inhibits colorectal cancer invasion and migration by repressing Wnt/ß-catenin signaling and predicts favorable prognosis.

Accumulating evidence reveals that long non-coding RNA (lncRNA) is essential for tumorigenesis and progression, but little is known about its roles and mechanisms in metastatic colorectal cancer (CRC). This study aimed to detect expression level and prognostic role of lncRNA­CTD903 in CRC patients, which was selected based on one microarray data. The effects on cell invasion, migration and proliferation were investigated after silencing or overexpression of CTD903 in CRC cell lines. We also observed the EMT (epithelial-mesenchymal transition) phenomenon and effect on cell adhesion. The associations between CTD903 and EMT markers, such as E-cadherin, N-cadherin, ß-catenin, ZEB1, ZO-1, Snail, and Twist, were determined by western blotting. Our results showed lncRNA-CTD903 expression was strongly upregulated in 115 CRC patients, comparing to adjacent normal tissues. CTD903 was proven to be an independent predicted factor of favorable prognosis in CRC patients by using multivariate Cox proportional hazards model. After knockdown of CTD903 in RKO and SW480, both cell invasion and migration increased, and cells exhibited EMT-like appearance, along with reduced adhering ability. Moreover, overexpression of CTD903 in DLD1 and HCT116 reversed these phenotypes. Furthermore, downregulation of CTD903 enhanced Wnt/ß-catenin activation and subsequently increased transcription factors (Twist and Snail) expression, along with increased mesenchymal marker Vimentin and decreased epithelial marker ZO-1 level, while overexpressed CTD903 confirmed these associations. In conclusion, this study shows that LncRNA-CTD903 acts as a tumor suppressor in CRC and can inhibit cell invasion and migration through repressing Wnt/ß-catenin signaling, which plays important roles in EMT and CRC metastasis.

Novel and Effective Almagate Enema for Hemorrhagic Chronic Radiation Proctitis and Risk Factors for Fistula Development.

Radiation proctitis is a common complication after radiotherapy for pelvic malignant tumors. This study was conducted to assess the efficacy of novel almagate enemas in hemorrhagic chronic radiation proctitis (CRP) and evaluate risk factors related to rectal deep ulcer or fistula secondary to CRP. All patients underwent a colonoscopy to confirm the diagnosis of CRP and symptoms were graded. Typical endoscopic and pathological images, risk factors, and quality of life were also recorded. A total of 59 patients were enrolled. Gynecological cancers composed 93.1% of the primary malignancies. Complete or obvious reduction of bleeding was observed in 90% (53/59) patients after almagate enema. The mean score of bleeding improved from 2.17 to 0.83 (P<0.001) after the enemas. The mean response time was 12 days. No adverse effects were found. Moreover, long-term successful rate in controlling bleeding was 69% and the quality of life was dramatically improved (P=0.001). The efficacy was equivalent to rectal sucralfate, but the almagate with its antacid properties acted more rapidly than sucralfate. Furthermore, we firstly found that moderate to severe anemia was the risk factor of CRP patients who developed rectal deep ulcer or fistulas (P= 0.015). We also found abnormal hyaline-like thick wall vessels, which revealed endarteritis obliterans and the fibrosis underlying this disease. These findings indicate that almagate enema is a novel effective, rapid and well-tolerated method for hemorrhagic CRP. Moderate to severe anemia is a risk factor for deep ulceration or fistula.

Increased urothelial cancer associated 1 is associated with tumor proliferation and metastasis and predicts poor prognosis in colorectal cancer.

Long non-coding RNA, urothelial cancer associated 1 (UCA1), is reported to play a critical role in progression of carcinogenesis. In the present study, we identified differential expression of UCA1 in colorectal cancer (CRC) and paired peritumoral tissues using gene expression microarray analyses. qPCR analysis confirmed that UCA1 was upregulated in CRC (p<0.001) and the expression of UCA1 was statistically correlated with lymph node metastasis (P=0.040), distant metastasis (P=0.043) and tumor stage (P=0.010). Kaplan-Meier analysis indicated that patients with high UCA1 expression had a poor prognosis. Moreover, multivariate analysis identified UCA1 overexpression as an independent predictor for CRC. We also found that knockdown of UCA1 significantly suppressed cell proliferation and metastasis in CRC cells. Flow cytometry assays showed UCA1 silencing induced G0/G1 growth arrest and apoptosis of CRC cells. To further investigate the regulatory mechanisms of UCA1, we identified that Ets-2 bound to the UCA1 core promoter using luciferase assays. Collectively, our findings suggested that UCA1 might be an important prognostic indicator in CRC and may be a potential target for diagnosis and gene therapy.