Normal Intestinal Epithelial Cells Research Articles

MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC): Phase I, first-in-human study in patients (pts) with advanced gastrointestinal (GI) malignancies expressing GCC.

TPS3646 Background: MLN0264, an investigational ADC that targets GCC, consists of a fully human monoclonal antibody conjugated to the cytotoxic microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker. MMAE and the linker technology are licensed from Seattle Genetics. GCC is a cell surface protein expressed by normal intestinal epithelial cells, ~95% of metastatic colorectal cancer (mCRC), and subsets of gastric and pancreatic cancers. In normal tissue, GCC is expressed on the apical side of epithelial cell tight junctions; therefore, systemically delivered GCC-targeting agents are expected to be preferentially delivered to tumor tissue in which cell polarity is disrupted. MLN0264 has demonstrated antitumor activity in mouse xenograft models of GCC-expressing tumors (Veiby et al. EORTC-NCI-AACR, 2012), supporting the scientific rationale for this first-in-human study. Methods: This study (NCT01577758) was designed to evaluate MLN0264 in ~60 adult pts with GI malignancies expressing GCC (≥1+), measurable disease by RECIST, and ECOG PS 0/1. This study is being conducted at 4 centers in the US and EU and is estimated to last ~36–42 months. Pts receive IV MLN0264 on day 1 of 21-day cycles for up to 17 cycles or until disease progression/unacceptable MLN0264-related toxicity. Dose escalation is proceeding via an adaptive Bayesian continual reassessment model approach based on dose-limiting toxicities in cycle 1. Following determination of the maximum tolerated dose (MTD), up to 14 pts each will be enrolled to mCRC and gastric carcinoma expansion groups to further characterize the safety, tolerability, and pharmacokinetics (PK) of MLN0264 and evaluate its clinical activity. The primary objectives are to evaluate the safety and tolerability, determine the MTD, and describe the PK of IV MLN0264. Secondary objectives are to evaluate the antitumor activity and immunogenicity of MLN0264. An exploratory objective is to examine the relationship between levels of GCC protein expression in tumor tissue and clinical response. Enrollment as of Jan 2013 was 12 pts. Clinical trial information: NCT01577758.

Monoallelic Chromatin Conformation Flanking Long-Range Silenced Domains in Cancer-Derived and Normal Cells

Epigenetic inactivation of chromatin plays an important role in determining cell phenotype in both normal and cancer cells, but our knowledge is still incomplete with respect to any potential monoallelic nature of the phenomenon. We have genotyped DNA isolated from chromatin of two colorectal cancer-derived lines and a culture of normal human intestinal epithelial cells (HIEC), which was immunoprecipitated with antibodies to acetylated vs. methylated histone H3K9, and presented the data as B allele frequency differences over multiple single-nucleotide polymorphism (SNP) moving window averages. [B allele is an arbitrary term defined as one of the two alleles at any given SNP, named A and B]. Three different validation tests confirmed that peaks exhibiting differences represented monoallelic domains. These complementary tests confirmed the following: 1) genes in the regions of high B allele frequency difference were expressed monoallelically; 2) in normal cells all five imprinting control regions which carried heterozygous SNPs were characterized by B allele difference peaks; and 3) the haplotypes in the B allele difference peaks were faithfully maintained in the chromatin immunoprecipitated with the respective antibodies. In both samples most of the monoallelic domains were found at the boundaries between regions of open and closed chromatin. With respect to the cancer line, this supports the established concept of conformation spreading, but the results from the normal cells were unexpected. Since these cells were polyclonal, the monoallelic structures were probably not determined by random choice as occurs in X-inactivation, so we propose that epigenetic inactivation in some domains may be heritable and polymorphic in normal human cells.

Sa1681 Inhibition of the Prostaglandin EP1 Receptor Suppresses Colon Cancer Cell Invasion and Metastasis.

Background: Kruppel-like factor 5 (KLF5) is a zinc-finger transcription factor that is highly expressed in the crypt cells of the intestinal epithelia and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. We previously showed that KLF5 is a critical mediator for intestinal tumorigenesis resulted from either ApcMin mutation or oncogenic KRAS activation (McConnell et al. [2009] Cancer Res. 69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively). KLF5 is regulated at the transcriptional, translational, and post-translational level. Previous studies indicate that the protein kinase GSK3β and E3 ubiquitin ligase FBW7 facilitate proteosomal degradation of KLF5 protein via phosphorylation (GSK3β) and recognition (FBW7) of a phosphodegron sequence surrounding Ser303 in human KLF5. A recent genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence (Sjoblom et al. [2006] Science 314:268). Aim: To investigate the impact of P301S mutation on KLF5 protein stability. Methods: Plasmids containing human wildtype (WT) and mutant (S303A, P301S, P301A, and P301E) KLF5 under the control of the CMV promoter were transiently transfected into HEK293 cells. The effect of FBW7 on the stability of KLF5 produced from the various constructs was investigated by cycloheximide chase and Western blot analysis. Transcriptional activity of various KLF5 constructs was examined by luciferase reporter assay with Cyclin D1 and CDC2 reporter plasmids. Results: All four KLF5 mutants examined are more resistant to FBW7-mediated proteosomal degradation as compared to WT KLF5. Proteasome inhibition using MG132 resulted in the accumulation of WT KLF5 but had little impact on P301S KLF5 protein levels. Co-immunoprecipitation experiments show that, in contrast to WT KLF5, P301S and S303A KLF5 do not interact with FBW7. Concomitantly, the P301S KLF5 mutant displayed reduced levels of phosphorylation at Ser303 in comparison with WT KLF5. Furthermore, luciferase assays using Cyclin D1 and CDC2 promoter constructs demonstrate significantly elevated transcriptional activity of P301S KLF5 as compared to WT KLF5. Conclusion: Our data indicate that residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence. Furthermore, we show that the colon cancer-derived P301S mutation in KLF5 inhibits FBW7 recognition of the phosphodegron, yielding a degradation-resistant KLF5 protein, elevated protein levels, and enhanced KLF5 transcriptional activity. These results suggest that the P301S mutation in KLF5 is potentially oncogenic in colon cancer.

Sa1993 Claudin-3 Expression Associates With Colonic Epithelial Differentiation and Loss of Its Expression Correlates With Colon Carcinogenesis and Poor Patient Survival

G A A b st ra ct s CDC5L expression was found in the nuclei of normal murine intestinal epithelial cells. Areas of the colonic epithelium in AhCre+Apcfl/fl and ApcMin/+ mice which showed evidence of Wnt signalling by nuclear localisation of beta catenin showed increased nuclear expression of SFRS2 and cytoplasmic localisation of CDC5L. CDC5L knockdown in HCT116 and HT29 cells resulted in significant inhibition of cell proliferation by both SRB and clonogenic assays (p,0.05). SFRS2 knockdown also resulted in increased nuclear expression of CDC5L in these cell lines. The malignant colonic lesions that developed in ApcMin/+Pten-/mice showed loss of nuclear beta catenin expression and reversal of the phenotype observed in ApcMin/+ mice, with nuclear CDC5L localisation once again. Conclusion Apc inactivation results in increased nuclear expression of SFRS2 in colonic epithelial cells in vivo. This coincides with translocation of CDC5L from the nucleus into the cytoplasm, which limits its ability to regulate the cell cycle and splice pre-mRNA. Acquisition of an additional mutation in Pten alters Wnt signalling further, causing reduced nuclear accumulation of beta catenin and allowing CDC5L to translocate back into the nucleus, where it possibly stabilises the tumor phenotype.

Abstract 176: Thymoquinone attenuates tumor development in a mouse model of Lynch syndrome.

Abstract Background: Hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) is caused by germline mutations of DNA mismatch repair proteins leading to microsatellite instability (MSI). Nigella sativa oil has known antitumorigenic effects and was identified in an in vitro screen for compounds that reduce microsatellite instability (C. Campregher, unpublished data). Here we used a mouse model for HNPCC (Villin-Cre Msh2loxP/loxP) to test the effect of thymoquinone (TQ), the active ingredient of Nigella sativa oil, on intestinal tumor development. Methods: 60 mice (20 per group) were treated for 10 months with chow containing TQ-low (37.5 mg/kg diet), TQ-high (375 mg/kg diet) or regular mouse chow. Serial tissue sections of paraffin embedded Swiss rolls of the entire intestine were H&E stained and analyzed for tumor multiplicity, incidence and size. As tumor multiplicity in these mice is generally low (1-2 tumors/mouse) serial sections were prepared from the upper-, middle- and lower part of the paraffin block. Tumor size was scored as small, medium or large according to its presence on 1, 2 or all 3 sections of the block. DNA was isolated from microdissected normal intestinal and tumor tissue and tested for MSI by fragment analysis using a panel of six markers (Kabbarah 2003). Data were quantitated as nucleotide shift per marker (NSPM) and compared to germline DNA from mouse tails. Results: Treatment with TQ-low and TQ-high resulted in a significant reduction of tumor incidence from 94.4% to 57.9% (p<0.01) and 55.0% (p<0.006), respectively. TQ-low and TQ-high also reduced tumor multiplicity from 3.1 to 1.4 (p=0.003) and to 0.9 (p<0.001), respectively. The number of small and medium sized tumors was reduced in both treatment groups. The number of large tumors was not affected. Fragment analysis demonstrated global MSI in normal intestinal epithelial cells (on average 1.8 NSPM). TQ-low and TQ-high reduced the global MSI in normal intestinal epithelial cells to 0.9 and 0.9 NSPM, respectively. Tumor DNA displayed generally higher MSI than normal intestinal epithelial cells (2.7, 2.5, and 1.5 NSPM for control mice, TQ-low, and TQ-high, respectively) and was only reduced by TQ-high. Conclusion: In this mouse model of HNPCC TQ reduces the tumor incidence and tumor multiplicity by increasing replication fidelity in the Msh2-deficient intestinal epithelial cells. We propose TQ as a candidate compound for prevention of colorectal cancer in carriers of HNPCC mutations. Citation Format: Benedikt Kortüm, Christoph Campregher, Matthias Pinter, Michaela Lang, Rayko Evstatiev, Vineeta Khare, Melanie H. Kucherlapati, Winfried Edelmann, Christoph Gasche. Thymoquinone attenuates tumor development in a mouse model of Lynch syndrome. [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 176. doi:10.1158/1538-7445.AM2013-176

Abstract 1852: Pyruvate dehydrogenase kinase expression and metabolic changes following dichloroacetate exposure in anoxia in human colorectal cancer cells.

Abstract Introduction: While glycolysis is a suboptimal metabolic strategy used by cells under hypoxic conditions, many cancer cells preferentially utilize glycolysis for energy production in the presence of abundant oxygen, a phenomenon known as the “Warburg Effect”. Dichloroacetate (DCA) is a small molecule metabolic modulator that shifts cellular metabolism to glucose oxidation by inhibition of pyruvate dehydrogenase kinase (PDK), an enzymatic inhibitor of pyruvate dehydrogenase (PDH). Furthermore, DCA has been shown to induce apoptosis in cancer but not normal cells. Our previous work showed that some human colorectal cancer (CRC) cells are protected from anoxia-induced apoptosis upon exposure to DCA, through as yet unclear mechanisms. Hypothesis: It is hypothesized that differential expression of PDK isoforms will correlate with the ability of different cancer cells to withstand the apoptotic effects of DCA in anoxia. Methods: SW480, LS174T (CRC) and IEC-18 (normal intestinal epithelial) cell lines were exposed to 10 mM DCA in either normoxic (20% O2) or anoxic (< 0.1% O2) conditions for 24 hours. Following treatment, expression of different PDK isoforms was determined through qRT-PCR and western blotting. Phosphorylation of PDH at Ser293 and Ser300 was assessed through western blotting. Glucose consumption and lactate production was determined using a colorimetric-based assay. Changes in mitochondrial function were quantified through Mitotracker staining and subsequent flow cytometry. Cell viability was assessed using the neutral red assay. Results: In all three cell lines: A) PDK isoforms 1 and 3 are highly expressed relative to isoforms 2 and 4, B) DCA and/or anoxic treatment induced a greater effect on PDH phosphorylation at Ser300 than Ser293, and C) DCA had minimal effect on glucose consumption in either normoxia or anoxia. Conclusions: Altered PDK expression and PDH activation may not fully explain the differences in resistance to DCA-induced apoptosis observed in human CRC cells. Investigating the mechanisms by which certain cancer cells are protected from DCA-induced apoptosis will lead to a better understanding of cancer metabolism and thus provide better therapeutic opportunities. Citation Format: Nelson Ho, Brenda L. Coomber. Pyruvate dehydrogenase kinase expression and metabolic changes following dichloroacetate exposure in anoxia in human colorectal cancer cells. [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 1852. doi:10.1158/1538-7445.AM2013-1852

Epithelial Cell Adhesion Molecule (EpCAM) Regulates Claudin Dynamics and Tight Junctions

Epithelial cell adhesion molecule (EpCAM) (CD326) is a surface glycoprotein expressed by invasive carcinomas and some epithelia. Herein, we report that EpCAM regulates the composition and function of tight junctions (TJ). EpCAM accumulated on the lateral interfaces of human colon carcinoma and normal intestinal epithelial cells but did not co-localize with TJ. Knockdown of EpCAM in T84 and Caco-2 cells using shRNAs led to changes in morphology and adhesiveness. TJ formed readily after EpCAM knockdown; the acquisition of trans-epithelial electroresistance was enhanced, and TJ showed increased resistance to disruption by calcium chelation. Preparative immunoprecipitation demonstrated that EpCAM bound tightly to claudin-7. Co-immunoprecipitation documented associations of EpCAM with claudin-7 and claudin-1 but not claudin-2 or claudin-4. Claudin-1 associated with claudin-7 in co-transfection experiments, and claudin-7 was required for association of claudin-1 with EpCAM. EpCAM knockdown resulted in decreases in claudin-7 and claudin-1 proteins that were reversed with lysosome inhibitors. Immunofluorescence microscopy revealed that claudin-7 and claudin-1 continually trafficked into lysosomes. Although EpCAM knockdown decreased claudin-1 and claudin-7 protein levels overall, accumulations of claudin-1 and claudin-7 in TJ increased. Physical interactions between EpCAM and claudins were required for claudin stabilization. These findings suggest that EpCAM modulates adhesion and TJ function by regulating intracellular localization and degradation of selected claudins.

Anti cancer effects of Cnidium officinale Makino extract mediated through Apoptosis and Cell cycle arrest in Human Colorectal cancer cell line (HT‐29)

Natural products have recently attracted many researchers due to their potential use as therapeutic agent. Cnidium officinale Makino (COM) has multiple anti‐cancer properties, but its molecular targets and mode of action on human colorectal cancer cells are unknown. Through cell counting kit assay (CCK‐8), dose dependent cytotoxic effects were shown in HT‐29 cells treated with various concentrations of COM. This cytotoxic effect was not observed when COM was used to treat normal intestinal epithelial cells (IEC‐6). Flow cytometry analysis of the cell cycle after treatment with COM clearly showed that COM inhibited cellular proliferation of HT‐29 cells via G1 phase arrest of the cell cycle. The apoptotic effect of COM on HT‐29 cells was confirmed by annexin V‐propidium iodide apoptosis test. An increase in the expression of p53, pro‐apoptotic factor Bax and a decrease in the expression of anti‐apoptotic factor Bcl‐2 were also observed in a dose‐dependent manner. COM also down regulated the expression of pro‐caspase‐3 and pro‐caspase‐9 in a dose dependent manner. These data suggest a possible mechanism whereby COM could induce apoptosis and cell cycle arrest. This property of COM also suggests that COM could have possible therapeutic potential against human colon adenocarcinoma.

The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53

Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner(;) however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.

Protein kinase CK2 is a critical regulator of epithelial homeostasis in chronic intestinal inflammation.

Molecular mechanisms that restore intestinal epithelial homeostasis during colitis are incompletely understood. Here, we report that during intestinal inflammation, multiple inflammatory cytokines promote the activity of a master regulator of cell proliferation and apoptosis, serine/threonine kinase CK2. Enhanced mucosal CK2 protein expression and activity were observed in animal models of chronic colitis, particularly within intestinal epithelial cells. In-vitro treatment of intestinal epithelial cell lines with cytokines resulted in increased CK2 expression and nuclear translocation of its catalytic α subunit. Similarly, nuclear translocation of CK2α was a prominent feature observed in colonic crypts from individuals with ulcerative colitis and Crohn's disease. Further invitro studies revealed that CK2 activity promotes epithelial restitution, and protects normal intestinal epithelial cells from cytokine-induced apoptosis. These observations identify CK2 as a key regulator of homeostatic properties of the intestinal epithelium that serves to promote wound healing, in part through inhibition of apoptosis under conditions of inflammation.

Mangiferin aglycone attenuates radiation‐induced damage on human intestinal epithelial cells

Recent studies suggest that mangiferin aglycone (norathyriol) has great potential as a novel radioprotector without any known toxic side effects. In this study, we assessed the protective effects of mangiferin aglycone against radiation-induced injuries on normal human intestinal epithelial cells (HIECs), while using mangiferin as a reference compound. The in vitro experiments showed that pretreatment of either mangiferin aglycone or mangiferin could inhibit cytotoxic effects of ionizing irradiation (IR) on HIECs. Cellular changes were estimated by measuring cell viability, clonogenic surviving rate, and apoptotic rate. Compared to mangiferin, we found mangiferin aglycone had greater radioprotective effects of mangiferin aglycone on HIECs. It has been demonstrated that the cytotoxicity of ionizing radiation relates to its capacity to induce DNA damage. In view of this, we monitored DNA double-strand breaks (DSBs) using γH2AX foci formation to test whether mangiferin aglycone and mangiferin could modulate genotoxic effects of radiation. It shows that mangiferin aglycone could eliminate 46.8% of the total DSBs of the cells exposed to 2 Gy IR, which is significantly better than mangiferin. Complementing earlier results from our group, it appears possible to conclude that mangiferin aglycone presents potential useful effects on IR-induced damage and may be a better radioprotective agent than mangiferin therapeutically.

Investigation of nuclear nano-morphology marker as a biomarker for cancer risk assessment using a mouse model

The development of accurate and clinically applicable tools to assess cancer risk is essential to define candidates to undergo screening for early-stage cancers at a curable stage or provide a novel method to monitor chemoprevention treatments. With the use of our recently developed optical technology--spatial-domain low-coherence quantitative phase microscopy (SL-QPM), we have derived a novel optical biomarker characterized by structure-derived optical path length (OPL) properties from the cell nucleus on the standard histology and cytology specimens, which quantifies the nano-structural alterations within the cell nucleus at the nanoscale sensitivity, referred to as nano-morphology marker. The aim of this study is to evaluate the feasibility of the nuclear nano-morphology marker from histologically normal cells, extracted directly from the standard histology specimens, to detect early-stage carcinogenesis, assess cancer risk, and monitor the effect of chemopreventive treatment. We used a well-established mouse model of spontaneous carcinogenesis--Apc(Min) mice, which develop multiple intestinal adenomas (Min) due to a germline mutation in the adenomatous polyposis coli (Apc) gene. We found that the nuclear nano-morphology marker quantified by OPL detects the development of carcinogenesis from histologically normal intestinal epithelial cells, even at an early pre-adenomatous stage (six weeks). It also exhibits a good temporal correlation with the small intestine that parallels the development of carcinogenesis and cancer risk. To further assess its ability to monitor the efficacy of chemopreventive agents, we used an established chemopreventive agent, sulindac. The nuclear nano-morphology marker is reversed toward normal after a prolonged treatment. Therefore, our proof-of-concept study establishes the feasibility of the SL-QPM derived nuclear nano-morphology marker OPL as a promising, simple and clinically applicable biomarker for cancer risk assessment and evaluation of chemopreventive treatment.

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

Abstract 4420: Serum regulates reovirus-mediated cytopathy in K-Ras activated colorectal cancer and intestinal epithelial cell lines

Abstract Background: The oncolytic potency of replication competent REOvirus has been demonstrated in various cancers. The activity is most pronounced in Kras mutant cancer cells, which account for 30-40 % of all cancers. Herein we simultaneously utilized isogenic human derived colorectal cancer cell lines that differ only by the presence of mutant Kras and normal rat intestinal epithelial cells with inducible Kras to evaluate whether the presence of oncogenic Kras alters the sensitivity of colon cancer cells to reovirus. Methods: Reovirus was obtained from Oncolytics Biotech Inc. Rat IEC's harboring IPTG inducible mutant Ras oncogene (IEC-iKras) were treated with reovirus ± IPTG. The infectivity was maintained at a multiplicity of infection (MOI) of 2. MTT assay was performed at 72 hours to determine cell vability (CV). Similar experiments were also performed with HCT116 (Kras mut) and its isogenic derivative Hke3 [(Kras wild type (WT)]. Cells were cultured and treated in both serum rich (SR) and serum free (SF) media to determine the effect of growth factors on sensitivity to reovirus. Alterations in gene expression were determined by real time PCR and protein expression by western blot. Results: The activity of reovirus was observed in all cell lines studied. Reduction in CV was greater in Kras mutant HCT116 compared to WT Hke3 cells. Consistently, induction of Kras in IEC cells increased the potency of reovirus. Furthermore, this effect was more prominent in cells cultured in SF suggesting that growth factors in the serum may attenuate the effect of mutant Kras on reovirus sensitivity. Expression of the cell cycle regulator, p21 was preferentially increased upon reovirus infection in Kras mutant compared to WT cells, and in cells cultured in SF compared to SR conditions. Conclusion: Oncogenic Kras mutations increase the sensitivity of colon cancer cells to Reovirus. Clinical trials are underway testing reovirus in patients with Kras mutant metastatic colorectal cancer. 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 4420. doi:1538-7445.AM2012-4420

Abstract 4169: The Wnt/β-catenin pathway is involved in KRAS-induced intestinal epithelial cell transformation

Abstract Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). The APC protein is an integral part of the destruction complex that controls cytoplasmic β-catenin levels by promoting ubiquitin-mediated degradation of β-catenin. Loss-of-function mutations in APC are commonly found in CRC, leading to constitutively high levels of β-catenin which then associates with TCF4, inducing expression of target genes that are important in cell growth. The KRAS/BRAF/MEK/ERK signaling pathway is also a major transforming growth regulatory signaling pathway. Indeed, gain-of-function mutations of KRAS and BRAF are detected in up to 60% of CRCs. Although both the Wnt/β-catenin and the ERK pathways are major pathways for tumorigenesis of intestinal epithelial cells (IECs), no significant interactions between these pathways have been identified. Methods. Retrovirus encoding the HA-tagged wild-type MEK1 (wtMEK) or activated MEK1 (MEK1S218D/S222D, caMEK) or oncogenic form of KRASG12V were used to infect and transform normal IECs in culture. The transcriptional activity of β-catenin/TCF4 complex was analyzed by using the luciferase reporter systems TOPFLASH and c-myc promoter (mutated or not for TCF4 binding elements). Expression levels of Wnt/β-catenin targets axin2, lef-1 and c-myc were evaluated by qPCR and protein localisation by immunofluorescence. A dominant-negative form of TCF4 (TCF4-DN) was used to verify the contribution of β-catenin/TCF4 complex in transformation induced by oncogenic activation of KRAS or MEK1. β-catenin transcriptional activity was analyzed in human CRC cells exhibiting APC mutation, treated or not with the MEK inhibitor U0126. Results. 1- Phase-contrast microscopy revealed that KRASG12V and caMEK-expressing IECs underwent a morphological change from an epithelial morphology to an elongated morphology. Cells clearly loose cell-to-cell contacts when compared to control cells. E-cadherin and β-catenin proteins disappeared from cell-cell junctions. 2- β-catenin/TCF4 transcriptional activity as well as mRNA levels of c-myc, axin2 and lef1 were markedly increased in IECs transformed by oncogenic MEK1 or KRAS. 3- Expression of the dominant-negative form of TCF4 severely attenuated IEC transformation induced by oncogenic MEK1 or KRAS. 4- Treatment of human CRC cell lines with U0126 significantly reduced axin2, lef-1 and c-myc mRNA levels, indicating that expression of these β-catenin target genes is likely dependent on MEK/ERK activity. Accordingly, β-catenin/TCF4 transcriptional activity was inhibited following MEK inhibition by U0126 or ERK dephosphorylation by DUSP4 expression. Conclusion. Our data indicate that the oncogenic activation of the KRAS/ERK signalling pathway in IECs up-regulates β-catenin/TCF4 transcriptional activity which contributes to the tumorigenic potential of this signaling. 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 4169. doi:1538-7445.AM2012-4169

Differential expression of insulin receptor isoforms A and B in highly proliferative stem and tumor cells vs. differentiated intestinal epithelial cells

Little is known about expression and roles of insulin receptor (IR) in the intestinal epithelium despite emerging links between obesity, hyperinsulinemia, and intestinal dysfunction or cancer risk. IR exists as an IR‐B isoform that mediates insulin's metabolic actions and an IR‐A isoform implicated in fetal growth and recently, cancer. This study used Sox9‐EGFP reporter mice, ApcMin/+ mice, and human colorectal cancer cell lines (CRC) to test the hypothesis that IR‐A and IR‐B have distinct expression profiles in highly proliferative vs differentiated normal intestinal epithelial cells (IEC) or CRC.ResultsIR‐A predominates in highly proliferative intestinal epithelial stem cells and progenitors, while IR‐B predominates in differentiated lineages. In ApcMin/+ mice, the IR‐B:IR‐A ratio is dramatically decreased in tumor vs non‐tumor tissue in small intestine (0.65 ± 0.02 vs 1.3 ± 0.03) and colon (0.63 ± 0.1 vs 1.27 ± 0.9). The IR‐B:IR‐A ratio is higher in CRC with the ability to differentiate vs undifferentiated, aggressively growing CRC. The ratio of IR‐B:IR‐A is significantly increased in differentiated vs undifferentiated Caco2 cells (2.2 ± 0.2 vs 1.5 ± 0.1). We conclude that relative expression levels of IR‐B:IR‐A may be critical determinants of normal IEC or CRC differentiation. Grants: F31AG040943 (SFB) and NIH DK040247‐19 (PKL).

Oncogenic KRAS and BRAF activation of the MEK/ERK signaling pathway promotes expression of dual-specificity phosphatase 4 (DUSP4/MKP2) resulting in nuclear ERK1/2 inhibition

Gain-of-function mutations in KRAS and BRAF genes are found in up to 50% of colorectal cancers. These mutations result in the activation of the BRAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. Upon activation, ERK1/2 translocate from the cytoplasm to the nucleus. This process has been shown to be required for the induction of many cellular responses, although the molecular mechanisms regulating ERK nuclear function, especially under oncogenic stimulation, remain to be explored. Herein, we examined the spatiotemporal regulation of ERK1/2 activity upon oncogenic activation of KRAS(G12V) and BRAF(V600E) in normal intestinal epithelial crypt cells (IECs). Results demonstrate that expression of these oncogenes markedly stimulated ERK1/2 activities and morphologically transformed IECs. Importantly however, ERK phosphorylation was not observed in the nucleus, but restricted to the cytoplasm of KRAS(G12V)- and BRAF(V600E)-transformed IECs. The absence of nuclear ERK phosphorylation was due to a vanadate-sensitive phosphatase activity. Nuclear ERK dephosphorylation was found to be tightly correlated with the rapid expression of DUSP4 phosphatase induced in an MEK-dependent manner. In addition, MEK-dependent phosphorylation of T361, T363, S390 and S395 residues highly stabilized DUSP4 protein. Finally, in human colorectal cancer cells, ERK1/2 activities were also confined to the cytoplasm and treatment with pervanadate reactivated ERK1/2 in the nucleus. Accordingly, DUSP4 mRNAs were found to be highly expressed, in an MEK-dependent manner, in all colorectal cancer cells analyzed. These findings indicate that DUSP4 functions as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during intestinal tumorigenesis.

Selective radiosensitization of p53 mutant pancreatic cancer cells by combined inhibition of Chk1 and PARP1

We have recently shown that inhibition of HRR (homologous recombination repair) by Chk1 (checkpoint kinase 1) inhibition radiosensitizes pancreatic cancer cells and others have demonstrated that Chk1 inhibition selectively sensitizes p53 mutant tumor cells. Furthermore, PARP1 [poly (ADP-ribose) polymerase-1] inhibitors dramatically radiosensitize cells with DNA double strand break repair defects. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 [via olaparib (AZD2281)] would selectively sensitize p53 mutant pancreatic cancer cells to radiation. We also used 2 isogenic p53 cell models to assess the role of p53 status in cancer cells and intestinal epithelial cells to assess overall cancer specificity. DNA damage response and repair were assessed by flow cytometry, γH2AX, and an HRR reporter assay. We found that the combination of AZD7762 and olaparib produced significant radiosensitization in p53 mutant pancreatic cancer cells and in all of the isogenic cancer cell lines. The magnitude of radiosensitization by AZD7762 and olaparib was greater in p53 mutant cells compared with p53 wild type cells. Importantly, normal intestinal epithelial cells were not radiosensitized. The combination of AZD7762 and olaparib caused G2 checkpoint abrogation, inhibition of HRR, and persistent DNA damage responses. These findings demonstrate that the combination of Chk1 and PARP1 inhibition selectively radiosensitizes p53 mutant pancreatic cancer cells. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRCA1/2 ‘deficient-like’ phenotype in p53 mutant tumor cells, while sparing normal tissue.

Colorectal cancer stem cell: a potential therapeutic target

The discovery of cancer stem cells has improved our understanding of tumour occurrence and development. Colorectal cancer stem cells may be derived from mutations in normal intestinal epithelial stem cells. CD133+ and aldehyde dehydrogenase 1 (ALDH1)+ cells have strong tumorigenic capacities and may represent different subpopulations of colorectal cancer stem cells. Multiple signalling pathways, especially the Wnt pathway, are important in colorectal cancer occurrence and development, and maintaining the stemness of colorectal cancer stem cells. Identifying colorectal cancer stem cells and understanding the related signalling pathways are important for developing new targeted interventions for colorectal cancer.

Overexpression of survivin is correlated with increased invasion and metastasis of colorectal cancer

The aim of this study was to investigate the association of survivin expression with metastasis of colorectal cancer (CRC). RT-PCR and Western blot assays were performed to detect survivin expression in CRC cells and normal intestinal epithelial cell. The expression of survivin gene was also detected in 15 CRC tissues, surrounding and adjacent colon tissues. Moreover, survivin expression in 48 CRC tissues with or without lymph node metastasis was analyzed. Multivariate analysis for lymph node metastasis was performed using logistic regression model. RNA interference was used to inhibit survivin expression in CRC cells and analyze its effect on invasion and metastasis of CRC cells. The expression levels of survivin mRNA and protein were higher in CRC cells than in normal intestinal epithelial cell line. The average levels of survivin mRNA and protein were higher in CRC tissues than surrounding or adjacent colon tissues (P < 0.05). High survivin expression was an independent factor for predicting lymph node metastasis of CRC (P = 0.043). RNAi-mediated survivin knockdown could significantly inhibit in vitro invasion and in vivo metastasis of CRC cells, which might be inactivation of matrix metalloproteinases. Targeting survivin will be a potential strategy to suppress metastasis of CRC.