Mónica Gomes

Functional polymorphisms of Toll-like receptors 2 and 4 alter the risk for colorectal carcinoma in Europeans

BACKGROUND Colon carcinogenesis is associated with increased expression levels of Toll-like receptor 2 and Toll-like receptor 4. AIM To determine in a Caucasian population the role of Toll-like receptor 2 and Toll-like receptor 4 polymorphisms in colorectal cancer development. METHODS Hospital based multicentre case control study involving 193 colorectal cancer patients and 278 healthy individuals. DNA samples were extracted from blood cells and genotyping of TLR2+597T>C, TLR2-4760T>C, TLR4-3745A>G, TLR2Arg753Gln, TLR4Asp299Gly was performed. Functionality of risk polymorphisms was evaluated through production of TNF-α in cell culture and Toll-like receptors levels quantified by real-time RT-PCR. RESULTS TLR2+597CC homozygous had 5-fold decreased risk (odds ratio (OR)=0.21, 95% CI: 0.09-0.50, p<0.001) and TLR4 299Gly homozygous 3-fold increased risk of colorectal cancer (OR=3.30, 95% CI: 1.18-9.28, p=0.015). In stratified analysis, TLR2+597CC genotype protective effect was even higher in overweight individuals (OR=0.17, 95% CI: 0.06-0.53, p<0.001) and in never smokers (OR=0.11, 95% CI: 0.02-0.51, p=0.001). Also, the increased risk effect for TLR4 299Gly homozygous genotype was higher in overweight individuals (OR=8.67, 95% CI: 1.11-87.85, p=0.011). TLR2+597T>C polymorphism conferred 41% less (p=0.03) and TLR4Asp299Gly 65% more TNF-α production (p=0.02) with no differences in Toll-like receptors levels. CONCLUSION Functional Toll-like receptor 2 and Toll-like receptor 4 polymorphisms significantly alter the risk to have colorectal cancer. Obesity and smoking may influence the risk for colorectal cancer in individuals presenting these genetic profiles.

The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma

Non-Hodgkin lymphoma (NHL) has been associated with immunological defects, chronic inflammatory and autoimmune conditions. Given the link between immune dysfunction and NHL, genetic variants in toll-like receptors (TLRs) have been regarded as potential predictive factors of susceptibility to NHL. Adequate anti-tumoral responses are known to depend on TLR9 function, such that the use of its synthetic ligand is being targeted as a therapeutic strategy. We investigated the association between the functional rs5743836 polymorphism in the TLR9 promoter and risk for B-cell NHL and its major subtypes in three independent case–control association studies from Portugal (1160 controls, 797 patients), Italy (468 controls, 494 patients) and the US (972 controls, 868 patients). We found that the rs5743836 polymorphism was significantly overtransmitted in both Portuguese (odds ratio (OR), 1.85; P=7.3E−9) and Italian (OR, 1.84; P=6.0E−5) and not in the US cohort of NHL patients. Moreover, the increased transcriptional activity of TLR9 in mononuclear cells from patients harboring rs5743836 further supports a functional effect of this polymorphism on NHL susceptibility in a population-dependent manner.

Improvement of a Predictive Model of Castration-Resistant Prostate Cancer: Functional Genetic Variants in TGFβ1 Signaling Pathway Modulation

Prostate cancer (PC) is the most frequently diagnosed cancer in men. The acquisition of castration-resistant (CR) phenotype is associated with the activation of signaling pathways mediated by growth factors. The TGFβ1 and its receptors have an important role in tumor progression, being the pro-apoptotic function modulated by the expression of TGFBR2. A single nucleotide polymorphism -875 G > A in TGFBR2 gene has been described, which may influence the expression levels of the receptor. Our purpose was to investigate the potential role of TGFBR2-875G>A in PC risk and in the response to androgen deprivation therapy (ADT). TGFBR2-875G>A polymorphism was studied by allelic discrimination using real-time polymerase chain reaction (PCR) in 891 patients with PC and 874 controls. A follow-up study was undertaken to evaluate response to ADT. The TGFBR2 and SMAD7 mRNA expression were analyzed by a quantitative real-time PCR. We found that TGFBR2-875GG homozygous patients present lower expression levels of TGFBR2 mRNA (AA/AG: 2-ΔΔCT =1.5, P=0.016). GG genotype was also associated with higher Gleason grade (OR=1.51, P=0.019) and increased risk of an early relapse after ADT (HR=1.47, P=0.024). The concordance (c) index analysis showed that the definition of profiles that contains information regarding tumor characteristics associated with genetic information present an increased capacity to predict the risk for CR development (c-index model 1: 0.683 vs model 2: 0.736 vs model 3: 0.746 vs model 4: 0.759). The TGFBR2-875G>A contribution to an early relapse in ADT patients, due to changes in mRNA expression, supports the involvement of TGFβ1 pathway in CRPC. Furthermore, according to our results, we hypothesize the potential benefits of the association of genetic information in predictive models of CR development.

Restoring TGFβ1 pathway-related microRNAs: possible impact in metastatic prostate cancer development

In developed countries, prostate cancer (PC) is the neoplasia more frequently diagnosed in men. The signaling pathway induced by the transforming growth factor β1 (TGFβ1) has an important role in cell growth, differentiation, and development, the downregulation of this pathway being associated with cancer development. In PC, the activation of this signaling pathway is lost, resulting in favoring of tumor growth, proliferation, and evasion of apoptosis. Several studies have shown that microRNAs (miRNAs), small non-coding RNA, are closely associated with the development, invasion, and metastasis, suggesting that they have a critical role in cancer development. Recently, Smad proteins, the signal transducers of the TGFβ1 signaling pathway, were found to regulate miRNA expression, through both transcriptional and posttranscriptional mechanisms. In this review, we summarize the mechanisms underlying Smad-mediated regulation of miRNA biogenesis and the effects on cancer development, particularly in PC. We identify that TGFβ1-related miR-143, miR-145, miR-146a, and miR-199a may have a key role in the development of prostate cancer metastasis and the restoration of their expression may be a promising therapeutic strategy for PC treatment.

Influence of functional genetic polymorphism (−590C/T) in non-small cell lung cancer (NSCLC) development: The paradoxal role of IL-4

Lung cancer is the leading cause of death by cancer in the world, originating about 17.5% of total deaths from cancer (1.18 million). Inflammation plays an important role in the pathogenesis of lung cancer. IL-4 is an anti-inflammatory cytokine, which reduces the production of proinflammatory cytokines by monocytes and with direct antiproliferative effects in some tumors. The polymorphism -590C/T SNP is a C to T transition in the -590 position of the promoter region of the IL-4 gene. The aim of this study was to evaluate the influence of this polymorphism in the susceptibility to NSCLC. DNA was extracted from peripheral blood of 1060 individuals (391 patients diagnosed with NSCLC and a control group of 669 individuals without cancer). The characterization of IL-4 -590C/T genotypes was performed by PCR-RFLP (BsmFI). The -590C/T polymorphism genotypes were classified as low (CC) and high expression (TT). The frequencies obtained for the CC and TT genotypes were 90.1% and 9.9%, respectively, in the control group and 92.9% and 7.1%, respectively, in the case group. The analysis of the TT and CC genotype frequencies in the two groups showed a statistically significant difference in its distribution, indicating a protection of 80% for the development of NSCLC, type epidermoid in individuals with the TT genotype when compared with individuals with CC genotype (P=0.024, OR=0.221: 95% CI=0.053-0.928). We present for the first time that increased expression of IL-4 associated with the TT genotype may contribute to immune surveillance during NSCLC development.

Combined Influence of EGF+61G>A and TGFB+869T>C Functional Polymorphisms in Renal Cell Carcinoma Progression and Overall Survival: The Link to Plasma Circulating MiR-7 and MiR-221/222 Expression

The epidermal growth factor (EGF) is responsible for the activation of intracellular signal transducers that act on cell-cycle progression, cell motility, angiogenesis and inhibition of apoptosis. However, cells can block these effects activating opposite signaling pathways, such as the transforming growth factor beta 1 (TGFβ1) pathway. Thus changes in expression levels of EGF and TGFB1 in renal cells might modulate the renal cell carcinoma (RCC) development, in consequence of changes in regulatory elements of signaling networks such as the microRNAs (miRNAs). Our purpose was to investigate the synergic role of EGF+61G>A and TGFB1+869T>C polymorphisms in RCC development. Genetic polymorphisms were studied by allelic discrimination using real-time PCR in 133 RCC patients vs. 443 healthy individuals. The circulating EGF/EGFR-MAPK-related miR-7, miR-221 and miR-222 expression was analyzed by a quantitative real-time PCR in plasma from 22 RCC patients vs. 27 healthy individuals. The intermediate/high genetic proliferation profile patients carriers present a significantly reduced time-to-progression and a higher risk of an early relapse compared with the low genetic proliferation profile carriers (HR = 8.8, P = 0.038) with impact in a lower overall survival (Log rank test, P = 0.047). The RCC patients presented higher circulating expression levels of miR-7 than healthy individuals (6.1-fold increase, P<0.001). Moreover, the intermediate/high genetic proliferation profile carriers present an increase in expression levels of miR-7, miR-221 and miR-222 during the RCC development and this increase is not observed in low genetic proliferation profile (P<0.001, P = 0.004, P<0.001, respectively). The stimulus to angiogenesis, cell-cycle progression and tumoral cells invasion, through activation of EGFR/MAPK signaling pathway in intermediate/high proliferation profile carriers is associated with an early disease progression, resulting in a poor overall survival. We also demonstrated that the intermediate/high proliferation profile is an unfavorable prognostic factor of RCC and miR-7, miR-221 and miR-222 expressions may be useful phenotype biomarkers of EGFR/MAPK activation.

Angiogenesis in NSCLC: is vessel co-option the trunk that sustains the branches?

The critical role of angiogenesis in tumor development makes its inhibition a valuable new approach in therapy, rapidly making anti-angiogenesis a major focus in research. While the VEGF/VEGFR pathway is the main target of the approved anti-angiogenic molecules in NSCLC treatment, the results obtained are still modest, especially due to resistance mechanisms. Accumulating scientific data show that vessel co-option is an alternative mechanism to angiogenesis during tumor development in well-vascularized organs such as the lungs, where tumor cells highjack the existing vasculature to obtain its blood supply in a non-angiogenic fashion. This can explain the low/lack of response to current anti-angiogenic strategies. The same principle applies to lung metastases of other primary tumors. The exact mechanisms of vessel co-option need to be further elucidated, but it is known that the co-opted vessels regress by the action of Angiopoietin-2 (Ang-2), a vessel destabilizing cytokine expressed by the endothelial cells of the pre-existing mature vessels. In the absence of VEGF, vessel regression leads to tumor cell loss and hypoxia, with a subsequent switch to a neoangiogenic phenotype by the remaining tumor cells. Unravelling the vessel co-option mechanisms and involved players may be fruitful for numerous reasons, and the particularities of this form of vascularization should be carefully considered when planning anti-angiogenic interventions or designing clinical trials for this purpose. In view of the current knowledge, rationale for therapeutic approaches of dual inhibition of Ang-2 and VEGF are swiftly gaining strength and may serve as a launchpad to more successful NSCLC anti-vascular treatments.

CYP2D6*4 polymorphism: A new marker of response to hormonotherapy in male breast cancer?

BACKGROUND Tamoxifen remains the standard hormonotherapy for Male breast cancer patients (MBC). Previous studies, in women, tried to evaluate the impact of CYP2D6 polymorphisms in tamoxifen efficacy with conflicting results. Herein we analyze the relation between CYP2D6*4 polymorphism and survival in MBC patients. PATIENTS AND METHODS Fifty-three patients, proposed to tamoxifen in adjuvant setting, were enrolled. Clinical information was collected from records and histological revision with additional immunochemistry analysis was done to better characterize the tumors. Comprehensive CYP2D6*4 genotyping from blood or tumor tissue was performed and translated into two predicted metabolic activity groups. RESULTS Patients included in the two CYP2D6*4 groups did not differ concerning to age, histological characteristics, and primary treatments performed. Median age at diagnosis was 63 years-old and patients were submitted at least to mastectomy and adjuvant hormonotherapy. Recurrence was observed in 7 patients (13.2%) and 13 patients (25.5%) died with a 5-year disease-free survival of 86.2%. The poorer metabolizer group had a high risk for recurrence (p = 0.034) and this outcome effect remains in different subgroups: in tumors larger than 2 cm (p < 0.001), nodal status, N0 vs N+ (p = 0.04) and in advanced stage, stage III (p < 0.001). Poorer metabolizer patients had also a worse overall survival when tumors were larger than 2 cm (p = 0.03). CONCLUSIONS In our series, there was an association between CYP2D6*4 polymorphism and a probability of recurrence, with a consistent effect in risk groups defined by classic prognostic factors. Multicentric studies with larger samples are needed to validate these results.

Cyclin D1 polymorphism in non-small cell lung cancer in a Portuguese population

Cyclin D1 (CCND1) is a key regulatory protein of the cell cycle. The purpose of our study was to assess the role of CCND1 genetic variants influencing the genetic susceptibility of non-small cell lung cancer (NSCLC). We conducted a study of 1234 individuals, including 892 controls and 342 cases. Individuals carrying two G-alleles have a 2-fold increased risk for the development of NSCLC and the waiting time for onset of NSCLC in these patients was 2 years earlier in comparison with other individuals. Our results may be important in contributing to the knowledge of the mechanisms involved in lung carcinogenesis.

Combined Ang-2 and VEGF serum levels: holding hands as a new integral biomarker in non-small-cell lung cancers

AIM Evaluate if serum levels of VEGF and Ang-2 are correlated in non-small-cell lung cancers (NSCLCs) and its implications in the diagnostic and prognostic of the disease. PATIENTS & METHODS Unselected cohort of 145 NSCLC patients and 30 control individuals. The serum levels of Ang-2 and VEGF of each patient were measured by ELISA prior to treatment. RESULTS & CONCLUSIONS Serum levels of Ang-2 and VEGF are correlated (p < 0.0001). High serum levels of Ang-2 and VEGF isolated and both combined (high(Ang-2/VEGF)) correlate with likelihood of presenting NSCLC (p = 0.016; p = 0.003; p < 0.0001, respectively). Serum levels of Ang-2 and high(Ang-2/VEGF) but not VEGF alone are independent prognostic factors (p = 0.001; p = 0.619; p = 0.005). High(Ang-2/VEGF) serum levels could be exploited as a new valuable integral biomarker in NSCLC.