Effect of BRAF-mediated PI3K/Akt/mTOR pathway on biological characteristics and chemosensitivity of NSCLC A549/DDP cells

The present study aimed to explore the biological characteristics of non-small cell lung cancer (NSCLC) cells and the mechanism of chemosensitivity through the role of the PI3K/Akt/mTOR signaling pathway mediated by BRAF gene silencing. Following cell transfection and grouping, an MTT assay detected the activity of NSCLC cells, a scratch wound test assessed the migration ability, flow cytometry using PI staining detected the cell cycle phase, TUNEL and flow cytometry through Annexin V-PI staining assessed the apoptosis, and colony formation was used to detect the sensitivity of lung cancer cells to cisplatin chemotherapy. Furthermore, the relative expression levels of BRAF, PTEN, PI3K, mTOR mRNA were assessed by RT-qPCR, and the protein expression levels of BRAF, PTEN, PI3K, phosphorylated (p)-PI3K, Akt, p-Akt, mTOR, p-mTOR, cisplatin resistance-related enzymes ERCC1 and BRCA1, apoptotic proteins Bax and Bcl-2 were assessed by western blotting. Compared with the control group and NC group, there were differences in decreased BRAF mRNA expression levels in the small interfering (si)BRAF group and siBRAF + IGF-1 group (both P<0.05). In addition, compared with the control group, the siBRAF, NVP-BEZ235 and siBRAF + NVP-BEZ235 groups had significant decreased cell viability at 2–6 days, decreased migration ability, shortened proportion of S-phase cells, increased proportion of G₁/G₀-phase cells, increased apoptosis rate, decreased number of colony-forming cells, decreased mRNA expression of PI3K, Akt and mTOR, increased PTEN mRNA expression, decreased protein expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, ERCC1, BRCA1 and Bcl-2, and increased protein expression levels of PTEN and Bax (all P<0.05); and more obvious trends were revealed in the siBRAF + NVP-BEZ235 group (all P<0.05); whereas opposite results were detected in the siBRAF + IGF-1 group when compared with the siBRAF group and NVP-BEZ235 group (all P<0.05). Silencing of BRAF gene expression to inhibit the activation of the PI3K/Akt/mTOR signaling pathway exerted a synergistic effect decreasing cell viability, inhibiting the cell cycle and migration, increasing the apoptosis rate, decreasing the number of colony-forming cells and increasing chemosensitivity of NSCLC. Activation of the PI3K/Akt/mTOR signaling pathway may reverse the role of silencing of BRAF gene expression, providing a potential approach for improving the chemosensitivity of NSCLC. The present study for the first time, to the best of our knowledge, clarified the possible mechanism of NSCLC cell biological characteristic changes and chemosensitivity from the perspective of BRAF gene silencing and PI3K/Akt/mTOR signaling pathway activation, providing a potential reference for suppressing tumor aggravation and improving the therapeutic outcomes of NSCLC at the genetic level.     

Ppp6c haploinsufficiency accelerates UV-induced BRAF(V600E)-initiated melanomagenesis

According to TCGA database, mutations in PPP6C (encoding phosphatase PP6) are found in c. 10% of tumors from melanoma patients, in which they coexist with BRAF and NRAS mutations. To assess PP6 function in melanoma carcinogenesis, we generated mice in which we could specifically induce BRAF(V600E) expression and delete Ppp6c in melanocytes. In these mice, melanoma susceptibility following UVB irradiation exhibited the following pattern: Ppp6c semi-deficient (heterozygous) > Ppp6c wild-type > Ppp6c-deficient (homozygous) tumor types. Next-generation sequencing of Ppp6c heterozygous and wild-type melanoma tumors revealed that all harbored Trp53 mutations. However, Ppp6c heterozygous tumors showed a higher Signature 1 (mitotic/mitotic clock) mutation index compared with Ppp6c wild-type tumors, suggesting increased cell division. Analysis of cell lines derived from either Ppp6c heterozygous or wild-type melanoma tissues showed that both formed tumors in nude mice, but Ppp6c heterozygous tumors grew faster compared with those from the wild-type line. Ppp6c knockdown via siRNA in the Ppp6c heterozygous line promoted the accumulation of genomic damage and enhanced apoptosis relative to siRNA controls. We conclude that in the presence of BRAF(V600E) expression and UV-induced Trp53 mutation, Ppp6c haploinsufficiency promotes tumorigenesis.     

Recent advances in subtyping tumors of the central nervous system using molecular data

Introduction: Primary brain tumors account for substantial morbidity and mortality. They often infiltrate the brain diffusely, continue growing, and cause adverse events, such as headaches, seizures, and neurological deficits. The classification of primary brain tumors, based for decades on histology, has been fundamentally changed by the World Health Organization in 2016 by incorporation of molecular data.

Areas covered: Literature from glioblastomas, high- and low-grade astrocytic, oligodendroglial, glioneuronal and ependymal tumors from the last five years were reviewed. Results from comprehensive molecular profiling of neoplasms and impact of recent molecular subtyping on neuropathological diagnosis are presented.

Expert commentary: The identification of frequent acquired mutations shows that adult and pediatric glioblastomas have divergent biology with differing prognoses. Astrocytoma and oligodendroglioma are more closely related than previously thought. Molecular profiling now enables the precise classification of most diffuse gliomas into three clinically and therapeutically different subtypes according to the presence or absence of IDH mutation and 1p/19q codeletion. New subgroups with different clinical outcomes and anatomic locations have emerged in ependymomas and pediatric embryonal tumors.     

Therapy response monitoring of the early effects of a new BRAF inhibitor on melanoma xenograft in mice: evaluation of 18F‐FDG‐PET and 18F‐FLT‐PET

Inhibition of the V600E mutated BRAF kinase gene (BRAF^V600E) is an important and effective approach to treating melanomas. A new specific small molecule inhibitor of BRAF^V600E, PLX3603, showed potent melanoma growth‐inhibiting characteristics in preclinical studies and is currently under clinical investigation. In this study we investigated the feasibility of ¹⁸F‐FDG and ¹⁸F‐FLT‐PET to monitor the early effects of the BRAF^V600E inhibitor in mice with melanoma xenografts. SCID/beige mice with subcutaneous (s.c.) A375 melanoma xenografts, expressing BRAF^V600E, received the BRAF^V600E inhibitor twice daily orally (0, 25, 50 and 75 mg/kg). At 1, 3 and 7 days after start of therapy, the uptake of ¹⁸F‐FDG and ¹⁸F‐FLT in the tumor and normal tissues was determined in ex vivo tissue samples. Serial ¹⁸F‐FDG and ¹⁸F‐FLT‐PET scans were acquired of animals at 1 day before and 1, 3 and 7 days after start of treatment with 75 mg/kg BRAF^V600E inhibitor. A dose‐dependent decrease in ¹⁸F‐FDG uptake in the A375 tumors was observed by ex vivo biodistribution analysis. Administration of 75 mg/kg BRAF inhibitor for 1, 3 and 7 days resulted in a significantly decreased ¹⁸F‐FDG uptake in A375 tumors (41, 35 and 51%, respectively). ¹⁸F‐FLT uptake in the A375 tumors was low at baseline and no significant changes in ¹⁸F‐FLT uptake were observed at any of the doses administered. These effects were corroborated by serial in vivo ¹⁸F‐FDG and ¹⁸F‐FLT‐PET imaging. These data demonstrate that ¹⁸F‐FDG‐PET can be used as an imaging biomarker to noninvasively evaluate the early effects of PLX3603. Copyright © 2014 John Wiley & Sons, Ltd.     

Traitement systémique des métastases cérébrales de mélanome

Melanomas have a high rate of brain metastases. Both the functional prognosis and the overall survival are poor in these patients. Until now, surgery and radiotherapy represented the two main modalities of treatment. Nevertheless, due to the improvement in the management of the extracerebral melanoma, the systemic treatment may be an option in patients with brain metastases. Immunotherapy with anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) – ipilimumab – or BRAF (serine/threonine-protein kinase B-raf) inhibitors – vemurafenib, dabrafenib – has shown efficacy in the management of brain metastases in a- or pauci-symptomatic patients. Studies are ongoing with anti-PD1 (programmed cell death 1) and combinations of targeted therapies associating anti-RAF (raf proto-oncogene, serine/threonine kinase) and anti-MEK (mitogen-activated protein kinase kinase).     

Time to re‐consider the meaning of BRAF V600E mutation in papillary thyroid carcinoma

The BRAF V600E mutation, resulting from the BRAFT1799A transversion, is the most common genetic mutation in papillary thyroid carcinoma (PTC), with a mean frequency close to 50% among all cases. A large number of studies in the past decade have tried to dissect the relevance and the function of the V600E mutation in controlling oncogenesis and progression of thyroid cancer. However, several works published in the latest years have provided new evidence, in partial conflict with the previous knowledge, suggesting the need of reconsidering the meaning of the BRAF V600E mutation in PTC. In this work, we attempt to discuss some of the most recent molecular, preclinical and clinical evidence to construct a more exhaustive model of function for the BRAF V600E in development, progression and therapeutic approach of thyroid cancer.     

Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer

NRAS mutations occur in 3–5% of colorectal cancer. Differently from KRAS and BRAF mutations, the role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer (mCRC) has been investigated to a lesser extent. A retrospective series suggested the role of NRAS mutations as predictors of resistance to anti‐EGFR monoclonal antibodies (MoAbs) in chemo‐refractory patients with mCRC. In our study, KRAS codons 12, 13, 61 and BRAF codon 600 mutational status were evaluated in mCRCs referred to our Institution from 2009 to 2012. NRAS codons 12, 13 and 61 mutational status was analyzed in KRAS/BRAF wt patients. We collected pathological and clinical features in the overall population and outcome data in a subset of NRAS mutated chemo‐refractory patients treated with anti‐EGFR MoAbs in advanced lines. NRAS was mutated in 47/786 (6%) mCRCs. NRAS and KRAS mutated tumors did not show significant differences in terms of clinical and pathological characteristics, except for a lower prevalence of mucinous histology (p = 0.012) and lung metastases (p = 0.012) among NRAS mutated tumors. In the uni‐ and multivariate model, NRAS mutations were associated with shorter overall survival (OS) compared to all wt patients (median OS 25.6 vs 42.7 months; univ: HR = 1.91, 95% CI 1.39–3.86, p = 0.0013; multiv: HR = 1.75, 95% CI 1.1.3–2.72, p = 0.013). None of the chemo‐refractory NRAS mutated patients evaluable for response to anti‐EGFRs achieved response. In conclusion, NRAS mutations have a relevant incidence in patients with mCRC and showed an association with specific clinical and pathological features. NRAS mutations affect mCRC patients' prognosis and predict lack of response to anti‐EGFRs.