GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

BACKGROUND: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. OBJECTIVES: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. METHODS: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. RESULTS: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. CONCLUSIONS: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.

Laryngeal sequelae secondary to surgical treatment for recurrent respiratory papillomatosis in children.

OBJECTIVE: to describe the frequency and predictive factors associated with laryngeal scarring caused by surgical treatment of recurrent respiratory papillomatosis (RRP) in children. INTRODUCTION: RRP is an important cause of hoarseness and respiratory obstruction in children. The current standard of care for RRP is the systematic and repetitive surgical interventions. The repetitive surgeries may increase the risk of sequelae. A larger number of surgeries, the surgical technique used, and disease severity are related to an increased risk of scarring. MATERIAL AND METHODS: A retrospective, descriptive review of the medical charts of patients with RRP younger than 18 years was conducted. Between 2014 and 2017, 79 patients were identified; five patients were excluded. Demographic and clinical data were recorded and analyzed. The patients were divided into two groups, one with and the other without surgical sequelae, for comparison to identify sequela-associated factors. RESULTS: 75 patients, 40 (53.3%) male, were analyzed. Age at symptom onset ranged from 2 months to 13 years. Median age at the time of diagnosis was 42 months. Overall, 44% presented with disseminated disease. A median of nine (range, 1 to 86) surgeries were performed in each patient with a median of two (range, 0.6 to 10) of the average number of surgeries per year per patient. 29 patients (38.7%) had laryngeal sequelae. When comparing the patients with and without sequelae, statistically significant differences were found in the variables of dissemination during the course of the disease, overall number of surgeries and mean number of surgeries per year, history and number of previous surgeries at an outside institution, urgent surgeries, and CO2 laser use. Patients who underwent more than 10 surgeries or who had a history of previous surgeries at an outside institution had a higher frequency of laryngeal scarring in multivariate analysis. CONCLUSION: Scarring secondary to surgical treatment for RRP is common. Surgery-related variables seem to be predisposing factors. A greater number of surgeries and surgeries performed at less specialized centers are strongly related to this complication. Future studies with a larger sample size are necessary to determine whether other factors are involved.

Laryngeal reconstruction in children with recurrent respiratory papillomatosis.

OBJETIVES: To describe our experience in reconstructive laryngeal surgery in patients with recurrent respiratory papillomatosis (RRP). INTRODUCTION: RRP is a rare laryngeal disease requiring multiple surgical endoscopic interventions during its course. These interventions may cause secondary lesions that may compromise airway patency. Open larynx reconstructive surgery, as tracheostomy, is a procedure considered to potentially favor extralaryngeal papilloma dissemination. In patients with RRP, the use of endoscopic posterior cricoid split and rib grafting has not been previously described. METHODS: The clinical charts of 230 patients with RRP seen between 1996 and 2017 were reviewed. All patients who underwent airway expansion procedures either by open or endoscopic approach were included in the study. RESULTS: Four patients with RRP underwent laryngeal surgery for laryngeal stenosis were included. A double-stage open approach was used in two patients and a single-stage endoscopic approach in the remaining two. The two tracheostomized patients were decannulated while tracheostomy was avoided in the two patients who underwent a single-stage endoscopic procedure. Two patients had active papillomatous lesions limited to the larynx at the time of surgery; no dissemination was observed during follow-up (cases 1 and 3). One patient had extralaryngeal disseminated papilomatosis; surgery did not lead to an increased lesion load compared to presurgical lesions (case 4). The patient who did not have active lesions did not have recurrence (case 2). CONCLUSIONS: Reconstructive laryngeal surgery is a safe and effective option in the management of stenotic sequelae resulting from the surgical treatment of RRP, allowing for decannulation or avoiding tracheostomy.

Targeting of the adaptor protein Tab2 as a novel approach to revert tamoxifen resistance in breast cancer cells.

Pharmacological resistance is a serious threat to the clinical success of hormone therapy for breast cancer. The antiproliferative response to antagonistic drugs such as tamoxifen (Tam) critically depends on the recruitment of NCoR/SMRT corepressors to estrogen receptor alpha (ERα) bound to estrogen target genes. Under certain circumstances, as demonstrated in the case of interleukin-1ß (IL-1ß) treatment, the protein Tab2 interacts with ERα/NCoR and causes dismissal of NCoR from these genes, leading to loss of the antiproliferative response. In Tam-resistant (TamR) ER-positive breast cancer cells, we observed that Tab2 presents a shift in mobility on sodium dodecyl sulfate--PAGE (SDS-PAGE) similar to that seen in MCF7 wt upon stimulation with IL-1ß, suggesting constitutive activation. Accordingly, TamR treatment with Tab2-specific short interfering RNA, restored the antiproliferative response to Tam in these cells. As Tab2 is known to directly interact with the N-terminal domain of ERα, we synthesized a peptide composed of a 14-aa motif of this domain, which effectively competes with ERα/Tab2 interaction in pull-down and co-immunoprecipitation experiments, fused to the carrier TAT peptide to allow internalization. Treatment of TamR cells with this peptide resulted in partial recovery of the antiproliferative response to Tam, suggesting a strategy to revert pharmacological resistance in breast cancer. Silencing of Tab2 in TamR cells by siRNA caused modulation of a gene set related to the control of cell cycle and extensively connected to BRCA1 in a functional network. These genes were able to discern two groups of patients, from a published data set of Tam-treated breast cancer profiles, with significantly different disease-free survival. Altogether, our data implicate Tab2 as a mediator of resistance to endocrine therapy and as a potential new target to reverse pharmacological resistance and potentiate antiestrogen action.

From single gene to integrative molecular concept MAPS: pitfalls and potentials of microarray technology.

Microarray experiments have a large variety of applications and several important achievements have been obtained by means of this technology, especially within the field of whole genome expression profiling, which undoubtedly is the most diffused world-wide. Nevertheless, care must be taken in unconditionally applying such high-throughput techniques and in extracting/interpreting their results. Both the validity and the reproducibility of microarray-based clinical research have recently been challenged. Pitfalls and potentials of the microarray technology for gene expression profiling are critically reviewed in this paper.