Progression Of Colorectal Carcinoma Research Articles

Aberration of Nrf2‑Bach1 pathway in colorectal carcinoma; role in carcinogenesis and tumor progression

Nrf2 and Bach1 are important transcriptional factors that protect against reactive oxygen species (ROS). Although aberration of these molecules was associated with malignant transformation and progression, their aberration pattern in colorectal carcinoma (CRC) is not yet fully studied.In this study, Nrf2 and Bach1 were immunohistochemically examined in 93 formalin-fixed paraffin-embedded blocks of colonic tumors (65 carcinoma with their corresponding surgical margins and 28 adenomas).Nrf2 expression was gradually increased in the apparently normal mucosa (57 ± 41)-adenoma (90 ± 36)-carcinoma (198 ± 78) direction and only showed significant higher mean of expression in CRC with brisk inflammatory peritumoral response. The mean of Bach1 expression was highest in apparently normal colonic mucosa (267 ± 16), lowest in adenoma (53 ± 31) and high in carcinoma tissues (194 ± 93). Significant higher mean of expression was detected in carcinoma with: LN metastasis (p = 0.04), lymphovascular invasion (p = 0.024); perineural invasion (p = 0.03) and advanced pathological stage (p < 0.001). Significant higher mean of expression of Nrf2 and Bach1 was detected in adenoma specimens with high grade dysplasia (p = 0.016 and p = 0.024) respectively.In conclusion, Nfr2 and Bach1 expression are altered in CRC but in different way. Nrf2 is gradually increasing from normal mucosa to adenoma and was highest in carcinoma but was not associated with features of tumor invasiveness. Bach1 was highest in normal mucosa; less in adenoma then increased in carcinoma and was associated with features of tumor invasiveness and metastasis. This may indicate a possible role of Nrf2 in CRC carcinogenesis and a role of Bach1 in CRC progression.

SCC-S2 Facilitates Tumor Proliferation and Invasion via Activating Wnt Signaling and Depressing Hippo Signaling in Colorectal Cancer Cells and Predicts Poor Prognosis of Patients.

SCC-S2 overexpression has been implicated in several human cancers, its correlation with prognosis and the mechanism how it reserved biological roles are still uncertain. The current study demonstrated that, in 142 archived colorectal carcinoma (CRC) tissue samples, SCC-S2 expression was significantly correlated with higher histological grade ( p=0.001), tumor invasion ( p=0.001), advanced Dukes staging ( p=0.002), positive regional lymph node metastasis ( p=0.024), and poor overall survival ( p<0.001). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Transwell assays showed that SCC-S2 significantly promoted the proliferation and invasion. SCC-S2 expression was also accompanied by the overexpression CyclinD1, matrix metalloproteinase-7 (MMP-7), active-β-catenin, yes-associated protein (YAP), and connective tissue growth factor (CTGF), as well as the depression of p-large tumor suppressor kinase 1 (p-LATS1) and p-YAP. Moreover, SCC-S2 interacted and colocalized with LATS1, the interaction may interrupt Hippo signaling and thereafter activate canonical Wnt signaling. In conclusion, our data suggested that SCC-S2 was associated with the progression and unfavorable prognosis of CRCs. Meanwhile, SCC-S2 facilitated canonical Wnt signaling and its downstream effectors (CyclinD1, MMP-7) and promoted tumor proliferation and invasion, which depended on the inhibition of Hippo signaling induced by SCC-S2-LATS1 interaction. These results indicated that SCC-S2 might be used as a novel target for the prevention and treatment of colorectal cancer.

MicroRNA-202-5p functions as a tumor suppressor in colorectal carcinoma by directly targeting SMARCC1

Recently, microRNAs (miRNAs) have been emerged as critical regulators for human diseases and as prognostic markers in several tumors, including colorectal carcinoma (CRC). Herein, we identified a tumor-suppressive miRNA, miR-202-5p, which may suppress CRC tumorigenesis. SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily c member 1 (SMARCC1) is a susceptibility gene in CRC. However, the role of SMARCC1 in CRC tumorigenesis has not been elucidated. In our present study, we demonstrated that miR-202-5p was a tumor-suppressive miRNA in CRC progression. We found that expression of miR-202-5p was obviously decreased in CRC tissues. Down-regulation of miR-202-5p was associated with postoperative survival. Overexpression of miR-202-5p inhibited the growth and metastasis of CRC cells. The SMARCC1 was a direct target of miR-202-5p and promoted the growth and metastasis of CRC cells. Further study showed that SMARCC1 could reverse the inhibitory effect of miR-202-5p on growth and metastasis of CRC cells. In conclusion, our data highlight the key role of miR-202-5p in the progression of CRC. Thus, miR-202-5p may be a potential prognostic marker and of treatment relevance for CRC progression intervention.

Long noncoding RNA kcna3 inhibits the progression of colorectal carcinoma through down-regulating YAP1 expression

Long non-coding RNAs (lncRNAs) regulate diverse cellular processes, and their anomalous expression exert an essential role in the progression of many kinds of cancers, including colorectal carcinoma (CRC). The objective of this study was to investigate the role of lncRNA kcna3 and its underlying mechanism in CRC progression. The expression of lncRNA kcna3 in human CRC tissues and the adjacent non-tumor tissues was evaluated by RT-PCR. The correlations between lncRNA kcna3 expression levels and the overall survival (OS), as well as the clinicopathological features of CRC patients were analyzed. Gain-of-function and loss-of-function experiments were used to evaluate the effects of lncRNA kcna3 on the proliferation, apoptosis, migration, invasion and tumorigenesis of colon cancer SW620 cells. We found that lncRNA kcna3 was lowly expressed in CRC tissues, and its low expression was closely associated with patients’ higher TNM grade and the higher occurrence rate of lymphatic metastasis and distant metastasis, as well as shorter OS. Enhanced expression of lncRNA kcna3 inhibited SW620 cells’ proliferation, migration and invasion, and induced cell apoptosis in vitro, and repressed CRC tumor growth in vivo. Whereas knockdown of lncRNA kcna3 showed the opposite results. Mechanistically, up-regulation of lncRNA kcna3 decreased YAP1 protein expression and accelerated its degradation. The effects of lncRNA kcna3 overexpression on cell growth and tumorigenesis inhibition and apoptosis promotion were weakened when the expression of YAP1 was up-regulated. In conclusion, this study revealed that lncRNA kcna3 exerts a tumor-inhibit role in CRC progression through down-regulating YAP1 expression, indicating that lncRNA kcna3/YAP1 might be served as a new prognostic biomarker and therapeutic target for CRC.

Hsp27 regulates epithelial mesenchymal transition, metastasis and proliferation in colorectal carcinoma.

The primary factor associated with poor survival rate in patients with colorectal carcinoma (CRC) is the presence of metastasis. The underlying molecular mechanisms of CRC metastasis are yet to be fully elucidated. The present study investigated the function of heat shock protein 27 (Hsp27) on the invasion and proliferation of CRC cells. The clinical significance of Hsp27 was evaluated using tissue microarray analysis (n=81). Invasion and metastasis assays were used to determine the function of Hsp27 in CRC metastasis in vitro and in vivo using RNA interference and the ectopic expression of Hsp27. The upregulation of Hsp27 has been frequently identified in CRC tissues. Patients with CRC and a high expression level of Hsp27 have a reduced overall survival rate. Silencing Hsp27 inhibited the growth and invasion of CRC cells in vitro and in vivo, whereas ectopic overexpression of Hsp27 promoted the proliferation and invasion of CRC cells in vitro. Furthermore, depletion of Hsp27 expression inhibited the epithelial-to-mesenchymal transition (EMT), whilst ectopic overexpression of Hsp27 induced EMT. The results of the present study indicated that Hsp27 serves an important function in the aggressiveness of CRC through inducing EMT. Hsp27 suppression may represent a potential therapeutic option for the suppression of CRC progression.

Immunohistochemical evaluation of the epithelial–mesenchymal transition profile

ObjectivesTo evaluate the role of the epithelial–mesenchymal transition (EMT) profile, Twist, E-cadherin, and vimentin in the progression of colorectal carcinoma (CRC).BackgroundMetastases are the most common cause of death in cancer patients. EMT is one of the central mechanisms that induces invasi

STAT3 induces colorectal carcinoma progression through a novel miR-572-MOAP-1 pathway.

PurposeColorectal carcinoma (CRC) is among the most common causes of death. Recent studies have shown that both STAT3 and miR-572 contribute to CRC progression. STAT3 plays an important role in miRNA expression. Moreover, MOAP-1, which is a pro-apoptotic protein that induces cell death or apoptosis, has a direct correlation with miRNA. Therefore, the current study is designed to explore whether miR-572 and STAT3 are involved in a common pathway and the role of MOAP-1 in this process.Patients and methodsThe expressions of STAT3, miR-572, and MOAP-1 in human CRC tissues and multiple cell lines were estimated by qRT-PCR or Western blot. MTT, transwell migration, and invasion assays were used to assess cell growth, migration, and invasion, respectively. Dual-luciferase reporter assay was applied to examine the association between miR-572 and MOAP-1.ResultsElevated STAT3 levels were accompanied by increased miR-572 and decreased MOAP-1 levels in primary CRC specimens and cell lines. STAT3 promoted CRC cell growth, migration, and invasion via the upregulated expression of miR-572. Subsequently, miR-572 inhibited MOAP-1 protein expression through an interaction with its 3′UTR.ConclusionOur study proposes a novel STAT3-miR-572-MOAP-1 pathway involved in the process of CRC progression, which might be a potential target for the development of new preventive and therapeutic approaches against human colorectal cancer.

STAT1-mediated upregulation of lncRNA LINC00174 functions a ceRNA for miR-1910-3p to facilitate colorectal carcinoma progression through regulation of TAZ

Several of the thousands of human long noncoding RNAs (lncRNAs) have been functionally characterized, yet their potential involvement in colorectal carcinoma (CRC) remains poorly understood. In present study, we aim to investigate the role of lncRNA LINC00174 in CRC carcinogenesis. We observed that increased expression of LINC00174 in CRC tissues and cells in comparison to their corresponding controls. Moreover, the aberrant overexpression of LINC00174 indicated the poor prognosis of CRC patients. Silence of LINC00174 was able to repress CRC cell growth in vitro and in vivo. We first reported that transcription factor STAT1 mediated LINC00174 expression in CRC. In addition, rescue assay was performed to further confirm that LINC00174 contributed to CRC progression by regulating miR-1910-3p/TAZ signal pathway. Taken together, our study discovered the oncogenic role of LINC00174 in clinical specimens and cellular experiments, showing the potential LINC00174/miR-1910-3p/TAZ pathway. This results and findings provide a novel insight for CRC tumorigenesis.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC.

KIF4A facilitates cell proliferation via induction of p21-mediated cell cycle progression and promotes metastasis in colorectal cancer

Kinesin family member 4A (KIF4A) was found to be implicated in the regulation of chromosome condensation and segregation during mitotic cell division, which is essential for eukaryotic cell proliferation. However, little is known about the role of KIF4A in colorectal carcinoma (CRC). This study explored the biological function of KIF4A in CRC progression and investigated the potential molecular mechanisms involved. Here, we found that KIF4A was remarkably upregulated in primary CRC tissues and cell lines compared with paired non-cancerous tissues and normal colorectal epithelium. Elevated expression of KIF4A in CRC tissues was significantly correlated with clinicopathological characteristics in patients as well as with shorter overall and disease-free cumulative survival. Multivariate Cox regression analysis revealed that KIF4A was an independent prognostic factor for poor survival in human CRC patients. Functional assays, including a CCK-8 cell proliferation assay, colony formation analysis, cancer xenografts in nude mice, cell cycle and apoptosis analysis, indicated that KIF4A obviously enhanced cell proliferation by promoting cell cycle progression in vitro and in vivo. Furthermore, gene set enrichment analysis, Luciferase reporter assays, and ChIP assays revealed that KIF4A facilitates cell proliferation via regulating the p21 promoter, whereas KIF4A had no effect on cell apoptosis. In addition, Transwell analysis indicated that KIF4A promotes migration and invasion in CRC. Taken together, these findings not only demonstrate that KIF4A contributes to CRC proliferation via modulation of p21-mediated cell cycle progression but also suggest the potential value of KIF4A as a clinical prognostic marker and target for molecular treatments.

LIM and SH3 protein 1 knockdown suppresses proliferation and metastasis of colorectal carcinoma cells via inhibition of the mitogen-activated protein kinase signaling pathway.

LIM and SH3 protein 1 (Lasp-1), a focal adhesion protein, serves a critical role in the regulation of cell proliferation and migration. The role of Lasp-1, as well as the intracellular signaling pathways involved in metastasis and progression of colorectal carcinoma, remains unclear. In the present study, the regulatory effect of Lasp-1 and the underlying molecular mechanism involved in migration and invasion of colorectal carcinoma were investigated. RNA interference and overexpression in SW480 cells were performed to elucidate the role of Lasp-1. Reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence were conducted to determine the mRNA and protein levels of Lasp-1 and extracellular-signal-regulated kinase 1/2 (ERK1/2) in SW480 cells as well as tumor and adjacent normal tissues obtained from 20 patients with colorectal carcinoma. Furthermore, a cell proliferation assay, flow cytometric analysis, and cell migration and invasion assays were performed to examine the effect of Lasp-1 on cell growth. The results of the present study demonstrated that Lasp-1 and ERK1/2 were upregulated in tumor tissue compared with adjacent normal colorectal mucosa. Cell-based in vitro assays demonstrated that Lasp-1 knockdown suppressed the expression and activation of ERK1/2, whereas Lasp-1 overexpression resulted in ERK1/2 upregulation. Additionally, Lasp-1 knockdown inhibited cell proliferation, migration, and invasion and induced cellular apoptosis and G0/G1 cell-cycle arrest. The results of the present study indicate that Lasp-1 serves a critical role in the progression of colorectal carcinoma regulating the activation of the mitogen-activated protein kinase signaling pathway.

Preliminary Study of the Role F-Box Protein 32 (FBXO32) in Colorectal Neoplasms Through the Transforming Growth Factor beta (TGF-β)/Smad4 Signalling Pathway

BackgroundF-box protein 32 (FBXO32) (also known as atrogin-1), a member of the F-box protein family, was recently shown to be a transforming growth factor beta (TGF-β)/Smad4 target gene involved in regulating cell survival. It can be transcriptionally silenced by epigenetic mechanisms in some cancers, but its role in colorectal carcinoma (CRC) is unclear. We investigated the role of FBXO32 in CRC and determined its prognostic significance.Material/MethodsWe used real-time quantitative PCR, Western blot, and immunohistochemistry to elucidate the role of FBXO32 in clinical specimens and primary CRC cell lines. Differences in patient survival were determined by the Kaplan-Meier method and log-rank test.ResultsWe found that the FBXO32 and SMAD4 levels were higher in normal tissues than in CRC tissues, but PAI-1 and VEGF levels showed the opposite pattern. The expressions of FBXO32 and SMAD4 were related to clinicopathological parameters in CRC. Kaplan-Meier analyses showed that the 5-year overall survival of the low-FBXO32 expression group was significantly shorter than that of the high-FBXO32 expression group (p=0.010).ConclusionsThe fbxo32 gene is a novel tumor suppressor that inhibits CRC progression by inducing differentiation. Elevated expression of FBXO32 predicts longer survival in CRC patients.

Cdc37 facilitates cell survival of colorectal carcinoma via activating the CDK4 signaling pathway.

Cell division cycle 37 (Cdc37) is an important partner for heat shock protein 90 (HSP90), assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Given its influence on cell growth pathways, Cdc37 has been discussed as a potential intermediate in carcinogenesis. However, to date, the potential functional roles and molecular mechanisms by which Cdc37 regulates cell survival in colorectal carcinoma (CRC) remain unclear. Here, we investigated the expression of Cdc37 and its clinical significance in CRC, and systematically explored the role and the underlying mechanism of Cdc37 in CRC cell survival both in vitro and in vivo. Our results showed that Cdc37 was remarkably up‐regulated in CRC, which facilitated cell survival mainly by promoting cell proliferation, G1‐S transition, and inhibiting cell apoptosis. Our data further indicated that Cdc37 increased the stability of cyclin‐dependent kinase 4 (CDK4) to activate the retinoblastoma 1 (RB1) signaling pathway, followed by increased expression of Bcl‐2 and Bcl‐xL, which ultimately promoted cell survival in CRC. Moreover, knockdown of CDK4 reversed the Cdc37‐mediated effect in promoting the progression of CRC. Our findings showed that Cdc37 played a critical role in promoting CRC cell survival by increasing CDK4 stability to activate the RB1 signaling pathway. Thereby, Cdc37 might serve as a potential therapeutic target in CRC patients.

Overexpression of GRK6 associates with the progression and prognosis of colorectal carcinoma.

There are a limited number of studies reporting on the expression of G protein-coupled receptor kinase 6 (GRK6) in colorectal carcinoma (CRC). The aim of the present study was to investigate and examine the clinical value of GRK6 expression in human CRC. The expression of the GRK6 protein was determined in CRC tissues (n=83) and in normal colorectal tissues (n=19) by immunohistochemical (IHC) analysis. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to investigate GRK6 mRNA levels in matched pairs of cancerous and non-cancerous fresh frozen tissues from 19 patients with CRC. Furthermore, GRK6 protein levels were evaluated in matched pairs of cancerous and non-cancerous fresh frozen tissues from 19 other patients with CRC by western blot analysis. The expression of GRK6 was significantly upregulated in patients with CRC as indicated by IHC analysis (P=0.028). The results of RT-qPCR and western blotting confirmed that GRK6 mRNA and protein levels were upregulated in CRC tissues compared with matched adjacent non-cancerous tissues (P<0.05). Additionally, GRK6 protein expression was significantly associated with histological differentiation (P=0.001), lymph node invasion (P=0.45), venous invasion (P=0.009), depth of invasion (P=0.026), distant metastasis (P<0.0001) and TNM stages (P=0.020). Survival analysis using the Kaplan-Meier method indicated that patients with high GRK6 expression levels exhibited lower overall survival rates compared with patients with low GRK6 expression. Multivariate analysis using the Cox proportional hazards model indicated that the expression levels of GRK6 (P=0.003) were independent prognostic factors for overall survival in patients. The overexpression of GRK6 in patients with CRC may serve as an independent predictor of patient outcome.

Membranous or Cytoplasmic HER2 Expression in Colorectal Carcinoma: Evaluation of Prognostic Value Using Both IHC & BDISH

ABSTRACTBackground: Human epidermal growth factor recptor-2 (HER2) was identified as a driver gene in several types of cancers with both prognostic and predictive value. However, the molecular association of HER2 gene mutation with HER2 gene amplification and/or protein expression in cancer tissues has not been clearly defined. Moreover, there is little information available on HER2 status role in tumor progression and metastasis in colorectal carcinoma (CRC) compared to other solid tumors. The aim of this study was to evaluate both HER2 amplification and protein expression profiles using immunohistochemistry (IHC) and bright-field dual in situ hybridization (BDISH) techniques, respectively. Patients and methods: Tissue microarray (TMA) was constructed to accommodate a total of 243 CRC formalin-fixed paraffin embedded (FFPE) samples of consent patients and stained by IHC and BDISH methods. The expression patterns of HER2 protein status were evaluated and correlated to HER2 gene amplification status and then assessed for its prognostic value. Results: The expression profile of 58% samples showed cytoplasmic expression patterns of different categories. Interestingly, only 1% showed strong (+3) membranous expression pattern of HER2 with perfect match with their corresponding gene amplification status (>2). However, the cytoplasmic HER2 protein status did not show significant correlation with most clinicopathological features and survival outcomes except with age (p = 0.04) and tumor size (p = 0.03). Conclusion: We demonstrated that the membranous HER2 gene/protein status is infrequent, while the main fraction of HER2 overexpression was cytoplasmic and lacking prognostic value. This cytoplasmic HER2 overexpression was induced through a gene-amplification independent pathway, making the HER2 gene status evaluation approach in those cases not worthy. Further investigations about the molecular pathways of the cytoplasmic HER2 protein in CRC and its associations with survival outcomes are required to allow either a breakthrough in CRC management; or to confirm the hypothesis of a marginal role in CRC onset and progression.

Down-regulation of B-Cell Translocation Gene 1 by Promoter Methylation in Colorectal Carcinoma.

B-cell translocation gene 1 (BTG1) acts as a tumour suppressor in human malignancies. However, the precise mechanism of BTG1 down-regulation in colorectal carcinoma (CRC) remains unclear. We analyzed BTG1 expression in CRC cell lines and tissues and investigated the mechanism underlying the observed alterations. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to analyze BTG1 expression in CRC cell lines. The methylation status of the BTG1 promoter region in cell lines was determined by methylation-specific PCR, and the effect of demethylation on BTG1 expression was explored with 5-aza-deoxycytidine treatment. BTG1 protein expression in CRC tissue samples was evaluated using immunostaining. CRC cell lines and tissue samples expressed lower levels of BTG1 compared to controls, and BTG1 levels were significantly lower in metastatic than primary CRC. In BTG1-down-regulated CRC cell lines, the BTG1 promoter was highly methylated, and 5-aza-deoxycytidine significantly restored BTG1 expression. BTG1 down-regulation in CRC occurs through epigenetic repression, which is involved in the development and progression of CRC.

Fecal Galectin-3: A New Promising Biomarker for Severity and Progression of Colorectal Carcinoma.

Background and Objectives The aim of the study was to determine systemic and fecal values of galectin-3 and pro- and anti-inflammatory cytokines in patients with CRC and the relationship with clinicopathological aspects. Methods Concentrations of galectin-3, TNF-α, TGF-β, IL-10, and IL-1β were analyzed in samples of blood and stool of 60 patients with CRC. Results Systemic concentration of TNF-α was significantly lower in patients with severe diseases (advanced TNM stage, nuclear grade, and poor histological differentiation) as in patients with more progressive CRC (lymph and blood vessel invasion, presence of metastasis). Fecal values of anti-inflammatory cytokines TGF-β and IL-10 were increased in patients with severe stadium of CRC. Fecal concentration of Gal-3 was enhanced in CRC patients with higher nuclear grade, poor tumor tissue differentiation, advanced TNM stage, and metastatic disease. Gal-3/TNF-α ratio in sera and feces had a higher trend in patients with severe and advanced diseases. Positive correlation between fecal Gal-3 and disease severity, tumor progression, and biomarkers AFP and CEA, respectively, was also observed. Conclusions Predomination of Gal-3 in patients with advanced diseases may implicate on its role in limiting ongoing proinflammatory processes. The fecal values of Gal-3 can be used as a valuable marker for CRC severity and progression.

Stemness Underpinning All Steps of Human Colorectal Cancer Defines the Core of Effective Therapeutic Strategies

Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. Here we show how tumor-initiating, human CRC stem-like cells (CCSCs) can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, circulating and metastatic CCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. Funding Statement: This work was supported by grants from AIRC (IG-14368) and MIUR (GR-2011-02351534, RC1703IC36). Declaration of Interests: The authors declare no competing interests. A.L. Vescovi has ownership interest in Hyperstem SA. Ethics Approval Statement: Fresh human colorectal cancer and blood specimens were collected from consenting patients at IRCCS “Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori” (IRST) and IRCCS “Casa Sollievo della Sofferenza”. All samples were obtained under protocols approved by the Research Ethics Boards (B063 and 94/CE). All animal procedures were carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committees (IACUCs) at the University of Milan-Bicocca.

The role and gene expression profile of SOCS3 in colorectal carcinoma.

SOCS3 has been postulated to play a role in the occurrence and progression of malignancies. However, the relationship of SOCS3 with colorectal carcinoma remains poorly understood. The purpose of the study was to explore the role of SOCS3 in colorectal carcinoma and its underlying mechanisms. Protein and mRNA expression of SOCS3 in colorectal carcinoma and normal colorectal mucosa was detected using immunohistochemistry and real-time quantitative PCR. SOCS3 expression was significantly lower in colorectal carcinoma tissue than in normal colorectal mucosa, and was negatively correlated with tumor invasion depth, lymph node metastasis, differentiation degree, and TNM stage. A stably transfected colorectal carcinoma cell line (8348SOCS3) with high expression of SOCS3 was established. The effects of SOCS3 overexpression on the growth, proliferation, invasion and tumor formation of colorectal carcinoma cells were examined by CCK-8 assay, transwell method and tumorigenicity assays in nude mice. Then we found SOCS3 overexpression significantly decreased proliferation and invasion capability of 8348 cells in vitro and in vivo. Furthermore, the effect of SOCS3 overexpression on the gene expression profile of colorectal carcinoma cells was analyzed using human genome arrays. The results revealed 369 genes that were differentially expressed in 8348SOCS3 cells. 193 genes was significantly increased and 176 genes was significantly decreased. Bioinformatics analysis demonstrated that high SOCS3 expression affected multiple signaling pathways in colorectal carcinoma including TGF-β/Smads, NF-κB, and HIF-MAPK pathways. Especially for the TGF-β/Smads pathways, high SOCS3 expression could inhibit TGF-β1 expression and activate Smad4 expression. These data suggested that low expression of SOCS3 was associated with the occurrence and progression of colorectal carcinoma. SOCS3 protein may be a useful indicator for malignancy and prognosis of colorectal carcinoma and also a new target for gene therapy.

Down-regulation of Inositol Polyphosphate 4-Phosphatase Type II Expression in Colorectal Carcinoma.

Aberrant expression of survival signaling pathways causes deregulation of cellular proliferation and resistance to apoptosis, and plays a crucial role in the development, progression and metastasis of cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and has a tumor-suppressive role in several human malignancies. We analyzed the expression levels of INPP4B mRNA and protein in colorectal carcinoma (CRC) cell lines and tissue samples using western blot, quantitative real-time reverse-transcriptase polymerase chain reaction, and immunohistochemical staining. Western blot analysis revealed that the CRC cell lines HCT 116, SW620, DLD-1, and WiDr expressed significantly lower levels of INPP4B protein than the normal colonic epithelial cell lines CCD 841 CoTr and FHC. Consistent with these results, INPP4B mRNA expression in the CRC cell lines was significantly lower than in the normal colonic epithelial cells. Immunohistochemical staining revealed that normal colonic mucosa displayed uniform and strong-to-moderate INPP4B immunoreactivity, whereas 60.7% (71/117; p<0.001) and 76.5% (62/81; p<0.001) of the primary and metastatic CRC tissue samples exhibited reduced INPP4B expression, respectively. Our results indicate that INPP4B is down-regulated in CRC and that INPP4B is involved in the development and progression of CRC.