Differential Immunoexpression of BRAF/V600E, Senescence Markers, PTEN, and T-type Calcium Channels in Acquired Naevi According to their Histopathological and Dermoscopic Classification.

BRAF/V600E mutation and other cell growth/growth-control mechanisms are involved in naevogenesis and melanomagenesis. Immunoexpression of BRAF/V600E and other molecules (p16, phosphatase and tensin homologue (PTEN), Ki67, hTERT and Cav3.1 and 3.2 calcium channels) were investigated in 80 histopatho-logically and dermoscopically classified acquired naevi. Regarding BRAF/V600E, dysplastic naevi showed lower immunostaining than common naevi, which was significant in comparison with intradermal naevi, which showed the highest BRAF/V600E histoscore. Junctional naevi showed the lowest BRAF/V600E levels. Globular/cobblestone and reticular dermoscopic patterns were consistently associated with high and low BRAF/V600E immunoexpression, respectively, but Zalaudek's peripheral globule pattern (CR/PG) showed the highest BRAF/V600E immunoexpression. Among global patterns, the previously not investigated multicomponent pattern showed the lowest BRAF/V600E immunoexpression. Regarding the remaining biomarkers, new immunohistochemical features were found, in particular p16 and PTEN low expression in multicomponent pattern; and Ki67, hTERT and Cav.3.1 high expression in CR/PG. In conclusion, histopathology and dermoscopy provide complementary information regarding the biology of melanocytic naevi.

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics.

BACKGROUND: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. METHODS: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. RESULTS: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. CONCLUSIONS: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

T-Type Calcium Channels: A Potential Novel Target in Melanoma.

T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAFV600E-mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic option.

Evaluation of Tumor Interstitial Fluid-Extraction Methods for Proteome Analysis: Comparison of Biopsy Elution versus Centrifugation.

The analysis of tumor interstitial fluid (TIF) composition is a valuable procedure to identify antimetastatic targets, and different laboratories have set up techniques for TIF isolation and proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force (10 000g) was required to obtain TIF from cSCC by centrifugation. Second, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77-80%) than among samples (50%). These results suggest that there exists an elevated consistence of TIF composition independently of the method used. However, we observed a 3-fold increase of extracellular proteins in nonoverlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies.

T-Type Calcium Channels as Potential Therapeutic Targets in Vemurafenib-Resistant BRAFV600E Melanoma.

Melanoma is a malignant neoplasia that is highly resistant to chemotherapy and radiotherapy and is associated with poor prognosis in advanced stage. Targeting melanoma that harbors the common BRAFV600E mutation with kinase inhibitors, such as vemurafenib, reduces tumor burden, but these tumors frequently acquire resistance to these drugs. We previously proposed that T-type calcium channel (TTCC) expression may serve as a biomarker for melanoma progression and prognosis, and we showed that TTCC blockers reduce migration and invasion rates because of autophagy blockade only in BRAFV600E-mutant melanoma cells. Here, we demonstrated that high expression of the TTCC Cav3.1 isoform is related to autophagic status in vemurafenib-resistant BRAFV600E-mutant melanoma cells and human biopsies, and in silico analysis revealed an enrichment of Cav3.1 expression in post-treatment melanomas. We also demonstrated that the TTCC blocker mibefradil induces apoptosis and impairs migration and invasion via inhibition of autophagy in resistant melanoma cells and mouse xenograft models. Moreover, we identified an association between PTEN status and Cav3.1 expression in these cells as a marker of sensitivity to combination therapy in resistant cells. Together, our results suggest that TTCC blockers offer a potential targeted therapy in resistant BRAFV600E-mutant melanoma and a therapeutic strategy to reduce progression toward BRAF inhibitor resistance.

In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.

Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

Tumor suppressive function of E2F-1 on PTEN-induced serrated colorectal carcinogenesis.

Many human cancers present Phosphatase and tensin homolog (PTEN) deficiency and between 20 and 30% of colorectal tumors show PTEN loss. The transcription factor, E2 promoter binding factor 1 (E2F-1), exhibits tumor promoter or suppressive functions depending on cellular type and tissue context, but its role in the progression and development of colorectal carcinogenesis was largely unknown. Here, using a tamoxifen-inducible PTEN knockout mouse model, we have demonstrated that loss of PTEN leads to the development of colorectal tumorigenesis through the serrated pathway. Next, we studied PTEN loss-driven colorectal lesions in the context of E2F-1 deficiency in vivo. Our results revealed that monoallelic and biallelic absence of E2F-1 led to an increased incidence and progression of serrated tumorigenesis induced by PTEN loss. Finally, we investigated the mechanisms by which double PTEN/E2F-1 deficiency leads to enhanced tumorigenesis. We found that colorectal tumors from PTEN/E2F-1 double knockout mice and the human colorectal carcinoma cell line HT29 with shRNA-mediated downregulation of PTEN and E2F-1 exhibit hyperactivation of the RAS-MAPK pathway, accumulation of DNA damage and resistance to apoptosis. To date, this is the first preclinical study evaluating the effect of genetic deletion of E2F-1 in colorectal malignancies driven by PTEN deficiency. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

T-type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1.

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).