Fibroblast Growth Factor 22 Is Not Essential for Skin Development and Repair but Plays a Role in Tumorigenesis

PMCID: PMC3380851This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells

Upregulation of functional voltage-gated Na+ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the 'neonatal' splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by similar to 90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (similar to 30%), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33%, and significantly increased the cells' sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43%, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42%, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa

Alternative Splicing of the Cardiac Sodium Channel Creates Multiple Variants of Mutant T1620K Channels

Alternative splicing creates several Nav1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Nav1.5 splice variants have been discovered. Four of them, namely Nav1.5a, Nav1.5c, Nav1.5d, and Nav1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Nav1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3) and isolated cardiac conduction disease (CCD). When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNav1.5, hNav1.5a, and hNav1.5c. In contrast, the hNav1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNav1.5e, the neonatal variant of hNav1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNav1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNav1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies

Cytochrome 450 1B1 (CYP1B1) polymorphisms associated with response to docetaxel in Castration-Resistant Prostate Cancer (CRPC) patients

BACKGROUND: The selection of patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in Castration-Resistant Prostate Cancer (CRPC) patients. Functional polymorphisms within the cytochrome P450 1B1 (CYP1B1) gene have been associated with alterations in enzymatic expression and activity and may change sensitivity to the widely used docetaxel regimen. METHODS: CYP1B1 genotyping was performed on blood samples of 60 CRPC patients treated with docetaxel, using TaqMan probes-based assays. Association between CYP1B1-142C>G (leading to the 48ArgGly transition), 4326C>G (432LeuVal), and 4390A>G (453AsnSer) polymorphisms and treatment response, progression-free-survival (PFS) and overall-survival (OS) was estimated using Pearson χ2 test, Kaplan-Meier curves and Log-rank test. RESULTS: Patients carrying the CYP1B1-432ValVal genotype experienced a significantly lower response-rate (P = 0.014), shorter progression-free-survival (P = 0.032) and overall-survival (P < 0.001). Multivariate analyses and correction for multiple comparisons confirmed its prognostic significance for OS. No significant associations were found among other polymorphisms and both response and clinical outcome. CONCLUSIONS: CYP1B1-4326C>G (432LeuVal) polymorphism emerged as possible predictive marker of response and clinical outcome to docetaxel in CRPC patients and may represent a potential new tool for treatment optimization. Larger prospective trials are warranted to validate these findings, which might be applied to the future practice of CRPC treatment

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naive cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGF beta/SMAD (transforming growth factor-beta/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naive cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.Fapesp-grant number 2012/04194-1, 2013/05172-4, 2014/24400-0 and 2015/10821-7, CNPq-grant number 150447/2013-2 and 471512/2013-3 and PRODOC-grant no 3193-32/2010. Work in the lab of KS Smalley was supported by the National Institutes of Health grants R01 CA161107, R21 CA198550, and Skin SPORE grant P50 CA168536info:eu-repo/semantics/publishedVersio

Organotypic modelling as a means of investigating epithelial-stromal interactions during tumourigenesis

The advent of co-culture approaches has allowed researchers to more accurately model the behaviour of epithelial cells in cell culture studies. The initial work on epidermal modelling allowed the development of reconstituted epidermis, growing keratinocytes on top of fibroblasts seeded in a collagen gel at an air-liquid interface to generate terminally differentiated 'skin equivalents'. In addition to developing ex vivo skin sheets for the treatment of burns victims, such cultures have also been used as a means of investigating both the development and repair of the epidermis, in more relevant conditions than simple two-dimensional culture, but without the use of animals. More recently, by varying the cell types used and adjusting the composition of the matrix components, this physiological system can be adapted to allow the study of interactions between tumour cells and their surrounding stroma, particularly with regards to how such interactions regulate invasion. Here we provide a summary of the major themes involved in tumour progression and consider the evolution of the approaches used to study cancer cell behaviour. Finally, we review how organotypic models have facilitated the study of several key pathways in cancer development and invasion, and speculate on the exciting future roles for these models in cancer research

Evidence that the 5p12 Variant rs10941679 Confers Susceptibility to Estrogen Receptor-Positive Breast Cancer through FGF10 and MRPS30 Regulation

Genome-wide association studies (GWASs) have revealed increased breast cancer risk associated with multiple genetic variants at 5p12. Here, we report the fine mapping of this locus using data from 104,660 subjects from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). With data for 3,365 genotyped and imputed SNPs across a 1 Mb region (positions 44,394,495–45,364,167; NCBI build 37), we found evidence for at least three independent signals: the strongest signal, consisting of a single SNP rs10941679, was associated with risk of estrogen receptor-positive (ER+) breast cancer (per-g allele OR ER+ = 1.15; 95% CI 1.13–1.18; p = 8.35 × 10−30). After adjustment for rs10941679, we detected signal 2, consisting of 38 SNPs more strongly associated with ER-negative (ER−) breast cancer (lead SNP rs6864776: per-a allele OR ER− = 1.10; 95% CI 1.05–1.14; p conditional = 1.44 × 10−12), and a single signal 3 SNP (rs200229088: per-t allele OR ER+ = 1.12; 95% CI 1.09–1.15; p conditional = 1.12 × 10−05). Expression quantitative trait locus analysis in normal breast tissues and breast tumors showed that the g (risk) allele of rs10941679 was associated with increased expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in ∼10% of human breast cancers, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis