Poor Prognosis In Colorectal Cancer Patients Research Articles

Abstract 5226: Tiam1 is a key modulator of chemoresistance in colorectal cancer

Abstract Purpose: Emerging evidence indicates that a small subpopulation of cancer cells with stem cell-like properties are responsible for acquisition of drug resistance in colorectal cancer (CRC). Intriguingly, the acquisition of such a stem-like state and subsequent drug resistance has been shown to be induced by the tumor microenvironment. In particular, cancer associated fibroblasts (CAFs) have been identified as a key component of tumor microenvironment which promote cancer stemness. Wnt signaling is a well-recognized pathway that drives self-renewal, and recently one of the Wnt-responsive genes, Tiam1, a guanine nucleotide exchange factor specific for Rac1, was shown to be overexpressed in CRC and associated with an aggressive cancer phenotype. Herein, we have investigated Tiam1 as a key modulator of chemoresistance in CRC. Methods: Tiam1 expression was assessed in resected CRC tissues from 300 patients who did or did not respond to chemotherapy. We used siRNA and CRISPR/CAS9 approach in three cell lines and animal models to examine whether inhibition of Tiam1 affects chemosensitivity in CRC. In addition, we established CRC patient derived-CAFs and examined whether the drug sensitivity of CRC cells was altered by supplementation of CAF-derived conditioned medium. Results: High Tiam1 expression significantly correlated with poor prognosis in CRC patients (P=0.032), and emerged as an independent prognostic factor (HR 5.06, 95% CI 1.10-23.2, P=0.038). In addition, Tiam1 overexpression is associated with patients who did not respond to chemotherapy (P=0.0005). We demonstrated that siRNA-mediated inhibition of Tiam1 enhanced sensitivity to three chemotherapeutic drugs (5-fluorouracil, oxaliplatin and irinotecan), and reduced tumor invasiveness in HCT116, SW480 and SW620 cell lines. Silencing of Tiam1 resulted in downregulation of stemness-related genes and spheroid formation ability. Furthermore, we validated that Tiam1-knockdown enhanced drug sensitivity and decreased the expression of stemness-related genes in xenograft tumors. Finally, we illustrated that the conditioned medium (CM) derived from CAFs led to increased stemness and chemoresistance in CRC cells through Tiam1 overexpression, and silencing of Tiam1 in CAFs resulted in reversal of stemness and chemoresistance properties. From a mechanistic standpoint, the target genes of Wnt signaling were also upregulated during co-culture with CM in CRC cells. Conclusion: We have firstly demonstrated that Tiam1 is overexpressed in CRC patients who did not respond to chemotherapeutic drugs; and furthermore, high Tiam1 expression emerged as an independent prognostic factor in CRC. Mechanistically, CAFs enhanced CRC chemoresistance through Tiam1 overexpression. Collectively, these results suggest that Tiam1 suppression in stroma may sensitize CRC cells to chemotherapeutic agents, suggesting that Tiam1 may be an attractive therapeutic target in colorectal cancer. Citation Format: Daisuke Izumi. Tiam1 is a key modulator of chemoresistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5226.

Abstract 5178: MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer

Abstract Objective: Colorectal cancer (CRC) is a leading cause of cancer mortality affecting over a million people every year. Biological markers for early detection and distant metastatic disease in patients with CRC are not well defined. We have identified transmembrane mucin MUC13, which is expressed in normal colon mucosa to be highly expressed in colorectal cancer, but the underlying pathways and the signaling mechanisms involved in CRC pathogenesis are not known. Our studies suggest MUC13 correlation with demographic and clinicopathologic characteristics. Materials and Methods: Retrospective institutional tumor registry was reviewed to identify patients with resected colon adenocarcinoma. Archived FFPE tissue blocks were reviewed by an experienced pathologist. Selected representative tissue blocks were serially sectioned at 4 µm. IHC staining was performed using an in-house MAb for MUC13. Slides were digitally scanned and analyzed qualitatively as well as quantitatively using a modified H-score based on the intensity of expression and percentage of stained cells. MUC13 was correlated with disease stage, aggressiveness and inflammatory markers, indicating poor prognosis in CRC patients. MUC13 splice variants were probed using novel designed probes. MUC13 SNPs were identified using unbiased approach by analyzing dpGAP database. Results: 196 tissues, of which 56.1% were female, 52% were white, and the median age at resection was 70. 38 (19.4%) were stage I, 64 (32.7%) stage II, 84 (42.9%) stage III, and 10 (5.1%) stage IV. 100% of colon adenocarcinoma tissues stained positively for MUC13, including definitive tumor epithelial staining, little-to-no background stromal staining, and mild staining of adjacent normal colon mucosa. Typical colon adenocarcinoma cells exhibited strong apical membranous staining with varying degrees of cytoplasmic staining. Advanced stage tumors were noted to more frequently exhibit basolateral and/or circumferential membranous staining compared to early stage tumors which more frequently displayed apical membrane staining alone. Five MUC13 transcript variants were identified by database analysis. Two of the protein coding MUC13 variants (long and short) showed differential expression in aggressive cell lines and patient tissues. Cyclic turnover between the short and long isoforms were observed during Anoikis resistance, cortisol, alcohol and BAP treatments. SNPs have been associated with increased risk of cancer incidence or fatality. Relevant MUC13 SNPs have been identified through systematic analysis of patient databases (dpGAP database). Verification and correlation of identified SNPs with patient disease stage and prognosis will also be presented. Conclusion: This is the first study to correlate MUC13 expression with disease outcome. Alternative transcripts and single nucleotide polymorphisms gave genetic insight of the role of MUC13 in CRC pathogenesis. Citation Format: Manish K. Tripathi, Chidi Zacheaus, Kyle Doxtater, Zachary Stiles, Fatemeh Keramatnia, Nadeem Zafar, Mahul Amin, Meena Jaggi, Subhash Chauhan. MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5178.

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.

CircRNA circ_0026344 as a prognostic biomarker suppresses colorectal cancer progression via microRNA-21 and microRNA-31

Recently, circular RNA (circRNA) is identified as a novel class of noncoding RNA with important roles in human diseases, such as cancer. However, how circRNA participates in colorectal cancer (CRC) remains unclear. In this study, we aimed to illustrate the role of circ_0026344 in CRC progression. We showed that circ_0026344 expression was downregulated in CRC tissues compared to adjacent normal tissues. Moreover, the level of circ_0026344 was inversely correlated with CRC advance and lymphoid node metastasis. Additionally, circ_0026344 low expression predicted poor prognosis in CRC patients. We identified circ_0026344 as a miRNA sponge for microRNA-21 (miR-21) and microRNA-31 (miR-31) whose expression levels were elevated in CRC tissues. And the level of circ_0026344 was reversely correlated with both miR-21 and miR-31 levels in CRC tissues. Functionally, we found that ectopic expression of circ_0026344 decreased the growth and invasion of CRC cells while promoting apoptosis in vitro. The xenograft experiment indicated that circ_0026344 overexpress led to CRC growth inhibition in vivo. Rescue assays further demonstrated that circ_0026344 exerted biological functions by sponging miR-21 and miR-31 in CRC. In conclusion, this study revealed that circ_0026344/miR-21/miR-31 regulatory signaling was implicated in CRC progression.

MicroRNA-302c represses epithelial-mesenchymal transition and metastasis by targeting transcription factor AP-4 in colorectal cancer.

MicroRNAs (miRNAs) contribute to tumorigenesis and progression via acting as tumor suppressors or oncogenes in human cancer. Aberrant expression of miR-302c has been reported in various types of cancer except colorectal cancer (CRC). Thus, our study was aimed to verify the expression of miR-302c and its functional role in CRC. We found a significant reduced expression of miR-302c in CRC tissues compared to tumor-adjacent tissues. Low miR-302c level was remarkably correlated with deeper tumor invasion, lymph node metastasis and advanced TNM stage. Importantly, low miR-302c expression was identified as an independent indicator for poor prognosis of CRC patients. Overexpression of miR-302c repressed migration and invasion capacities of SW620 and SW480 cells in vitro. Mechanistically, miR-302c inversely regulated transcription factor AP4 (TFAP4) abundance in both SW620 and SW480 cells, and it negatively correlated with TFAP4 mRNA expression in CRC samples. Herein, TFAP4, a regulator of epithelial-mesenchymal transition (EMT), was recognized as a direct target gene of miR-302c in CRC. Otherwise, miR-302c overexpression increased E-cadherin expression and reduced the levels of Vimentin and SNAI1, suggesting an inhibitory effect of miR-302c on EMT of CRC cells. Notably, our findings established that the EMT and metastasis of Caco-2 cells were enhanced by miR-302c knockdown, and subsequently reversed by TFAP4 silencing. Collectively, these data indicate that miR-302c represses EMT and CRC metastasis possibly by targeting TFAP4, and it may serve as a potential prognostic factor and therapeutic target for CRC.

STX2 promotes colorectal cancer metastasis through a positive feedback loop that activates the NF-\u03baB pathway

Metastatic progression is the main contributor to the poor prognosis of colorectal cancer (CRC). Thus, identifying the determinants of CRC metastasis will be of great significance. Based on our previous bioinformatics analysis, Syntaxin2 (STX2) may be upregulated and correlated with the poor prognosis of CRC patients. In this study, we found that STX2 expression was associated with CRC invasion and metastasis and poor patient survival. Gain- and loss-of-function analyses demonstrated that STX2 functioned as a key oncogene by promoting CRC invasion and metastasis. Mechanistically, STX2 selectively interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and activated the nuclear transcription factor-κB (NF-κB) signaling pathway. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that NF-κB directly bound to the STX2 promoter and drove STX2 transcription. Therefore, STX2 activated the NF-κB pathway, and in turn, NF-κB increased STX2 expression, forming a positive signaling loop that eventually promoted CRC metastasis. Collectively, our results reveal STX2 as a crucial modulator of the aggressive CRC phenotype and highlight STX2 as a potential prognostic biomarker and therapeutic target for combating CRC metastasis.

A miR-124/ITGA3 axis contributes to colorectal cancer metastasis by regulating anoikis susceptibility

Metastasis is the major cause for the death of patients with colorectal cancer (CRC). Anoikis resistance enhances the survival of cancer cells during systemic circulation, thereby facilitating secondary tumor formation in distant organs. miR-124 is a pleiotropically tumor suppressive small non-coding molecule. However, its role and mechanism in the regulation of cancer cell anoikis are still unknown. Here, we found that overexpression of miR-124 promotes anoikis of CRC cells in vitro and in vivo. In silico analysis and the experimental evidence supported that ITGA3 is a bona fide target of miR-124. Moreover, we identifies that ITGA3 plays a critical role in the regulation of anoikis sensitivity in CRC cells. Finally, our analysis in TCGA datasets demonstrates that high levels of ITGA3 are closely associated with poor prognosis in CRC patients. Collectively, we establish a functional link between miR-124 and anoikis susceptibility and provide that a miR-124/ITGA3 axis could be a potential target for the treatment of metastatic CRC.

Prognostic and Clinicopathological Significance of SATB1 in Colorectal Cancer: A Meta-Analysis.

Background: A large number of studies have reported the aberrant expression of special AT-rich sequence binding protein 1 (SATB1) in colorectal cancer (CRC). However, the role of SATB1 in CRC is still controversial. Therefore, we performed this meta-analysis to elucidate the prognostic and clinical value of SATB1 in CRC patients.Methods: We searched Web of Science, EMBASE and PubMed entirely in January 2018 to identify related articles. Pooled Hazard ratio (HR) was adopted to evaluate the prognostic value of SATB1 in CRC and odd ratio (OR) was used to assess the clinicopathological significance of SATB1 in CRC.Results: Ten eligible studies containing 7 on prognosis and 9 on clinicopathological characteristics were finally included in the present meta-analysis. Results revealed that patients with high expression of SATB1 tended to have shorter overall survival (OS) (pooled HR: 1.64, 95% CI: 1.04–2.57). Besides, we also discovered that the expression of SATB1 was associated with histologic grade (OR = 1.88, 95% CI: 1.06–3.34), distant metastasis (OR = 1.43, 95% CI: 1.11–1.85) and lymph node metastasis (OR = 1.50, 95% CI: 1.03–2.19).Conclusion: Broadly speaking, our meta-analysis demonstrated that high expression level of SATB1 was related to poor prognosis in CRC patients.

MicroRNA-520b Functions as a Tumor Suppressor in Colorectal Cancer by Inhibiting Defective in Cullin Neddylation 1 Domain Containing 1 (DCUN1D1).

MicroRNAs (miRs), a class of small noncoding RNAs, are important regulators for gene expression through directly binding to the 3′-untranslated region (3′-UTR) of their target mRNA. Recently, downregulation of miR-520b has been observed in several common human cancers. However, the exact role of miR-520b in colorectal cancer (CRC) has not previously been studied. In this study, our data showed that miR-520b was significantly downregulated in CRC and cell lines when compared with adjacent normal tissues and a normal intestinal epithelial cell line. Low expression of miR-520b was notably associated with the malignant progress and a shorter survival time for CRC patients. Restoration of miR-520b inhibited cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT) in CRC cells. Defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was then identified as a novel target gene of miR-520b in CRC cells. The expression of DCUN1D1 was significantly increased in CRC, with a negative correlation to miR-520b expression in CRC tissues. Moreover, a high expression of DCUN1D1 was significantly associated with the malignant progress and a poor prognosis for CRC patients. Furthermore, overexpression of DCUN1D1 rescued the miR-520b-mediated malignant phenotypes and EMT in CRC cells. The data demonstrate that miR-520b functions as a tumor suppressor in CRC through targeting DCUN1D1, suggesting that miR-520b may become a potential therapeutic target for the treatment of CRC.

Forkhead box D1 promotes proliferation and suppresses apoptosis via regulating polo-like kinase 2 in colorectal cancer

Transcription factor forkhead box D1 (FOXD1), a member of forkhead box family, has been recognized as a caner-related gene. Aberrant expression of FOXD1 is observed in glioma, lung cancer and breast cancer. However, the clinical significance of FOXD1 and its role in colorectal cancer (CRC) are unknown. Here, we found that FOXD1 displayed a higher expression in CRC tissues compared to tumor-adjacent tissues. Upregulation of FOXD1 was further confirmed in CRC tissues compared to normal tissues based on data from three GSE cohorts. Our clinical data indicated that high FOXD1 level was associated with tumor size (≥5 cm) and TNM tumor stage (III + IV). Moreover, both our data and TCGA data found that high expression of FOXD1 predicted poor prognosis of CRC patients. Next, we revealed that FOXD1 knockdown suppressed proliferation, cell cycle progression and induced apoptosis of SW480 cells in vitro. In accordance, FOXD1 overexpression promoted proliferation and reduced apoptosis of HT29 cells. Interestingly, polo-like kinase 2 (Plk2) expression was elevated and it positively correlated with FOXD1 expression in CRC tissues. FOXD1 promoted the expression of Plk2 mRNA and protein in CRC cells. Notably, Plk2 restoration abolished the effect of FOXD1 knockdown on proliferation and apoptosis of SW480 cells. Plk2 knockdown resulted in decreased proliferation and increased apoptosis of FOXD1-overexpressing HT29 cells. Altogether, we demonstrate for the first time that FOXD1 functions as an oncoprotein and a potential prognostic biomarker in CRC.

Colorectal cancer patients with different C-reactive protein levels and 5-year survival times can be differentiated with quantitative serum proteomics.

Over 1.4 million people are diagnosed with colorectal cancer (CRC) each year, making it the third most common cancer in the world. Increased screening and therapeutic modalities including improved combination treatments have reduced CRC mortality, although incidence and mortality rates are still increasing in some areas. Serum-based biomarkers are mainly used for follow-up of cancer, and are ideal due to the ease and minimally invasive nature of sample collection. Unfortunately, CEA and other serum markers have too low sensitivity for screening and preoperative diagnostic purposes. Increasing interest is focused on the possible use of biomarkers for predicting treatment response and prognosis in cancer. In this study, we have performed mass spectrometry analysis (UPLC-UDMSE) of serum samples from 19 CRC patients. Increased levels of C-reactive protein (CRP), which occur during local inflammation and the presence of a systemic inflammatory response, have been linked to poor prognosis in CRC patients. We chose to analyze samples according to CRP values by dividing them into the categories CRP <30 and >30, and, separately, according to short and long 5-year survival. The aim was to discover differentially expressed proteins associated with poor prognosis and shorter survival. We quantified 256 proteins and performed detailed statistical analyses and pathway analysis. We discovered multiple proteins that are up- or downregulated in patients with CRP >30 as compared to CRP <30 and in patients with short as compared to long 5-year survival. Pathways that were enriched include LXR/RXR activation, FXR/RXR activation, complement and coagulation cascades and acute phase signaling response, with some of the proteins we identified having roles in these pathways. In this study, we have identified multiple proteins, of which a few have been previously identified as potential biomarkers, and others that have been identified as potential biomarkers for CRC for the first time, to the best of our knowledge. While these proteins still need to be validated in larger patient series, this pilot study will pave the way for future studies aiming to provide better biomarkers for patients with CRC.

Association between KRAS gene mutations and clinicopathological characteristics and prognosis of colorectal cancer patients

Objective To investigate the association between KRAS gene mutations and clinico-pathological characteristics and prognosis of colorectal cancer (CRC) patients. Methods The retrospective case-control study was conducted. The clinicophathological data of 315 patients who underwent radical resection of CRC in the Yangpu Hospital Affiliated to Tongji University between January 2007 and July 2011 were collected. Next-generation sequencing was performed to identify KRAS gene mutations from surgical specimens. Observation indicators: (1) detection of KRAS gene; (2) association between KRAS gene mutations and clinicopathological characteristics of CRC patients; (3) follow-up and survival situations; (4) multivariate analysis of KRAS gene mutations in the prognosis of CRC patients. Follow-up using outpatient examination and telephone interview was performed to detect postoperative overall survival up to August 2016. Comparisons of count data were analyzed using the chi-square test. Measurement data with skewed distribution were described as M (interquartile range), and comparison between groups was analyzed using the nonparametric test. The survival rate was calculated using the Kaplan-Meier method, and survival was compared using the Log-rank test. The multivariate analysis was done using the COX regression model. Results (1) Detection of KRAS gene: all the 315 patients finished gene detection of surgical specimens, including 172 in wide-type mutations and 143 in mutant-type mutations (mutations at codon 12 and 13 of KRAS exon 2 and other mutant points were respectively detected in 80, 24 and 40 patients, and 1 patient had simultaneous mutations at codon 12 and 13 of KRAS exon 2; missense and nonsense mutations were respectively detected in 141 and 2 patients). The major point mutations were at p. G12D and p. G13D. (2) Association between KRAS gene mutations and clinicophathological characteristics of CRC patients: tumors located in the proximal colon, distal colon and rectum were respectively detected in 34, 48, 90 patients with wild-type mutation and in 44, 27, 72 patients with mutant-type mutation, with a statistically significant difference (χ2=0.038, P<0.05). (3) Follow-up and survival situation: 315 patients were followed up for 3-115 months, with a median time of 78 months. The postoperative overall survival rate was 41.0% in 172 patients with wild-type KRAS mutations, 27.4% in 80 patients with KRAS codon 12 mutations, 26.3% in 24 patients with KRAS codon 13 mutations and 48.2% in 40 patients with other KRAS mutations, showing a statistically significant difference (χ2=0.040, P<0.05). (4) Multivariate analysis of KRAS gene mutations in the prognosis of CRC patients: the results of multivariate analysis showed that mutations at codon 12 of KRAS exon 2 was an independent factor affecting poor prognosis of CRC patients (Hazard ratio=1.543, 95% confidence interval: 1.050-2.265, P<0.05). Conclusions Most KRAS mutations of CRC patients are at codon 12 and 13 of KRAS exon 2, and the major point mutations are at p. G12D and p. G13D. KRAS gene mutations may be associated with tumor location. Mutations at codon 12 of KRAS exon 2 is an independent factor affecting poor prognosis of CRC patients. Key words: Colonic neoplasms; Rectal neoplasms; KRAS gene; Genetic mutations; Surgical procedures, operative; Prognosis

Association of RAS mutations with race in metastatic colorectal cancer: CALGB/SWOG 80405 (ALLIANCE).

638 Background: Colorectal cancer (CRC) is a heterogeneous disease with distinct molecular subtypes in part based on RAS mutational status. It is plausible that RAS mutations are differentially distributed between CC and AA and may contribute to poor outcomes in AAs with CRC. Methods: We did a retrospective analysis of CALGB/SWOG 80405 trial patients. We divided the entire cohort into 2 groups: a) Common RAS: mutation in KRAS exon 2, codon 12 or 13; b) Extended RAS: any NRAS mutations or mutation in KRAS except those listed above. We then analyzed these two subgroups for association between RAS mutations and race (3 categories: Caucasian, AA, Others) using chi-square test for univariate analyses and logistic regression for multivariate analysis. We also analyzed the effect of extended RAS testing on prognosis of metastatic CRC by estimating the overall survival (OS) using Kaplan-Meier method and 95% confidence interval (CI). Cox proportional-hazard model was used for multivariate analyses. Results: There were 1729 CRC patients in common RAS group of which 357 (20.6%) had mutations present. Extended RAS group had 621 patients of which 95 (15.5%) had mutations present. There was no significant difference in the rate of common RAS mutations between CC and AA (20.5% vs. 24%, p=0.22). However, extended RAS mutations were significantly more in AA as compared to CC (25% vs. 14%, p=0.02). Multivariate analysis adjusted for age, gender, prior adjuvant chemotherapy and pelvic radiation confirmed higher odds of extended RAS mutation in AA compared to CC (adjusted OR 1.12; 95% CI 1.01-1.23; p=0.02). The median OS in patients with an extended RAS mutation was shorter as compared to those without extended RAS mutation (25.3 vs. 31.9 months; HR 1.26; 95% CI 0.99-1.62; p=0.05). Multivariate analyses adjusted for age, gender, race, prior adjuvant chemotherapy and pelvic radiation showed a trend towards longer OS in patients without extended RAS mutation as compared those with extended RAS mutation (adjusted HR= 1.24, 95% CI, 0.97-0.1.58, p=0.08). Conclusions: Extended RAS mutations are significantly more common in AA as compared to CC. Additionally, presence of extended RAS mutation may confer a poor prognosis in CRC patients.

Annexin A9 promotes invasion and metastasis of colorectal cancer and predicts poor prognosis.

AnnexinA9 (ANXA9), a member of annexin family, has been reported be associated with colorectal cancer (CRC) carcinogenesis. However, the clinical significance of ANXA9 in CRC, particularly its correlation to invasion and metastasis remains ambiguous. The aim of the present study was to investigate the significance of ANXA9 in CRC and understand the molecular mechanism of ANXA9 in CRC invasion and metastasis. Expression levels of the ANXA9 protein in CRC tissues were detected using immunohistochemistry (IHC), and the clinical and prognostic value of ANXA9 was investigated. ANXA9‑siRNA was utilized to investigate the effect and molecular mechanism of ANXA9 in HCT116 cells. The IHC result demonstrated that the positivity rate of the ANXA9 protein in CRC tissue was significantly higher than that in adjacent mucosa (P<0.05), which was consistent with the western blot results. ANXA9 protein expression levels are associated with invasion depth and lymphatic metastasis. Furthermore, patients with ANXA9‑positive expression demonstrated a poor prognosis and ANXA9 was an independent risk factor for survival (P<0.05). After inhibiting ANXA9 in HCT116 cells, the activity and metastatic and invasion capacity of cells decreased significantly, and expression levels of ADAM metallopeptidase domain17 and matrix metallopeptidase 9 were significantly downregulated, while the expression levels of tissue inhibitors of metalloproteinases‑1 and E‑cadherin were upregulated (P<0.05). Thus, positive ANXA9 expression may present as a novel marker for predicting poor prognosis in CRC patients, and ANXA9 may promote the invasion and metastasis of CRC by regulating invasion and metastasis‑associated genes.

Nuclear division cycle 80 promotes malignant progression and predicts clinical outcome in colorectal cancer.

Colorectal cancer (CRC) is a common human malignancy worldwide and increasing studies have attributed its malignant progression to abnormal molecular changes in cancer cells. Nuclear division cycle 80 (NDC80) is a newly discovered oncoprotein that regulates cell proliferation and cycle in numerous malignancies. However, its clinical significance and biological role in CRC remain unclear. Therefore, in this study, we firstly analyze its expression in a retrospective cohort enrolling 224 CRC patients and find its overexpression is significantly correlated with advanced tumor stage and poor prognosis in CRC patients. In addition, our result reveals it is an independent adverse prognostic factor affecting CRC‐specific and disease‐free survival. The subgroup analysis indicates NDC80 expression can stratify the clinical outcome in stage II and III patients, but fails in stage I and IV patients. In cellular assays, we find knockdown of NDC80 dramatically inhibits the proliferative ability, apoptosis resistance, cell cycle progression, and clone formation of CRC cells in vitro. Using xenograft model, we further prove knockdown of NDC80 also inhibits the tumorigenic ability of CRC cells in vivo. Finally, the microarray analysis is utilized to preliminarily clarify the oncogenic molecular mechanisms regulated by NDC80 and the results suggest it may promote CRC progression partly by downregulating tumor suppressors such as dual specificity phosphatase 5 and Forkhead box O1. Taken together, our study provides novel evidences to support that NDC80 is not only a promising clinical biomarker but also a potential therapeutical target for CRC precise medicine.

Chronic oxymatrine treatment induces resistance and epithelial‑mesenchymal transition through targeting the long non-coding RNA MALAT1 in colorectal cancer cells.

A major reason for colorectal cancer (CRC) chemoresistance is the enhanced migration and invasion of cancer cells, such as the cell acquisition of epithelial-mesenchymal transition (EMT). Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been considered as a pro-oncogene in multiple cancers. However, the precise functional mechanism of lncRNA MALAT1 in chemoresistance and EMT is not well known. In the present study, we focused on the effect of oxymatrine on CRC cells and further investigated the role of MALAT1 in oxymatrine-induced resistance and EMT process. The human CRC cell line HT29 was exposed to increasing doses of oxymatrine to establish stable cell lines resistant to oxymatrine. The established HT29 oxymatrine resistant cells showed an EMT phenotype including specific morphologic changes, enhanced migratory and invasive capacity, and downregulation of E-cadherin protein expression. Subsequently, high-throughput HiSeq sequencing and RT-qPCR showed that lncRNA MALAT1 was significantly upregulated in the oxymatrine resistant cells (P<0.01), while knockdown of MALAT1 partially reversed the EMT phenotype in HT29 resistant cells. Furthermore, oxymatrine treatment suppressed the migration and invasion ability of CRC cells, however, this effect was significantly reversed by overexpression of MALAT1. Finally, we investigated the clinical role of MALAT1 and found that high lncRNA MALAT1 expression level is associated with poor prognosis in CRC patients receiving oxymatrine treatment (P<0.01). In conclusion, we demonstrate that lncRNA MALAT1 is a stimulator for oxymatrine resistance in CRC and it may provide therapeutic and prognostic information for CRC patients.

MicroRNA-1296 Facilitates Proliferation, Migration And Invasion Of Colorectal Cancer Cells By Targeting SFPQ.

MicroRNAs (miRNAs) are involved in cancer genesis and progression via acting as tumor suppressors or oncogenes. Previous studies report that miR-1296 shows upregulation in both colorectal cancer (CRC) tissues and plasma samples. However, the accurate clinical significance of miR-1296 and its role in CRC have not been well investigated. The aim of the present study was to disclose the aberrant expression, clinical significance, and the relevant biological function of miR-1296 in CRC. We found a marked upregulation of miR-1296 expression in CRC tissues compared to tumor-adjacent tissues. MiR-1296 overexpression was detected in five CRC cell lines (HCT116, Caco2, HT29, SW620 and SW480). High miR-1296 level was remarkably correlated with tumor size (>5cm), lymph node metastasis and TNM stage (III+IV). Notably. High miR-1296 expression was identified as a predictive factor for poor prognosis of CRC patients by survival analysis. MiR-1296 knockdown inhibited proliferation, migration, invasion capacities of HCT116 and SW480 cells in vitro. Moreover, miR-1296 silencing restrained the growth of CRC cells in vivo. Splicing factor proline and glutamine rich (SFPQ), a novel RNA binding protein, was identified as a direct target gene of miR-1296 in CRC. Downregulation of SFPQ expression was inversely associated with miR-1296 expression in CRC tissues. The Cancer Genome Atlas (TCGA) data revealed the prognostic value of dysregulated SFPQ in CRC patients. Interestingly, our findings established that the oncogenic role of miR-1296 was at least partially mediated by SFPQ in CRC cells. Collectively, these data indicate that miR-1296 accelerates CRC progression possibly by targeting SFPQ and may serve as a potential predictive factor and therapeutic target for CRC.

Cell migration–inducing hyaluronan-binding protein is regulated by miR-140-3p and promotes the growth and invasion of colorectal cancer cells

Dysregulation of cell migration–inducing hyaluronan-binding protein (CEMIP) is associated with the growth and metastasis of multiple malignancies. But, the underlying mechanism by which CEMIP contributes to colorectal cancer (CRC) remains undocumented. The association of CEMIP or miR-140-3p expression with clinicopathological characteristics and prognosis in CRC patients was analyzed by the tissue microarray and TCGA dataset. MiR-140-3p-specific binding with CEMIP was confirmed by luciferase report assay. In vitro experiments were conducted to assess the effects of CEMIP on the growth and invasion of CRC cells. Consequently, we found that CEMIP expression was dramatically elevated in CRC tissues and associated with a poor prognosis in CRC patients. The upregulation of CEMIP was attributable to the dysregulation of miR-140-3p rather than its genetic and epigenetic alterations. Ectopic expression of CEMIP facilitated the cell viability, colony formation, and invasive potential, but silencing of CEMIP reversed these effects. Furthermore, CEMIP was identified as a direct target of miR-140-3p and attenuated miR-140-3p-induced anti-proliferation effects by regulating c-Myc, E-cadherin, and Twist-1 expression. MiR-140-3p indicated a negative correlation with CEMIP expression and was an independent prognostic factor of tumor recurrence in CRC patients. Taken together, CEMIP is regulated by miR-140-3p and promotes the growth and invasion of CRC cells. MiR-140-3p/CEMIP axis may represent the potential markers for CRC patients.

The clinicopathological significance of HES1 promoter hypomethylation in patients with colorectal cancer.

Hairy/enhancer of split 1 (HES1) is a basic helix–loop–helix transcriptional repressor. Aberrant demethylation has been considered a common mechanism of tumor promoter gene activation. In the current study, we aimed to investigate the methylation status of the HES1 promoter and correlations with clinicopathological parameters and prognosis in colorectal cancer (CRC). The expression of HES1 in 50 paired CRC specimens and adjacent normal tissues was determined by using quantitative real-time polymerase chain reaction and immunohistochemical analysis. Moreover, DNA methylation status was evaluated through methylation-specific polymerase chain reaction and bisulfite sequencing. The correlation of methylation status with HES1 expression level and clinicopathological parameters was statistically analyzed in CRC patients. Our data showed that the methylation level of HES1 was significantly decreased and negatively correlated with HES1 expression in CRC tissues. Moreover, HES1 hypomethylation was associated with a poor histological grade, Dukes’ classification, lymph node metastasis, and clinical stages (P<0.05). Furthermore, survival analyses revealed that a decreased methylation status of HES1 was linked to poor prognosis of CRC patients. In conclusion, promoter hypomethylation upregulates HES1 expression and plays a critical role in the progression and prognosis of CRC patients.

Long non-coding RNA SOX21-AS1 sponges miR-145 to promote the tumorigenesis of colorectal cancer by targeting MYO6

Emerging evidences have proved that long non-coding RNAs (lncRNAs) act as important molecular regulator in the tumor progression, including colorectal cancer (CRC). LncRNA SOX21-AS1 has been verified as oncogenic molecular in other cancers and tumorigenesis. In present study, our team investigates the clinical characteristic and molecular function in CRC carcinogenesis. Results showed that lncRNA SOX21-AS1 expression was significantly over-expressed in CRC tissue samples and cells. The aberrant over-expression of SOX21-AS1 indicated poor prognosis of CRC patients. In vitro and in vivo validation experiments, SOX21-AS1 silencing inhibited the proliferation, invasion, and decreased the tumor growth of CRC cells. Moreover, miR-145 was proved to be the target of SOX21-AS1, besides, myosin VI (MYO6) was found to be one of the targets of miR-145 using bioinformatics prediction programs and rescue confirmation experiments. In summary, our study reveals the tumorigenic effect of lncRNA SOX21-AS1 in CRC cells via targeting miR-145/MYO6, providing a novel insight for CRC carcinogenesis.