Tumor Suppressor In Colorectal Cancer Research Articles

HOTAIRM1 as a potential biomarker for diagnosis of colorectal cancer functions the role in the tumour suppressor.

Recent studies have revealed many different long noncoding RNAs (lncRNA), however, the investigation for their function and clinical value as tumour biomarkers has scarcely begun. Here, we found that expression of HOTAIRM1 was reduced in colorectal cancer (CRC) tissues compared with matched normal tissues, and plasma HOTAIRM1 levels in CRC patients were less than in controls. The cut‐off point was chosen as 0.003 with a sensitivity of 64.00% and a specificity of 76.50% in the validation set. The performance of HOTAIRM1 was highly comparable to carcinoembryonic antigen (CEA), and better than CA19‐9 and CA125. The combined assay of HOTAIRM1 and CEA raised the sensitivity and specificity to 84.00%. HOTAIRM1 knockdown resulted in obvious changes in expression of the cell proliferation related to genes and promoted cell proliferation. HOTAIRM1 plays a role of tumour suppressor in CRC; Down‐regulation of HOTAIRM1 can serve as a biomarker for CRC, and combined HOTAIRM1 and CEA assay might provide a promising diagnosis for CRC.

TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3.

Dysregulation of the Wnt pathway leading to accumulation of β-catenin (CTNNB1) is a hallmark of colorectal cancer (CRC). Nuclear CTNNB1 acts as a transcriptional coactivator with TCF/LEF transcription factors, promoting expression of a broad set of target genes, some of which promote tumor growth. However, it remains poorly understood how CTNNB1 interacts with different transcription factors in different contexts to promote different outcomes. While some CTNNB1 target genes are oncogenic, others regulate differentiation. Here, we found that TCF7L1, a Wnt pathway repressor, buffers CTNNB1/TCF target gene expression to promote CRC growth. Loss of TCF7L1 impaired growth and colony formation of HCT116 CRC cells and reduced tumor growth in a mouse xenograft model. We identified a group of CTNNB1/TCF target genes that are activated in the absence of TCF7L1, including EPHB3, a marker of Paneth cell differentiation that has also been implicated as a tumor suppressor in CRC. Knockdown of EPHB3 partially restores growth and normal cell cycle progression of TCF7L1-Null cells. These findings suggest that while CTNNB1 accumulation is critical for CRC progression, activation of specific Wnt target genes in certain contexts may in fact inhibit tumor growth.

The tumor suppressor miR-138-5p targets PD-L1 in colorectal cancer.

microRNAs (miRNAs) play critical roles in cancer development and progression. This study investigated the effects of miR-138-5p in human colorectal cancer (CRC) development. miR-138-5p was frequently downregulated in CRC tissues and was associated with advanced clinical stage, lymph node metastasis and poor overall survival. We found that miR-138-5p decreased expression of programmed cell death ligand 1 (PD-L1) through interaction with its PD-L1 3′ untranslated region. miR-138-5p also dramatically suppressed CRC cell growth in vitro and inhibited tumorigenesis in vivo. PD-L1 and miR-138-5p levels were inversely correlated in human CRC tumors, and miR-138-5p inhibited PD-L1 expression in tumor models. These results suggest that miR-138-5p is a tumor suppressor in CRC, and its effects are exerted at least partially through PD-L1 downregulation. Low miR-138-5p and high PD-L1 levels correlated with shorter overall CRC patient survival, indicating that miR-138-5p and PD-L1 may serve as CRC biomarkers for risk group assignment, optimal therapy selection and clinical outcome prediction. Targeting PD-L1, possibly by administering miR-138-5p mimics, might be a clinically effective anti-CRC therapeutic strategy.

MiR-139-5p sensitizes colorectal cancer cells to 5-fluorouracil by targeting NOTCH-1

Multidrug resistance (MDR), a phenomenon that often occurs with drug treatment and is characterized by relapse or attenuation of drug efficacy, is almost unavoidable in colorectal cancer (CRC) patients receiving 5-fluorouracil (5-FU)-based chemotherapy. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression. Our previous study has identified miR-139-5p as a potential tumor suppressor in CRC, but its role in chemoresistance of CRC has not been elucidated. In this study, we demonstrated that miR-139-5p was down-regulated either in CRC tumors receiving chemotherapy or in 5-FU-resistant CRC cell lines (HCT-8/5-FU and HCT-116/5-FU). Ectopic expression of miR-139-5p sensitized CRC cells to 5-FU by increasing 5-FU-induced apoptosis. In addition, miR-139-5p inhibited the expression of the miR-139-5p target gene NOTCH-1 and its downstream molecules MRP-1 and BCL-2, two key MDR-associated genes. Furthermore, silencing NOTCH-1 expression promoted the chemotherapeutic effects of 5-FU, and up-regulation of NOTCH-1 abrogated miR-139-5p-mediated sensitization to 5-FU in LoVo and HCT-116 cells. Taken together, our data indicate a new role of miR-139-5p/NOTCH-1 pathway in the drug resistance of CRC cells to 5-FU, which may be a promising therapeutic target for the anti-MDR treatment of CRC.

Processed Aloe vera gel inhibits colitis-related colon carcinogenesis by inhibiting both chronic inflammation and cell cycle progression in the colon

Abstract The processed Aloe vera gel (PAG) contains immunomodulatory polysaccharides, which consists of a mixture of polymer chains of b-(1,4)-linked acetylated galactomannan with different lengths. PAG was shown to inhibit azoxymethane(AOM)-initiated and dextran sodium sulfate(DSS)-promoted colon carcinogenesis model in mice. In the present study, we examined the molecular mechanisms for the inhibition of colitis-associated colon carcinogenesis by PAG. PAG administration significantly inhibited the production of pro-inflammatory cytokines and mediators in the colon. Western blot analysis using the colon extracts showed that PAG administration reduced the activation of NF-kB resulting in the inhibition of iNOS and COX-2 expression. PAG appeared to inhibit NF-kB activation through the PPARγ activation. PAG administration also inhibited the expression and phosphorylation of STAT3, which was known to connect inflammation and cancer. PAG administration also inhibited cell cycle progression-related transcription factors such as the proliferating cell nuclear antigen (PCNA), ERK, cyclin-dependent kinase (CDK) 4 and cyclin D1. In contrast, PAG administration increased the expression of CDX2, which was known to be a tumor suppressor in colorectal cancers. These findings show that PAG suppresses colitis-related colon carcinogenesis by inhibiting both chronic inflammation and cell cycle progression in the colon.

MiRs-134 and -370 function as tumor suppressors in colorectal cancer by independently suppressing EGFR and PI3K signalling.

Growth factor receptor signalling plays a central and critical role in colorectal cancer. Most importantly, the EGFR signalling cascade involving PI3K/AKT/mTOR and Raf/MEK/ERK pathways are particularly relevant, since they are commonly activated in several cancer entities, including colorectal cancer. In this study, we show that miRs-134 and -370 are both capable of regulating these pathways by targeting EGFR and PIK3CA. In three different colorectal cancer cell lines (DLD1, HCT-116 and RKO), suppression of EGFR and PIK3CA through the enhanced expression of miR-134 or -370 led to a suppression of the key molecules of the PI3K/AKT/mTOR pathway. Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis. Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins. Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

Overexpression of long non-coding RNA-CTD903 inhibits colorectal cancer invasion and migration by repressing Wnt/β-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 lncRNA‑CTD903 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, β-catenin, ZEB1, ZO-1, Snail, and Twist, were determined by western blotting. Our results showed lncRNA-CTD903 expression was strongly upregulated in 115CRC 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/β-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/β-catenin signaling, which plays important roles in EMT and CRC metastasis.

MiR-381 functions as a tumor suppressor in colorectal cancer by targeting Twist1.

MiR-381 has been reported to be dysregulated in several human cancers. However, the function and mechanism of miR-381 in colorectal cancer (CRC) remains unclear. In the present study, the miR-381 expression was assessed in a cohort of 113 CRC specimens using real-time quantitative polymerase chain reaction (RTq-PCR), which demonstrated that miR-381 was significantly downregulated in CRC and correlated with distant metastasis and tumor, node, and metastasis (TNM) stage. Functional study revealed that restoration of miR-381 significantly inhibited the invasion, migration, and epithelial–mesenchymal transition (EMT) of CRC cells. Luciferase reporter assay validated that Twist1, an important EMT inducer, was a direct target of miR-381, and rescued Twist1 attenuated the function of miR-381 in CRC cells. Correlation analysis also revealed an inverse correlation between miR-381 and Twist1 expression levels in CRC specimens. Taken together, our results highlight the significance of miR-381/Twist1 interaction in the development and progression of CRC, and suggest that restoration of miR-381 may be a potential therapeutic strategy for the patients with CRC.

MiR-152 functions as a tumor suppressor in colorectal cancer by targeting PIK3R3.

Accumulating evidence showed that microRNA-152 (miR-152) was frequently downregulated, and functioned as tumor suppressor in many cancers, but little is known about its biological role and intrinsic regulatory mechanisms in colorectal cancer (CRC). Here, we explored the potential role of miR-152 in CRC and the possible molecular mechanisms. Our results proved that miR-152 expression was downregulated in CRC cell lines and tissue samples, and its expression was inversely correlated with advanced tumor-node-metastasis (TNM) stage (P < 0.01) and lymph node metastasis (P < 0.01). Function assays demonstrated that restoring the expression of miR-152 in CRC cells dramatically reduced the cell proliferation and cell migration and invasion and promoted apoptosis and caspase-3 activity in vitro, as well as suppressed tumor growth in vivo. Mechanistic investigations defined phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) as a direct and functional downstream target of miR-152. In addition, we also found that PIK3R3 expression was upregulated and was inversely correlated with miR-152 expression in clinical CRC tissues. Downregulation of PIK3R3 mimicked the tumor-suppressive effects of miR-152 overexpression in CRC cells. Taken together, these results elucidated the function of miR-152 in CRC progression and suggested that miR-152 might function as tumor suppressor in CRC by targeting PIK3R3.

MiR-422a is an independent prognostic factor and functions as a potential tumor suppressor in colorectal cancer.

To determine the expression of miR-422a in colorectal cancer (CRC) tissues and to further explore the prognostic value and function of miR-422a in CRC carcinogenesis. miR-422a expression was analyzed in 102 CRC tissues and paired normal mucosa adjacent to carcinoma by quantitative real-time PCR. The relationship of miR-422a expression with clinicopathological parameters was also analyzed. Kaplan-Meier analysis and Cox multivariate analysis were performed to estimate the potential role of miR-422a. Cell proliferation, migration, and invasion were used for in vitro functional analysis of miR-422a. The levels of miR-422a were dramatically reduced in CRC tissues compared with normal mucosa (P < 0.05), and significantly correlated with local invasion (P = 0.004) and lymph node metastasis (P < 0.001). Kaplan-Meier survival and Cox regression multivariate analyses revealed that miR-422a expression (HR = 0.568, P = 0.015) and clinical TNM stage (HR = 2.942, P = 0.003) were independent prognostic factors for overall survival in CRC patients. Furthermore, in vitro experiments showed that overexpression of miR-422a inhibited the proliferation, migration, and invasion of SW480 and HT-29 cells. Down-regulation of miR-422a may serve as an independent prognosis factor in CRC. MiR-422a functions as a tumor suppressor and regulates progression of CRC.

Decreased miR-452 expression in human colorectal cancer and its tumor suppressive function.

MicroRNA-452 (miR-452) is dysregulated in some human malignancies, and is correlated with tumor progression. However, its expression and function in human colorectal cancer (CRC) remain unclear. The aim of our study was to explore the effects of miR-452 in CRC tumorigenesis and development. Using reverse transcription quantitative real-time polymerase chain reaction, we detected miR-452 expression in CRC cell lines and primary tumor tissues. We also examined the association between miR-452 expression and clinicopathological factors. We then investigated the effects of miR-452 on the biological behavior of CRC cells. miR-452 expression was significantly downregulated in CRC compared with the adjacent noncancerous tissues. A low level of miR-452 was associated with larger tumor size, deeper invasion depth, and advanced TNM stage. Multivariate Cox regression analysis identified decreased miR-452 expression as an independent factor predicting poor prognosis for CRC patients. In addition, in vitro functional analysis showed that overexpression of miR-452 in HCT116 cells reduced cell proliferation, promoted cell apoptosis, and inhibited cell invasion and migration. These findings indicate that miR-452acts as a tumor suppressor in CRC, and would serve as a novel molecular therapeutic agent for the treatment of the disease.

Decreased expression of miR-874 and its tumor suppressive function in human colorectal cancer.

Dysregulation of miRNAs is associated with cancer development and progression. For example, aberrant expression of miR-874 has been found in some types of cancer. However, miR-874 expression and its clinical significance in colorectal cancer (CRC) have not yet been explored. The aim of the current study was to explore the effects of miR-874 in CRC tumorigenesis and development. Quantitative reverse-transcription PCR was performed to evaluate miR-874 levels in CRC cell lines and in 135 pairs of primary human CRC specimens and adjacent noncancerous tissues. The association of miR-874 expression with clinicopathological factors and prognosis was also analyzed. Furthermore, the effects of miR-874 on the biological behavior of CRC cells in vitro were investigated. Our results revealed that miR-874 expression was significantly downregulated in CRC cancer tissues and cell lines. Decreased miR-874 expression was significantly associated with larger tumor size, deeper invasion depth, and advanced TNM stage in vivo. Additionally, low miR-874 expression in CRC was an independent predictor of poor survival. Moreover, overexpression of miR-874 inhibited cell proliferation, invasion, and migration, and promoted cell apoptosis of the SW620 CRC cell line in vitro. Taken together, these findings indicate that miR-874 may act as a tumor suppressor in CRC, and may serve as a novel therapeutic target for miR-based therapy.

Potential role of TRIM3 as a novel tumour suppressor in colorectal cancer (CRC) development

ABSTRACTObjective Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in the United States. Recent cancer genome-sequencing efforts and complementary functional studies have led to the identification of a collection of candidate ‘driver’ genes involved in CRC tumorigenesis. Tripartite motif (TRIM3) is recently identified as a tumour suppressor in glioblastoma but this tumour-suppressive function has not been investigated in CRC. Material and methods In this study, we investigated the potential role of TRIM3 as a tumour suppressor in CRC development by manipulating the expression of TRIM3 in two authentic CRC cell lines, HCT116 and DLD1, followed by various functional assays, including cell proliferation, colony formation, scratch wound healing, soft agar, and invasion assays. Xenograft experiment was performed to examine in vivo tumour-suppressive properties of TRIM3. Results Small-interfering RNA (siRNA) mediated knockdown of TRIM3 conferred growth advantage in CRC cells. In contrast, overexpression of TRIM3 affected cell survival, cell migration, anchorage independent growth and invasive potential in CRC cells. In addition, TRIM3 was found to be down-regulated in human colon cancer tissues compared with matched normal colon tissues. Overexpression of TRIM3 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that TRIM3 can regulate p53 protein level through its stabilisation. Conclusions TRIM3 functions as a tumour suppressor in CRC progression. This tumour-suppressive function is exerted partially through regulation of p53 protein. Therefore, this protein may represent a novel therapeutic target for prevention or intervention of CRC.

MicroRNA-490-3p inhibits colorectal cancer metastasis by targeting TGFβR1.

BackgroundColorectal cancer (CRC) is one of the most common malignances worldwide. Metastasis is responsible for the rapid recurrence and poor prognosis of CRC. However, the underlying molecular mechanism of CRC metastasis remains largely unclear. In this study we purposed to investigate the expression and biological functions of miR-490-3p in CRC metastasis, as well as to identify its downstream target genes and influenced pathway.MethodsThe expression level of miR-490-3p in CRC cell lines, CRC adjacent normal tissues, non-metastasis and metastasis tissues were assessed by quantitative real-time PCR. Patient survivals were follow-up up to 7 years. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by transfection of miR-490-3p mimics or inhibitors respectively. The molecular targets of miR-490-3p were computationally identified and experimentally verified by dual-luciferase reporter assay and western blot. Functional rescue was also conducted to confirm miR-490-3p inhibits CRC metastasis by targeting TGF-β signaling pathway.ResultsmiR-490-3p expression was persistently downregulated during CRC malignant progression, as well as in CRC cell lines. Artificially overexpression miR-490-3p in CRC cell lines inhibited cell migration and invasion abilities while knockdown miR-490-3p expression caused the reverse effects. TGFβR1 and MMP2/9 were the downstream targets of miR-490-3p in CRC. Inhibition of TGFβR1 could partially recover the tumor suppression effect of miR-490-3p.ConclusionmiR-490-3p is downregulated during CRC malignant progression. miR-490-3p represses CRC cell migration and invasion abilities, partially by targeting to the TGF-β signaling pathway. Taken together, miR-490-3p is acting as a tumor suppressor in CRC.

MicroRNA-206 functions as a tumor suppressor in colorectal cancer by targeting FMNL2.

Colorectal cancer (CRC) is one of the most common cancers in the world. MicroRNAs play important roles in the progression of CRC. This study aimed to investigate the role of miR-206 and its novel mechanism in the invasion and metastasis of CRC. Real-time RT-PCR or Western blotting was used to detect the expressions of miR-206, FMNL2 and c-MET in CRC cell lines and tissues. Luciferase reporter assays were conducted to detect the associations between miR-206 and 3'UTRs of FMNL2 and c-MET. A series of loss-of-function and gain-of-function assays were performed to evaluate the effect of miR-206 on the proliferation, invasion and metastasis of CRC cells. miR-206 was significantly down-regulated in CRC tissues and correlated closely with differentiation, lymphatic metastasis and serosal invasion. miR-206 suppressed CRC cell proliferation by arresting CRC cells in the G1/G0 phase and accelerating apoptosis. miR-206 also inhibited cell invasion and lung metastasis in CRC cells. Mechanically, FMNL2 and c-MET were identified as direct targets of miR-206. And FMNL2 rescued the suppression of miR-206 in the proliferation and invasion of CRC cells. This study revealed functional and mechanistic links between miR-206 and oncogene FMNL2 and c-MET in the progression of CRC. miR-206 functioned as a tumor suppressor in the progression of CRC by targeting FMNL2 and c-MET. Restoration of miR-206 expression may represent a promising therapeutic approach for targeting malignant CRC.

Abstract 3116: MiR-215 is a tumor suppressor in colorectal cancer in vitro and in vivo

Abstract Introduction: Colorectal cancer (CRC) is one of the most common types of cancers worldwide and it is the third leading cause of cancer-related death. Therefore, several efforts have been made to identify not only new diagnostic, prognostic and predictive biomarkers, but also therapeutic targets. One of the promising approaches is the characterization of the tumors using microRNAs (miRNAs). Patients and Methods: We have analyzed expression profiles of 667 miRNAs in 8 patients diagnosed for CRC and 8 paired adjacent non-tumoral tissues using TaqMan Low Density miRNA arrays. We have found miR-215 to be one of the most deregulated miRNAs, therefore its expression was further validated on independent cohort of 250 paired samples and correlated with clinicopathological features of the patients. Consequently, involvement of this miRNA in CRC pathogenesis was investigated using numerous in vitro and in vivo assays. Results: By use of miRNA expression profiling we have identified miR-215 to be highly down-regulated in tumor tissue of CRC patients (P &amp;lt; 0,0001). Subsequent validation on independent cohort of 250 paired samples of tumor tissues and adjacent non-tumor tissues confirmed this result, moreover, correlation between low expression of this miRNA and clinical stage (P &amp;lt; 0.0001) and lymph node metastasis (P &amp;lt; 0.0001) has been observed. In addition, significantly reduced levels of miR-215 have been detected in metastatic tissue (P &amp;lt; 0.0001) compared to primary tumor tissue and low expression of miR-215 was associated with shorter disease free survival (P = 0.0403). Subsequently, in vitro analyses have been performed using stable colorectal cancer cell lines in which the level of miR-215 was increased using transient or stable transfection. Higher levels of miR-215 lead to the cell cycle arrest in G1 phase of HCT-116+/+ cells (P = 0.01) and in G2/M phase of DLD-1 cells (P = 0.05), HCT-116-/- cells (P &amp;lt; 0.001) and HT-29 cells (P = 0.01), increased apoptosis of HCT-116+/+ cells (P &amp;lt; 0.001), reduced migration of DLD-1 cells (P &amp;lt; 0.001) and HCT-116+/+ cells (P &amp;lt; 0.001) and decreased proliferation activity of all analyzed cell lines (P &amp;lt; 0.001). Using qRT-PCR we have validated several predicted targets of miR-215 including XIAP, CD164, ALCAM or HOXB9. Finally, we have established human tumor xenografts in immunodeficient mice. We have observed that HCT-116+/+ transduced with vector containing precursor of miR-215 were associated with the slower growth of the tumors in vivo (N = 5; P = 0.04). Conclusions: The reduced expression of miR-215 has been observed in several cancers, therefore we presume that this miRNA functions as an important tumor suppressor. The results of our study indicate that miR-215 could serve as a new diagnostic and prognostic biomarker as well as potential therapeutic target for the treatment of CRC patients. The work has been supported by IGA MZCR NT13549-4/2012, NT13860-4/2012, NT13547-4/2012 and NT13514-4/2012. Citation Format: Petra Vychytilova-Faltejskova, Jana Merhautova, Jitka Mlcochova, Lenka Radova, Robert Iliev, Marek Svoboda, Rostislav Vyzula, Ondrej Slaby. MiR-215 is a tumor suppressor in colorectal cancer in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3116. doi:10.1158/1538-7445.AM2015-3116

Abstract 2044: Characterization of a novel tumor suppressor PRSS8 in colorectal cancer

Abstract PRSS8, a glycosylphosphatidylinositol-anchored epithelial extracellular membrane serine protease prostasin, is expressed in normal epithelia and is involved in terminal epithelial differentiation. Aberrantly expression of PRSS8 was observed in human prostate, breast and gastric cancers, and the reduction might be linked to promoter hypermethylation in vitro. However, the expression of PRSS8 in colorectal cancer and its significance in colorectal carcinogenesis and progression are largely unknown. In this study, the expression levels of PRSS8 were firstly determined in four human colorectal cancer cell lines and in 47 paired human colorectal cancer tissues (cancers and their adjacent non-cancer tissues) by real-time PCR, Western blotting and immunohistochemistry. We found that PRSS8 was significantly reduced in colorectal cancer tissues, compared to adjacent non-cancer tissues, and was in very low levels in the colorectal cancer cell lines. Interestingly, the reduced PRSS8 was associated with histopathological stages and differentiation: PRSS8 was lower in poor differentiated cancers and was higher in well differentiated cancers. In addition, the expression levels of PRSS8 were also determined in esophageal cancer, prostate cancer, breast cancer, liver cancer and lung cancer using tissue microarray (TMA) by immunohistochemical staining. Consistent with those in colorectal cancer, PRSS8 protein levels were reduced in all the five cancers and the reduction was associated with cancer differentiation. Further analysis revealed that the patients with lower expression of PRSS8 in esophageal cancer were likely to have a shorter survival time. Functional studies by gain- and loss-expression of PRSS8 showed that increased PRSS8 expression inhibited colon cancer cell proliferation, promoted apoptosis, attenuated cell migration and epithelial-mesenchymal transition (EMT). Mechanistic study showed tumor suppression of PRSS8 was linked to Wnt/beta-catenin signaling pathways. Moreover, PRSS8 interacted with Sphk1 to target S1P and affect EMT/metastasis. Lastly, PRSS8 inhibited colorectal cancer cell growth in tumor-bearing mice, and purified PRSS8 protein showed therapeutic effects in vitro and nude mice. Taken together, this study has identified PRSS8 as a tumor suppressor in colorectal cancer and acts as a key regulator in colorectal cancer formation, progression and outcomes. Therefore, PRSS8 could be a useful biomarker for monitoring colorectal carcinogenesis and progression, and also could have a therapeutic potential for cancers. Note: This abstract was not presented at the meeting. Citation Format: Yonghua Bao, Kai Li, Qian Wang, Yongchen Guo, Rongfei Han, Zhiguo Chen, Zexin Li, Jianguo Wang, Weixing Zhao, Wenliang Han, Jiaqi Wang, Huijuan Zhang, Pan He, Wancai Yang. Characterization of a novel tumor suppressor PRSS8 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2044. doi:10.1158/1538-7445.AM2015-2044

SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression.

Colorectal cancer (CRC) is the third most common cancer in the world, and second leading cause of cancer deaths in the US. Although, anti-EGFR therapy is commonly prescribed for CRC, patients harboring mutations in KRAS or BRAF show poor treatment response, indicating an ardent demand for new therapeutic targets discovery. SPINK1 (serine peptidase inhibitor, Kazal type 1) overexpression has been identified in many cancers including the colon, lung, breast and prostate. Our study demonstrates the functional significance of SPINK1 in CRC progression and metastases. Stable knockdown of SPINK1 significantly decreases cell proliferation, invasion and soft agar colony formation in the colon adenocarcinoma WiDr cells. Conversely, an increase in these oncogenic phenotypes was observed on stimulation with SPINK1-enriched conditioned media (CM) in multiple benign models such as murine colonic epithelial cell lines, MSIE and YAMC (SPINK3-negative). Mechanistically, SPINK1 promotes tumorigenic phenotype by activating phosphatidylinositol 3-kinase (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways, and the SPINK1-positive WiDr cells are sensitive to AKT and MEK inhibitors. Importantly, SPINK1 silencing mediated upregulation of various Metallothionein isoforms, considered as tumor suppressors in CRC, confer sensitivity to doxorubicin, which strengthens the rationale for using the combinatorial treatment approach for the SPINK1-positive CRC patients. Furthermore, in vivo studies using chicken chorioallantoic membrane assay, murine xenograft studies and metastasis models further suggest a pivotal role of SPINK1 in CRC progression and metastasis. Taken together, our study demonstrates an important role for the overexpressed SPINK1 in CRC disease progression, a phenomenon that needs careful evaluation towards effective therapeutic target development.

Abstract 4959: Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response

Abstract Aberrant activation of the canonical Wnt signal transduction pathway is implicated in the development and progression of colorectal cancer (CC). The key effector protein of this pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We have previously identified klotho as a potent tumor suppressor in breast and pancreatic cancer, and recent data indicate it as a potent tumor suppressor in a wide array of malignancies, including CC. The activities of klotho include inhibition of the insulin like growth factor (IGF)-1 and the Wnt pathways. We aimed to decipher the role of klotho as a tumor suppressor and modulator of the Wnt pathway in CC. We first tested the effect of klotho on proliferation of CC cells and noted that overexpression of klotho inhibited colony formation of the HCT 116, SW480, HT-29, Colo-320 and RKO CC cells. We next examined the effects of klotho in vivo, using the azoxymethane (AOM) model, and found that daily administration of klotho to mice inhibited formation of colon polyps induced by AOM. While we did not see an effect of klotho on the IGF-1 signaling pathway, klotho did affect the Wnt pathway. Klotho reduced Wnt3A and β-catenin protein levels as measured by Western blotting, and inhibited transcriptional activity of the Wnt pathway as evidenced in luciferase assay. Moreover, this transcriptional inhibition was abrogated by overexpression of a constitutively active β-catenin. We, therefore, suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. As the inhibitory effect of klotho on colony formation of CC cells was only partially rescued by transfection with constitutively active β-catenin, we concluded that additional mechanisms may mediate the effects of klotho. In order to elucidate these mechanisms, we conducted a cDNA expression microarray and involvement of klotho in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) was observed. Taken together, our data indicate klotho as a potent tumor suppressor in CC and suggest its role already at the early stages of polyp formation. Klotho effects in CC are partially mediated by the Wnt pathway however additional mechanisms, including regulation of ER stress response, are involved. Citation Format: Tammi Rubinstein, Shiri Shahmoon, Gil Har Zahav, Nir Skalka, Tal Etan, Rina Arbesfeld, Metsada Pasmanik-Chor, Tami Rubinek, Ido Wolf. Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2015-4959

Abstract 3932: Using Sleeping Beauty transposon mutagenesis to identify CRC driver genes

Abstract In order to better understand the initiation and development of colorectal cancer (CRC), Sleeping Beauty (SB) transposon mutagenesis screens were performed in mice with mutations in genes that play important roles in differen stages of CRC. Functional validation assays, including invasion assay and xenograft assay, were carried out for the candidate genes that were selected by their potentials in tumor progression and/or human CRC. We identified six genes driving malignant tumor progression, and a new human CRC tumor suppressor gene, ZNF292, that may also function in other types of cancer. More detailed investigations are being done for those identified genes, including experiments using knockout mice. Our study provides a resource for the development of new CRC therapies. Citation Format: Zhubo Wei, Haruna Takeda, Neal G. Copeland, Nancy A. Jenkins. Using Sleeping Beauty transposon mutagenesis to identify CRC driver genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3932. doi:10.1158/1538-7445.AM2015-3932