lithium-chloride and Colorectal-Neoplasms

Cellular plasticity, the ability of cells to switch from an epitheial phenotype to a mesenchymal one and vice versa, plays a crucial role in tumour progression and metastases development. In 20-25% of patients with colon cancer and in 18% of patients with rectal cancer, metastases are present at the time of the first diagnosis. They are the first cause of colorectal cancer (CRC)-related mortality, defining stage IV CRC, which is characterized by a relatively short overall survival. We previously isolated two primary colon adenocarcinoma cell cultures that had undergone epithelial-mesenchymal transition (EMT), one with a high microsatellite instability phenotype (T88) and one with a chromosomal instability phenotype (T93). The aim of this study was to establish a model with which to study EMT, stemness features and cell plasticity in cancer progression and to examine the effects of incubation with lithium chloride (LiCl), a specific glycogen synthase kinase 3 β (GSK-3β) inhibitor, on these cellular processes. Indeed, GSK3β is an important regulator of cell survival, which promotes tumourigenesis in colon cells by facilitating the crosstalk between colorectal cancer pathways. Thus, we further characterized our system of adherent primary mesenchymal colon cancer cells and their paired tumourspheres by examining the expression and localisation of a panel of markers, including E- and N‑cadherin, CD133, CD44v6, aldehyde dehydrogenase 1 (ALDH1) and leucine-rich repeat‑containing G-protein coupled receptor 5 (LGR5). We also characterised the molecular features of these tumourspheres and examined their response to LiCl. Furthermore, we explored the effects of LiCl on cell motility and plasticity. We demonstrated that LiCl reduced cell migration, stemness features and cell plasticity. We also observed the atypical nuclear localisation of membrane proteins, including N‑cadherin, CD133 and CD44v6 in mesenchymal tumour cells. Of note, CD133 and CD44v6 appeared to localise at the plasma membrane in cells with a more epithelial phenotype, suggesting that the cytoplasmic/nuclear localisation of these proteins could favour and characterize cell plasticity in colorectal cancer progression.

Topics: Biomarkers, Tumor; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Plasticity; Colorectal Neoplasms; Disease Progression; Epithelial-Mesenchymal Transition; Genomic Instability; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Mesenchymal Stem Cells; Neoplasm Metastasis; Spheroids, Cellular

Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3β (GSK-3β), a kinase that is involved in several cellular processes related to cancer progression. GSK-3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK-3β-independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment.

Topics: Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HCT116 Cells; HT29 Cells; Humans; Lithium Chloride; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transcriptional Activation

Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP's role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation (p < 0.001) and increased apoptosis (p < 0.01) coincided with 23-fold increase (p < 0.025) in the expression of the NCWP ligand, Wnt9A. LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation.

Topics: Adult; Antimanic Agents; Antineoplastic Agents; Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Colon; Colorectal Neoplasms; Humans; Lithium Chloride; Middle Aged; Rectum; Wnt Proteins; Wnt Signaling Pathway

Many epithelial cancers, particularly gastrointestinal tract cancers, remain poor prognosis diseases, due to resistance to cytotoxic therapy and local or metastatic recurrence. We have previously shown that apoptosis incompetent esophageal cancer cells induce autophagy in response to chemotherapeutic agents and this can facilitate their recovery. However, known pharmacological inhibitors of autophagy could not enhance cytotoxicity. In this study, we have examined two well known, clinically approved autophagy inducers, rapamycin and lithium, for their effects on chemosensitivity in apoptosis incompetent cancer cells. Both lithium and rapamycin were shown to induce autophagosomes in esophageal and colorectal cancer cells by western blot analysis of LC3 isoforms, morphology and FACS quantitation of Cyto-ID or mCherry-GFP-LC3. Analysis of autophagic flux indicates inefficient autophagosome processing in lithium treated cells, whereas rapamycin treated cells showed efficient flux. Viability and recovery was assessed by clonogenic assays. When combined with the chemotherapeutic agent 5-fluorouracil, rapamycin was protective. In contrast, lithium showed strong enhancement of non-apoptotic cell death. The combination of lithium with 5-fluorouracil or oxaliplatin was then tested in the syngenic mouse (balb/c) colorectal cancer model--CT26. When either chemotherapeutic agent was combined with lithium a significant reduction in tumor volume was achieved. In addition, survival was dramatically increased in the combination group (p < 0.0001), with > 50% of animals achieving long term cure without re-occurrence (> 1 year tumor free). Thus, combination treatment with lithium can substantially improve the efficacy of chemotherapeutic agents in apoptosis deficient cancer cells. Induction of compromised autophagy may contribute to this cytotoxicity.

Topics: Animals; Antineoplastic Agents; Autophagy; Cell Line, Tumor; Colorectal Neoplasms; Drug Synergism; Esophageal Neoplasms; Female; Fluorouracil; Genes, Reporter; Humans; Lithium Chloride; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Organoplatinum Compounds; Oxaliplatin; Sirolimus; Transplantation, Heterologous

Activating mutations in the Wnt signaling pathway account for the initiation of greater than 90% of all colorectal cancers and this pathway has been implicated in numerous other diseases. Therefore, identifying small molecule inhibitors of this pathway is of critical importance towards identifying clinically relevant drugs. Numerous screens have been employed to identify therapeutic reagents, but none have made it to advanced clinical trials, suggesting that traditional screening methods are ineffective at identifying clinically relevant targets. Here, we describe a novel in vivo screen to identify small molecule inhibitors of the Wnt pathway. Specifically, treatment of zebrafish embryos with LiCl inhibits GSK3 kinase function, resulting in hyperactivation of the signaling pathway and an eyeless phenotype at 1 day post fertilization. Using the small molecule XAV939, a known inhibitor of Wnt signaling, we rescued the LiCl induced eyeless phenotype, confirming efficacy of the screen. We next tested our assay with 400 known small molecule kinase inhibitors, none of which should inhibit Wnt signaling below the level of GSK3 based on their known targets. Accordingly, none of these small molecules rescued the eyeless phenotype, which demonstrates the stringency of the assay. However, several of these small molecule kinase inhibitors did generate a non-Wnt phenotype in accordance with the kinase they targeted. Therefore, combining the efficacy, sensitivity, and stringency of this preliminary screen, this model will provide an alternative to the traditional in vitro screen, generating potentially clinical relevant drugs in a rapid and cost-effective way.

Topics: Animals; Colorectal Neoplasms; Drug Evaluation, Preclinical; Embryo, Nonmammalian; Embryonic Development; Gene Expression Regulation, Developmental; Glycogen Synthase Kinase 3; Heterocyclic Compounds, 3-Ring; Lithium Chloride; Phenotype; Wnt Signaling Pathway; Zebrafish

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, has been regarded as a potential therapeutic target for multiple human cancers. In addition, oxidative stress is closely related to all aspects of cancer. We sought to determine the biological function of lithium, one kind of GSK-3β inhibitors, in the process of reactive oxygen species (ROS) production in colorectal cancer. In this study, we analyzed the cell apoptosis and proliferation by cell viability, EdU, and flow cytometry assays through administration of LiCl. We used polymerase chain reaction and Western blotting to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Results showed administration of LiCl increased apoptosis and the level of ROS in colorectal cancer cells. Furthermore, the underlying mechanisms could be mediated by the reduction of NF-κB expression and NF-κB-mediated transcription. Taken together, our results demonstrated that therapeutic targeting of ROS/GSK-3β/NF-κB pathways may be an effective way for colorectal cancer intervention, although further preclinical and clinical testing are desirable.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Colorectal Neoplasms; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Inhibitor of Apoptosis Proteins; Lithium Chloride; NF-kappa B; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Survivin

Aberrant activation of the canonical Wnt/beta-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Phopholipase D (PLD) has been implicated in progression of colorectal carcinoma However, an understanding of the targets and regulation of this important pathway remains incomplete and besides, relationship between Wnt signaling and PLD is not known.. Here, we demonstrate that PLD isozymes, PLD1 and PLD2 are direct targets and positive feedback regulators of the Wnt/beta-catenin signaling. Wnt3a and Wnt mimetics significantly enhanced the expression of PLDs at a transcriptional level in HCT116 colorectal cancer cells, whereas silencing of beta-catenin gene expression or utilization of a dominant negative form of T cell factor-4 (TCF-4) inhibited expression of PLDs. Moreover, both PLD1 and PLD2 were highly induced in colon, liver and stomach tissues of mice after injection of LiCl, a Wnt mimetic. Wnt3a stimulated formation of the beta-catenin/TCF complexes to two functional TCF-4-binding elements within the PLD2 promoter as assessed by chromatin immunoprecipitation assay. Suppressing PLD using gene silencing or selective inhibitor blocked the ability of beta-catenin to transcriptionally activate PLD and other Wnt target genes by preventing formation of the beta-catenin/TCF-4 complex, whereas tactics to elevate intracellular levels of phosphatidic acid, the product of PLD activity, enhanced these effects. Here we show that PLD is necessary for Wnt3a-driven invasion and anchorage-independent growth of colon cancer cells.. PLD isozyme acts as a novel transcriptional target and positive feedback regulator of Wnt signaling, and then promotes Wnt-driven anchorage-independent growth of colorectal cancer cells. We propose that therapeutic interventions targeting PLD may confer a clinical benefit in Wnt/beta-catenin-driven malignancies.

Topics: Animals; Base Sequence; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Lithium Chloride; Male; Mice; Neoplasm Invasiveness; Phospholipase D; Promoter Regions, Genetic; Signal Transduction; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein; Transcription, Genetic; Up-Regulation; Wnt Proteins

Inappropriate activation of the Wnt/APC/beta-catenin signaling pathways plays a critical role at early stages in a variety of human cancers. However, their respective implication in tumor cell invasion is still hypothetical. Here, we show that two activators of the canonical Wnt/beta-catenin transcription pathway, namely Dvl-2, the Axin 501-560 fragment binding glycogen synthase kinase -3beta (GSK-3beta), and the negative Wnt regulator wt-Axin did not alter cell invasion into type I collagen. In addition, both Dvl-2 and Axin 501-560 exerted a permissive action on the proinvasive activity of HGF and intestinal trefoil factor. Upstream activation of Wnt signaling by the Wnt-2 and Wnt-3a ligands, stable overexpression of Wnt-2, as well as GSK-3beta inhibition by lithium, SB216763, and GSK-3beta dominant negative forms (K85R and R96E) conferred the invasive phenotype through several proinvasive pathways. Induction of the matrix metalloprotease MMP-7 (matrilysin) gene and protein by Wnt-2 was abolished by inactivation of the AP-1 binding site in the promoter. Accordingly, invasion induced by Wnt-2 was prevented by soluble FRP-3 and FRP-1, sequestration of Gbetagamma subunits, depletion of the GSK-3beta protein by RNA interference, the c-Jun dominant negative mutant TAM67 and was not reversed by wt-Axin. Thus, the proinvasive activity of Wnt-2 is mediated by a noncanonical Wnt pathway using GSK-3beta and the AP-1 oncogene. Our data provide a potential clue for our understanding of the action and crosstalk between Wnt activators and other proinvasive pathways, in relation with matrix substrates and proteases in human cancers.

Topics: Animals; Antibodies, Monoclonal; Axin Protein; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Dogs; Epithelial Cells; Genetic Vectors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Heterotrimeric GTP-Binding Proteins; HT29 Cells; Humans; Indoles; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Neoplasms; Ligands; Lithium Chloride; Maleimides; Matrix Metalloproteinase 7; Neoplasm Invasiveness; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Repressor Proteins; Retroviridae; Signal Transduction; Transcription Factor AP-1; Transcription, Genetic; Wnt Proteins; Wnt2 Protein

Wnt-targeted gene therapy has been proposed as a treatment for human colorectal cancer (CRC). The Cre-Lox system consists of methodology for enhancing targeted expression from tissue-specific or cancer-specific promoters. We analyzed the efficiency of Wnt-specific promoters as drivers of the Cre-mediated activity of a luciferase reporter gene or cell death effector gene in CRC cell lines in the presence and absence of two modulators of Wnt activity, sodium butyrate and lithium chloride. Butyrate is present in the colonic lumen after digestion of fiber-rich foods, whereas the colonic lumen is readily accessible to lithium chloride. In both SW620 and HCT-116 CRC cells, a physiologically relevant concentration of butyrate upregulated reporter and effector activity and altered the Wnt-specific expression pattern. Lithium chloride markedly enhanced Cre-Lox-mediated Wnt-specific reporter expression only in APC wild-type CRC cells. Possibilities for genetic modulation of the proposed CRC therapy included Wnt-specific expression of a floxed Lef1-VP16 fusion that enhanced Wnt-specific cell death and of a floxed dominant-negative Tcf4 that specifically downregulated endogenous Wnt activity. These findings demonstrated that the Cre-Lox system, in combination with pharmacological and genetic modulators, represents effective methodology for enhancing Wnt-targeted gene therapy.

Topics: Apoptosis; Butyrates; Cell Line, Tumor; Colorectal Neoplasms; Cytomegalovirus; Diphtheria Toxin; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation; Genes, Reporter; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; Herpes Simplex Virus Protein Vmw65; Humans; Integrases; Isobutyrates; Lithium Chloride; Luciferases; Luminescent Proteins; Lymphoid Enhancer-Binding Factor 1; Peptide Fragments; Promoter Regions, Genetic; Proto-Oncogene Proteins; Recombinant Fusion Proteins; Transcription Factors; Viral Proteins; Wnt Proteins; Zebrafish Proteins