BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.     

Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7

Expression of an oncogene in a primary cell can, paradoxically, block proliferation by inducing senescence or apoptosis through pathways that remain to be elucidated. Here we perform genome-wide RNA-interference screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we find a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi, and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.     

Selective BRAFV600E inhibitor PLX4720, requires TRAIL assistance to overcome oncogenic PIK3CA resistance

Documented sensitivity of melanoma cells to PLX4720, a selective BRAFV600E inhibitor, is based on the presence of mutant BRAF(V600E) alone, while wt-BRAF or mutated KRAS result in cell proliferation. In colon cancer appearance of oncogenic alterations is complex , since BRAF, like KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. When PLX4720 was used to treat colon tumours, results were not encouraging and herein we attempt to understand the cause of this recorded resistance and discover rational therapeutic combinations to resensitize oncogene driven tumours to apoptosis. Treatment of two genetically different BRAF(V600E) mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKO(BRAFV600E/PIK3CAH1047) cells. In contrast, for the same level of apoptosis in HT29(BRAFV600E/PIK3CAP449T) cells, TRAIL was combined with 17-AAG, an Hsp90 inhibitor. For cells where PLX4720 was completely ineffective, 17-AAG was alternatively used to target mutant BRAF(V600E). TRAIL dependence on the constitutive activation of BRAF(V600E) is emphasised through the overexpression of BRAF(V600E) in the permissive genetic background of colon adenocarcinoma Caco-2 cells. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CA(MT) as the inhibitory factor. Another rational combination includes 17-AAG synergism with TRAIL in a BRAF(V600E) mutant dependent manner to commit cells to apoptosis, through DR5 and the amplification of the apoptotic pathway. We have successfully utilised combinations of two chemically unrelated BRAF(V600E) inhibitors in combination with TRAIL in a BRAF(V600E) mutated background and provided insight for new anti-cancer strategies where the activated PI3KCA mutation oncogene should be suppressed.     

Resistance to vemurafenib resulting from a novel mutation in the BRAFV600E kinase domain

Resistance to the BRAF inhibitor vemurafenib poses a significant problem for the treatment of BRAFV600E‐positive melanomas. It is therefore critical to prospectively identify all vemurafenib resistance mechanisms prior to their emergence in the clinic. The vemurafenib resistance mechanisms described to date do not result from secondary mutations within BRAFV600E. To search for possible mutations within BRAFV600E that can confer drug resistance, we developed a systematic experimental approach involving targeted saturation mutagenesis, selection of drug‐resistant variants, and deep sequencing. We identified a single nucleotide substitution (T1514A, encoding L505H) that greatly increased drug resistance in cultured cells and mouse xenografts. The kinase activity of BRAFV600E/L505H was higher than that of BRAFV600E, resulting in cross‐resistance to a MEK inhibitor. However, BRAFV600E/L505H was less resistant to several other BRAF inhibitors whose binding sites were further from L505 than that of PLX4720. Our results identify a novel vemurafenib‐resistant mutant and provide insights into the treatment for melanomas bearing this mutation.     

甲状腺乳头状癌合并桥本氏甲状腺炎的BRAF基因表达及侵袭性

目的:比较甲状腺乳头状癌合并桥本氏甲状腺炎与不合并桥本氏甲状腺炎的BRAFV600E基因表达以及侵袭性的区别。方法:2011年9月到2013年9月四川大学华西医院手术治疗并有BRAFV600E基因测定的甲状腺乳头状癌患者226名,均有病理证实。其中合并桥本氏甲状腺炎者50例为研究组,同期随机抽取50例不合并桥本氏甲状腺炎者作为对照组。比较两组性别、年龄、肿瘤大小、数量、BRAFV600E基因表达以及甲状腺外侵犯和淋巴结转移与侵袭性相关的因素的区别。结果:甲状腺乳头状癌合并桥本氏甲状腺炎在男女性别,发病年龄、肿瘤大小上和对照组相比无差异(P0.05);BRAFV600E突变率、甲状腺外侵犯和淋巴结转移都较对照组更低(P0.05)。BRAF基因突变阳性组甲状腺外侵犯和淋巴结转移率较BRAFV600E基因突变阴性组更高(P0.05)。结论:BRAFV600E基因突变的甲状腺乳头状癌患者有更高的甲状腺腺外侵犯和淋巴结转移。甲状腺乳头状癌合并桥本氏甲状腺炎较不合并桥本氏甲状腺炎有着更低的BRAFV600E突变率,更低的甲状腺外侵犯和淋巴结转移。     

The BRAFT1799A mutation confers sensitivity of thyroid cancer cells to the BRAFV600E inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF(V600E), as a result of the BRAF(T1799A) mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF(V600E)-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF(T1799A) mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF(T1799A) mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC(50) values (0.115-1.156μM) in BRAF(V600E) mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC(50) values (56.674-1349.788μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF(T1799A) mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF(T1799A) mutation-selective therapeutic agent for thyroid cancer.     

A Further Investigation of Combined Mismatch Repair and BRAFV600E Mutation Specific Immunohistochemistry as a Predictor of Overall Survival in Colorectal Carcinoma

Mutation specific immunohistochemistry (IHC) is a promising new technique to detect the presence of the BRAFV600E mutation in colorectal carcinoma (CRC). When performed in conjunction with mismatch repair (MMR) IHC, BRAFV600E IHC can help to further triage genetic testing for Lynch Syndrome. In a cohort of 1426 patients undergoing surgery from 2004 to 2009 we recently demonstrated that the combination of MMR and BRAFV600E IHC holds promise as a prognostic marker in CRC, particularly because of its ability to identify the poor prognosis MMR proficient (MMRp) BRAFV600E mutant subgroup. We attempted to validate combined MMR and BRAFV600E IHC as a prognostic indicator in a separate cohort comprising consecutive CRC patients undergoing surgery from 1998 to 2003. IHC was performed on a tissue microarray containing tissue from 1109 patients with CRC. The 5 year survivals stratified by staining patterns were: MMRd/BRAFwt 64%, MMRd/BRAFV600E 64%, MMRp/BRAFwt 60% and MMRp/BRAFV600E 53%. Using the poor prognosis MMRp/BRAFV600E phenotype as baseline, univariate Cox regression modelling demonstrated the following hazard ratios for death: MMRd/BRAFwt HR = 0.71 (95%CI = 0.40–1.27), p = 0.31; MMRd/BRAFV600E HR = 0.74 (95%CI = 0.51–1.07), p = 0.11 and MMRp/BRAFwt HR = 0.79 (95%CI = 0.60–1.04), p = 0.09. Although the findings did not reach statistical significance, this study supports the potential role of combined MMR and BRAF IHC as prognostic markers in CRC.     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

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).     

Dabrafenib; Preclinical Characterization,Increased Efficacy when Combined with Trametinib,while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAF^V600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G₁ cell cycle arrest, followed by cell death. In a BRAF^V600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.     

Role of BMPs in controlling the spatial and temporal origin of GFAP astrocytes in the embryonic spinal cord

In the vertebrate central nervous system (CNS), astrocytes are the most abundant and functionally diverse glial cell population. However, the mechanisms underlying their specification and differentiation are still poorly understood. In this study, we have defined spatially and temporally the origin of astrocytes and studied the role of BMPs in astrocyte development in the embryonic chick spinal cord. Using explant cultures, we show that astrocyte precursors started migrating out of the neuroepithelium in the mantle layer from E5, and that the dorsal-most level of the neuroepithelium, from the roof plate to the dl3 level, did not generate GFAP-positive astrocytes. Using a variety of early astrocyte markers together with functional analyses, we show that dorsal-most progenitors displayed a potential for astrocyte production but that dorsally-derived BMP signalling, possibly mediated through BMP receptor 1B, promoted neuronal specification instead. BMP treatment completely prevented astrocyte development from intermediate spinal cord explants at E5, whereas it promoted it at E6. Such an abrupt change in the response of this tissue to BMP signalling could be correlated to the onset of new foci of BMP activity and enhanced expression of BMP receptor 1A, suggesting that BMP signalling could promote astrocyte development in this region.     

Vemurafenib and concomitant stereotactic radiation for the treatment of melanoma with spinal metastases: A case report

A 56-year-old man with BRAFV600E melanoma and spinal metastases treated with vemurafenib and stereotactic radiation showed a partial response without neurological, skin or mucosal toxicity, 8 months after completion of this combination. This case suggests that stereotactic radiation spares normal tissues and might be safer than conventional fractionated radiation with vemurafenib.     

Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord.

The role of the hyaluronate receptor, CD44, is well known in adult mammal astrocytes where it modulates neuron-glia interactions. However, no data exist regarding its expression in other vertebrates during their development. In order to detect the expression of CD44 in the chicken and its possible involvement in glial precursor migratory patterns during spinal cord development, a monoclonal antibody (MoAb) against the mammalian standard isoform, CD44-H, was used in immunohistochemical and immunoblot assays. With these methods, CD44 hyaluronate receptors were found on mature astrocyte membranes of adult chicken spinal cord. Astrocytes were identified using a MoAb against GFAP. During development, small clusters of CD44 labelled cells were seen lining the central canal starting from embryonic stage E10. These labelled cells were dispersed in the dorsal, lateral and ventral funiculi of the spinal cord in the subsequent stages. After stage E15, the CD44 labelled cells were identified as astrocytes because of their GFAP immunoreactivity. We conclude that CD44 receptors on immature astrocyte precursors should be considered as early astrocyte markers which have a possible role during cell migratory dispersal.     

BRAF V600E protect from cell death via inhibition of the mitochondrial permeability transition in papillary and anaplastic thyroid cancers

BRAF T1799A mutation is the most common genetic variation in thyroid cancer, resulting in the production of BRAF V600E mutant protein reported to make cells resistant to apoptosis. However, the mechanism by which BRAF V600E regulates cell death remains unknown. We constructed BRAF V600E overexpression and knockdown 8505C and BCPAP papillary and anaplastic thyroid cancer cell to investigate regulatory mechanism of BRAF V600E in cell death induced by staurosporine (STS). Induced BRAF V600E expression attenuated STS‐induced papillary and anaplastic thyroid cancer death, while BRAF V600E knockdown aggravated it. TMRM and calcein‐AM staining showed that opening of the mitochondrial permeability transition pore (mPTP) during STS‐induced cell death could be significantly inhibited by BRAF V600E. Moreover, our study demonstrated that BRAF V600E constitutively activates mitochondrial ERK (mERK) to inhibit GSK‐3‐dependent CypD phosphorylation, thereby making BRAF V600E mutant tumour cells more resistant to mPTP opening. In the mitochondria of BRAF V600E mutant cells, there was an interaction between ERK1/2 and GSKa/ß, while upon BRAF V600E knockdown, interaction of GSKa/ß to ERK was decreased significantly. These results show that in thyroid cancer, BRAF V600E regulates the mitochondrial permeability transition through the pERK‐pGSK‐CypD pathway to resist death, providing new intervention targets for BRAF V600E mutant tumours.     

The extracellular matrix molecule tenascin C modulates expression levels and territories of key patterning genes during spinal cord astrocyte specification

The generation of astrocytes during the development of the mammalian spinal cord is poorly understood. Here, we demonstrate for the first time that the extracellular matrix glycoprotein tenascin C regulates the expression of key patterning genes during late embryonic spinal cord development, leading to a timely maturation of gliogenic neural precursor cells. We first show that tenascin C is expressed by gliogenic neural precursor cells during late embryonic development. The loss of tenascin C leads to a sustained generation and delayed migration of Fgfr3-expressing immature astrocytes in vivo. Consistent with an increased generation of astroglial cells, we documented an increased number of GFAP-positive astrocytes at later stages. Mechanistically, we could demonstrate an upregulation and domain shift of the patterning genes Nkx6.1 and Nkx2.2 in vivo. In addition, sulfatase 1, a known downstream target of Nkx2.2 in the ventral spinal cord, was also upregulated. Sulfatase 1 regulates growth factor signalling by cleaving sulphate residues from heparan sulphate proteoglycans. Consistent with this function, we observed changes in both FGF2 and EGF responsiveness of spinal cord neural precursor cells. Taken together, our data implicate Tnc in the regulation of proliferation and lineage progression of astroglial progenitors in specific domains of the developing spinal cord.