Melanoma cells with acquired resistance to vemurafenib have decreased autophagic flux and display enhanced ability to transfer resistance.

Over the last years, the incidence of melanoma, the deadliest form of skin cancer, has risen significantly. Nearly half of the melanoma patients exhibit the BRAFV600E mutation. Although the use of BRAF and MEK inhibitors (BRAFi and MEKi) showed an impressive success rate in melanoma patients, durability of response remains an issue because tumor quickly becomes resistant. Here, we generated and characterized Lu1205 and A375 melanoma cells resistant to vemurafenib (BRAFi). Resistant cells (Lu1205R and A375R) exhibit higher IC50 (5-6 fold increase) and phospho-ERK levels and 2-3 times reduced apoptosis than their sensitive parents (Lu1205S and A375S). Moreover, resistant cells are 2-3 times bigger, display a more elongated morphology and have a modulation of migration capacity. Interestingly, pharmacological inhibition of sphingosine kinases, that prevents sphingosine-1-phosphate production, reduces migration of Lu1205R cells by 50 %. In addition, although Lu1205R cells showed increased basal levels of the autophagy markers LC3II and p62, they have decreased autophagosome degradation and autophagy flux. Remarkably, expression of Rab27A and Rab27B, which are involved in the release of extracellular vesicles are dramatically augmented in resistant cells (i.e. 5-7 fold increase). Indeed, conditioned media obtained from Lu1205R cells increased the resistance to vemurafenib of sensitive cells. Hence, these results support that resistance to vemurafenib modulates migration and the autophagic flux and may be transferred to nearby sensitive melanoma cells by factors that are released to the extracellular milieu by resistant cells.

Comprehensive transcriptomic analysis of heat shock proteins in the molecular subtypes of human breast cancer.

BACKGROUND: Heat Shock Proteins (HSPs), a family of genes with key roles in proteostasis, have been extensively associated with cancer behaviour. However, the HSP family is quite large and many of its members have not been investigated in breast cancer (BRCA), particularly in relation with the current molecular BRCA classification. In this work, we performed a comprehensive transcriptomic study of the HSP gene family in BRCA patients from both The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohorts discriminating the BRCA intrinsic molecular subtypes. METHODS: We examined gene expression levels of 1097 BRCA tissue samples retrieved from TCGA and 1981 samples of METABRIC, focusing mainly on the HSP family (95 genes). Data were stratified according to the PAM50 gene expression (Luminal A, Luminal B, HER2, Basal, and Normal-like). Transcriptomic analyses include several statistical approaches: differential gene expression, hierarchical clustering and survival analysis. RESULTS: Of the 20,531 analysed genes we found that in BRCA almost 30% presented deregulated expression (19% upregulated and 10% downregulated), while of the HSP family 25% appeared deregulated (14% upregulated and 11% downregulated) (|fold change| > 2 comparing BRCA with normal breast tissues). The study revealed the existence of shared HSP genes deregulated in all subtypes of BRCA while other HSPs were deregulated in specific subtypes. Many members of the Chaperonin subfamily were found upregulated while three members (BBS10, BBS12 and CCTB6) were found downregulated. HSPC subfamily had moderate increments of transcripts levels. Various genes of the HSP70 subfamily were upregulated; meanwhile, HSPA12A and HSPA12B appeared strongly downregulated. The strongest downregulation was observed in several HSPB members except for HSPB1. DNAJ members showed heterogeneous expression pattern. We found that 23 HSP genes correlated with overall survival and three HSP-based transcriptional profiles with impact on disease outcome were recognized. CONCLUSIONS: We identified shared and specific HSP genes deregulated in BRCA subtypes. This study allowed the recognition of HSP genes not previously associated with BRCA and/or any cancer type, and the identification of three clinically relevant clusters based on HSPs expression patterns with influence on overall survival.

TP73 DNA methylation and upregulation of ΔNp73 are associated with an adverse prognosis in breast cancer.

AIM: Accumulated evidence suggests that aberrant methylation of the TP73 gene and increased levels of ΔNp73 in primary tumours correlate with poor prognosis. However, little is known regarding the transcriptional and functional regulation of the TP73 gene in breast cancer. The aim of the present study was to determine the expression of the ΔNp73 isoform, its relationship with DNA methylation of TP73 and their clinical prognostic significance in breast cancer patients. METHODS: TP73 gene methylation was studied in TCGA datasets and in 70 invasive ductal breast carcinomas (IDCs). The expression of p73 isoforms was evaluated by immunohistochemistry (IHC) and Western blot and correlated with clinicopathological variables and clinical outcome. RESULTS: We observed that the methylation of diverse CpG islands of TP73 differed significantly between molecular subtypes. An inverse correlation was found between p73 protein expression and the methylation status of the TP73 gene. The expression of exon 3' of p73 (only expressed in ΔNp73) was significantly higher in patients with wild-type p53. Immunohistochemical analysis revealed that all p73 isoforms were localised in both the nuclear and cytoplasmic compartments. We confirmed a positive association between the expression of ∆Np73 and high histological grade. CONCLUSIONS: Our findings suggest that high expression of ΔNp73 could be used to determine the aggressiveness of IDCs and could be incorporated in the pathologist's report.

HER2 and ß-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

In human breast cancer, ß-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of ß-catenin/HER2 has been reported, in the present study we have explored whether ß-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed ß-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of ß-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different ß-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in ß-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and ß-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated ß-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.

Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae.

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5-/- cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.

Autophagy induction favours the generation and maturation of the Coxiella-replicative vacuoles.

Pathogens evolved mechanisms to invade host cells and to multiply in the cytosol or in compositionally and functionally customized membrane-bound compartments. Coxiella burnetii, the agent of Q fever in man is a Gram-negative gamma-proteobacterium which multiplies in large, acidified, hydrolase-rich and fusogenic vacuoles with phagolysosomal-like characteristics. We reported previously that C. burnetii phase II replicative compartments are labelled by LC3, a protein specifically localized to autophagic vesicles. We show here that autophagy in Chinese hamster ovary cells, induced by amino acid deprivation prior to infection with Coxiella increased the number of infected cells, the size of the vacuoles, and their bacterial load. Furthermore, overexpression of GFP-LC3 or of GFP-Rab24 - a protein also localized to autophagic vacuoles - likewise accelerated the development of Coxiella-vacuoles at early times after infection. However, overexpression of mutants of those proteins that cannot be targeted to autophagosomes dramatically decreased the number and size of the vacuoles in the first hours of infection, although by 48 h the infection was similar to that of non-transfected controls. Overall, the results suggest that transit through the autophagic pathway increases the infection with Coxiella by providing a niche more favourable to their initial survival and multiplication.