Andres I. Roig

Immortalized Epithelial Cells Derived From Human Colon Biopsies Express Stem Cell Markers and Differentiate In Vitro

BACKGROUND & AIMS Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsy-derived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability. METHODS We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture. RESULTS The actively proliferating HCECs expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining. CONCLUSIONS We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis.

Toll-like Receptor 4 Variant D299G Induces Features of Neoplastic Progression in Caco-2 Intestinal Cells and Is Associated With Advanced Human Colon Cancer

BACKGROUND & AIMS The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions. METHODS We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage. RESULTS Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. CONCLUSIONS TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.

MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells

Background/Aim Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. Methods HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay. Results Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10−4) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. Conclusions MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.

CDDO-Me Protects against Space Radiation-Induced Transformation of Human Colon Epithelial Cells

Abstract Radiation-induced carcinogenesis is a major concern both for astronauts on long-term space missions and for cancer patients being treated with therapeutic radiation. Exposure to radiation induces oxidative stress and chronic inflammation, which are critical initiators and promoters of carcinogenesis. Many studies have demonstrated that non-steroidal anti-inflammatory drugs and antioxidants can reduce the risk of radiation-induced cancer. In this study, we found that a synthetic triterpenoid, CDDO-Me (bardoxolone methyl), was able to protect human colon epithelial cells (HCECs) against radiation-induced transformation. HCECs that were immortalized by ectopic expression of hTERT and cdk4 and exhibit trisomy for chromosome 7 (a non-random chromosome change that occurs in 37% of premalignant colon adenomas) can be transformed experimentally with one combined exposure to 2 Gy of protons at 1 GeV/nucleon followed 24 h later by 50 cGy of 56Fe ions at 1 GeV/nucleon. Transformed cells showed an increase in proliferation rate and in both anchorage-dependent and independent colony formation ability. A spectrum of chromosome aberrations was observed in transformed cells, with 40% showing loss of 17p (e.g. loss of one copy of p53). Pretreatment of cells with pharmacological doses of CDDO-Me, which has been shown to induce antioxidative as well as anti-inflammatory responses, prevented the heavy-ion-induced increase in proliferation rate and anchorage-dependent and independent colony formation efficiencies. Taken together, these results demonstrate that experimentally immortalized human colon epithelial cells with a non-random chromosome 7 trisomy are valuable premalignant cellular reagents that can be used to study radiation-induced colorectal carcinogenesis. The utility of premalignant HCECs to test novel compounds such as CDDO-Me that can be used to protect against radiation-induced neoplastic transformation is also demonstrated.

Functional Parsing of Driver Mutations in the Colorectal Cancer Genome Reveals Numerous Suppressors of Anchorage-Independent Growth

Landmark cancer genome resequencing efforts are leading to the identification of mutated genes in many types of cancer. The extreme diversity of mutations being detected presents significant challenges to subdivide causal from coincidental mutations to elucidate how disrupted regulatory networks drive cancer processes. Given that a common early perturbation in solid tumor initiation is bypass of matrix-dependent proliferation restraints, we sought to functionally interrogate colorectal cancer candidate genes (CAN-genes) to identify driver tumor suppressors. We have employed an isogenic human colonic epithelial cell (HCEC) model to identify suppressors of anchorage-independent growth by conducting a soft agar-based short hairpin RNA (shRNA) screen within the cohort of CAN-genes. Remarkably, depletion of 65 of the 151 CAN-genes tested collaborated with ectopic expression of K-RAS(V12) and/or TP53 knockdown to promote anchorage-independent proliferation of HCECs. In contrast, only 5 of 362 random shRNAs (1.4%) enhanced soft agar growth. We have identified additional members of an extensive gene network specifying matrix-dependent proliferation, by constructing an interaction map of these confirmed progression suppressors with approximately 700 mutated genes that were excluded from CAN-genes, and experimentally verifying soft agar growth enhancement in response to depletion of a subset of these genes. Collectively, this study revealed a profound diversity of nodes within a fundamental tumor suppressor network that are susceptible to perturbation leading to enhanced cell-autonomous anchorage-independent proliferative fitness. Tumor suppressor network fragility as a paradigm within this and other regulatory systems perturbed in cancer could, in large part, account for the heterogeneity of somatic mutations detected in tumors.

Two- and Three-Dimensional Models for Risk Assessment of Radiation-Enhanced Colorectal Tumorigenesis

Abstract Roig, A. I., Hight, S. K. and Shay, J. W. Two- and Three-Dimensional Models for Risk Assessment of Radiation-Enhanced Colorectal Tumorigenesis. Radiat. Res. 171, 33–40 (2009). Astronauts may be at an increased risk for developing colorectal cancer after a prolonged interplanetary mission given the potential for greater carcinogenic effects of radiation to the colon. In addition, with an increase in age, there is a greater incidence of premalignant colon adenomas with age. In the present study, we have compared the effects of radiation on human colon epithelial cells in two-dimensional (2D) monolayer culture, in three-dimensional (3D) culture, and in intact human colon tissue biopsies. Immortalized colon epithelial cells were irradiated at the NASA Space Radiation Laboratory (NSRL) with either 1 Gy 1 GeV/nucleon 56Fe particles or 1 Gy 1 GeV/nucleon protons and were stained at various times to assess DNA damage and repair responses. The results show more persisting damage at 24 h with iron-particle radiation compared to protons. Similar results were seen in 3D colon epithelial cell cultures in which 56Fe-particle-irradiated specimens show more persisting damage at 24 h than those irradiated with low-LET γ rays. We compared these results to those obtained from human colon tissue biopsies irradiated with 1 Gy γ rays or 1 Gy 1 GeV 56Fe particles. Observations of radiation-induced DNA damage and repair in γ-irradiated specimens revealed more pronounced early DNA damage responses in the epithelial cell compartment compared to the stromal cell compartment. After low-LET irradiation, the damage foci mostly disappeared at 24 h. Antibodies to more than one type of DNA repair factor display this pattern of DNA damage, and staining of nonirradiated cells with nonphosphorylated DNA-PKcs shows a predominance of epithelial staining over stromal cells. Biopsy specimens irradiated with high-LET radiations also show a pattern of predominance of the DNA damage response in the highly proliferative epithelial cell compartment. Persistent unrepaired DNA damage in colon epithelial cells and the differing repair responses between the epithelial and mesenchymal compartments in tissues may enhance tumorigenesis by both stem cell transformation and alterations in the radiation-induced permissive tissue microenvironment that may potentiate cancer progression.

DNA damage intensity in fibroblasts in a 3-dimensional collagen matrix correlates with the Bragg curve energy distribution of a high LET particle.

Purpose: The DNA double-strand break (DSB) damage response induced by high energy charged particles on lung fibroblast cells embedded in a 3-dimensional (3-D) collagen tissue equivalents was investigated using antibodies to the DNA damage response proteins gamma-histone 2AX (γ-H2AX) and phosphorylated DNA-PKcs (p-DNA-PKcs). Materials and methods: 3-D tissue equivalents were irradiated in positions across the linear distribution of the Bragg curve profiles of 307.7 MeV/nucleon, 556.9 MeV/nucleon, or 967.0 MeV/nucleon 56Fe ions at a dose of 0.30 Gy. Results: Patterns of discrete DNA damage streaks across nuclei or saturated nuclear damage were observed, with saturated nuclear damage being more predominant as samples were positioned closer to the physical Bragg peak. Quantification of the DNA damage signal intensities at each distance for each of the examined energies revealed a biological Bragg curve profile with a pattern of DNA damage intensity similar to the physical Bragg curve for the particular energy. Deconvolution microscopy of nuclei with streaked or saturated nuclear damage pattern revealed more details of the damage, with evidence of double-strand breaks radially distributed from the main particle track as well as multiple discrete tracks within saturated damage nuclei. Conclusions: These 3-D culture systems can be used as a biological substrate to better understand the interaction of heavy charged particles of different energies with tissue and could serve as a basis to model space-radiation-induced cancer initiation and progression.

Abstract 3712: p21-activated kinase-1 in the prevention of inflammation-driven colon cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chronic gut inflammation in inflammatory bowel diseases (IBD) predisposes to the development of colorectal cancer, causing increased mortality. Use of mesalamine in the treatment and remittance of ulcerative colitis (UC) modulates the risk of neoplastic progression. We have previously reported (Khare V, et al. Biochem Pharm 2012) that p-21 activated kinase-1 (PAK1) mediates mesalamine activity by orchestrating MAPK signaling; Wnt-β catenin pathway and cell adhesion; all implicated in the colon carcinogenesis. Since mesalamine has primarily an anti-inflammatory function, we evaluated the role of PAK1, its target, in IBD and in colitis-associated cancer (CAC). PAK1 expression was scored by immunohistochemistry in human samples from IBD, CAC and normal mucosa. Compared to normal mucosa and Crohns disease, a higher PAK1 expression was detected in UC which further increased in CAC. PAK1 expression was also elevated in the inflamed mucosa and carcinomas in IL-10−/− mouse model of colitis. The efficacy of mesalamine in reducing inflammation and tumor incidence in these mice has been previously reported (Brown JB et al, Gastroenetrology 2010). The consequence of PAK1 overexpression was further investigated in vitro. Normal diploid colon epithelial cells (HCEC-1CT) showed higher proliferation (46 ± 3.1% increase) upon overexpression of wildtype (wt) PAK1 while dominant negative (dn) PAK1 overexpression reduced proliferation (25% ± 2.5 decrease) compared to control. Apoptosis (Annexin V staining) was reduced in HCEC-1CT overexpressing wtPAK1 (1.2% ± 0.40) compared to dnPAK1 (11.6% ± 0.3) and control (16% ± 0.83). Mesalamine treatment (20mM) was effective in counteracting the effect of PAK1 overexpression by reducing cell proliferation and inducing apoptosis. Treatment of cells with specific inhibitors (PD98059/LY294002/Rapamycin) of signaling pathways (MEK/PI3K/mTOR) demonstrated that mesalamine regulated PAK1 expression by the MEK, PI3K and mTOR signaling pathways. Our data indicate that (1) PAK1 is upregulated in UC and in CAC, (2) PAK1 may be utilized as a predictive marker for the development of CAC, and (3) PAK1 is a viable target for chemoprevention in the setting of colitis. Mesalamine counteracts PAK1 expression and its effect on cell proliferation and apoptosis. In the longterm management of UC mesalamine may protect from CAC through inhibition of PAK1. Citation Format: Vineeta Khare, Kyle Dammann, Manuela Jambrich, Andres Roig, Jerry Shay, Christoph Gasche. p21-activated kinase-1 in the prevention of inflammation-driven colon cancer. [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 3712. doi:10.1158/1538-7445.AM2013-3712

Abstract 2207: Functional interrogation of the colorectal cancer genome identifies numerous suppressors of anchorage-independent growth

INTRODUCTION: High throughput cancer genome sequencing efforts are leading to the identification of frequently mutated genes in cancer. Unfortunately, the extreme diversity of lesions being detected presents a challenge to segment causal from coincidental mutations and to elucidate how causal lesions disrupt regulatory networks to drive cancer processes. AIMS AND METHODS: Given that a common early perturbation in solid tumor initiation is bypass of matrix-dependent proliferation restraints, we used immortalized human colonic epithelial cells (HCECs) to identify suppressors of anchorage-independent growth by conducting a soft-agar based shRNA screen within frequently mutated colorectal cancer (CRC) genes. RESULTS AND CONCLUSIONS: Remarkably, depletion of 65 of the 151 frequently CRC-mutated genes tested collaborated with K-RASV12 or TP53 knockdown to promote anchorage-independent proliferation of HCECs. These candidates fall under a variety of different signaling pathways, but in particular JNK signaling was found to be a master suppressor of anchorage-independent growth in normal HCECs. We have identified additional members of an extensive gene network specifying matrix-dependent proliferation, by constructing an interaction map of these confirmed progression suppressors with the ∼700 rare CRC-mutated genes and experimentally verifying soft-agar growth enhancement in response to depletion of these genes. Collectively, this study reveals a profound diversity of fragile nodes within a fundamental tumor suppressor network, perturbation of which leads to enhanced cell-autonomous proliferative fitness. These studies establish the importance of functionally annotating cancer genomes using biologically relevant assays. 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 2207. doi:10.1158/1538-7445.AM2011-2207