The Epithelial to Mesenchymal Transition in Colorectal Cancer Progression: The Emerging Role of Succinate Dehydrogenase Alterations and Succinate Accumulation.

Colorectal cancer (CRC) stands as the third most significant contributor to cancer-related mortality worldwide. A major underlying reason is that the detection of CRC usually occurs at an advanced metastatic stage, rendering therapies ineffective. In the progression from the in situ neoplasia stage to the advanced metastatic stage, a critical molecular mechanism involved is the epithelial-to-mesenchymal transition (EMT). This intricate transformation consists of a series of molecular changes, ultimately leading the epithelial cell to relinquish its features and acquire mesenchymal and stem-like cell characteristics. The EMT regulation involves several factors, such as transcription factors, cytokines, micro RNAs and long noncoding RNAs. Nevertheless, recent studies have illuminated an emerging link between metabolic alterations and EMT in various types of cancers, including colorectal cancers. In this review, we delved into the pivotal role played by EMT during CRC progression, with a focus on highlighting the relationship between the alterations of the tricarboxylic acid cycle, specifically those involving the succinate dehydrogenase enzyme, and the activation of the EMT program. In fact, emerging evidence supports the idea that elucidating the metabolic modifications that can either induce or inhibit tumor progression could be of immense significance for shaping new therapeutic approaches and preventative measures. We conclude that an extensive effort must be directed towards research for the standardization of drugs that specifically target proteins such as SDH and SUCNR1, but also TRAP1, PDH, ERK1/2, STAT3 and the HIF1-α catabolism.

Inhibition of Interleukin-6-Induced Matrix Metalloproteinase-2 Expression and Invasive Ability of Lemon Peel Polyphenol Extract in Human Primary Colon Cancer Cells.

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.

Phenotypic Effects of Homeodomain-Interacting Protein Kinase 2 Deletion in Mice.

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates various transcriptional and chromatin regulators, thus modulating numerous important cellular processes, such as proliferation, apoptosis, DNA damage response, and oxidative stress. The role of HIPK2 in the pathogenesis of cancer and fibrosis is well established, and evidence of its involvement in the homeostasis of multiple organs has been recently emerging. We have previously demonstrated that Hipk2-null (Hipk2-KO) mice present cerebellar alterations associated with psychomotor abnormalities and that the double ablation of HIPK2 and its interactor HMGA1 causes perinatal death due to respiratory failure. To identify other alterations caused by the loss of HIPK2, we performed a systematic morphological analysis of Hipk2-KO mice. Post-mortem examinations and histological analysis revealed that Hipk2 ablation causes neuronal loss, neuronal morphological alterations, and satellitosis throughout the whole central nervous system (CNS); a myopathic phenotype characterized by variable fiber size, mitochondrial proliferation, sarcoplasmic inclusions, morphological alterations at neuromuscular junctions; and a cardiac phenotype characterized by fibrosis and cardiomyocyte hypertrophy. These data demonstrate the importance of HIPK2 in the physiology of skeletal and cardiac muscles and of different parts of the CNS, thus suggesting its potential relevance for different new aspects of human pathology.

A Potential Role of IL-6/IL-6R in the Development and Management of Colon Cancer.

Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second greatest cause of cancer deaths. About 75% of all CRCs are sporadic cancers and arise following somatic mutations, while about 10% are hereditary cancers caused by germline mutations in specific genes. Several factors, such as growth factors, cytokines, and genetic or epigenetic alterations in specific oncogenes or tumor-suppressor genes, play a role during the adenoma-carcinoma sequence. Recent studies have reported an increase in interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) levels in the sera of patients affected by colon cancer that correlate with the tumor size, suggesting a potential role for IL-6 in colon cancer progression. IL-6 is a pleiotropic cytokine showing both pro- and anti-inflammatory roles. Two different types of IL-6 signaling are known. Classic IL-6 signaling involves the binding of IL-6 to its membrane receptor on the surfaces of target cells; alternatively, IL-6 binds to sIL-6R in a process called IL-6 trans-signaling. The activation of IL-6 trans-signaling by metalloproteinases has been described during colon cancer progression and metastasis, involving a shift from membrane-bound interleukin-6 receptor (IL-6R) expression on the tumor cell surface toward the release of soluble IL-6R. In this review, we aim to shed light on the role of IL-6 signaling pathway alterations in sporadic colorectal cancer and the development of familial polyposis syndrome. Furthermore, we evaluate the possible roles of IL-6 and IL-6R as biomarkers useful in disease follow-up and as potential targets for therapy, such as monoclonal antibodies against IL-6 or IL-6R, or a food-based approach against IL-6.

Implications of Splicing Alterations in the Onset and Phenotypic Variability of a Family with Subclinical Manifestation of Peutz-Jeghers Syndrome: Bioinformatic and Molecular Evidence.

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant pre-cancerous disorder caused in 80-90% of cases by germline mutations in the tumor suppressor gene STK11. We performed a genetic test of the STK11 gene in two Italian young sisters suspected of PJS, since they showed pathognomonic café au lait spots in absence of other symptoms and familiarity. Sequencing of all exons of STK11 gene and other 8 genes, suggested to be involved in hamartomatous syndromes, (PTEN, BMPR1A, SDHB, SDHD, SMAD4, AKT1, ENG, PIK3CA) led to the identification in both the probands of a novel germline silent mutation named c.597 G>A, hitting the last nucleotide of exon 4. Interestingly, genetic testing of the two probands' parents showed that their unaffected father was carrier of this mutation. Moreover, he carried a second intronic substitution named c.465-51 T>C (rs2075606) which was not inherited by his daughters. We also observed that all the family members carrying the c.597 G>A mutation presented an aberrant splice variant of STK11 mRNA lacking exon 4. Furthermore, in silico analysis of c.465-51 T>C substitution showed that it may activate an Enhancer Splicing Element. Finally, qRT-PCR analysis of STK11 expression levels showed a slight downregulation of the wild type allele in the father and a 2-fold downregulation in the probands compared to the unaffected mother. Our results have led the hypothesis that the c.465-51 T>C intronic variant, which segregates with the wild type allele, could increase the splicing effectiveness of STK11 wild-type allele and compensate the side effect of the c.597 G>A splicing mutation, being responsible for the phenotypic variability observed within this family. This finding highlight the importance of RNA analysis in genetic testing, remarking that silent DNA variant can often be splicing variant involved in disease onset and progression. The identification of these variants has a crucial role to ensure an appropriate follow-up and cancer prevention in at-risk individuals.

Nitrodi thermal water downregulates protein S­nitrosylation in RKO cells.

Balneotherapy and spa therapy have been used in the treatment of ailments since time immemorial. Moreover, there is evidence to suggest that the beneficial effects of thermal water continue for months following the completion of treatment. The mechanisms through which thermal water exerts its healing effects remain unknown. Both balneological and hydroponic therapy at 'the oldest spa in the world', namely, the Nitrodi spring on the Island of Ischia (Southern Italy) are effective in a number of diseases and conditions. The aim of the present study was to investigate the molecular basis underlying the therapeutic effects of Nitrodi spring water in low­grade inflammation and stress­related conditions. For this purpose, an in vitro model was devised in which RKO colorectal adenocarcinoma cells were treated with phosphate­buffered saline or phosphate­buffered saline prepared with Nitrodi water for 4 h daily, 5 days a week for 6 weeks. The RKO cells were then subjected to the following assays: 3­(4,5­Dimethylthiazol­2­yl)­2,5­diphenyl­2H­tetrazolium bromide assay, Transwell migration assay, western blot analysis, the fluorimetric detection of protein S­nitrosothiols and S­nitrosylation western blot analysis. The results revealed that Nitrodi spring water promoted cell migration and cell viability, and downregulated protein S­nitrosylation, probably also the nitrosylated active form of the cyclooxygenase (COX)­2 protein. These results concur with all the previously reported therapeutic properties of Nitrodi spring water, and thus reinforce the concept that this natural resource is an important complementary therapy to traditional medicine.

Sporadic pediatric severe familial adenomatous polyposis: A case report.

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary precancerous condition caused by germline pathogenetic variants in the tumor suppressor adenomatous polyposis coli (APC) gene. Patients with FAP develop multiple gastrointestinal adenomatous polyps usually at the age of ~20 years, which, if untreated, become cancerous in 100% of cases. Genotype-phenotype associations have been extensively described; however, inter- and intra-familial variability exists. It is crucial to characterize the causative pathogenetic variant in each pedigree in order to develop a cancer prevention program and follow-up strategy for at-risk families. The present report describes a severe case of sporadic FAP that was diagnosed when the patient was ~2 years old. The patient was a carrier of the de novo pathogenic c.4132 C>T (p.Gln1378X) variant. Additionally, the patient was a carrier of the homozygous c.5465 T>A (p.Asp1822Val) polymorphism, inherited from both parents. However, it remains unclear whether or not this polymorphism is involved in the phenotypic manifestation. This case highlights the need to extend molecular screening to very young children when they show iron-deficiency, anaemia and/or rectal bleeding, even in the absence of a familial history of disease.

Lithium chloride increases sensitivity to photon irradiation treatment in primary mesenchymal colon cancer cells.

Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. It is also the second most common cause of cancer­associated mortality; it accounted for about 9.2% of all cancer deaths in 2018, most of which were due to resistance to therapy. The main treatment for CRC is surgery, generally associated with chemotherapy, radiation therapy and combination therapy. However, while chemo­radiotherapy kills differentiated cancer cells, mesenchymal stem­like cells are resistant to this treatment, and this can give rise to therapy­resistant tumors. Our previous study isolated T88 primary colon cancer cells from a patient with sporadic colon cancer. These cells exhibited mesenchymal and epithelial features, high levels of epithelial­to­mesenchymal transition transcription factors, and stemness markers. In addition, it was revealed that lithium chloride (LiCl), a specific glycogen synthase kinase (GSK)­3ß inhibitor, induced both the mesenchymal­to­epithelial transition and differentiation, and also reduced cell migration, stemness features and cell plasticity in these primary colon cancer cells. The aim of the present study was to investigate the effect of LiCl treatment on the viability of primary colon cancer cells exposed to 7 Gy delivered by high­energy photon beams, which corresponds to 6 megavolts of energy. To achieve this aim, the viability of irradiated T88 cells was compared with that of irradiated T88 cells pre­treated with LiCl. As expected, it was observed that LiCl sensitized primary colon cancer cells to high­energy photon irradiation treatment. Notably, the decrease in cell viability was greater with combined therapy than with irradiation alone. To explore the molecular basis of this response, the effect of LiCl on the expression of Bax, p53 and Survivin, which are proteins involved in the apoptotic mechanism and in death escape, was analyzed. The present study revealed that LiCl upregulated the expression of pro­apoptotic proteins and downregulated the expression of proteins involved in survival. These effects were enhanced by high­energy photon irradiation, suggesting that LiCl could be used to sensitize colon cancer cells to radiation therapy.

Promising Colorectal Cancer Biomarkers for Precision Prevention and Therapy.

Colorectal cancer (CRC) has been ranked as the third most prevalent cancer worldwide. Indeed, it represents 10.2% of all cancer cases. It is also the second most common cause of cancer mortality, and accounted for about 9.2% of all cancer deaths in 2018. Early detection together with a correct diagnosis and staging remains the most effective clinical strategy in terms of disease recovery. Thanks to advances in diagnostic techniques, and improvements of surgical adjuvant and palliative therapies, the mortality rate of CRC has decreased by more than 20% in the last decade. Cancer biomarkers for the early detection of CRC, its management, treatment and follow-up have contributed to the decrease in CRC mortality. Herein, we provide an overview of molecular biomarkers from tumor tissues and liquid biopsies that are approved for use in the CRC clinical setting for early detection, follow-up, and precision therapy, and of biomarkers that have not yet been officially validated and are, nowadays, under investigation.

Mesenchymal Stem/Stromal Cells in Stromal Evolution and Cancer Progression.

The study of cancer biology has mainly focused on malignant epithelial cancer cells, although tumors also contain a stromal compartment, which is composed of stem cells, tumor-associated fibroblasts (TAFs), endothelial cells, immune cells, adipocytes, cytokines, and various types of macromolecules comprising the extracellular matrix (ECM). The tumor stroma develops gradually in response to the needs of epithelial cancer cells during malignant progression initiating from increased local vascular permeability and ending to remodeling of desmoplastic loosely vascularized stromal ECM. The constant bidirectional interaction of epithelial cancer cells with the surrounding microenvironment allows damaged stromal cell usage as a source of nutrients for cancer cells, maintains the stroma renewal thus resembling a wound that does not heal, and affects the characteristics of tumor mesenchymal stem/stromal cells (MSCs). Although MSCs have been shown to coordinate tumor cell growth, dormancy, migration, invasion, metastasis, and drug resistance, recently they have been successfully used in treatment of hematopoietic malignancies to enhance the effect of total body irradiation-hematopoietic stem cell transplantation therapy. Hence, targeting the stromal elements in combination with conventional chemotherapeutics and usage of MSCs to attenuate graft-versus-host disease may offer new strategies to overcome cancer treatment failure and relapse of the disease.

Clinical relevance of thyroid cell models in redox research.

BACKGROUND: Thyroid-derived cell models are commonly used to investigate the characteristics of thyroid cancers. It is noteworthy that each in vitro single cell model system imitates only a few characteristics of thyroid cancer depending on e.g. source of cells or oncogene used to transform the cells. METHODS: In the current work we utilized rat thyroid cancer cell models to determine their clinical relevance in redox gene studies by comparing in vitro expression data to thyroid Oncomine microarray database. To survey the cell lines we analyzed mRNA expression of genes that produce superoxide anion (nox family), genes that catalyze destruction of superoxide anion to hydrogen peroxide (sod family), and genes that remove hydrogen peroxide from cellular environment (catalase, gpx family and prdx family). RESULTS: Based on the current results, rat thyroid PC Cl3, PC PTC1, PC E1A, or FRLT5 cell models can be used to study NOX2, NOX4, SOD2, SOD3, CATALASE, GPX1, GPX2, GPX5, PRDX2, and PRDX3 gene expression and function. CONCLUSIONS: Redox gene expression in rat originated single cell model systems used to study human thyroid carcinogenesis corresponds only partly with human redox gene expression, which may be caused by differences in redox gene activation stimulus. The data suggest careful estimation of the data observed in rat thyroid in vitro models.

Ras oncogene-mediated progressive silencing of extracellular superoxide dismutase in tumorigenesis.

Extracellular superoxide dismutase (SOD3) is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reaction that results in the formation of hydrogen peroxide. Both of these reactive oxygen species affect growth signaling in cells. Although SOD3 has growth-supporting characteristics, the expression of SOD3 is downregulated in epithelial cancer cells. In the current work, we studied the mechanisms regulating SOD3 expression in vitro using thyroid cell models representing different stages of thyroid cancer. We demonstrate that a low level of RAS activation increases SOD3 mRNA synthesis that then gradually decreases with increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells. Our data indicate that SOD3 regulation can be divided into two classes. The first class involves RAS-driven reversible regulation of SOD3 expression that can be mediated by the following mechanisms: RAS GTPase regulatory genes that are responsible for SOD3 self-regulation; RAS-stimulated p38 MAPK activation; and RAS-activated increased expression of the mir21 microRNA, which inversely correlates with sod3 mRNA expression. The second class involves permanent silencing of SOD3 mediated by epigenetic DNA methylation in cells that represent more advanced cancers. Therefore, the work suggests that SOD3 belongs to the group of ras oncogene-silenced genes.

Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3-induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3-driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.