How do prolonged anchorage-free lifetimes strengthen non-small-cell lung cancer cells to evade anoikis? - A link with altered cellular metabolomics.

BACKGROUND: Malignant cells adopt anoikis resistance to survive anchorage-free stresses and initiate cancer metastasis. It is still unknown how varying periods of anchorage loss contribute to anoikis resistance, cell migration, and metabolic reprogramming of cancerous cells. RESULTS: Our study demonstrated that prolonging the anchorage-free lifetime of non-small-cell lung cancer NCI-H460 cells for 7 days strengthened anoikis resistance, as shown by higher half-life and capability to survive and grow without anchorage, compared to wild-type cells or those losing anchorage for 3 days. While the prolonged anchorage-free lifetime was responsible for the increased aggressive feature of survival cells to perform rapid 3-dimensional migration during the first 3 h of a transwell assay, no significant influence was observed with 2-dimensional surface migration detected at 12 and 24 h by a wound-healing method. Metabolomics analysis revealed significant alteration in the intracellular levels of six (oxalic acid, cholesterol, 1-ethylpyrrolidine, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, ß-alanine, and putrescine) among all 37 identified metabolites during 7 days without anchorage. Based on significance values, enrichment ratios, and impact scores of all metabolites and their associated pathways, three principal metabolic activities (non-standard amino acid metabolism, cell membrane biosynthesis, and oxidative stress response) offered potential links with anoikis resistance. CONCLUSIONS: These findings further our insights into the evolution of anoikis resistance in lung cancer cells and identify promising biomarkers for early lung cancer diagnosis.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles.

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines.

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.

Stable and high expression of Galectin-8 tightly controls metastatic progression of prostate cancer.

Two decades ago, Galectin-8 was described as a prostate carcinoma biomarker since it is only expressed in the neoplastic prostate, but not in the healthy tissue. To date, no biological function has been attributed to Galectin-8 that could explain this differential expression. In this study we silenced Galectin-8 in two human prostate cancer cell lines, PC3 and IGR-CaP1, and designed a pre-clinical experimental model that allows monitoring the pathology from its early steps to the long-term metastatic stages. We show for the first time that the natural and conserved expression of Gal-8 in tumour cells is responsible for the metastatic evolution of prostate cancer. In fact, Gal-8 controls the rearrangement of the cytoskeleton and E-Cadherin expression, with a major impact on anoikis and homotypic aggregation of tumour cells, both being essential processes for the survival of circulating tumour cells during metastasis. While localized prostate cancer can be cured, metastatic and advanced disease remains a significant therapeutic challenge, urging for the identification of prognostic markers of the metastatic process. Collectively, our results highlight Galectin-8 as a potential target for anti-metastatic therapy against prostate cancer.

Timp1 Promotes Cell Survival by Activating the PDK1 Signaling Pathway in Melanoma.

High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and ß1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generated under phosphatidylinositol-3-kinase (PI3K) activation, enables the recruitment and activation of protein kinase B (PKB/AKT) and phosphoinositide-dependent kinase 1 (PDK1) at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC) inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased anoikis resistance, clonogenicity, dacarbazine resistance, and in vivo tumor growth and lung colonization. In metastatic cells, pAKTThr308 is highly expressed, contributing to anoikis resistance. We showed that PDK1Ser241 and PKCßIISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.

HaCaT anchorage blockade leads to oxidative stress, DNA damage and DNA methylation changes.

Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.