Effect of ZEB1 Associated with microRNAs on Tumor Stem Cells in Head and Neck Cancer.

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

Use of histone methyltransferase inhibitors in cancer treatment: A systematic review.

Histone modifications are an epigenetic mechanism, and the dysregulation of these proteins is known to be associated with the initiation and progression of cancer. In the search for the development of new and more effective drugs, histone modifications were identified as possible therapeutic targets. Histone methyltransferase (HMT) inhibitors correspond to the third generation of epigenetic drugs capable of writing or deleting epigenetic information. This systematic review summarized the development and prospect for the use of different HMT inhibitors in cancer therapy. An electronic search was applied across CENTRAL, Clinical Trials, Embase, LILACS, LIVIVO, Open Gray, PubMed, Scopus, and Web of Science. Based on the title and abstracts, two authors independently selected eligible studies. After the complete reading of the articles, based on the eligibility criteria, 11 studies were included in the review. Different inhibitors of HMT have been explored in multiple clinical studies, and have shown considerable anti-tumor effects. However, few phase 2 studies have been completed and/or have available results. The most advanced clinical trials mainly include tazemetostat, an Enhancer of zeste homolog 2 (EZH2) inhibitor approved for follicular lymphoma (FL). The use of HMT inhibitors has presented, so far, concise results in the treatment of hematological cancers, moreover, the adverse effects presented after the use of these medicines (alone or in combination) did not show a high level of risk for the patient. These findings, in addition to ongoing clinical studies, can represent a promising future regarding the use of HMT inhibitors in treating different types of cancer.

Treatment effects of the EGFR pathway drugs on head and neck cancer stem cells.

(1) Head and neck cancer (HNC) is the sixth most common cancer worldwide and show low survival rates and drug resistance, which can be due to the presence of cancer stem cells (CSCs), a small cell population with metastatic potential, invasion and self-renewal ability. (2) Here, seven tumor cells were sorted as CD44+/CD117+/CD133+ or ALDH+, considered as HNC stem cells (HNCSCs), and as CD44-/CD117-/CD133- or ALDH-, considered non-HNCSCs after both cells sorted criteria was compared to evaluate cell migration, invasion, and colony forming assays. These subpopulations were treated with Cetuximab, Paclitaxel, or a combination of both drugs and evaluated for cell viability. Quantitative PCR and western blot were performed to evaluate EGFR, TRKB, KRAS and HIF-1α gene and protein expression. (3) HNCSCs presented more colonies and appeared to be more sensitive to the drug combination when compared with non-HNCSCs, regardless cells sorted criteria and primary tumor subsite. The EGFR, TRKB, KRAS and HIF-1α genes and proteins were upregulated in CSCs compared with non-HNCSCs, thus explaining the drug resistance. (4) This study contributes to the better development of specific therapeutic protocols based on Cetuximab and Paclitaxel drugs in the treatment of HNC in the presence of CSCs and cell proliferation biomarkers.

Regulation of VEGFA, KRAS, and NFE2L2 Oncogenes by MicroRNAs in Head and Neck Cancer.

Mutations and alterations in the expression of VEGFA, KRAS, and NFE2L2 oncogenes play a key role in cancer initiation and progression. These genes are enrolled not only in cell proliferation control, but also in angiogenesis, drug resistance, metastasis, and survival of tumor cells. MicroRNAs (miRNAs) are small, non-coding regulatory RNA molecules that can regulate post-transcriptional expression of multiple target genes. We aimed to investigate if miRNAs hsa-miR-17-5p, hsa-miR-140-5p, and hsa-miR-874-3p could interfere in VEGFA, KRAS, and NFE2L2 expression in cell lines derived from head and neck cancer (HNC). FADU (pharyngeal cancer) and HN13 (oral cavity cancer) cell lines were transfected with miR-17-5p, miR-140-5p, and miR-874-3p microRNA mimics. RNA and protein expression analyses revealed that miR-17-5p, miR-140-5p and miR-874-3p overexpression led to a downregulation of VEGFA, KRAS, and NFE2L2 gene expression in both cell lines analyzed. Taken together, our results provide evidence for the establishment of new biomarkers in the diagnosis and treatment of HNC.

Association between folate metabolism polymorphisms and breast cancer: a case-control study.

We investigated the association between methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthetase (MTR A2756G), and methionine synthase reductase (MTRR A66G) polymorphisms involved in folate pathway and breast cancer risk, and the interaction between these polymorphisms and tobacco and alcohol consumption. Furthermore, we evaluated the association between these polymorphisms and clinicopathological variables. This case-control study included 606 Brazilian women, comprising 128 patients with breast cancer and 478 controls. MTHFR and MTR polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and MTRR polymorphisms using real-time PCR. Age ≥50 years (odds ratio [OR]: 2.65; 95% confidence interval [CI]: 1.65-4.26; p<0.001) and alcohol consumption (OR: 1.76; 95% CI: 1.0-2.85; p=0.021) were associated with an increased risk of breast cancer. For MTHFR A1298C, we observed a reduced risk of developing breast cancer in the codominant model (genotype CC-OR: 0.22; 95% CI: 0.06-0.74; p=0.014), recessive model (OR: 0.22; 95% CI: 0.07-0.76 p=0.004), and log-additive model (OR: 0.70; 95% CI: 0.49-0.98; p=0.035). Women aged ≥50 years and those who are alcohol consumers had increased susceptibility to breast cancer, and MTHFR A1298C modulated the risk for this disease. This is the first study to evaluate the association between polymorphisms in folate metabolism and breast cancer in the northwest region of São Paulo State, Brazil.

Evaluation of molecular markers GSTM1 and GSTT1 and clinical factors in breast cancer: case-control study and literature review.

The study was conducted to evaluate the frequency of polymorphisms in GSTM1 and GSTT1 genes in patients with breast cancer compared with individuals without history of cancer, and the association of these polymorphisms with clinical/epidemiological parameters.There were evaluated 752 women (219 patients and 533 controls). Molecular analysis was performed by the Polymerase Chain Reaction (PCR). Statistical analysis was used multiple logistic regression and descriptive statistics.Age ≥ 50 years (OR = 3.22, 95% CI = 2.30-4.51, p < 0.001) and alcohol consumption (OR = 1.60, 95% CI = 1.13-2.27, p = 0.008) were associated to the development of breast cancer, while smoking and null genotypes GSTM1 and GSTT1 presented no association. GSTM1 and GSTT1 polymorphisms presented no relationship with the clinical and histopathological parameters or molecular subtypes of breast cancer. Ninety-two percent of tumours were invasive ductal, 66% were grade II, 65% were larger than 2 cm, the stages II (35.3%) and III (31.2%) were the most prevalent, and 47.7% were molecular subtype luminal B.Individuals aged ≥ 50 years and alcohol consumers have more chance to developing breast cancer. GSTM1 and GSTT1 polymorphisms are not associated to the risk of breast cancer.

Polymorphisms in xenobiotic metabolism-related genes in patients with hepatocellular carcinoma: a case-control study.

This study was performed to investigate the relationship between polymorphisms in microsomal epoxide hydrolase (mEH; Tyr113His and His139Arg substitution) and glutathione S-transferase (GST; GSTM1 deletion, GSTT1 deletion, and GSTP1.Ala114Val substitution) and their correlation with clinico-histopathological features in hepatocellular carcinoma (HCC).We evaluated environmental risk factors and genetic alterations in 556 individuals (86 cases and 470 controls). PCR multiplex for GSTM1 and GSTT1, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) for GSTP1, and real-time PCR for mEH were performed. Statistical analyses were performed using multiple logistic regression tests.Age over 48 years (p < 0.001) and alcohol consumption (p = 0.021) were the predictors of increased risk of developing HCC. GSTP1.Ala114Val for all regression models (p < 0.05), except the recessive model, and the GSTT1 null genotype (odds ratio [OR] = 0.43, 95% confidence interval [CI] = 0.21-0.87, p = 0.019) were predictors of an increased risk of developing HCC. Polymorphic GSTT1, GSTM1, GSTP1.Ala114Val, and mEH.His139Arg and wild-type mEH.Tyr113His (OR = 5.04; 95% CI = 1.59-16.04; p = 0.006) were associated with HCC.Age over 48 years, alcohol consumption, and the presence of polymorphic variants of GSTP1 and GSTT1 were associated with the risk of developing HCC.

MicroRNAs as regulators of VEGFA and NFE2L2 in cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in post-transcriptional regulation of various genes, and their deregulation can lead to tumorigenesis. They may play the role of oncogenes or tumor suppressors by regulating different genes involved in cellular processes. One of the genes regulated by the miRNAs is the vascular endothelial growth factor A (VEGFA), which is responsible for angiogenesis. Angiogenesis is the process of formation of new blood vessels from pre-existing ones. This process plays an important role in tumor development, since it is responsible for the transport of nutrients required for tumor growth. Several studies have shown an increased expression of VEGFA in various cancers. Another gene regulated by miRNAs, the nuclear factor erythroid 2-like-2 (NFE2L2/NRF2), has a cytoprotective function and regulates cellular defense against oxidative stress. The NFE2L2 is the major regulator of cytoprotective agents and their oxidative damage to cells, which is down-regulated by Kelch-like ECH-associated protein 1 (KEAP1) at the post-transcriptional level. Regulation of the VEGFA and NFE2L2 by miRNAs has been observed in hepatocellular carcinoma and breast, lung, esophageal, endometrial, gastric, and ovarian cancer. This review highlights the role of miRNAs in the regulation of VEGFA and NFE2L2 and their relevance as therapeutic targets in various cancers.

Clinical, Epidemiological and Histopathological Aspects in Patients with Hepatocellular Carcinoma Undergoing Liver Transplantation

Background: Hepatocellular Carcinoma (HCC) is the primary liver cancer with high incidence and mortality rates. Currently one of the major etiologies for liver disease, HCC and liver transplantation is nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the epidemiological, histopathological and clinical aspects of HCC transplant patients, with emphasis on NAFLD etiology. Methods: This study included all HCC patients submitted to liver transplantation from 2010 to 2016 of the University Reference Center. The analyzed variables were age, gender, ethnicity, causes that led to liver transplantation, alpha-fetoprotein (AFP) dosage, histological aspects, recurrence, survival and NAFLD. Results: A total of 60 patients were included in the study being 80% men with a mean age of 58.3 ± 10.6 years. All patients were cirrhotic. The causes that led to the transplantation were the presence of the hepatitis C virus (HCV) (56.6% of the patients), an association of the virus with alcohol (20%), the presence of the hepatitis B virus (HBV) (20%), alcoholic liver disease (ALD) (50.9%) and NAFLD (25%). Of the latter, eight were diagnosed pre-transplantation and seven were NAFLD carriers without a previous diagnosis. Regarding the Edmondson-Steiner histological classification, 58.5% of the patients were classified as grade ≤ II. Conclusions: There is predominance of male patients with a mean age of 58.3 years. Degree ≤ II is the most frequent to the Edmondson-Steiner histological classification in the evaluated casuistic. HCV, ALD and NAFLD is the most common etiological agents found in the study. The (high) underestimated prevalence of NAFLD in the pre-transplanted patients is due to the fact that all patients presented cirrhosis, masking NAFLD signals.

Relationship between CD44high/CD133high/CD117high cancer stem cells phenotype and Cetuximab and Paclitaxel treatment response in head and neck cancer cell lines.

Recent evidence suggests that cancer stem cells (CSCs), a small population of cancer cells that are highly tumourigenic, capable of self-renewal and have the ability to differentiate into cells that constitute the tumor, are the "drivers" of local recurrence and metastatic spread and may be associated with resistant to conventional therapy. The objectives of the study are to identify and characterize two head and neck cancer cell lines with regard CD44high/CD133high/CD117high profile (CSCs) and CD44low/CD133low/CD117low profile (Non-CSCs); to investigate the influence of chemotherapy treatment in CSCs and compare with Non-CSCs; to evaluate CD44 and EGFR gene expression in CSCs. Fluorescent-activated cell sorting (FACS) using specific cell surface marker combination (CD44, CD117 and CD133) was performed to isolate CSCs of Non-CSCs from cell lines. The Wound Healing assay was performed to confirm the presence of CSCs. After, the CSCs subpopulation and Non-CSCs were cultured and exposed for 24 h to Cetuximab and Paclitaxel treatment, separately. Cell proliferation was determined by MTS assay. CD44 and EGFR gene expression was quantified by quantitative real time PCR (qPCR) using TaqMan® Assay in both subpopulations. CSCs subpopulation untreated were considered as relative expression control. We firstly characterized CSCs in HN13 and HEP-2 cell lines with CD44, CD133 and CD117 biomarkers. We treated CSCs and Non-CSCs subpopulations with Cetuximab and Paclitaxel treatment and found that CSCs subpopulations demonstrated more resistance to Paclitaxel chemoterapy, when compared with Non-CSCs subpopulations of oral cancer cell line. These CSCs subpopulations presented up-regulation of CD44 gene and down-regulation of EGFR gene in oral cancer cell line, and down-regulation of CD44 gene and up-regulation of EGFR gene in laryngeal cancer cell line when compared with Non-CSCs subpopulations. We conclude that the combination of CD44, CD133 and CD117 biomarkers have stem cell properties in both cell lines. CSCs has ability to resist to Paclitaxel treatment in oral cancer cell line. CSCs present high expression of CD44 gene and down expression of EGFR gene in oral cancer cell line. CSCs in laryngeal cell line present down expression of CD44 gene and high expression of EGFR gene when compared with cells without characteristics of cancer stem cells.

Hepatocellular Carcinoma: a Comprehensive Review of Biomarkers, Clinical Aspects, and Therapy

Hepatocellular carcinoma (HCC) is a cause of several deaths related to cancer worldwidely. In early stage, curative treatments such as surgical resection, liver transplant and local ablation can improve the patient ´s survival. However, the disease is detected in advanced stage; moreover some available therapies are restricted to palliative care and local treatment. Early detections of HCC and adequate therapy are crucial to increase survival as well as to improve the patient´s quality of life. Therefore, researchers have been investigating molecular biomarkers with high sensibility and reliability as Golgi 73 protein (GP73), Glypican-3 (GPC3), Osteopontin (OPN), microRNAs and others. MicroRNAs can regulate important pathways on carcinogenesis, as tumor angiogenesis and progression. So, they can be considered as possible markers of prognosis in HCC, and therapeutic target for this tumor type. In this review, we discuss the recent advances related to the cause (highlighting the main risk factors), treatment, biomarkers, clinic aspects, and outcome in hepatocellular carcinoma.

Polymorphisms of folate metabolism genes in patients with cirrhosis and hepatocellular carcinoma.

AIM: To evaluated the association of the risk factors and polymorphisms in MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G genes. METHODS: Patients with cirrhosis (n = 116), hepatocellular carcinoma (HCC) (n = 71) and controls (n = 356) were included. Polymerase chain reaction followed by enzymatic digestion and allelic discrimination technique real-time PCR techniques were used for analysis. MINITAB-14.0 and SNPstats were utilized for statistical analysis. RESULTS: Showed that age ≥ 46 years (OR = 10.31; 95%CI: 5.66-18.76; P < 0.001) and smoking (OR = 0.47; 95%CI: 0.28-0.78; P = 0.003) were associated with cirrhosis. Age ≥ 46 years (OR = 16.36; 95%CI: 6.68-40.05; P < 0.001) and alcohol habit (OR = 2.01; 95%CI: 1.03-3.89; P = 0.039) were associated with HCC. MTHFR A1298C in codominant model (OR = 3.37; 95%CI: 1.52-7.50; P = 0.014), recessive model (OR = 3.04; 95%CI: 1.43-6.47; P = 0.0051) and additive model (OR = 1.71; 95%CI: 1.16-2.52; P = 0.0072) was associated with HCC, as well as MTR A2756G in the additive model (OR = 1.68; 95%CI: 1.01-2.77; P = 0.047), and MTRR A66G in the codominant model (OR = 3.26; 95%CI: 1.54-6.87; P < 0.001), dominant model (OR = 2.55; 95%CI: 1.24-5.25; P = 0.007) and overdominant model (OR = 3.05; 95%CI: 1.66-5.62; P < 0.001). MTR A2756G in the additive model (OR = 1.54; 95%CI: 1.02-2.33; P = 0.042) and smokers who presented at least one polymorphic allele for MTRR A66G (OR = 1.71; 95%CI: 0.77-3.82; P = 0.0051) showed increased risk for cirrhosis. There was no association between clinical parameters and polymorphisms. CONCLUSION: Age ≥ 46 years, alcohol habit and MTR A2756G, MTHFR A1298C and MTRR A66G polymorphisms are associated with an increased risk of HCC development; age ≥ 46 years, tobacco habit and the MTR A2756G polymorphism are associated with cirrhosis.