PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells.

Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3ß (GSK-3ß), a kinase that is involved in several cellular processes related to cancer progression. GSK-3ß 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-3ß-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.

Progeny from irradiated colorectal cancer cells acquire an EMT-like phenotype and activate Wnt/ß-catenin pathway.

Radiotherapy remains a major approach to adjuvant therapy for patients with advanced colorectal cancer, however, the fractionation schedules frequently allow for the repopulation of surviving tumors cells, neoplastic progression, and subsequent metastasis. The aim of the present study was to analyze the transgenerational effects induced by radiation and evaluate whether it could increase the malignant features on the progeny derived from irradiated parental colorectal cancer cells, Caco-2, HT-29, and HCT-116. The progeny of these cells displayed a differential radioresistance as seen by clonogenic and caspase activation assay and had a direct correlation with survivin expression as observed by immunoblotting. Immunofluorescence showed that the most radioresistant progenies had an aberrant morphology, disturbance of the cell-cell adhesion contacts, disorganization of the actin cytoskeleton, and vimentin filaments. Only the progeny derived from intermediary radioresistant cells, HT-29, reduced the E-cadherin expression and overexpressed ß-catenin and vimentin with increased cell migration, invasion, and metalloprotease activation as seen by immunoblotting, wound healing, invasion, and metalloprotease activity assay. We also observed that this most aggressive progeny increased the Wnt/ß-catenin-dependent TCF/LEF activity and underwent an upregulation of mesenchymal markers and downregulation of E-cadherin, as determined by qRT-PCR. Our results showed that the intermediate radioresistant cells can generate more aggressive cellular progeny with the EMT-like phenotype. The Wnt/ß-catenin pathway may constitute an important target for new adjuvant treatment schedules with radiotherapy, with the goal of reducing the migratory and invasive potential of the remaining cells after treatment.

Ouabain-induced alterations of the apical junctional complex involve α1 and ß1 Na,K-ATPase downregulation and ERK1/2 activation independent of caveolae in colorectal cancer cells.

Studies have reported that Na,K-ATPase interacts with E-cadherin to stabilize (AJs) and regulate the expression of claudins, the main proteins present in the tight junction (TJ) in epithelial cells containing caveolae. However, the role of this ATPase in the regulation of the AJ and TJ proteins in colorectal cancer cells as well as the molecular events underlying this event in a caveolae-independent system remain undefined. In the present study, we used ouabain, a classic drug known to inhibit Na,K-ATPase, and Caco-2 cells, which are a well-established human colorectal cancer model that does not exhibit caveolae. We demonstrated that ouabain treatment resulted in a reduction of the ß1 Na,K-ATPase protein and cell redistribution of the AJ proteins E-cadherin and ß-catenin, as well as the α1 Na,K-ATPase subunit. Furthermore, ouabain increased claudin-3 protein levels, impaired the TJ barrier function and increased cell viability and proliferation during the early stages of treatment. Additionally, the observed ouabain-induced events were dependent on the activation of ERK1/2 signaling; but in contrast to previous studies, this signaling cascade was caveolae-independent. In conclusion, our findings strongly suggest that α1 and ß1 Na,K-ATPase downregulation and ERK1/2 activation induced by ouabain are interlinked events that play an important role during cell-cell adhesion loss, which is an important step during the tumor progression of colorectal carcinomas.

Crosstalk between PI3K/Akt and Wnt/ß-catenin pathways promote colorectal cancer progression regardless of mutational status.

The PI3K/Akt and Wnt/ß-catenin pathways play an important role in the acquisition of the malignant phenotype in cancer. However, there are few data regarding the role of the interplay between both pathways in colorectal cancer (CRC) progression. The mutational status and the clinicopathological characteristics of PI3K/Akt and Wnt/ß-catenin pathways were accessed by bioinformatic analysis whereas that the impact of the interplay between the activity of both pathways to explain tumorigenic potential was performed in vitro using IGF-1 and Wnt3a treatments in CRC cell models. The mutational status of these pathways did not influence the survival of CRC patients, but an association between clinicopathological characteristics in patients with mutations in one, but not in both pathways was observed. A potentiating effect on the activation of both pathways and enhanced cellular migration and proliferation was observed when both pathways were activated simultaneously with IGF-1 and Wnt3a. In addition, these effects were hindered after pretreatment with LY294002, a specific PI3K inhibitor, suggesting some dependence between these two signaling cascades. Our findings show that, regardless of mutational status, there is an interplay between the activity of PI3K/Akt and Wnt/ß-catenin pathways that contributes to events related to CRC progression and that the reversal of such events using a PI3K inhibitor highlights the value of targeting these pathways for potential directed therapies in CRC patients.

Docosahexaenoic acid promotes cell cycle arrest and decreases proliferation through WNT/ß-catenin modulation in colorectal cancer cells exposed to γ-radiation.

The effects of radiation are known to be potentiated by N-3 polyunsaturated fatty acids, which modulate several signaling pathways, but the molecular mechanisms through which these fatty acids enhance the anticancer effects of irradiation in colorectal cancer (CRC) treatment remain poorly elucidated. Here, we aimed to ascertain whether the fatty acid docosahexaenoic acid (DHA) exerts a modulating effect on the response elicited by radiation treatment (RT). Two CRC cell lines, Caco-2 and HT-29, were exposed to RT, DHA, or both (DHA + RT) for various times, and then cell viability, proliferation, and clonogenicity were assessed. Moreover, cell cycle, apoptosis, and necrosis were analyzed using flow cytometry, and the involvement of WNT/ß-catenin signaling was investigated by immunofluorescence to determine nuclear ß-catenin, GSK3ß phosphorylation status, and TCF/LEF-activity reporter. DHA and RT applied separately diminished the viability of both HT-29 and Caco-2 cells, and DHA + RT caused a further reduction in proliferation mainly in HT-29 cells, particularly in terms of colony formation. Concomitantly, our results verified cell cycle arrest in G0/G1 phase, a reduction of cyclin D1 expression, and a decrease in GSK3ß phosphorylation after the combined treatment. Furthermore, immunofluorescence quantification revealed that nuclear ß-catenin was increased in RT-exposed cells, but this effect was abrogated in cells exposed to DHA + RT, and the results of TCF/LEF-activity assays confirmed that DHA attenuated the increase in nuclear ß-catenin activity induced by irradiation. Our finding shows that DHA applied in combination with RT enhanced the antitumor effects of irradiation on CRC cells, and that the underlying mechanism involved the WNT/ß-catenin pathway. © 2018 BioFactors, 45(1):24-34, 2019.

Blockade of irradiation-induced autophagosome formation impairs proliferation but does not enhance cell death in HCT-116 human colorectal carcinoma cells.

This work was undertaken to gain further information on the molecular mechanisms underlying autophagosome formation and its relation with tumor cell survival in response to radiation in colon cancer. A human colon cancer cell line, HCT-116, was examined with respect to cell survival after blockade of irradiation-induced autophagosome formation by pharmacological interference. Autophagosome formation was confirmed using a kinetic study with incorporated bovine serum albumin gold-conjugate (BSA-Au) analyzed by electron microscopy and an autophagosome-associated LC3B antibody measured by immunofluorescence and Western blotting. Annexin V/PI double staining was used to monitor cell death by apoptosis, and cell cycle profiles by flow cytometry. Ionizing radiation (IR) promoted autophagosome formation in the HCT-116 IR-surviving cells. Pharmacological interference showed that PI3K/Akt and Src were involved in early stages of autophagosome formation. IR alone decreased cell proliferation by arresting cells in the G2/M phase, and pharmacological interference of autophagosome formation decreased proliferation, but did not affect cell survival. Also, our data suggest that decreased proliferation caused by PI3K and Src inhibitors could be through S phase cell cycle delay. Our results clearly indicate that blockade of IR-induced autophagosome formation impairs proliferation but does not enhance cell death in colon cancer cells.

Lithium reduces tumorigenic potential in response to EGF signaling in human colorectal cancer cells.

Lithium is a specific inhibitor of GSK3-ß, and hence, an activator of the Wnt/ß-catenin pathway, whereas the epidermal growth factor (EGF) has been linked to malignant transformation in epithelial cancer cells. Both pathways are aberrantly activated in most colorectal cancers (CRCs). However, the relationship between them in modulating events related to the progression of this cancer type remains to be defined. In this study, we investigated whether the Wnt/ß-catenin and EGFR pathways converge to modulate the malignant potential of CRC. We used Caco-2 cells, a well-established model used to study CRC, and treatments with lithium chloride, as a modulator of the Wnt/ß-catenin pathway, and with EGF as an inducer of EGFR signaling. We found that both agents altered the subcellular distribution of claudin-1 and ß-catenin, two important proteins of the apical junctional complex, but not their abundance in the cell. Nuclear stabilization of ß-catenin, a marker of Wnt pathway activation, was observed after treatment with both compounds. However, lithium, but not EGF, inhibited GSK3-ß, indicating that these agents modulate this enzyme in a differential fashion. Furthermore, EGF treatment increased the proliferative and migratory capacity but did not alter the colony formation potential of these cells. Surprisingly, lithium, known to activate the Wnt/ß-catenin pathway, inhibited the increased proliferation by arresting cells in the G2/M phase as well as the cell migration promoted by EGF, as demonstrated by the combined treatment with these agents. Lithium treatment alone reduced the cell colony formation. Thus, our findings suggest that lithium plays an important role in regulating cellular events related to tumor progression in CRC.

Inhibition of N-linked glycosylation by tunicamycin induces E-cadherin-mediated cell-cell adhesion and inhibits cell proliferation in undifferentiated human colon cancer cells.

PURPOSE: Aberrant protein glycosylation and disassembly of E-cadherin-mediated cell-cell adhesion are characteristics of epithelial cancer. However, the relationship between these two events in colorectal cancer remains to be defined. In this study, we analyzed whether N-glycan expression is crucial for the loss of E-cadherin-mediated cell-cell adhesion in human colorectal cancer cells. METHODS: Differentiated Caco-2 and undifferentiated HCT-116 colon cancer cells were used as models of stable and unstable adherens junctions (AJs), respectively. Complex-type N-glycans were detected using the lectins E-PHA (Phaseolus vulgaris E.) and L-PHA (Phaseolus vulgaris L.). To study E-cadherin-mediated AJ assembly, we examined the effects of swainsonine, an inhibitor of α-mannosidase II, and tunicamycin, a drug that inhibits the biosynthesis of N-glycans, via western blot, immunofluorescence, differential extraction in Triton X-100, and electron microscopy. Cell proliferation and apoptosis were examined by crystal violet staining and flow cytometry, respectively. RESULTS: We observed positive labeling for E-PHA and L-PHA lectins in both cell lines; however, HCT-116 cells had increased E-cadherin-linked complex-type N-glycans. Interestingly, tunicamycin, but not swainsonine, was able to induce functional E-cadherin-mediated cell-cell adhesion in undifferentiated HCT-116 cells, as shown by the increased association of E-cadherin with the actin cytoskeleton. Moreover, in HCT-116 cells, tunicamycin also induced the formation of tight cell-cell contacts, and it inhibited cell proliferation without triggering apoptosis. CONCLUSIONS: Collectively, our results demonstrate for the first time that altered N-glycan expression plays an important role in the loss of AJ stability in undifferentiated colorectal cancer cells and that this loss may be associated with the progression of colorectal cancer.

PI3K/Akt and GSK-3ß prevents in a differential fashion the malignant phenotype of colorectal cancer cells.

PURPOSE: During colorectal cancer progression, the loss of differentiation and cell-cell adhesion as well as a higher migratory potential are well-defined features; however, the signaling mechanism governing these events is not fully elucidated. The aim of this study was to investigate the role that PI3K and downstream effectors play in controlling colon cancer malignant phenotypes. METHODS: HCT-116 cells, a human model of colon cancer, which are highly metastatic and undifferentiated, were treated with LY294002, a specific inhibitor of PI3K. Cell differentiation and apical junctional complex (AJC) formation were monitored using alkaline phosphatase and electron microscopy analysis. Immunofluorescence and Western blotting were used to accompany the subcellular localization of AJC proteins. PI3K downstream molecules were analyzed by western blotting, and proliferation, wound healing, and colony formation techniques to determine malignant phenotype alterations. RESULTS: PI3K inhibition increased alkaline phosphatase activity, led to an enterocyte-like growth and formed a functional AJC. LY294002 treatment was able to recruit E-cadherin, ß-catenin, claudin-3, and ZO-1 to the cell-cell contact region, and this effect was essential for AJC assembly and association of these proteins to the cytoskeleton. Furthermore, we provided evidence that PI3K inhibition leads to a decrease in p-Akt and p-GSK-3ß and increased p-ß-catenin levels, which in turn controlled cell proliferation, motility, and colony formation. CONCLUSION: Our results demonstrate that PI3K/Akt and GSK-3ß prevents in a differential fashion the malignant phenotype of HCT-116 colorectal cancer cells, which could constitute a potential therapeutic target for treatment of this cancer type.