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.

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.

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.

Anti-EGFR treatment effects on laryngeal cancer stem cells.

Laryngeal cancer (LC) is one of the common head and neck neoplasms and is characterized by resistance to conventional therapy and poor prognosis. This may result from the presence of cancer stem cells (CSCs), which form a small population in tumors with metastatic potential, high invasive capacity, self-renewal, and differentiation. This study aimed to evaluate the effectiveness of 5-fluorouracil and cisplatin individually, as well as the combination of cetuximab and paclitaxel in a CSC subpopulation separated with biomarkers related to tumoral growth (CD44, CD117, and CD133). In addition, expression of TrkB, KRAS, HIF-1α, and VEGF-A genes and proteins related to cell proliferation were evaluated in this subpopulation. The CD44, CD133, and CD117 biomarkers were used to analyze the identification and separation of both subpopulations using FACSAria Fusion. Subpopulations positive for CD44, CD133, and CD117 or lacking these biomarkers were classified as laryngeal cancer stem cells (LCSCs) or laryngeal cancer non-stem cells (non-LCSCs), respectively. Matrigel invasion and colony forming assays were performed to confirm CSC presence. Subpopulations were cultured and exposed to 5-fluorouracil, cisplatin, and cetuximab/paclitaxel drugs for 24 h. Cell proliferation was determined using MTS assay. KRAS and TrkB gene expression levels were evaluated using quantitative real time PCR with TaqMan® Assay in both subpopulations. The non-LCSC subpopulation was considered as the control for relative expression. We found that the LCSC subpopulation demonstrated more resistance to cetuximab and paclitaxel combination chemotherapy when compared with the non-LCSC subpopulation of the cell line. These LCSC subpopulations presented up-regulated expression of KRAS, HIF-1α, and VEGF-A genes and proteins and no TrkB gene expression, but TrkB protein expression was up-regulated in the LC cell line when compared to the non-CSC subpopulation. "In conclusion, the combination of CD44, CD133, and CD117 biomarkers has stem cell properties. Moreover, LCSCs, are capable of resisting treatment and present high KRAS, HIF-1α, and VEGF-A gene expression".

Role of Tropomyosin-related kinase B receptor and brain-derived neurotrophic factor in cancer.

The tropomyosin-related kinase B (TrkB) receptor is a member of the neurotrophic tyrosine kinase receptors family and, together with the brain-derived neurotrophic factor (BDNF), plays an important role in the development of breast cancer, lung cancer, neuroblastoma, colorectal cancer, leukemia, cervical cancer, gallbladder cancer, gastric cancer, kidney cancer, Ewing's sarcoma, esophageal cancer, and head and neck cancer. Overexpression of these two factors has been associated with increased processes involved in carcinogenesis, such as invasion, migration, epithelial-mesenchymal transition (EMT), angiogenesis, metastasis, cell proliferation, resistance to apoptosis, resistance to cell death due to loss of adhesion (anoikis), activation of cell proliferation pathways, regulation of tumor suppressor genes, and drug resistance, and is related to advanced clinical stage. Inhibition of the TrkB/BDNF axis using drugs in phase 1 studies, approved drugs, and small interfering RNA (siRNA) are promising strategies for the treatment of various malignant tumors in addition to increasing the sensitivity of cells resistant to chemotherapy, improving the effectiveness of drugs without increasing toxicity. Another factor related to poor cancer prognosis is the presence of cancer stem cells, having effects similar to the high expression of the TrkB/BDNF axis, on cancer. This review aimed to show the role of the TrkB/BDNF axis in several types of cancer, its possible use as a prognostic biomarker, the effects of inhibiting this axis, and its role in the cancer stem cells.

Molecular evaluation of glutathione S transferase family genes in patients with sporadic colorectal cancer.

AIM: To evaluate the association between polymorphisms in glutathione S transferases (GSTs) and the risk of sporadic colorectal cancer (SCRC), tumor progression and the survival of patients. METHODS: A case-control study of 970 individuals from the Brazilian population was conducted (232 individuals from the case group with colorectal cancer and 738 individuals from the control group without a history of cancer). PCR multiplex and PCR-RFLP techniques were used to genotype the GST polymorphisms. The tumors were categorized according to the TNM classification: tumor extension (T), affected lymph nodes (N), and presence of metastasis (M). Logistic regression, multiple logistic regression and survival analysis were used to analyze the data. The results are presented in terms of odds ratio (OR) and 95% confidence interval (CI). The level of significance was set at 5% (P ≤ 0.05). RESULTS: Age equal to or over 62 years (OR = 8.79; 95%CI: 5.90-13.09, P < 0.01) and female gender (OR = 2.91; 95%CI: 1.74-4.37; P < 0.01) were associated with increased risk of SCRC. Analysis of the polymorphisms revealed an association between the GSTM1 polymorphisms and a risk of SCRC (OR = 1.45; 95%CI: 1.06-2.00; P = 0.02), as well as between GSTT1 and a reduced risk of the disease (OR = 0.65; 95%CI: 0.43-0.98; P = 0.04). An interaction between the presence of the wild-type allele of GSTP1 Ile105Val polymorphism and tobacco consumption on risk of SCRC (OR = 2.33; 95%CI: 1.34-4.05; P = 0.05) was observed. There was an association between the GSTM1 null genotype and the presence of advanced tumors (OR = 2.33; 95%CI: 1.23-4.41; P = 0.009), as well as increased risk of SCRC in the presence of a combination of GSTT1 non-null/GSTM1 null genotypes (OR = 1.50; 95%CI: 1.03-2.19; P = 0.03) and GSTT1 non-null/GSTM1 null/GSTP1 Val* (OR = 1.85; 95%CI: 1.01-3.36, P = 0.04). Combined GSTT1 non-null/GSTM1 null genotypes (OR = 2.40; 95%CI: 1.19-4.85; P = 0.01) and GSTT1 non-null/GSTM1 null/GSTP1 Val* (OR = 2.92; 95%CI: 1.05-8.12; P = 0.04) were associated with tumor progression. Polymorphisms were not associated with the survival of patients with SCRC. CONCLUSION: Females aged 62 years or older are more susceptible to SCRC. Polymorphisms of GSTT1 and GSTM1 null genotypes modulated the susceptibility to SCRC in the population studied.

Clinical and epidemiological evaluation of patients with sporadic colorectal cancer / Avaliação clínica e epidemiológica de pacientes com câncer colorretal esporádico

Background: This study aims to perform a survey on clinical data, sociodemographic and risk factors from patients with sporadic colorectal cancer (SCRC) treated between 2004 and 2008 in the Coloproctology Service of a teaching hospital in the North-western region of São Paulo. Methods: We analyzed 749 medical records. Of these, 460 were from colon cancer patients and 289 from rectal cancer patients. Most of the individuals had white skin and were aged over 62 years. The variables that were analyzed included gender, age, skin color, professional occupation, alcohol drinking and cigarette smoking, family history of cancer, and comorbidities. The identification of the clinical-sociodemographic profile and risk factors in a population with the SCRC the northwest region of São Paulo was performed to collaborate with prevention strategies. Results: The occurrence of SCRC did not differ much between genders. The most prevalent professional occupations were those related to household chores, agricultural and commercial activities. Among the comorbidities, hypertension and cholelithiasis were the most representative. The most common diagnosis method and treatment for the majority of patients were coloscopy and surgery, respectively. On average, the time of the disease progression was eight months. The median number of lymph nodes excised ranged between 11 and 14. The most common metastasis was hepatic. Conclusion: The occurrence of colorectal cancer is more frequent in men's white skin with aged over 62 years. Professional occupation seems to be more important for those exposed to carcinogenic agents. This type of tumor mostly affects the distal regions of the colon and rectum with the occurrence of liver metastasis. The affected individuals usually have low survival due to its high aggressiveness. (AU)

Experiência: O presente estudo tem como objetivo realizar um levantamento de dados clínicos e fatores sociodemográficos e de risco de pacientes com câncer colorretal esporádico (CCRE) tratados entre 2004 e 2008 no Serviço de Coloproctologia de um hospital-escola na região Noroeste de São Paulo. Métodos: Foram analisados 749 prontuários clínicos. Destes, 460 foram de pacientes com câncer de cólon e de 289 de pacientes com câncer retal. A maioria dos indivíduos era da raça branca, com mais de 62 anos de idade. As variáveis analisadas foram gênero, idade, cor da pele, ocupação profissional, consumo de álcool e tabagismo, história familiar de câncer e co-morbidades. A identificação do perfil clínico-sociodemográfico e dos fatores de risco em uma população com CCRE na região noroeste de São Paulo foi realizada para colaborar com as estratégias de prevenção. Resultados: A ocorrência de CCRE não diferiu muito entre gêneros. As ocupacões profissionais mais prevalentes foram as relacionadas aos afazeres domésticos, atividades agrícolas e comerciais. Entre as comorbidades, hipertensão e colelitíase foram as mais representativas. O método de diagnóstico e de tratamento mais comum para a maioria dos pacientes foi colonoscopia e cirurgia, respectivamente. Em média, o tempo de progressão da doença foi de oito meses. O número mediano de linfonodos extirpados variou entre 11 e 14. A metástase mais comum foi a hepática. Conclusão: A ocorrência de câncer colorretal é mais frequente em homens de pele branca com idade superior a 62 anos. A ocupação profissional parece ser mais importante para as pessoas expostas a agentes cancerígenos. Este tipo de tumor afeta principalmente as regiões distais do cólon e do reto, com a ocorrência de metástases no fígado. Geralmente, os indivíduos afetados exibem baixa sobrevida, devido à alta agressividade dessa neoplasia. (AU)