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.     

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.     

Expression of p21WAF1/CIP1 in bladder cancer: relation to schistosomiasis

Cell cycle regulation is mediated in part through expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Loss of p21WAF1/CIP1 expression may, therefore, contribute partially to schistosomal carcinogenesis in the urinary bladder. We compared p21WAF1/CIP1 expression in schistosomal and nonschistosomal bladder cancer to explore possible differences in p21WAF1/CIP1 expression between the two subtypes and the possible association between schistosomiasis and loss of p21WAF1/CIP1 expression. Tumor specimens were obtained from 130 patients who underwent transurethral biopsy or cystectomy. p21WAF1/CIP1 was determined by immunodot blot, Western blot, and enzyme immunoassay (EIA). We validated a highly sensitive quantitative EIA assay for determination of p21WAF1/CIP1 in cell lysates. Precision, analytical recovery, and linearity were all excellent. Our results did not show any correlation between p21WAF1/CIP1 expression and most clinicopathologic variables. Lower expression of p21WAF1/CIP1 was evident in squamous cell carcinoma (SCC) and schistosomal subtype than in transitional cell carcinoma and nonschistosomal tumors. Our data suggest a potential role for p21WAF1/CIP1 alteration in schistosomal carcinogenesis.     

Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

Regulation of cyclin-dependent kinase inhibitor p21<Superscript>WAF1/CIP1</Superscript> by protein kinase Cδ-mediated phosphorylation

Cyclin-dependent kinase (CDK) inhibitor p21^{WAF1/CIP1(-/-)}-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21^WAF1/CIP1 is unstable in HeLa cells treated with siRNA duplexes that target PKCδ. PKCδ phosphorylates p21^WAF1/CIP1 at a serine residue (¹⁴⁶Ser) located in its C-terminal domain. In cells treated with 12-O-tetradecanoylphorbol 13-acetate, the levels of both p21^WAF1/CIP1 and its ¹⁴⁶Ser-phosphorylated form increased significantly. We also show that a substitution, resulting from a single nucleotide polymorphism (SNP) at ¹⁴⁹Asp found in certain cancer patients, strongly compromises PKCδ-mediated phosphorylation at ¹⁴⁶Ser and results in cells that are relatively resistant to TNFα-induced apoptosis. Thus, post-translational phosphorylation of p21^WAF1/CIP1 is important from an apoptotic cell death, and may also have patho-physiological relevance for the development of human cancer.     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

The cyclin-dependent kinase inhibitor p21CIP/WAF is a positive regulator of insulin-like growth factor I-induced cell proliferation in MCF-7 human breast cancer cells

To study the role of IGF-I receptor signaling on cell cycle events we utilized MCF-7 breast cancer cells. IGF-I at physiological concentrations increased the level of p21CIP/WAF mRNA after 4has well as protein after 8hby 10- and 6-fold, respectively, in MCF-7 cells. This IGF-1 effect was reduced by 50% in MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression, demonstrating that IGF-1 receptor activation was involved in this process. Preincubation with the ERK1/2 inhibitor U0126 significantly reduced the IGF-1 effect on the amount of p21CIP/WAF protein in MCF-7 cells. These results were confirmed by the expression of a dominant negative construct for MEK-1 suggesting that the increase of the abundance of p21CIP/WAF in response to IGF-1 occurs via the ERK1/2 mitogen-activated protein kinase pathway. Using an antisense strategy, we demonstrated that abolition of p21CIP/WAF expression decreased by 2-fold the IGF-1 effect on cell proliferation in MCF-7. This latter result is explained by a delay in G1 to S cell cycle progression due partly to a reduction in the activation of some components of cell cycle including the induction of cyclin D1 expression in response to IGF-1. MCF-7 cells transiently overexpressing p21 showed increased basal and IGF-I-induced thymidine incorporation. Taken together, these results define p21CIP/WAF as a positive regulator in the cell proliferation induced by IGF-1 in MCF-7 cells.     

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor,in head and neck squamous carcinoma cells

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous-cell carcinomas of head and neck (SCCHN). ZD1839 ('Iressa') is an orally active, selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27(KIP1) and p21(CIP1/WAF1) cyclin-dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose-dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27(KIP1) and p21(CIP1/WAF1) proteins associated with CDK2-cyclin-E and CDK2-cyclin-A complexes. In addition, ZD1839-induced growth inhibition was significantly reduced in cell transfectants expressing p27(KIP1) or p21(CIP1/WAF1) antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27(KIP1) and p21(CIP1/WAF1) upregulation, suggest a mechanistic connection between these events.     

VCAM-1 Upregulation Contributes to Insensitivity of Vemurafenib in BRAF-Mutant Thyroid Cancer

Vemurafenib, an inhibitor of mutant BRAF activity, is a promising anticancer agent for patients with BRAF-mutant metastatic melanoma. However, it is less effective in BRAF-mutant thyroid cancer, and the reason for this discrepancy is not yet fully elucidated. By RNA sequencing analysis, we identified vascular cell adhesion molecular-1 (VCAM-1) to be highly upregulated in both time- and dose-dependent manners during BRAF inhibition (BRAFi) in a BRAF-mutant papillary thyroid cancer cell line (BCPAP). Cell cytotoxicity and apoptosis assays showed that knockdown of the induced VCAM-1 in BCPAP cells augmented the antitumor effects of vemurafenib, with decreased IC50 values of 1.4 to 0.8 μM. Meanwhile, overexpression of VCAM-1 in a BRAF-mutant anaplastic thyroid cancer cell line (FRO) reduced the sensitivity to vemurafenib, with increased IC50 values of 1.9 to 5.8 μM. Further investigation showed that PI3K-Akt-mTOR pathway was activated during BRAFi. Co-treatment with Akt signaling inhibitor MK2206 decreased the induced expression of VCAM-1 during BRAFi. This combination further improved the efficacy of vemurafenib. Moreover, VCAM-1 promoted migration and invasion in thyroid cancer cells in vitro, which was also indicated in thyroid cancer patients. The present study is the first to demonstrate that VCAM-1 is upregulated in thyroid cancer cells treated with vemurafenib and contributes to vemurafenib resistance in BRAF-mutant thyroid cancer cells. Targeting the PI3K-Akt-mTOR pathway–mediated VCAM-1 response may be an alternative strategy to sensitize BRAF-mutant thyroid cancers to vemurafenib.     

Oxidative stress response results in increased p21WAF1/CIP1 degradation in cystic fibrosis lung epithelial cells

Lung epithelium in cystic fibrosis (CF) patients is characterized by structural damage and altered repair due to oxidative stress. To gain insight into the oxidative stress-related damage in CF, we studied the effects of hyperoxia in CF and normal lung epithelial cell lines. In response to a 95% O2 exposure, both cell lines exhibited increased reactive oxygen species. Unexpectedly, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 protein was undetectable in CF cells under hyperoxia, contrasting with increased levels of p21WAF1/CIP1 in normal cells. In both cell lines, exposure to hyperoxia led to S-phase arrest. Apoptotic features including nuclear condensation, DNA laddering, Annexin V incorporation, and elevated caspase-3 activity were not readily observed in CF cells in contrast to normal cells. Interestingly, treatment of hyperoxia-exposed CF cells with two proteasome inhibitors, MG132 and lactacystin, restored p21WAF1/CIP1 protein and was associated with an increase of caspase-3 activity. Moreover, transfection of p21WAF1/CIP1 protein in CF cells led to increased caspase-3 activity and was associated with increased apoptotic cell death, specifically under hyperoxia. Taken together, our data suggest that modulating p21WAF1/CIP1 degradation may have the therapeutic potential of reducing lung epithelial damage related to oxidative stress in CF patients.     

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.     

Novel tricyclic pyrazole BRAF inhibitors with imidazole or furan central scaffolds

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-{5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl}-phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-{2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells.     

BRAF initiating mutations in the papillary thyroid carcinoma

Among genetic alterations most important for the initiation of papillary thyroid carcinoma (PTC) is mutation T1799A in the BRAF gene which is the most frequent event (54.5%) in this type of thyroid cancer. It is seen in all stages, from microcarcinoma through clinically overt disease to anaplastic cancer. It has been shown that BRAF mutation is correlated with PTC histotype. It is identified most frequently in classical PTC and in tall cell variant. Moreover, BRAF mutation is described more often in older patients, whereas in young patients RET/PTC rearrangements dominate. In PTC cases with BRAF mutation V600E the prognosis is poorer, with more cancer invasiveness, metastasis and recurrence. The presence of BRAF mutation is related to the specific gene expression signature, different than in cancer cases showing RET/PTC rearrangement or no known initiating mutation.