BID mediates selective killing of APC-deficient cells in intestinal tumor suppression by nonsteroidal antiinflammatory drugs.

Colorectal tumorigenesis is driven by genetic alterations in the adenomatous polyposis coli (APC) tumor suppressor pathway and effectively inhibited by nonsteroidal antiinflammatory drugs (NSAIDs). However, how NSAIDs prevent colorectal tumorigenesis has remained obscure. We found that the extrinsic apoptotic pathway and the BH3 interacting-domain death agonist (BID) are activated in adenomas from NSAID-treated patients. Loss of BID abolishes NSAID-mediated tumor suppression, survival benefit, and apoptosis in tumor-initiating stem cells in APC(Min/+) mice. BID-mediated cross-talk between the extrinsic and intrinsic apoptotic pathways is responsible for selective killing of neoplastic cells by NSAIDs. We further demonstrate that NSAIDs induce death receptor signaling in both cancer and normal cells, but only activate BID in cells with APC deficiency and ensuing c-Myc activation. Our results suggest that NSAIDs suppress intestinal tumorigenesis through BID-mediated synthetic lethality triggered by death receptor signaling and gatekeeper mutations, and provide a rationale for developing more effective cancer prevention strategies and agents.

Transcriptomic Analysis of Calcium Remodeling in Colorectal Cancer.

Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca2+ homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca2+ remodeling includes critical changes in store-operated Ca2+ entry (SOCE) and Ca2+ store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca2+ transport, a comprehensive view of Ca2+ remodeling in CRC is lacking. We have used Next Generation Sequencing (NGS) to investigate differences in expression of 77 selected gene transcripts involved in intracellular Ca2+ transport in CRC. To this end, mRNA from normal human colonic NCM460 cells and human colon cancer HT29 cells was isolated and used as a template for transcriptomic sequencing and expression analysis using Ion Torrent technology. After data transformation and filtering, exploratory analysis revealed that both cell types were well segregated. In addition, differential gene expression using R and bioconductor packages show significant differences in expression of selected voltage-operated Ca2+ channels and store-operated Ca2+ entry players, transient receptor potential (TRP) channels, Ca2+ release channels, Ca2+ pumps, Na⁺/Ca2+ exchanger isoforms and genes involved in mitochondrial Ca2+ transport. These data provide the first comprehensive transcriptomic analysis of Ca2+ remodeling in CRC.

Methionine adenosyltransferase 2B-GIT1 complex serves as a scaffold to regulate Ras/Raf/MEK1/2 activity in human liver and colon cancer cells.

Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth.

Chemoprevention of colorectal cancer by targeting APC-deficient cells for apoptosis.

Cancer chemoprevention uses natural, synthetic, or biological substances to reverse, suppress, or prevent either the initial phase of carcinogenesis or the progression of neoplastic cells to cancer. It holds promise for overcoming problems associated with the treatment of late-stage cancers. However, the broad application of chemoprevention is compromised at present by limited effectiveness and potential toxicity. To overcome these challenges, here we developed a new chemoprevention approach that specifically targets premalignant tumour cells for apoptosis. We show that a deficiency in the adenomatous polyposis coli (APC) gene and subsequent activation of beta-catenin lead to the repression of cellular caspase-8 inhibitor c-FLIP (also known as CFLAR) expression through activation of c-Myc, and that all-trans-retinyl acetate (RAc) independently upregulates tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors and suppresses decoy receptors. Thus, the combination of TRAIL and RAc induces apoptosis in APC-deficient premalignant cells without affecting normal cells in vitro. In addition, we show that short-term and non-continuous TRAIL and RAc treatment induce apoptosis specifically in intestinal polyps, strongly inhibit tumour growth, and prolong survival in multiple intestinal neoplasms C57BL/6J-Apc(Min)/J (Apc(Min)) mice. With our approach, we further demonstrate that TRAIL and RAc induce significant cell death in human colon polyps, providing a potentially selective approach for colorectal cancer chemoprevention by targeting APC-deficient cells for apoptosis.

A reciprocal shift in transient receptor potential channel 1 (TRPC1) and stromal interaction molecule 2 (STIM2) contributes to Ca2+ remodeling and cancer hallmarks in colorectal carcinoma cells.

We have investigated the molecular basis of intracellular Ca(2+) handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca(2+) stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca(2+) levels, agonist-induced Ca(2+) increases, store-operated Ca(2+) entry (SOCE), and store-operated currents (ISOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca(2+) stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca(2+) release-activated Ca(2+) currents (ICRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced ICRAC plus a nonselective ISOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca(2+) store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca(2+) store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca(2+) remodeling and tumor features in colon cancer.

Integrated chemical and toxicological investigation of UV-chlorine/chloramine drinking water treatment.

As the use of alternative drinking water treatment increases, it is important to understand potential public health implications associated with these processes. The objective of this study was to evaluate the formation of disinfection byproducts (DBPs) and cytotoxicity of natural organic matter (NOM) concentrates treated with chlorine, chloramine, and medium pressure ultraviolet (UV) irradiation followed by chlorine or chloramine, with and without nitrate or iodide spiking. The use of concentrated NOM conserved volatile DBPs and allowed for direct analysis of the treated water. Treatment with UV prior to chlorine in ambient (unspiked) samples did not affect cytotoxicity as measured using an in vitro normal human colon cell (NCM460) assay, compared to chlorination alone when toxicity is expressed on the basis of dissolved organic carbon (DOC). Nitrate-spiked UV+chlorine treatment produced greater cytotoxicity than nitrate-spiked chlorine alone or ambient UV+chlorine samples, on both a DOC and total organic halogen basis. Samples treated with UV+chloramine were more cytotoxic than those treated with only chloramine using either dose metric. This study demonstrated the combination of cytotoxicity and DBP measurements for process evaluation in drinking water treatment. The results highlight the importance of dose metric when considering the relative toxicity of complex DBP mixtures formed under different disinfection scenarios.

IL-1ß stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a.

Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/ß-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1ß-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1ß-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1ß-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1ß stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1ß-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1ß-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.

Methylselenol, a selenium metabolite, plays common and different roles in cancerous colon HCT116 cell and noncancerous NCM460 colon cell proliferation.

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenol's stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenol's anticancer action.

Hamamelitannin from witch hazel (Hamamelis virginiana) displays specific cytotoxic activity against colon cancer cells.

Hamamelis virginiana (witch hazel) bark is a rich source of condensed and hydrolyzable tannins reported to exert a protective action against colon cancer. The present study characterizes different witch hazel tannins as selective cytotoxic agents against colon cancer. To cover the structural diversity of the tannins that occur in H. virginiana bark, the hydrolyzable tannins, hamamelitannin and pentagalloylglucose, together with a proanthocyanidin-rich fraction (F800H4) were selected for the study. Treatment with these compounds reduced tumor viability and induced apoptosis, necrosis, and S-phase arrest in the cell cycle of HT29 cells, with hamamelitannin being the most efficient. Owing to polyphenol-mediated H(2)O(2) formation in the incubation media, the antiproliferative effect was determined in the presence and absence of catalase to rule out any such interference. The presence of catalase significantly changed the IC(50) only for F800H4. Furthermore, at concentrations that inhibit the growth of HT29 cells by 50%, hamamelitannin had no harmful effects on NCM460 normal colonocytes, whereas pentagalloylglucose inhibited both cancerous and normal cell growth. Using the TNPTM assay, we identified a highly reactive phenolic position in hamamelitannin, which may explain its efficacy at inhibiting colon cancer growth.

Prolonged sulforaphane treatment activates survival signaling in nontumorigenic NCM460 colon cells but apoptotic signaling in tumorigenic HCT116 colon cells.

Sulforaphane (SFN) is a naturally occurring chemopreventive agent; the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that SFN (15 µmol/L) exposure (72 h) inhibited cell proliferation by up to 95% in colon cancer cells (HCT116) and by 52% in normal colon mucosa-derived (NCM460) cells. Our data also showed that SFN exposure (5 and 10 µmol/L) led to the reduction of G1 phase cell distribution and an induction of apoptosis in HCT116 cells, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen-activated protein kinase (MAPK) signaling status revealed that SFN upregulated the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. In contrast, SFN enhanced the phosphorylation of stress-activated protein kinase (SAPK) and decreased cellular myelocytomatosis oncogene (c-Myc) expression in HCT116 cells but not NCM460 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may play a critical role in SFN's stronger potential of inhibiting cell proliferation in colon cancer cells than in normal colon cells.

Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.

CCK2 receptor expression transforms non-tumorigenic human NCM356 colonic epithelial cells into tumor forming cells.

Expression of gastrin and cholecystokinin 2 (CCK(2)) receptor splice variants (CCK(2)R and CCK(2i4sv)R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK(2) receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non-tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK(2i4sv)R or CCK(2)R. RT-PCR and intracellular Ca(2+) ([Ca(2+)](i)) imaging of RIE/CCK(2)R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK(2)R or CCK(2i4sv)R (71 and 81 fmol/mg, respectively) grew faster in vitro, and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK(2) receptor selective antagonist, YM022, partially inhibited the growth of both receptor-expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor-expressing cells. Vector-NCM356 cells did not form tumors in nude mice, whereas, either CCK(2) receptor-expressing cells formed large tumors. Autocrine activation CCK(2) receptor variants are sufficient to increase in vitro growth and tumorigenicity of non-transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.

Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.

Substance P (SP) regulates important intestinal functions, such as mucosal permeability, motility, chloride secretion, and inflammation via the neurokinin-1 receptor (NK-1R). Previous reports showed that vascularization and expression of angiogenic factors are evident in the colonic mucosa of rats with colitis and patients with inflammatory bowel disease. Here we determined whether SP is associated with angiogenesis. Human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and mice with dextran sodium sulfate-induced colitis were used. We found that expression of the angiogenic factor CCN1 was increased in the colons of patients with Crohn's disease and ulcerative colitis. Mucosal extracts from inflammatory bowel disease patients induced human intestinal microvascular endothelial cell migration that was inhibited by blockade of CCN1 and its receptor integrin alphavbeta3. Both the degree of angiogenesis and CCN1 expression were elevated in the colons of mice with dextran sodium sulfate-induced colitis, which was reduced by treatment with the NK-1R antagonist CJ-12255. SP also increased CCN1 expression in NCM460-NK-1R colonocytes. SP exposure to human intestinal microvascular endothelial cells co-cultured with NCM460-NK-1R cells induced angiogenic activity that was inhibited by CCN1 silencing. In addition, intracolonic overexpression of CCN1 induced angiogenesis in mouse colon. Thus, SP mediates angiogenesis via CCN1 during colitis.

gp96 is a human colonocyte plasma membrane binding protein for Clostridium difficile toxin A.

Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.

Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines.

Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell lines derived from the normal human colon, HCEC, NCM356 and NCM460, were grown for 32-34 days in media containing 25, 50, 75 or 150 nM folic acid, and the expression of genes involved in cell-cycle checkpoints, intracellular signaling, folate uptake and cell adhesion and migration was determined. Expression of Folate Receptor 1 was increased with decreasing media folate in all cell lines, as was p53, p21, p16 and beta-catenin. With decreasing folate, the expression of both E-cadherin and SMAD-4 was decreased in NCM356. APC was elevated in NCM356 but unchanged in the other lines. No changes in global methylation were detected. A significant increase in p53 exon 7-8 strand breaks was observed with decreasing folate in NCM460 cells. The changes observed are consistent with DNA damage-induced activation of cell-cycle checkpoints and cellular adaptation to folate depletion. Folate-depletion-induced changes in the Wnt/APC pathway as well as in genes involved in cell adhesion, migration and invasion may underlie observed relationships between folate status and cancer risk.

Inhibition of growth and induction of differentiation of colon cancer cells by peach and plum phenolic compounds.

The action of extracts from anthocyanin-enriched plums and peaches on growth and differentiation was studied with human colon cancer cells. Growth inhibitory effects were observed in Caco-2, SW1116, HT29 and NCM460 cells. In Caco-2 cells but not in the other cells studied there was evidence for increased differentiation as judged by increased activity of alkaline phosphatase and dipeptidyl peptidase. A differentiating effect on Caco-2 cells was not seen with cyanidin or cyanidin-3-glucoside but the action of the fruit extracts was additive with the action of butyrate and with the MEK1/2 inhibitor U0126. Fractionation using C18 indicated activity resided within a fraction containing anthocyanins but further fractionation using LH-20 suggested that most of the activity was in a fraction containing polyphenols other than anthocyanins. It was concluded that several peach and plum phenolic molecules can influence growth and differentiation in human colon cancer cells.

Induction of differentiation of colon cancer cells by combined inhibition of kinases and histone deacetylase.

The MAP kinase pathway inhibitor U0126 in combination with butyrate promotes differentiation in some colon cancer cell lines. We examined several inhibitors of histone deacetylase (HDAC) in combination with U0126 and other protein kinase inhibitors to see if these effects are general properties of HDAC inhibitors or butyrate alone. Alkaline phosphatase and peptidase activities were examined as markers for cellular differentiation in the human colon cancer cell lines Caco-2 and HT29 and the minimally transformed NCM460. Several HDAC inhibitors caused greater increases of alkaline phosphatase in the cancer cells than in NCM460, in which butyrate was the only HDAC inhibitor that caused a consistent increase. Unlike the JNK and PKC inhibitors examined, the MEK 1/2 inhibitor U0126 induced alkaline phosphatase activity in Caco-2 as a single agent and caused additive effects with HDAC inhibitors. The PI-3 kinase inhibitor LY294002 had little effect alone but enhanced the response of most HDAC inhibitors as did the raf inhibitor GW5074. In addition to butyrate, several HDAC inhibitors can induce differentiation in colon cancer cells and the responses may be enhanced by U0126, GW5074 and LY294002.

Mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells: reversal by non-steroidal anti-inflammatory drugs.

Tumor cells undergo a critical remodeling of intracellular Ca2+ homeostasis that contribute to important cancer hallmarks. Store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca2+-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca2+ due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca2+ uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention.

Upregulation of Cystathionine-ß-Synthase in Colonic Epithelia Reprograms Metabolism and Promotes Carcinogenesis.

The trans-sulfuration enzyme cystathionine-ß-synthase (CBS) and its product hydrogen sulfide (H2S) are aberrantly upregulated in colorectal cancers, where they contribute to tumor growth and progression by both autocrine and paracrine mechanisms. However, it is unknown whether the CBS/H2S axis plays a role in colorectal carcinogenesis. Here, we report upregulation of CBS in human biopsies of precancerous adenomatous polyps and show that forced upregulation of CBS in an adenoma-like colonic epithelial cell line is sufficient to induce metabolic and gene expression profiles characteristic of colorectal cancer cells. Differentially expressed metabolites (65 increased and 20 decreased) clustered into the glycolytic pathway, nucleotide sugars, intermediates of the pentose phosphate pathway, and lipogenesis, including primarily phospholipids, sphingolipids, and bile acids. CBS upregulation induced broad changes in the NCM356 cell transcriptome with over 350 differentially expressed genes. These genes overlapped significantly with gene sets related to glycolysis, hypoxia, and a colon cancer cell phenotype, including genes regulated by NF-κB, KRAS, p53, and Wnt signaling, genes downregulated after E-cadherin knockdown, and genes related to increased extracellular matrix, cell adhesion, and epithelial-to-mesenchymal transition. The CBS-induced switch to an anabolic metabolism was associated with increased NCM356 cell bioenergetics, proliferation, invasion through Matrigel, resistance to anoikis, and CBS-dependent tumorigenesis in immunocompromised mice. Genetic ablation of CBS in CBS heterozygous mice (CBS+/- ) reduced the number of mutagen-induced aberrant colonic crypt foci. Taken together, these results establish that activation of the CBS/H2S axis promotes colon carcinogenesis. Cancer Res; 77(21); 5741-54. ©2017 AACR.

Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells.

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil. For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU. Here, we demonstrated that MCT inhibition using CHC (α-cyano-4-hydroxycinnamic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing. These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.