A Preclinical Model of Cutaneous Melanoma Based on Reconstructed Human Epidermis

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) as a prognostic biomarker of poor outcomes in esophageal cancer patients

Background Esophageal cancer is one of the most aggressive malignancies, and is associated with multiple genetic mutations. At present, the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) gene mutation has been observed in esophageal cancer and is associated with poor prognosis. This study aimed to investigate the protein expression of BRAF in esophageal cancer and determine its effect on patient outcomes. Methods We used immunohistochemistry to detect the expression of BRAF via tissue microarrays in esophageal cancer samples, the Kaplan–Meier method to perform survival analysis, and the Cox proportional hazards regression model to explore the risk factors of esophageal cancer. The role of BRAF in the proliferation, invasion, and metastasis of esophageal cancer was studied by clone formation, scratch test, Transwell invasion and migration test. The tumor-bearing model of BRAF inhibitor was established using TE-1 cells, and corresponding negative control was set up to observe the growth rate of the two models. Results The results revealed that BRAF overexpression was significantly correlated with Ki67 (P < 0.05). Survival analysis showed that BRAF overexpression contributed to a shorter overall survival (P = 0.014) in patients with esophageal cancer. Univariate and multivariate regression analyses demonstrated that BRAF was a prognostic factor for poor esophageal cancer outcomes (P < 0.05). Small interfering RNA knockdown of BRAF significantly reduced the cell clone formation rate compared to the control group. Transwell assay analysis showed that the migration and invasion of cells in the experimental group were significantly inhibited relative to the control group, and the inhibition rates of the small interfering RNA group were 67% and 60%, respectively. In the scratch test, the wound healing ability of the BRAF knockdown group was significantly weaker than that of the control group. There were significant differences in tumor growth volume and weight between the two groups in nude mice. Conclusion BRAF overexpression may serve as an effective predictive factor for poor prognosis.

Alpha-Terpineol as Antitumor Candidate in Pre-Clinical Studies

Background: Alpha-terpineol is a monoterpene alcohol with anti-tumor activity against different tumor cell lines (lung, breast, leukemias and colorectal) through blockage of NF-kB expression, which play an important role in tumor cells growth. Objective: Evaluate the antitumor activity of alpha-terpineol in murine Sarcoma 180 cell line Methods: For the tests, different cytotoxic and genotoxic assays were used, including Trypan blue, cytokinesis-blocked micronucleus assay, comet assay, agarose gel DNA fragmentation, flow cytometry and cell viability using fluorescence. Ascitic fluid cells from sarcoma 180 were obtained from Mus musculus peritoneal cavity and Alpha-terpineol was tested at 100, 250 and 500 μg/mL. Doxorubicin and Cisplatin were used as positive controls. Results: Cytotoxic effects of alpha-terpineol were found in all concentrations tested, reducing cell viability in 50.9; 38.53; 30.82% at 100, 250 and 500 μg/mL, respectively. Alpha-terpineol induced genotoxic effects due to DNA fragmentation (increased frequency and index of damage), and was clastogenic by increased micronuclei formation, nucleoplasmic bridges and nuclear buds. DNA fragmentation and increased cell death indicated that alpha-terpineol can cause early, late, and necrotic apoptosis. Conclusion: Our data indicate that alpha-terpineol has antitumor activity revealed by cytogenetic mechanisms and / or loss of cell membrane integrity.

Into the Unknown-exploring a Rare BRAF K601E Mutation in a Rectal Adenocarcinoma

Aim: v-Raf murine sarcoma viral oncogene homolog B (BRAF) gene mutations are broadly divided as BRAF V600E and BRAF non-V600E. These groups exhibit differences in the clinical, histological, molecular and therapeutic mechanisms. Case Presentation and Discussion: The case described is of a middle aged female with an aggressive rectal adenocarcinoma diagnosed on biopsy and immunohistochemistry. A rare BRAF non-V600E mutation at codon 601, K601E was detected. This mutation has been described in very few cases of colonic adenocarcinoma. The tumor showed extensive local and peritoneal disease with lung metastasis at presentation and progression on the primary line of chemotherapy. The aggressive predisposition of the tumor was contrary to reports suggesting non-V600E tumors to connote a better prognosis. Conclusion: Active reporting of non-V600E BRAF variants in colorectal adenocarcinoma is essential in order to create a robust database which will help in assessing a biological behavior of these tumors and explore the role of targeted therapy.

A Rare p.T599dup BRAF Mutant NSCLC in a Non-Smoker

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) mutated non-small-cell lung cancer (NSCLC) is an exceptionally rare form of lung cancer, found only in one to two percent of patients with an NSCLC diagnosis. BRAF NSCLC traditionally affects former or active smokers. BRAF mutations have always been of special interest to the oncological community, as they offer potential for targeted therapies. BRAF mutation spectrum includes mutations that are of both V600 and non-V600 types. BRAF V600 is an activating mutation, which results in high kinase activity and overproduction of active oncoproteins such as rapidly accelerated fibrosarcoma (RAF). This makes them susceptible to targeted therapies with RAF inhibitors. There has been little evidence, however, regarding efficacy of RAF inhibitors towards non-activating mutations that have intermediate to low kinase activity, such as non-V600 BRAF mutations. While several approaches have been investigated to overcome the limitations of RAF inhibitors, such as use of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) inhibitors or combination of MEK and RAF inhibitors, none of them have been proven to have a superior efficacy for low kinase activity non-V600 BRAF tumors. We present a case of an extremely rare variant of NSCLC BRAF p.T599dup mutation in a non-smoker that responded to a targeted combination therapy with RAF and MEK inhibitors. The patient responded well to therapy that usually targets high kinase activity V600 mutations. Our hope is to bring more attention to non-V600 mutations and document their responses to existing and new therapies.

BRAF: A Two-Faced Janus

Gain-of-function of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) is one of the most frequent oncogenic mutations in numerous cancers, including thyroid papillary carcinoma, melanoma, colon, and lung carcinomas, and to a lesser extent, ovarian and glioblastoma multiforme. This mutation aberrantly activates the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, thereby eliciting metastatic processes. The relevance of BRAF mutations stems from its prognostic value and, equally important, from its relevant therapeutic utility as an actionable target for personalized treatment. Here, we discuss the double facets of BRAF. In particular, we argue the need to implement diagnostic molecular algorithms that are able to detect this biomarker in order to streamline and refine diagnostic and therapeutic decisions.

Tumor Microenvironment: Implications in Melanoma Resistance to Targeted Therapy and Immunotherapy

Antitumor therapies have made great strides in recent decades. Chemotherapy, aggressive and unable to discriminate cancer from healthy cells, has given way to personalized treatments that, recognizing and blocking specific molecular targets, have paved the way for targeted and effective therapies. Melanoma was one of the first tumor types to benefit from this new care frontier by introducing specific inhibitors for v-Raf murine sarcoma viral oncogene homolog B (BRAF), mitogen-activated protein kinase kinase (MEK), v-kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), and, recently, immunotherapy. However, despite the progress made in the melanoma treatment, primary and/or acquired drug resistance remains an unresolved problem. The molecular dynamics that promote this phenomenon are very complex but several studies have shown that the tumor microenvironment (TME) plays, certainly, a key role. In this review, we will describe the new melanoma treatment approaches and we will analyze the mechanisms by which TME promotes resistance to targeted therapy and immunotherapy.