Differentiation Of Colon Cancer Cells Research Articles

Exosome-derived lnc-HOXB8-1:2 induces tumor-associated macrophage infiltration to promote neuroendocrine differentiated colorectal cancer progression by sponging hsa-miR-6825-5p

IntroductionNeuroendocrine differentiation (NED) in colorectal cancer (CRC) cells has been known for decades, and our previous meta-analysis indicated that CRC patients with neuroendocrine differentiation have a lower 5-year survival rate. In recent years, an increasing number of studies have found that exosome-derived long non-coding RNAs (lncRNAs) play important roles in cancer progression and metastasis. However, the functions and mechanism of exosome-derived lncRNAs in CRC with neuroendocrine differentiation are not yet fully clear.Materials and methodsThe clinical significance of NED was assessed in a retrospective study of 105 patients. Next-generation sequencing and bioinformatics analysis were conducted to select lnc-HOXB8-1:2 for further study. Using immunohistochemistry, qRT–PCR, western blot, transwell assay, immunofluorescence assay, fluorescence in situ hybridization assay and dual-luciferase reporter assay, the oncogenic role of exosome-derived lnc-HOXB8-1:2 was determined in CRC with NED. The mechanism underlying the lnc-HOXB8-1:2/hsa-miR-6825-5p/CXCR3 axis was also explored.ResultsNED was a risk factor for the progression and mortality of CRC. lnc-HOXB8-1:2, derived from exosomes secreted by neuroendocrine differentiated colon cancer cells, was identified in our study. The proportion of M2 macrophages and the migration and invasion capacities of tumor-associated macrophages (TAMs) markedly increased after the addition of neuroendocrine differentiated CRC cell-derived exosomes. More excitingly, the expression of lnc-HOXB8-1:2 and the protein level of CXCR3 were also upregulated in TAMs. The lnc-HOXB8-1:2/hsa-miR-6825-5p/CXCR3 axis was predicted via miRanda software and confirmed by the dual-luciferase reporter assay. Furthermore, the increased expression of lnc-HOXB8-1:2 was accompanied by downregulation of hsa-miR-6825-5p expression and upregulation of CXCR3 protein levels. Overexpression of hsa-miR-6825-5p also reduced CXCR3 expression.Conclusionlnc-HOXB8-1:2 in exosomes derived from neuroendocrine differentiated CRC cells acted as a ceRNA competitively binding hsa-miR-6825-5p to upregulate CXCR3 expression and leading to TAM infiltration and M2 polarization, which promotes neuroendocrine differentiated CRC progression.

Identification of Subtypes and a Prognostic Gene Signature in Colon Cancer Using Cell Differentiation Trajectories.

Research on the heterogeneity of colon cancer (CC) cells is limited. This study aimed to explore the CC cell differentiation trajectory and its clinical implication and to construct a prognostic risk scoring (RS) signature based on CC differentiation-related genes (CDRGs). Cell trajectory analysis was conducted on the GSE148345 dataset, and CDRG-based molecular subtypes were identified from the GSE39582 dataset. A CDRG-based prognostic RS signature was constructed using The Cancer Genome Atlas as the training set and GSE39582 as the validation set. Two subsets with distinct differentiation states, involving 40 hub CDRGs regulated by YY1 and EGR2, were identified by single-cell RNA sequencing data, of which subset I was related to hypoxia, metabolic disorders, and inflammation, and subset II was associated with immune responses and ferroptosis. The CDRG-based molecular subtypes could successfully predict the clinical outcomes of the patients, the tumor microenvironment status, the immune infiltration status, and the potential response to immunotherapy and chemotherapy. A nomogram integrating a five-CDRG-based RS signature and prognostic clinicopathological characteristics could successfully predict overall survival, with strong predictive performance and high accuracy. The study emphasizes the relevance of CC cell differentiation for predicting the prognosis and therapeutic response of patients to immunotherapy and chemotherapy and proposes a promising direction for CC treatment and clinical decision-making.

Colon cancer cell differentiation by sodium butyrate modulates metabolic plasticity of Caco-2 cells via alteration of phosphotransfer network.

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state.

PRMT1 inhibition induces differentiation of colon cancer cells

Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.

Discovery of 4-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline as a novel c-myc inhibitor against colorectal cancer in vitro and in vivo

Proto-oncogene c-Myc plays an essential role in the development of colorectal cancer (CRC), since downregulation of c-Myc inhibits intestinal polyposis, which is the most cardinal pathological change in the development of CRC. Herein, a series of novel phenoxy-N-phenylaniline derivatives were designed and synthesized. The cytotoxicity activities of all the derivatives were measured by MTT assay in different colon cancer cells, 4-(3,5-dimethoxy-4-(((4-methoxyphenethyl)amino)methyl)phenoxy)-N-phenylaniline (42) was discovered, the lead compound 42 with excellent cytotoxicity activity of IC50 = 0.32 μM, IC50 = 0.51 μM, in HT29 and HCT 15 cells, respectively. Compound 42 had a good inhibitory activity of c-Myc/MAX dimerization and DNA binding. Besides, compound 42 could effectively induce apoptosis and induced G2/M arrest in low concentration and G0/G1 arrest in high concentration to prevent the proliferation and differentiation in colon cancer cells. Western blot analysis confirmed the 42 strongly down-regulated expression of c-Myc. Furthermore, during 30 days treatment 42 exhibited excellent efficacy in HT29 tumor xenograft model without causing significant weight loss and toxicity. Consequently, 42 could be a promising drug candidate for CRC therapy.

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

Low-intensity pulsed ultrasound affects growth, differentiation, migration, and epithelial-to-mesenchymal transition of colorectal cancer cells.

Ultrasound (US) offers potentially important opportunities from a therapeutic point of view. Thus, the study of the biological effects of US on cancer cells is important to understand the consequences of these changes on the malignant phenotype. This study aimed to investigate the effects of low-intensity ultrasound (LIPUS) on the phenotype of colorectal cancer cell lines. Cell proliferation was evaluated by viability test and by evaluation of pERK expression, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity. Epithelial mesenchymal transition was assessed by analyzing the expression of Vimentin and E-Cadherin. Release and uptake of extracellular vesicles (EVs) were evaluated by flow cytometry. LIPUS effects on the organization of cytoskeleton were analyzed by confocal microscopy and by evaluation of Rho GTPase expression. No alterations in vitality and clonogenicity were observed when the intermediate (0.4 MPa) and the lowest (0.035 MPa) acoustic intensities were administered while the treatment with high intensity (1 MPa) induced a reduction of both cell viability and clonogenicity in both cell lines in a frequency-dependent manner. LIPUS promoted the differentiation of colon cancer cells, affected epithelial-to-mesenchymal transition, promoted the closure of a wound as well as increased the release of EVs compared with untreated cells. LIPUS-induced increase in cell motility was likely due to a Rho GTPase-dependent mechanism. Overall, the results obtained warrant further studies on the potential combined effect of LIPUS with differentiating agents and on their potential use in a clinical setting.

Abstract 2997: 15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1

Abstract Prostaglandin E2 (PGE2) is a crucial regulator of colon cancer (CC), an inflammation associated cancer. Tumor suppressor 15-hydroxyprostglandin dehydrogenase (15-PGDH) is a PGE2 degrading enzyme but is often downregulated in CC. Interestingly, CC-patients with high levels of Cysteinyl leukotriene receptor 2 (CysLT2R) exhibit good prognoses. Previously, we have reported differentiation promoting role of 15-PGDH in CC cells when induced by LTC4, the CysLT2R ligand. However the mechanism is unclear. Hedgehog (Hh) signalling is involved in embryogenesis and development but hyper activated in colon carcinogenesis. LTC4-induced activation of 15-PGDH downregulated both mRNA and protein expression of GLI1 in HT-29 and Caco-2 colon cancer cells. Moreover, absence of Gli1 significantly reduced the differentiation promoting ability of 15-PGDH, with no change in the differentiation markers Sucrase isomaltase, Mucin-2 and Protocadherin-24. In addition, no noticeable change in the mRNA expression of WNT-specific target AXIN-2 coupled with restoration of the proliferation marker cMYC was observed. In HT-29-derived colonospheres depletion of mRNA expression of CC-CSC markers ALDH1A1 and DCLK1 remained regulated by Gli1. Elevation in Mucin-2 expression in zebrafish xenograft with HT-29 was also noted to be Gli1 dependent. GLI1 and MUCIN-2 mRNA expression in normal mucosa and matched tumor in colon cancer patient also exhibited higher co-relation along with concomitant elevation in DCLK1 expression in tumor in compared to normal mucosa. Tumor microarray (TMA) with low expression of CysLT2R and 15-PGDH exhibited higher Gli1 expression with no trace of Mucin-2. Therefore, restoring the expression of 15-PGDH in CC through activation of CysLT2R signalling and targeting Hh-Gli signalling would open new therapeutic possibilities with minimal collateral damage to the normal tissue. Citation Format: Shakti R. Satapathy, Lubna M. Mehdawi, Roger Olsson, Anita Sjolander. 15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2997.

Bazedoxifene as a novel GP130 inhibitor for Colon Cancer therapy

BackgroundInterleukin-11 (IL-11), a dominant IL-6 family cytokine, is involved in tumorigenesis, tumor progression and differentiation in colon cancer cells. IL-11 signaling has been recently identified as a potential therapeutic target in colon cancer. Bazedoxifene, a third- generation selective estrogen modulator approved by the Food and Drug Administration (FDA), is a novel inhibitor of IL-11/GP130 signaling discovered by docking modeling.MethodsIn this study, the inhibition efficacy of bazedoxifene in colon cancer cells and its potential mechanism were investigated in vitro and in vivo by using MTT cell viability assay, BrdU cell proliferation assay, colony formation assay, wound-healing/cell migration assay, immunofluorescence, western blot assay and the mouse xenograft tumor model.ResultsBazedoxifene inhibits phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) and its nuclear translocation induced by IL-11 in colon cancer cells. It also inhibits p-STAT3 induced by IL-6 and IL-11 but not by OSM or STAT1 phosphorylation induced by INF-γ in human colon cancer cells. In addition, bazedoxifene can significantly inhibit phosphorylation of AKT and STAT3 downstream targets. Furthermore, bazedoxifene alone or together with oxaliplatin can significantly induce apoptosis, inhibit cell viability, cell colony formation and cell migration in colon cancer cells. Knock-down of IL-11R can reduce the sensitivity of colon cancer cells to bazedoxifene. IL-11 can reduce the efficacy of oxaliplatin-mediated inhibition of cell viability. Consistent with in vitro findings, bazedoxifene alone also attenuated HCT-15 xenograft tumor burden and reduced p-STAT3, p-AKT and p-ERK in vivo. Its combination with oxaliplatin attenuated DLD-1 xenograft tumor burden and reduced p-STAT3 in vivo.ConclusionsTaken together, these results support bazedoxifene as a novel and effective therapeutic agent targeting IL-11/GP130 signaling for human colorectal cancer therapy.

VEGFR2 regulates endothelial differentiation of colon cancer cells

BackgroundRecent studies suggested that cancer stem-like cells contribute to tumor vasculogenesis by differentiating into endothelial cells. However, such process is governed by still undefined mechanism.MethodsAt varying differentiation levels, three representative colon cancer cells were cultured in endothelial-inducing conditioned medium: human colon cancer cells HCT116 (HCT116) (poorly differentiated), SW480 (moderately differentiated), and HT29 (well differentiated). We tested for expression of endothelial markers (cluster of differentiation (CD) 31, CD34, and vascular endothelial (VE)-cadherin and their ability to form tube-like structures in 3D culture. We also observed VEGF secretion and expressions of endothelial markers and VEGFRs in HCT116 cells under hypoxia to simulate physiological conditions. In in vitro and in xenotransplantation experiments, VE growth factor receptor 2 (VEGFR2) antagonist SKLB1002 was used to test effect of VEGFR2 in endothelial differentiation of HCT116 cells. Expression levels of VEGFR2 and VE-cadherin were assessed by immunohistochemistry of human colon cancer tissues to evaluate clinicopathological significance of VEGFR2.ResultsAfter culturing in endothelial-inducing conditioned medium, poorly differentiated HCT116 cells expressed endothelial markers and formed tube-like structure in vitro. HCT116 cells secreted more endogenous VEGF and expressed higher VEGFR2 under hypoxia. SKLB1002 impaired endothelial differentiation in vitro and xenotransplantation experiments, suggesting a VEGFR2-dependent mechanism. Increased expression of VEGFR2 correlated with differentiation, metastasis/recurrence, and poor prognosis in 203 human colon cancer samples. Positive correlation was observed between VEGFR2 and VE-cadherin expression.ConclusionsVEGFR2 regulates endothelial differentiation of colon cancer cell and may be potential platform for anti-angiogenesis cancer therapy.

Abstract 1386: Effects of histone deacetylase inhibitors on differentiation markers and growth of colon and bladder cancer cells

Abstract Alkaline phosphatase and dipeptidyl peptidase are markers of differentiation in colon cancer cells. In colon cancer cells differentiation can often be induced by treatment with inhibitors of histone deacetylase (HDAC) activity. In some colon cancer cells butyrate causes a several-fold induction of alkaline phosphatase but only modest effects on dipeptidyl peptidase. There is relatively little information on changes in activity of cell surface hydrolases in bladder cancer. However, there is some evidence for decreased alkaline phosphatase activity in bladder cancer. We tested the hypothesis that there may be retention of activity in more slowly growing cancer cells and the activity will be regulated by HDAC inhibitors in a manner similar to that in colon cancer. In our initial studies we screened seven human bladder cancer cell lines for alkaline phosphatase activity. We identified three cell lines (5637, HT1197 and HT1376) with activity comparable to that in more differentiated colon cancer cell lines. The response to incubation with the HDAC inhibitors butyrate and valproate was compared with effects in three colon cancer cell lines Caco-2, HT29 and SW1116. In all six cell lines there were growth inhibitory effects that tended to be a little greater with butyrate than with valproate. Induction of alkaline phosphatase activity was generally greater with butyrate than with valproate and was apparent even without normalizing the activity on a protein basis. After determining the activity per unit protein there were increases in dipeptidyl peptidase activity after treatment with the HDAC inhibitors that were smaller than for alkaline phosphatase. Growth inhibition was observed with all the HDAC inhibitors examined including LMK235, nicotinamide, RGFP966, romidepsin and tubacin but increased activity of alkaline phosphatase was not always seen. Activities of the enzymes were very low in CRL1790 human colonocytes of fetal origin and there was little or no induction with HDAC inhibitors. Although pyruvate and carnitine have been reported to be HDAC inhibitors, incubation with colon and bladder cancer cells did not cause induction of alkaline phosphatase activity. It was concluded that regulation of alkaline phosphatase and dipeptidyl peptidase by HDAC inhibitors can be similar in bladder and colon cancer cells. Citation Format: Michael A. Lea, Lauren Cué, Elizabeth Batista, Erik Lew, Charles desBordes. Effects of histone deacetylase inhibitors on differentiation markers and growth of colon and bladder cancer cells [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 1386. doi:10.1158/1538-7445.AM2017-1386

Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system,whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells.

A potential anti-tumor effect of leukotriene C4 through the induction of 15-hydroxyprostaglandin dehydrogenase expression in colon cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2, which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2, and is often down-regulated in cancer. Interestingly, CRC patients expressing high levels of the cysteinyl leukotriene 2 (CysLT2) receptor have a good prognosis; therefore, we investigated a potential link between CysLT2 signaling and the tumor suppressor 15-PGDH in colon cancer cells.We observed a significant up-regulation of 15-PGDH after treatment with LTC4, a CysLT2 ligand, in colon cancer cells at both the mRNA and protein levels, which could be reduced by a CysLT2 antagonist or a JNK inhibitor. LTC4 induced 15-PGDH promoter activity via JNK/AP-1 phosphorylation. Furthermore, we also observed that LTC4, via the CysLT2/JNK signaling pathway, increased the expression of the differentiation markers sucrase-isomaltase and mucin-2 in colon cancer cells and that down-regulation of 15-PGDH totally abolished the observed increase in these markers.In conclusion, the restoration of 15-PGDH expression through CysLT2 signaling promotes the differentiation of colon cancer cells, indicating an anti-tumor effect of CysLT2 signaling.

Decreased expression of MS4A12 inhibits differentiation and predicts early stage survival in colon cancer.

Lack of normal differentiation was considered as a common defect in cancer cells. MS4A12, a colon-specific gene, belongs to MS4A family that plays an important role in differentiation, proliferation and cell cycle regulation. The aim of the study was to investigate MS4A12 role in colon cancer cell differentiation and its prognostic value in colon cancer. We used sodium butyrate (BS) to set up differentiated model of colon cancer cell line LoVo. Cell differentiation was evaluated with ALP activity and E-cadherin. We used BS (4 mmol/L) inducing differentiation of LoVo cell and found after BS treated over 48h MS4A12 variant-1 (one of MS4A12 gene transcripts) as well as ALP and E-cadherin of LoVo cells were all increased significantly. When silence MS4A12 variant-1, the elevation of ALP and E-cadherin in BS-treated cells were all inhibited. Besides, after silence MS4A12 variant-1, the cells showed significant resistances to BS function of induction cell cycle arrest and apoptosis. Survival analysis used GEO datasets GSE39582 and GSE38832 that include 681 distinct colon cancer samples. Log-rank test and Cox's proportional hazards regression were applied to analyzing single and multiple prognostic variables, respectively. In early stage colon cancer, the patients with low MS4A12 expression had a poor survival (HR=1.72; p=0.036), while in advanced stage colon cancer MS412 had little prognostic value (HR=0.89; p=0.601). These results indicated MS4A12 might relate to colon cancer cell differentiation and supposed to be a risk classification marker for early stage colon cancer.

Non-canonical WNT5A signaling up-regulates the expression of the tumor suppressor 15-PGDH and induces differentiation of colon cancer cells

The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in prostaglandin E2 catabolism and is down-regulated in colorectal cancer (CRC) tissue. Canonical Wnt signaling is frequently elevated in colon cancers and has been shown to down-regulate 15-PGDH expression. Therefore, we have in the current study investigated if the non-canonical ligand WNT5A relates to increased expression of 15-PGDH in colon cancer cells. In the same cohort of patients, we demonstrated a parallel and significant loss of 15-PGDH and WNT5A protein expression in CRC tissues compared with matched normal colon tissues. Furthermore, patients with low 15-PGDH/WNT5A expression in their tumors showed reduced survival compared with patients with high 15-PGDH/WNT5A expression. To investigate if WNT5A signaling directly affects 15-PGDH expression, we performed in vitro analyses of colon cancer cells (HT-29 and Caco-2). Both cell lines, when treated with recombinant WNT5A (rWNT5A) or Foxy-5, a WNT5A-mimicking peptide, responded by increasing their expression of 15-PGDH mRNA and protein. Our investigations showed that rWNT5A and Foxy-5 induced this increased expression of 15-PGDH through reduced β-catenin signaling as well as increased JNK/AP-1 signaling in colon cancer cells. WNT5A signaling also induced increased 15-PGDH expression in a breast cancer cell line both in vitro and in vivo. In agreement, WNT5A signaling also increased the expression of the differentiation markers sucrose-isomaltase and mucin-2 in colon cancer cells. Our results show that WNT5A signaling regulates 15-PGDH expression, thus uncovering a novel mechanism by which WNT5A acts as a tumor suppressor and suggests that increased 15-PGDH expression could be used as an indicator of a positive response to Foxy-5 in patients treated with this WNT5A agonist.

FAM172A modulates apoptosis and proliferation of colon cancer cells via STAT1 binding to its promoter.

In our previous study, low expression of FAM172A protein was found in colon cancer tissues. This research was planned to explore the functions of FAM172A gene and examine the mechanisms of its transcriptional regulation. Firstly, flow cytometry showed that FAM172A inhibited proliferation and promoted apoptosis and differentiation of colon cancer cells. Then through continuous truncation, we identified the minimal functional promoter region of FAM172A. Subsequently, we found that STAT1, as a transcription factor, could bind to the minimal FAM172A promoter, as evaluated using Chromatin immunoprecipitation (ChIP) and Electrophoreticmobility shift assay (EMSA). The results of Western blot analysis and qRT-PCR indicated that STAT1 was able to upregulate the expression of FAM172A. Our results showed that FAM172A could suppress proliferation of colon cancer cells, and STAT1 could bind to the minimum promoter region of FAM172A and upregulated the expression of FAM172A. These results may provide advanced insights into the functions of FAM172A and its regulatory mechanisms.

Abstract 3363: Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3)

Abstract In human colon cancer cells, glycolytic activity shows a positive correlation with proliferation. PFKFB3 is an important enzyme regulating glycolysis in many tumor cells. We have studied the action of an established inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation in four colon cancer cell lines (Caco-2, HCT116, HT29 and SW1116). Glucose uptake and cell proliferation were inhibited by 3PO and butyrate and additive effects were seen with 10 µM 3PO and 1 mM butyrate. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The large induction of alkaline phosphatase activity by butyrate seen in most colon cancer cell lines was not observed with 3PO. Similar contrasting effects on induction of alkaline phosphatase activity were seen with rat Morris hepatoma 7777 cells where 2-3 fold induction was seen with 1 µM vorinostat or 1 mM butyrate but no significant effect was seen with 3PO in the range of 5-30 µM. The biguanides, metformin and phenformin, increase glycolysis but inhibit proliferation of colon cancer cells. After combination treatment with 3PO, glycolysis was inhibited, as judged by glucose uptake and acidification of the medium, and additive inhibitory effects on cell proliferation could be observed. For example, after a 72 hour incubation of Caco-2 cells with 10 µM 3PO, 25 µM phenformin or 1 mM metformin, as single agents, proliferation relative to controls was decreased 20%, 17% and 34%, respectively. For combinations of 3PO and phenformin or 3PO and metformin the decreases relative to controls were 48% and 58%, respectively. In conclusion, 3PO was an inhibitor of proliferation of colon cancer cells, especially the rapidly growing HCT116 and HT29 cell lines. 3PO may be a useful agent in combination with other drugs that inhibit colon cancer cell proliferation. Citation Format: Michael A. Lea, Raymond Geherty, Rachelle David, Charles desBordes. Inhibition of glycolysis and proliferation of colon cancer cells by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) an inhibitor of 6-phosphofructo-2-kinase (PFKFB3). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2014-3363

Abstract 2339: Transcriptional basis for loss of cellular differentiation in colon cancer

Abstract Background: Colon cancer is a leading cause of cancer related death. Colon cancer arises due to uncontrolled cell proliferation, failure of cells to undergo apoptosis and loss of cellular differentiation. While the pathways which drive the uncontrolled proliferation of colon cancer cells are relatively well understood, the mechanistic basis for loss of cell differentiation is less well characterized. The aim of this study was to identify novel transcription factors that regulate the differentiation status of colon cancer cells. Methods: Affymetrix microarrays were used to identify transcription factors which are significantly differentially expressed between well differentiated and poorly differentiated colon cancer cell lines. Differential gene expression was confirmed by q-RT-PCR in 40 colon cancer cell lines. The well differentiated colon cancer cell line SW948 was transfected with siRNAs using lipofectamine. Results: Microarray analysis of well and poorly differentiated colon cancer cell lines identified the EHF transcription factor to be significantly downregulated in expression in poorly differentiated cell lines. Q-RT-PCR analysis demonstrated that while both EHF variants were expressed in a panel of 40 colon cancer cells, variant 2 was the isoform differentially expressed between well and poorly differentiated cell lines. Basal expression of EHF correlated significantly with expression of the differentiation markers villin and KRT20 across the 40 cell line panel. Knockdown of EHF in the well differentiated colon cancer cell line SW948, reduced expression of villin and KRT20 by 82% and 64% respectively. Knockdown of EHF also reduces expression of Cdx1 suggesting it may be act upstream of this known differentiation regulator. Conclusion: This study identifies the transcription factor EHF as a novel regulator of cell differentiation in colon cancer. Citation Format: Ian Y. Luk, Hoanh Tran, Janson WT Tse, Anderly C. Chueh, Fiona Chionh, Nicholas J. Clemons, John M. Mariadason. Transcriptional basis for loss of cellular differentiation in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2014-2339

The Intestinal Epithelial Cell Differentiation Marker Intestinal Alkaline Phosphatase (ALPi) Is Selectively Induced by Histone Deacetylase Inhibitors (HDACi) in Colon Cancer Cells in a Kruppel-like Factor 5 (KLF5)-dependent Manner

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity.

Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF(V637E) knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.