Colorectal Cancer Cell Colony Formation Research Articles

Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression.

We aimed to explore the biological function of CPNE7 and determine the impact of CPNE7 on chemotherapy resistance in colorectal cancer (CRC) patients. According to the Gene Expression Profiling Interactive Analysis database and previously published data, CPNE7 was identified as a potential oncogene in CRC. RT-qPCR and Western blotting were performed to verify the expression of CPNE7. Chi-square test was used to evaluate the associations between CPNE7 and clinical features. Cell proliferation, colony formation, cell migration and invasion, cell cycle and apoptosis were assessed to determine the effects of CPNE7. Transcriptome sequencing was used to identify potential downstream regulatory genes, and gene set enrichment analysis was performed to investigate downstream pathways. The effect of CPNE7 on 5-fluorouracil chemosensitivity was verified by half maximal inhibitory concentration (IC50). Subcutaneous tumorigenesis assay was used to examine the role of CPNE7 in sensitivity of CRC to chemotherapy invivo. Transmission electron microscopy was used to detect autophagosomes. CPNE7 was highly expressed in CRC tissues, and its expression was correlated with T stage and tumour site. Knockdown of CPNE7 inhibited the proliferation and colony formation of CRC cells and promoted apoptosis. Knockdown of CPNE7 suppressed the expression of ATG9B and enhanced the sensitivity of CRC cells to 5-fluorouracil invitro and invivo. Knockdown of CPNE7 reversed the induction of the autophagy pathway by rapamycin and reduced the number of autophagosomes. Depletion of CPNE7 attenuated the malignant proliferation of CRC cells and enhanced the chemosensitivity of CRC cells to 5-fluorouracil.

PFKFB2 is a favorable prognostic biomarker for colorectal cancer by suppressing metastasis and tumor glycolysis.

This study was to investigate the biological effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) in colorectal cancer (CRC). PFKFB2 was selected by metabolism polymerase chain reaction (PCR) array from CRC cells under alkaline culture medium (pH 7.4) and acidic culture medium (pH 6.8). The expression of PFKFB2 mRNA and protein was detected by quantitative real-time PCR and immunohistochemistry in 70 paired fresh and 268 paired paraffin-embedded human CRC tissues, respectively, and then the prognostic value of PFKFB2 was investigated. The effects of PFKFB2 on CRC cells were also verified in vitro, which were through detecting the change of migration, invasion, sphere formation, proliferation, colony formation, and extracellular acidification rate of CRC cells after PFKFB2 knockdown in alkaline culture medium (pH 7.4) and overexpression in acidic culture medium (pH 6.8). PFKFB2 expression was downregulated in acidic culture medium (pH 6.8). In addition, we found PFKFB2 expression decreased in human CRC tissues compared with the adjacent normal tissues. Furthermore, the OS and DFS rate of CRC patients with low PFKFB2 expression was significantly shorter than those of patients with high PFKFB2 expression. Multivariate analysis indicated that low PFKFB2 expression was an independent prognostic factor for both OS and DFS in CRC patients. Moreover, the abilities of migration, invasion, spheroidizing ability, proliferation, and colony formation of CRC cells were significantly increased after depletion of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8) in vitro. Epithelial-mesenchymal transition (EMT) pathway was found and verified involved in the PFKFB2-mediated regulation of metastatic function in CRC cells. Further, glycolysis of CRC cells was significantly elevated after knockdown of PFKFB2 in alkaline culture medium (pH 7.4) and decreased after overexpression of PFKFB2 in acidic culture medium (pH 6.8). PFKFB2 expression is downregulated in CRC tissues and associated with worse survival for CRC patients. PFKFB2 could inhibit metastasis and the malignant progression of CRC cells by suppressing EMT and glycolysis.

Berberine suppresses colorectal cancer by regulation of Hedgehog signaling pathway activity and gut microbiota

BackgroundA growing body of evidence reveals that dysregulation of Hedgehog signaling pathway and dysbiosis of gut microbiota are associated with the pathogenesis of colorectal cancer (CRC). Berberine, a botanical benzylisoquinoline alkaloid, possesses powerful activities against various malignancies including CRC, with the underlying mechanisms to be illuminated. PurposeThe present study investigated the potencies of berberine on CRC and deciphered the action mechanisms in the context of Hedgehog signaling cascade and gut microbiota. MethodsThe effects of berberine on the malignant phenotype, apoptosis, cell cycle and Hedgehog signaling of CRC cells were examined in vitro. In azoxymethane/dextran sulfate sodium-caused mouse CRC, the efficacies of berberine on the carcinogenesis, pathological profile, apoptosis, cell cycle and Hedgehog signaling were determined in vivo. Also, the influences of berberine on gut microbiota in CRC mice were assessed by high-throughput DNA sequencing analysis of 16S ribosomal RNA of fecal microbiome in CRC mice. ResultsIn the present study, berberine was found to dampen the proliferation, migration, invasion and colony formation of CRC cells, without toxicity to normal colonic cells. Additionally, berberine induced apoptosis and arrested cell cycle at G0/G1 phase in CRC cells, accompanied by reduced Hedgehog signaling pathway activity in vitro. In mouse CRC, berberine suppressed tumor growth, ameliorated pathological manifestations, and potentially induced the apoptosis and cell cycle arrest of CRC, with lowered Hedgehog signaling cascade in vivo. Additionally, berberine decreased β-diversity of gut microbiota in CRC mice, without influence on α-diversity. Berberine also enriched probiotic microbes and depleted pathogenic microbes, and modulated the functionality of gut microbiota in CRC mice. ConclusionsOverall, berberine may suppress colorectal cancer, orchestrated by down-regulation of Hedgehog signaling pathway activity and modulation of gut microbiota.

Functional assessment of miR‑1291 in colon cancer cells.

miR-1291 exerts an anti-tumor effect in a subset of human carcinomas, including pancreatic cancer. However, its role in colorectal cancer (CRC) is largely unknown. In the present study, the expression and effect of miR-1291 in CRC cells was investigated. It was identified that miR-1291 significantly suppressed the proliferation, invasion, cell mobility and colony formation of CRC cells. Additionally, miR-1291 induced cell apoptosis. A luciferase reporter assay revealed that miR-1291 directly bound the 3′-untranslated region sequence of doublecortin-like kinase 1 (DCLK1). miR-1291 also suppressed DCLK1 mRNA and protein expression in HCT116 cells that expressed DCLK1. Furthermore, miR-1291 suppressed cancer stem cell markers BMI1 and CD133, and inhibited sphere formation. The inhibitory effects on sphere formation, invasion and mobility in HCT116 cells were also explored and verified using DCLK1 siRNAs. Furthermore, miR-1291 induced CDK inhibitors p21WAF1/CIP1 and p27KIP1 in three CRC cell lines, and the overexpression of DCLK1 in HCT116 cells led to a decrease of p21WAF1/CIP1 and p27KIP1. Intravenous administration of miR-1291 loaded on the super carbonate apatite delivery system significantly inhibited tumor growth in the DLD-1 xenograft mouse model. Additionally, the resultant tumors exhibited significant upregulation of the p21WAF1/CIP1 and p27KIP1 protein with treatment of miR-1291. Taken together, the results indicated that miR-1291 served an anti-tumor effect by modulating multiple functions, including cancer stemness and cell cycle regulation. The current data suggested that miR-1291 may be a promising nucleic acid medicine against CRC.

Knockdown of SFRS9 Inhibits Progression of Colorectal Cancer Through Triggering Ferroptosis Mediated by GPX4 Reduction

Ferroptosis, a newly discovered form of programmed cell death characterized by lipid peroxidation, crafts a new perspective on cancer treatment. Serine and arginine rich splicing factor 9 (SFRS9) is frequently described as a proto-oncogene in cervical and bladder cancer. However, the role of SFRS9 in colorectal cancer (CRC) and whether SFRS9 exerts its function associated with ferroptosis is largely unknown. Herein, we found that the expression of SFRS9 mRNA and protein in the CRC tissues was obviously higher than that in the paracancerous tissues. Function assays revealed that SFRS9 overexpression (SFRS9-OE) significantly promoted cell viability, cell cycle progression and colony formation of CRC cells. While SFRS9 knockdown by shRNAs transfection inhibited these progressions. Furthermore, cell death and lipid peroxidation induced by ferroptosis inducers erastin and sorafenib were suppressed by SFRS9-OE. Bioinformatics analysis indicated that SFRS9 can bind to peroxidase 4 (GPX4) mRNA which is a central regulator of ferroptosis. Western blot showed that GPX4 protein expression was clearly elevated upon SFRS9-OE, while it was decreased in SFRS9-inhibited CRC cells. RNA immunoprecipitation experiment was carried out in HCT116 cells to confirm the binding of SFRS9 and GPX4 mRNA specifically. SiGPX4 transfection reversed the inhibitory effects of SFRS9-OE on the erastin and sorafenib-induced ferroptosis. Consistent with our in vitro observations, SFRS9 promoted the growth of tumors while SFRS9 knockdown significantly inhibited tumor growth in nude mice. In conclusion, SFRS9 represents an obstructive factor to ferroptosis by upregulating GPX4 protein expression, and knocking down SFRS9 might be an effective treatment for CRC.

The inhibition of colorectal cancer growth by the natural product macrocarpal I

BackgroundPresently, few small molecule compounds are used as targeted therapy drugs in the treatment of colorectal cancer (CRC). It is important to identify new small molecule compounds, which can be used in the treatment of CRC. MethodsIn this study, we selected four protein molecules as drug targets: PRL-3 (Phosphatase of regenerating liver 3), CLIC4 (Chloride intracellular channel 4), THBS2 (Thrombospondin 2), and BGN (Biglycan). These protein molecules were associated with the growth and metastasis of CRC cells. Small molecular compounds were screened on the basis of their target structures. Thus, five small molecule compounds were screened from each target structure, and three small molecule compounds (macrocarpal I, sildenafil, and neoandrographolide) were found to bind with two drug targets at the same time. Further experiments revealed that the inhibition rate of macrocarpal I was the highest in CRC cells. Therefore, we determined the effects of macrocarpal I on proliferation, apoptosis, cytoskeleton of CRC cells, and subcutaneous tumorigenesis in nude mice. Furthermore, RNA-seq analysis was performed to determine the molecular mechanism through which macrocarpal I inhibited the progression of CRC. ResultsWe found that macrocarpal I could effectively inhibit proliferation, colony formation of CRC cells, and subcutaneous tumorigenesis in nude mice. Moreover, it also destroyed the cytoskeleton of CRC cells and promoted apoptosis. The effects on kinase activity, cytoskeleton, and DNA repair is the mechanism of macrocarpal I to inhibiting CRC growth. ConclusionMacrocarpal I is a small molecule compound that can effectively inhibit the progression of CRC. Thus, macrocarpal I is a therapeutic compound that shows promising results in the treatment of advanced CRC.

SIRT2, a direct target of miR-212-5p, suppresses the proliferation and metastasis of colorectal cancer cells.

The aberrant expression of human sirtuin 2 (SIRT2) has been detected in various types of cancer; however, the biological roles, underlying mechanisms and clinical significance of SIRT2 dysregulation in human colorectal cancer (CRC) remain unclear. The results of this study demonstrate that compared with paired normal tissues, SIRT2 expression is significantly decreased in CRC tissues. SIRT2 loss has been correlated with clinicopathological characteristics, including distant metastasis, lymph node metastasis and American Joint Committee on Cancer (AJCC) stage; this loss serves as an independent factor that indicates a poor prognosis for patients with CRC. Further gain‐ and loss‐of‐function analyses have demonstrated that SIRT2 suppresses CRC cell proliferation and metastasis both in vivo and in vitro. Mechanistically, miR‐212‐5p was identified to directly target the SIRT2 3′‐untranslated region (3′‐UTR), leading to SIRT2 down‐regulation. The ectopic expression of SIRT2 reverses the effect of miR‐212‐5p overexpression on CRC cell colony formation, invasion, migration and proliferation. Clinically, an inverse correlation was found between miR‐212‐5p and SIRT2 expression. High miR‐212‐5p expression has been found to result in a poor prognosis and aggressive clinicopathological characteristics in patients with CRC. Taken together, these results suggest that SIRT2, targeted by miR‐212‐5p, acts as a tumour suppressor in CRC and that the miR‐212‐5p/SIRT2 axis is a promising prognostic factor and potential therapeutic target in CRC.

Metformin blocks MYC protein synthesis in colorectal cancer via mTOR-4EBP-eIF4E and MNK1-eIF4G-eIF4E signaling.

The antidiabetic drug metformin has been associated with reduced colorectal cancer (CRC) risk and improved prognosis of CRC patients. However, the detailed mechanisms underlying such beneficial effects remain unknown. In this study, we aimed to evaluate metformin activity in CRC models and unveil the underlying molecular mechanisms. We showed that metformin inhibits CRC cell proliferation by arresting cells in the G1 phase of the cell cycle and dramatically reduces colony formation of CRC cells. We discovered that metformin causes a robust reduction of MYC protein level. Through the use of luciferase assay and coincubation with either protein synthesis or proteasome inhibitors, we demonstrated that regulation of MYC by metformin is independent of the proteasome and 3′ UTR‐mediated regulation, but depends on protein synthesis. Data from polysome profiling and ribopuromycylation assays showed that metformin induced widespread inhibition of protein synthesis. Repression of protein synthesis by metformin preferentially affects cell cycle‐associated proteins, by altering signaling through the mTOR‐4EBP‐eIF4E and MNK1‐eIF4G‐eIF4E axes. The inhibition of MYC protein synthesis may underlie metformin's beneficial effects on CRC risk and prognosis.

Physciosporin suppresses the proliferation, motility and tumourigenesis of colorectal cancer cells

BackgroundLichens, which represent symbiotic associations of fungi and algae, are potential sources of numerous natural products. Physciosporin (PHY) is a potent secondary metabolite found in lichens and was recently reported to inhibit the motility of lung cancer cells via novel mechanisms. PurposeThe present study investigated the anticancer potential of PHY on colorectal cancer (CRC) cells. MethodsPHY was isolated from lichen extract by preparative TLC. The effect of PHY on cell viability, motility and tumourigenicity was elucidated by MTT assay, hoechst staining, flow cytometric analysis, transwell invasion and migration assay, soft agar colony formation assay, Western blotting, qRT-PCR and PCR array in vitro as well as tumorigenicity study in vivo. ResultsPHY decreased the viability of various CRC cell lines (Caco2, CT26, DLD1, HCT116 and SW620). Moreover, PHY elicited cytotoxic effects by inducing apoptosis at toxic concentrations. At non-toxic concentrations, PHY dose-dependently suppressed the invasion, migration and colony formation of CRC cells. PHY inhibited the motility of CRC cells by suppressing epithelial-mesenchymal transition and downregulating actin-based motility markers. In addition, PHY downregulated β-catenin and its downstream target genes cyclin-D1 and c-Myc. Moreover, PHY modulated KAI1 C-terminal-interacting tetraspanin and KAI1 expression, and downregulated the downstream transcription factors c-jun and c-fos. Finally, PHY administration showed considerable bioavailability and effectively decreased the growth of CRC xenografts in mice without causing toxicity. ConclusionPHY suppresses the growth and motility of CRC cells via novel mechanisms.

MICAL2 Mediates p53 Ubiquitin Degradation through Oxidating p53 Methionine 40 and 160 and Promotes Colorectal Cancer Malignance

Molecule interacting with CasL2 (MICAL2), a microtubule-associated monooxygenase, is highly expressed in various cancers and is involved in cancer pathogenesis, but the mechanisms underlying its regulation in carcinogenesis are unclear. In this study, we aim to clarify the mechanism by which MICAL2 participates in colorectal cancer (CRC) and identify novel markers for predicting prognosis of CRC patients.Methods: The value of MICAL2 in CRC prognosis was determined by immunohistochemical analysis of a CRC biopsy array. A short hairpin RNA target MICAL2 (shMICAL2) was designed to knock down MICAL2 expression and observe MICAL2's function on CRC cell growth. mRNA expression array was used to screen target molecules of MICAL2. HCT116 p53+/+ and HCT116 p53-/- cells were used to confirm whether MICAL2 exerts its oncogenic effect through p53. The in vivo effect of MICAL2 on CRC growth was assessed by subcutaneously injecting MICAL2-knockout CRC cells into the dorsal flank of each mouse. Immunofluorescence was used to observe the effect of MICAL2 on p53 cellular location. Reverse-phase nano ESI-LCMS analysis was used to investigate if MICAL2 mediates p53 oxidation.Results: MICAL2 was found to be highly expressed in CRC tissues, and its expression was associated with CRC carcinogenesis and poor patient outcome. MICAL2-knockdown decreased growth and colony formation of CRC cells, which was linked with cell cycle arrest and apoptosis. MICAL2 physically interacted with p53 and retained p53 in the cytoplasm. MICAL2 shortened the half-life of p53, and ectopic MICAL2 expression decreased p53 protein stability through ubiquitin degradation. MICAL2 was also found to oxidize p53 at methionine 40 and 160, which mediated p53 ubiquitin degradation. MICAL2-promoted CRC growth in vivo was confirmed in nude mice.Conclusion: MICAL2 binds to p53, retains p53 in the cytoplasm and oxidizes it at Met 40 and 160, promotes p53 ubiquitination, and decreases p53 function. MICAL2-reduced p53 promotes CRC development.

Abstract 4507: Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer

Abstract Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor and as an inhibitor of the IGF-1 pathway. Recent data indicate klotho as a tumor suppressor in an array of malignancies, including colorectal cancer (CRC). We aimed to decipher the role of klotho as a tumor suppressor in CRC. Analysis of a public database (Oncomine) indicated reduced expression of klotho in CRC. Two models were employed to study the effect of klotho in vivo. Using the azoxymethane inflammation model, we discovered that klotho inhibited colon polyp formation. Using an orthotopic model consisting of direct injection of MC38 cells to the mice colons, we found that klotho inhibited colonic obstruction and tumor formation. Similarly, klotho inhibited colony formation and proliferation of CRC cell lines. Aberrant activation of the canonical Wnt pathway is implicated in CRC development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. While we did not see an effect of klotho on the IGF-1 pathway, it reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. Yet, the inhibitory effect of klotho on CRC cell colony formation was only partially rescued by a constitutively active β-catenin. These data indicate on additional mechanisms involved in the TS activity of klotho. Thus, we conducted an RNA expression array and observed involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Further studies showed that klotho induced elevation in XBP1 RNA levels, GRP78 protein levels, and eIF2α phosphorylation, all indicative of UPR regulation. Importantly, pharmacologic inhibition of ER stress and UPR overcame the growth suppression effect of klotho in CRC cells. Our data indicate klotho as a potent tumor suppressor in CRC, and suggest its role at very early stages of tumor formation, already at polyp formation. Our data also indicate, for the first time, klotho as a regulator of ER stress and UPR in cancer. These findings may pave the way for the development of novel therapeutic strategies based on enhancement of UPR. Citation Format: Tal Etan, Tammi Rubinstein, Tami Rubinek, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Arbesfeld, Iris Barshack, Ido Wolf. Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2017-4507

CUL4A promotes proliferation and metastasis of colorectal cancer cells by regulating H3K4 trimethylation in epithelial-mesenchymal transition.

Increasing evidence suggests that CUL4A, a ubiquitin ligase, is involved in the promotion of cancer malignancy and correlated with worse clinical prognosis in several kinds of human cancers. Although its effect and mechanism on the progression of colorectal cancer (CRC) remain unknown. Our clinical data show that CUL4A protein is overexpressed, positively associated with lymph nodes status, differentiation degree, tumor size, and poor prognosis in 80 CRC patients. CUL4A overexpression promotes cell proliferation and colony formation of CRC cells. Knockdown of CUL4A inhibits cell proliferation and migration. CUL4A can significantly promote the in vitro migration of CRC cells via induction of the epithelial–mesenchymal transition process. And the modulation of CUL4A expression altered the level of H3K4 trimethylation at the E-cadherin, N-cadherin, and vimentin gene promoters, which in turn transcriptionally regulated their expression. Moreover, knockdown of CUL4A also decreased the tumor volume and tumor weight in vivo. Together, our results reveal that CUL4A plays as an oncogene in CRC and may become a potential therapeutic target in the treatment of colorectal cancer.

Abstract 3679: Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response

Abstract Aberrant activation of the canonical Wnt pathway is implicated in colorectal cancer (CC) development and progression. β-catenin, the key effector of this pathway, 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 shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor (TS). Recent data indicate klotho as a TS in an array of malignancies, including CC. Among other activities, klotho inhibits the insulin-like growth factor (IGF)-1 and Wnt pathways. We aimed to decipher the role of klotho as a TS in CC. We first noted that klotho overexpression inhibited CC cell colony formation. Next, we examined in vivo effects using the azoxymethane mouse model, and found that klotho inhibited colon polyp formation. We then used an orthotopic model by injecting MC38 cells to mice colons, and found that klotho inhibited colonic obstruction and tumor formation. While we did not see an effect of klotho on the IGF-1 pathway, klotho did affect the Wnt pathway. Klotho reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we 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 CC cell colony formation was only partially rescued by a constitutively active β-catenin, additional mechanisms may be involved. Thus, we conducted a cDNA expression microarray and involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR) was observed. Further studies showed that klotho induced elevation in XBP1 RNA, GRP78 protein levels, and eIF2α phosphorylation, all indicative of a role klotho plays in UPR regulation. Our data indicate klotho as a potent TS in CC, and suggest its role at early stages of tumor formation. The Wnt pathway partially mediates klotho effects in CC, however additional mechanisms, including ER stress response regulation, are involved. Citation Format: Tami Rubinek, Tammi Rubinstein, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Absesfelf, Iris Barshack, Ido Wolf. Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3679.

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

Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells

IntroductionBcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.MethodsRT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.ResultsThe levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.ConclusionsTargeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors

Activation of Wnt signaling has been implicated in tumorigenesis, and epigenetic silencing of Wnt antagonist genes has been detected in various cancers. In the present study, we examined the expression and methylation of DICKKOPF (DKK) family genes in gastrointestinal cancer cell lines. We found that all known DKK genes were frequently silenced in colorectal cancer (CRC) cells (DKK1, 3/9, 33%; DKK2, 8/9, 89%; DKK3, 5/9, 56% and DKK4, 5/9, 56%), but not in normal colon mucosa. DKK1, -2 and -3 have 5' CpG islands, and show an inverse relation between expression and methylation. DKK methylation also was frequently observed in gastric cancer (GC) cell lines (DKK1, 6/16, 38%; DKK2, 15/16, 94% and DKK3, 10/16, 63%), but was seen less frequently in hepatocellular carcinoma and pancreatic cancer cell lines. DKKs also were frequently methylated in primary CRCs (DKK1, 7/58, 12%; DKK2, 45/58, 78% and DKK3, 12/58, 21%) and GCs (DKK1, 15/31, 48%; DKK2, 26/31, 84% and DKK3, 12/31, 39%). Against a background of CTNNB1 or APC mutations, Dickkopfs (Dkks) were less effective inhibitors of Wnt signaling than secreted frizzled-related proteins, though over-expression of Dkks suppressed colony formation of CRC cells with such mutations. Our results demonstrate that DKKs are frequent targets of epigenetic silencing in gastrointestinal tumors, and that loss of DKKs may facilitate tumorigenesis through beta-catenin/T-cell factor-independent mechanisms.