Differentiation Of Colon Cancer Cells Research Articles

Cell differentiation along multiple pathways accompanied by changes in histone acetylation status

Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4.

Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1

Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca2+ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca2+ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca2+ level after activation; time to reach peak cytosolic free Ca2+ and the EC50 of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca2+ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca2+ signaling.

The cysteinyl leukotriene 2 receptor contributes to all-transretinoic acid-induced differentiation of colon cancer cells

BackgroundCysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT1R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT2R) is lost. Further, our previous data indicate that patients with high CysLT1R and low CysLT2R expression have a poor prognosis. In this study, we examined whether the balance between CysLT1R and CysLT2R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA).MethodsTo determine the effect of ATRA on CysLT2R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions.ResultsATRA treatment induces CysLT2R mRNA and protein expression without affecting CysLT1R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT2R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT2R-specific inhibitor.ConclusionsThis study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.

Abstract 2991: Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). .

Abstract The HDAC-inhibitor sodium-butyrate, promotes differentiation of colon cancer cells as evidenced by induction of alkaline phosphatase (ALP) enzyme activity. Screening of a panel of 34 colon cancer cell lines identified cell lines responsive (44%) and refractory (56%) to butyrate-induced differentiation. Refractory cell lines were significantly enriched for those harbouring the CpG island methylator phenotype (CIMP) (P=0.016). The ALP gene family comprises 4 members - ALPi, ALPL, ALPP and ALPPL2 - each of which was induced by butyrate in responsive cell lines, demonstrating co-ordinate regulation of this gene family in response to HDACi treatment. Deletion analyses of the ALPi promoter identified a KLF/Sp1 cis element essential for butyrate-induction of ALPi promoter activity. Remarkably, we observed that a CpG dinucleotide within this site was preferentially methylated in refractory cell lines. Furthermore, the majority of cell lines with methylation of this site in the ALPi promoter also harboured methylation of similar sites within the ALPL and ALPP promoters, indicating coordinate methylation of this gene family in refractory cell lines. Frequent methylation and reduced expression of ALPi and ALPL were also observed in primary human colon cancers. Targeted inactivation of the DNA methyltransferases Dnmt1 and Dnmt3b (HCT116-DKO) and co-treatment with decitabine restored butyrate-inducibility of ALP in the methylated HCT116 cell line. However, butyrate-induction of an exogenous ALPi promoter-reporter paralleled induction of endogenous ALPi mRNA across the cell lines, suggesting ALPi promoter methylation may not be the primary determinant of ALP inducibility. We therefore explored whether the differential induction of the ALP gene family in colon cancer cells was due to selective induction of a common transcriptional regulator. To address this, responsive and refractory cell lines were treated with butyrate for 72h and transcription factors selectively induced in responsive cell lines identified by microarray. By focussing on factors which bind KLF/Sp1 regulatory elements, we identified KLF5 as selectively induced in responsive cell lines. KLF5 was also preferentially induced by butyrate in HCT116 DKO cells. Importantly, knockdown of KLF5 markedly attenuated butyrate-induction of ALP mRNA expression and enzyme activity. These findings demonstrate that the ALP gene family is co-ordinately inducible by butyrate in select colon cancer cell lines, in a KLF5-dependent manner. Cells lines refractory to butyrate-induction of the ALP gene family were enriched for CIMP high lines, and displayed coordinate promoter methylation. We propose that the coordinate methylation of this gene family in CIMP high colon cancers is likely linked to their coordinate regulation. Citation Format: John Mariadason, Azadeh Carr, Georgia A. Corner, Sheren Al-Obaidi, Anderly Chueh, Fiona Chionh, Lars Tögel, Naseem Ahmed, Diego Arango, Daniel Buchanan, Joanne Young, Leonard H. Augenlicht, Madhu S. Malo, Richard Hodin, Oliver M. Sieber, Thomas Weber, Joongho Shin. Coordinate methylation of the alkaline phosphatase gene family in colon cancer. Implications for understanding the manifestation of the CpG island methylator phenotype (CIMP). . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2991. doi:10.1158/1538-7445.AM2013-2991

Abstract 1855: Regulation of proliferation of colon cancer cells by compounds affecting glycolysis including 3-bromopyruvate, 2-deoxyglucose and biguanides.

Abstract In earlier work we found that incubation of colon cancer cells with 2-deoxyglucose had an additive inhibitory effect on proliferation with the biguanides, metformin and phenformin, and blocked the acidification of the medium caused by incubation of cells with biguanides. In the present work, studies were performed with Caco-2, HCT116, HT29 and SW1116 colon cancer cells. Metabolism of glucose and acidification of the medium was greater with the more rapidly proliferating cells being greatest with HCT116 cells and least with SW1116 cells. We have seen that, as with 2-deoxyglucose, 3-bromopyruvate can also prevent the lowering of medium pH caused by biguanide treatment. . Inhibition of glucose metabolism paralleled the decrease in acidification of the medium. 3-bromopyruvate inhibited colon cancer cell proliferation. Additive effects with metformin and 3-bromopyruvate were not observed in any of the four colon cancer cell lines but some additive effects were seen with phenformin and 3-bromopyruvate. An additive effect was more notable in combined treatment with 3-bromopyruvate and 2-deoxyglucose. Induction of alkaline phosphatase activity serves as a marker of differentiation in colon cancer cells. The induction of alkaline phosphatase activity by butyrate was not consistently affected by combination with other agents that modified glucose metabolism. The drug combinations that were examined inhibited proliferation in wild type and P53 null HCT116 cells and affected colon cancer lines with different growth rates. Although metformin and phenformin increase glucose metabolism by colon cancer cells while 3-bromopyruvate and 2-deoxyglucose inhibit glucose metabolism, all these compounds have potential inhibitory action on the proliferation of colon cancer cells. Citation Format: Michael A. Lea, Mehreen Qureshi, Michael Buxhoeveden, Nicolette Gengel, Jessica Kleinschmit, Charles desBordes. Regulation of proliferation of colon cancer cells by compounds affecting glycolysis including 3-bromopyruvate, 2-deoxyglucose and biguanides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1855. doi:10.1158/1538-7445.AM2013-1855

FTIR microspectroscopic analysis of sodium butyrate induced differentiation in colon cancer cells in a time‐dependent manner

Butyric acid is a product of dietary fiber fermentation in the colon. Previous studies showed that this fatty acid inhibited cell proliferation and caused cell differentiation and apoptosis. It has been known that there is a relation between the tumorigenesis of colon cancer and the poor differentiation of colonic epithelial cells. Although the effects of butyrate on cellular differentiation has been documented at molecular level, the butyrate‐induced changes in the content of cancer cell macromolecules such as saturated, unsaturated lipids, protein and glycogen are less understood. The objective of this study was to clarify these changes in colon cancer cells in a time dependent manner using Fourier Transform Infrared (FTIR) microscopy. In this study, CaCo‐2 cells were treated with 3mM sodium butyrate (NaB) and cultured for 12, 24 and 48h. The macromolecular structural and function alterations induced by NaB were determined from spectral analysis of chemical maps of the studied groups. An increase in saturated and unsaturated lipid and a decrease in the triglyceride, and protein content has been obtained in 48 h treated group with respect to the other groups. The variation in membrane fluidity and lipid order has been also determined for this treated group. This study demonstrated that FTIR imaging enables to determine the colon cancer differentiation at the cellular level. This work is supported by METU‐BAP 07.02.2012.004.

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier.

Characterization of Sodium Butyrate Induced Differentiation in Colon Cancer Cells by Fourier Transform Infrared Spectroscopy and Microscopy

Colon cancer is a major cause of morbidity and mortality throughout the world.The pathogenesis of colon cancer with respect to loss of cellular differentiation has not been clearly understood. To understand this loss in colon cancer and to differentiate the cancer cells, sodium butyrate (NaB), one of the differentiation inducer, is commonly used. NaB is produced in the colonic lumen as a consequence of bacterial fermentation of complex carbohydrates and it alters colon cancer cell morphology and reduces the cell growth and motility. Although, the differentiation process of colon cancers, which are stimulated with NaB, has been resolved at genetic level, the underlying mechanisms regarding structural and functional alterations regulating the differentiation of colon cancer cells have not been well characterized. Therefore, in this study, it was aimed to explore the NaB-induced macromolecular structural and functional changes in differentiation of colon cancer cells by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and FTIR microspectroscopy. These changes were determined from the spectral analysis of control and NaB treated colon cancer cell spectra and their chemical maps. Based on the spectral differences, cluster analysis was applied to discriminate the groups. The spectral analysis indicated the differences in saturated and unsaturated lipids, protein and nucleic acid content between the control and NaB treated cancer cells. The variation in membrane fluidity and lipid order was also determined in the NaB treated cells. Moreover, the successful discrimination between control and treated cells was obtained. The results of this study not only shed light on better understanding the loss of differentiation mechanisms in colon cancers but also strongly support the power of ATR-FTIR spectroscopy and FTIR microscopy as novel, simple, reagent-free methods for the identification of differentiated cancer cells.

STK31 Maintains the Undifferentiated State of Colon Cancer Cells

The expression of serine/threonine kinase (STK) family is frequently altered in human cancers. However, the functions of these kinases in cancer development remain elusive. Here, we report that STK31 is robustly and heterogeneously expressed in colon cancer tissues and plays a critical role in determining the differentiation state of colon cancer cells. Knockdown or overexpression of STK31 induced or inhibited differentiation of colon cancer cells, respectively. Deletion of the STK domain abolished the inhibiting effect of STK31. Associated with differentiation, knockdown of STK31 resulted in significant suppression of tumorigenicity both in vitro and in vivo. Genome microarray analysis showed that knockdown of STK31 altered the expression profile of genes that are known to be involved in germ cell and cancer differentiation. Taken together, these results suggest that STK31 is able to control the differentiation state of colon cancer cells, which critically depends on its STK domain. The present findings may shed light on the new therapeutic approach against cancer by targeting STK31 and cancer differentiation.

Abstract B4: Everolimus inhibits invasion and angiogenesis in a KRAS-mutated mesenchymal-differentiated colorectal cancer xenografts

Abstract Background: KRAS mutations accounting for 32% to 40% of colorectal cancers and epithelial-to-mesenchymal transition (EMT) have been associated with resistance to targeted therapies in colorectal carcinomas. The growth factor receptor- and/or oncogenic KRAS-dependent AKT-activations may lead mTOR to promote the expression of transcription factors involved in EMT activation in cancer cells. mTOR also plays an important role in survival of cancer cells in response to hypoxia during tumor angiogenesis. In this study, we evaluated the effects of everolimus on KRAS-mutated mesenchymal differentiated colon cancer cells in culture and in xenografts. Methods: The cellular and signaling effects of everolimus was investigated in parental COLO205-S cells (WT KRAS) that display an epithelial phenotype and their derived KRAS mutated (G13D/exon 2) mesenchymal counterparts, COLO205-R cells (Serova, 2010) using MTT assay and Western Blot. COLO205-S and COLO205-R were engrafted in nude mice treated with placebo or 10mg/kg everolimus for 3 weeks. Tumor invasion in mice was evaluated by counting tumor cells invading the skin. Angiogenesis and necrosis were evaluated using CD31 and HE staining, respectively. Results: Acquisition of KRAS mutation and EMT in COLO205-R cells was associated with constitutive AKT activation and resistance to several targeted therapies including lapatinib, and enzastaurin. Inhibition of mTOR with everolimus decreased the proliferation in COLO205-S and COLO205-R cells at high concentrations. KRAS mutation and EMT in COLO205-R tumors were associated with an increased tumor growth rate compared to COLO205-S tumors (mean tumor volume after 17 days of treatment: 1650mm3 vs. 953mm3, respectively, p<0.05). Pathological examinations also showed that COLO205-R cells were more invasive (number of invading cells in surrounding tumor tissues: 321 vs. 202) and more angiogenic than COLO205-S cells in tumors. In COLO205-S and COLO205-R xenografts everolimus significantly reduced tumor growth in nude mice compared to placebo (mean tumor growth inhibition: 35% and 34%, respectively). Everolimus also decreases the invasive potential of cancer cells in tissues surrounding the tumors in COLO205-R xenografts (number of invading cells per field: 63 vs. 321). The number and the size of vessels were significantly reduced (>2-fold) in everolimus treated tumors, suggesting antiangiogenic properties of this mTOR inhibitor. In addition, everolimus also induced larger necrosis area in tumors as compared with placebo (in COLO205-R xenografts: 59% vs. 28%, respectively, p<0.05; in COLO205-S xenografts: 31% vs 15%, respectively, p<0.05). Conclusions: Everolimus displayed effects on cancer cell proliferation and invasion and may also inhibit tumor angiogenesis in KRAS-mutated mesenchymal-differentiated colorectal cancer models. This suggested that odds antiangiogenic effects might be expected using everolimus despite adverse genetic and differentiation profiles of colon cancer cells.

Abstract 1334: Overexpression of claudin-1 induces Notch and Wnt signaling to regulate colon carcinogenesis

Abstract Under physiological conditions, colonic homeostasis is a carefully balanced interplay between stem cells, their progeny, differentiation and cell fate determination. Dysregulation of this harmony signifies susceptibility to neoplastic growth. Colonic epithelium undergoes continuous proliferation, differentiation, and apoptosis along the crypt (crypt bottom to top). Notch signaling is a key regulator of the absorptive/secretory lineage determination. Dysregulation of notch signaling disrupts the normal colonic differentiation and depletes the goblet cell population while immature colonocyte population increases. Importantly, emerging literature suggests that Notch- signaling helps regulate colon cancer growth and progression in association with Wnt signaling. We have previously demonstrated that claudin-1 expression is highly increased in colon cancer and is causally associated with the tumorigenic and metastatic abilities of colon cancer cells. We also reported an inverse relationship between claudin-1 expression and differentiation in colon cancer cells. Here, we show a significant decrease in the goblet cell population (as determined by PAS and Alcian Blue) and Muc-2 expression accompanied with an increase in Carbonic Anhydrase I (CAI) expression, a commonly used marker for colonocytes in Villin-claudin-1 transgenic (Cl-1TG) mice that overexpress claudin-1 in the intestinal epithelium. Further analysis to detect Ki-67 expression suggested increase in colonic proliferation in Cl-1TG mice vs. WT mice. Taken together, these data suggest a role for claudin-1 in the regulation of colonic epithelial differentiation. To further determine the role of claudin-1 overexpression in cancer progression, we crossed Cl-1TG mice with APCmin mice, most widely used mouse model of colon cancer. Notably, APCmin mice develop few colonic tumors (mean=0.8) and have a life span of ∼22.5-23 weeks. Importantly, we noted a ∼4fold increase in the colonic tumor burden in (mean=3.8 tumors) and decrease in life span in APCmin/Cl-1 mice vs. APCmin mice. Also, tumors in APCmin/Cl-1 mice were larger, exhibited high grade of dysplasia and increased nuclear β-catenin (activated Wnt signaling) compared to tumors from APCmin mice. Our further studies using SW480claudin-1 cells {SW480 cells stably overexpressing claudin-1} and SW620siRNA cells {SW620 cells with stable inhibition of claudin-1 expression} demonstrated activation of Notch and Wnt signaling in SW480 cells (APCmutant) overexpressing claudin-1 while inhibited on inhibition of claudin-1. Taken together, our data supported a novel role of claudin-1 in the regulation of Notch and Wnt-signaling and thus colon carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1334. doi:1538-7445.AM2012-1334

Abstract 5439: Inhibition of growth and induction of differentiation of colon cancer cells by extracts from okra (Abelmoschus esculentus) and drumstick (Moringa oleifera)

Abstract In previous studies we have observed that several polyphenolic molecules can inhibit growth and induce the synthesis of differentiation markers in colon cancer cells. In the present work we have sought to determine if similar activity can be observed with methanolic and ethanolic extracts from okra and drumstick. In okra, phenolic molecules and antioxidant activity were associated primarily with the seeds, whereas in drumstick the seeds had lower concentrations of phenolics and antioxidant activity than skin, pulp and seed coat. In studies with okra extracts we have concentrated on the effects of seed extracts because they were generally most inhibitory for cell proliferation as judged by protein yield, thymidine incorporation into DNA and tetrazolium salt reduction. With extracts from drumstick the greatest inhibition of growth was generally with seed coat extracts but anomalous results were obtained in assays of tetrazolium salt reduction. Inhibition was greater at low concentrations but at high concentrations the apparent effect was reversed. Studies in the absence of cells showed that a component of the drumstick seed-coat extract reacted directly to reduce the tetrazolium salt. Comparison with other parts of the drumstick indicated that this reduction was much greater with the seed coat extract. Small effects on colon cancer cell differentiation have been seen with extracts of okra seed and with pulp and seed extract from drumstick as judged by induction of alkaline phosphatase activity. In the case of okra seed extracts this effect was additive with the action of butyrate. Okra and drumstick have had a role in traditional medicine and the present data are encouraging for further studies on potential cancer chemopreventive action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5439. doi:1538-7445.AM2012-5439

Abstract LB-21: Regulation of Bmi1 by β-catenin and KLF4 and its implication in colon cancer

Abstract B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein that is important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and development of nervous systems. Recently, great attention is drawn upon the role of Bmi1 in stem cell regulation and cancer. It was reported that Bmi1 is a potential marker for the intestinal stem cells. Since Wnt signaling plays a key role in the intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression in colon cancer cell lines. Krüppel-like factor 4 (KLF4) is a zinc finger protein that is highly expressed in the gut and skin. We recently found that KLF4 crosstalks with Wnt/[[Unsupported Character - Symbol Font ]]-catenin in regulating intestinal homeostasis. In addition, KLF4 is one of the four factors that induce pluripotent stem cells, thus playing a crucial role in stem cell regulation. In this study, the effects of KLF4 on Bmi1 expression were analyzed as well. These studies will provide novel insights into the mechanisms of Bmi1 function and regulation in the intestinal stem cells and colorectal cancers. Since Bmi1 is a stem cell or progenitor cell marker, we analyzed Bmi1 expression in human colorectal cancers tissues by IHC. Bmi1 levels are significantly increased in the tumor tissues from colon cancer patients compared with adjacent normal tissues; and expression of Bmi1 is significantly associated with nuclear [[Unsupported Character - Symbol Font ]]-catenin. Bmi1 knockdown by lentivirus-mediated Bmi1-shRNA repressed growth of different colon cancer cell lines, inhibited xenograft tumor growth, and increased differentiation of colon cancer cells in mouse xenograft. We found that dominant negative TCF (DnTCF), which blocks Wnt signaling, only marginally inhibits Bmi1 expression. However, both Western blot and RT-PCR results demonstrated that the expression of Bmi1 was inhibited by KLF4 in LS174T colon cancer cells. Using luciferase reporter assay, we found that repression of Bmi1 promoter by KLF4 is independent of c-myc. Using ChIP assay, we confirmed that KLF4 binds the endogenous Bmi1 promoter in colon cancer cells. Finally, we found that KLF4 inhibits Bmi1-mediated histone ubiquitination but by regulating more than one protein in the Polycomb complex. We found that Bmi1 is required for colon cancer cell proliferation and tumor growth, and it is up-regulated in colon cancer. Wnt signaling weakly regulates Bmi1; KLF4 inhibits Bmi1 by directly binding to Bmi1 promoter, thus inhibiting Bmi1-mediated H2A ubiquitination. It is of great interest to further study the mechanisms of Bmi1 regulation and the function of Bmi1 in normal intestine and in colon tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-21. doi:1538-7445.AM2012-LB-21

Differential staining of γ synuclein in poorly differentiated compared to highly differentiated colon cancer cells

Synuclein α, β and γ are proteins usually found in neurodegenerative diseases. However, interestingly synucleins are expressed in cancer cells of several organs including ovary, mammary gland and colon. By immunocytochemistry using specific antibodies to γ synuclein (SNCG), we examined the distribution of this protein in poorly differentiated, compared to highly differentiated colon cancer cells. In poorly differentiated cancer cells tumors were very frequently stained intensely with antibodies to SNGG, suggesting high expression of this protein. In contrast, in highly differentiated cells, there was no labeling. Labeled cells could be found only at the edges or in between the lobules of the differentiated tumor cells. However, in moderately differentiated tumors, a weak cytoplasmic staining of SNCG was evident. Interestingly in cancer patients (stage II-IV) both poorly and highly differentiated tumor cells were often present in the same patient. Labeled cancer cells with SNCG were evident also in lymph nodes, around the wall of blood vessels and in fat tissue, where only poorly differentiated cancer cells were exclusively present. Since cancer cells with poor differentiation are believed to be aggressive with metastases formation it is suggested that SNCG can serve as a marker for the potential of the tumor cell for the rapid spreading and metastazing of the non-differentiated tumors.

Brush border myosin Ia has tumor suppressor activity in the intestine.

The loss of the epithelial architecture and cell polarity/differentiation is known to be important during the tumorigenic process. Here we demonstrate that the brush border protein Myosin Ia (MYO1A) is important for polarization and differentiation of colon cancer cells and is frequently inactivated in colorectal tumors by genetic and epigenetic mechanisms. MYO1A frame-shift mutations were observed in 32% (37 of 116) of the colorectal tumors with microsatellite instability analyzed, and evidence of promoter methylation was observed in a significant proportion of colon cancer cell lines and primary colorectal tumors. The loss of polarization/differentiation resulting from MYO1A inactivation is associated with higher tumor growth in soft agar and in a xenograft model. In addition, the progression of genetically and carcinogen-initiated intestinal tumors was significantly accelerated in Myo1a knockout mice compared with Myo1a wild-type animals. Moreover, MYO1A tumor expression was found to be an independent prognostic factor for colorectal cancer patients. Patients with low MYO1A tumor protein levels had significantly shorter disease-free and overall survival compared with patients with high tumoral MYO1A (logrank test P = 0.004 and P = 0.009, respectively). The median time-to-disease recurrence in patients with low MYO1A was 1 y, compared with >9 y in the group of patients with high MYO1A. These results identify MYO1A as a unique tumor-suppressor gene in colorectal cancer and demonstrate that the loss of structural brush border proteins involved in cell polarity are important for tumor development.

The Indolic Diet-Derivative, 3,3′-Diindolylmethane, Induced Apoptosis in Human Colon Cancer Cells through Upregulation of NDRG1

N-myc downstream regulated gene-1 participates in carcinogenesis, angiogenesis, metastases, and anticancer drug resistance. In the present study, we analyzed the expression pattern of N-myc downstream regulated gene-1 following treatment of human colonic cancer cell lines; HCT-116 (well differentiated with wild-type p53 gene) and Colo-320 (poorly differentiated with mutant p53 gene), with 3,3′-diindolylmethane, a well-established proapoptotic agent product derived from indole-3-carbinol. Treatment of Colo-320 and HCT-116 with 3,3′-diindolylmethane disclosed inhibition of cell viability in a dose-dependent manner, mediated through apoptosis induction. The increased expression of N-myc downstream regulated gene-1 was detected only in poorly differentiated colon cancer cells, Colo-320 cell line. Our results suggest that N-myc downstream regulated gene-1 expression is enhanced by 3,3′-diindolylmethane in poorly differentiated cells and followed by induction of apoptosis. 3,3′-diindolylmethane induced apoptosis may represent a new regulator of N-myc downstream regulated gene-1 in poorly differentiated colonic cancer cells.

Increased Expression of Insulin Receptor Isoform a (IR-a) and Insulin Receptor Substrate 1 (IRS1) in Poorly Differentiated Colon Cancer Cell Lines and APC MIN/+ Tumors

Increased Expression of Insulin Receptor Isoform a (IR-a) and Insulin Receptor Substrate 1 (IRS1) in Poorly Differentiated Colon Cancer Cell Lines and APC MIN/+ Tumors

Abstract 1136: Gene amplification and epigenetic modifications: Mechanisms behind unbalanced expression of the proto-oncogene CYP24A1 in colorectal tumors

Abstract There is an inverse association between colorectal cancer and pre-diagnostic Vitamin D levels (25-OHD3), underlining the importance of Vitamin D in the prevention of colon cancer. 1α,25(OH)2D3 (1,25D3) is the most active form of Vitamin D and exhibits anti-proliferative, pro-differentiating, anti-angiogenic functions. Both, calcitriol and the calcitriol precursor 25(OH)D3 can be catabolized by the 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1). CYP24A1 is expressed at very low basal levels in the colon but is strongly induced by 1,25D3. This induction is due to a complex formation of the 1,25D3 and the Vitamin D Receptor and binding of this complex to Vitamin D Responsive Elements (VDRE) located within the CYP24A1 promoter region. This feedback loop shortens the biological half-life of 1,25D3. A high expression of the Vitamin D catabolizing enzyme CYP24A1 implies faster degradation of 1,25D3, thereby restricting its anti-tumorigenic functions. A genomic copy number gain and an overexpression of CYP24A1 on mRNA level have been observed in different malignancies, such as colon and breast. Our aim is to test whether the observed overexpression of CYP24A1 is due to gene amplification and/or epigenetic modulations. In human colon cancer cell lines we found an increase in genomic copy number of CYP24A1 compared to human placenta using real time-PCR. In human colorectal cancer patients (n=89) we observed a gain of CYP24A1 copy number in approximately 40% of the tumors compared to their respective adjacent mucosa indicating a role of CYP24A1 gene amplification in tumorigenesis. Further, on mRNA level we could show a trend toward increased CYP24A1 expression in tumors compared with their adjacent mucosa using reverse transcription qRT-PCR. However, the effect of copy number gain can be impaired by epigenetic regulation. Aberrant DNA methylation is a characteristic of various tumors. The promoter region of the CYP24A1 gene contains several CpG islands. Therefore we have examined if there are any differences in the methylation status of the CYP24A1 promoter between the tumor tissue and the adjacent “normal” mucosa. First we have compared the methylation status in 4 cell lines. Bisulfit-sequencing of the genomic DNA of the highly differentiated colon cancer cell line Caco2Aq showed heavy methylation of the CYP24A1 promoter including the VDREs located within this promoter area. In comparison, the colon cell lines Coga1A, LS174, and Coga13 (derived from less differentiated tumors) appeared largely unmethylated. Using methylation specific PCR we found that human colorectal tumors are often less methylated than their adjacent mucosa. In conclusion, our data indicate that both gene amplification and DNA-hypomethylation could be mechanisms leading to the overexpression of CYP24A1 in colorectal tumor patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1136. doi:10.1158/1538-7445.AM2011-1136

Butyrate elicits a metabolic switch in human colon cancer cells by targeting the pyruvate dehydrogenase complex

Butyrate, a short-chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO(2) , they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate-stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC.