Role of Raf kinase in cancer: therapeutic potential of targeting the Raf/MEK/ERK signal transduction pathway

Improvements in our understanding of the molecular basis of cancer have led to the clinical development of protein kinase inhibitors, which target pivotal molecules involved in intracellular signaling pathways implicated in tumorigenesis and progression. These novel targeted agents have demonstrated activity against a wide range of solid tumors, are generally better tolerated than standard chemotherapeutics, and may revolutionize the management of advanced refractory cancer. The ubiquitous Raf serine/threonine kinases are pivotal molecules within the Raf/mitogen extracellular kinase (MEK)/extracellular signal-related kinase (ERK) signaling pathway, which regulates cellular proliferation and survival. Raf kinase isoforms (wild-type Raf-1 or the b-raf V600E oncogene) are overactivated in a variety of solid tumor types, including renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), melanoma, and papillary thyroid carcinoma. In this review, the role of Raf in normal cells and in cancer is discussed, and an overview is given of Raf inhibitors currently in development, focusing on sorafenib tosylate (BAY 43-9006 or sorafenib). Sorafenib is the first oral multi-kinase inhibitor to be developed that targets Raf kinases (Raf-1, wild-type B-Raf, and b-raf V600E), in addition to receptor tyrosine kinases associated with angiogenesis (vascular endothelial growth factor receptor [VEGFR]-2/-3, platelet-derived growth factor receptor [PDGFR]-beta) or tumor progression (Flt-3, c-kit). Preclinical and clinical sorafenib data that led to its recent approval for the treatment of advanced RCC are summarized, along with current thinking on sorafenib's mechanism of effect on the tumor and tumor vasculature in melanoma and RCC.     

Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer therapy

The identification of intracellular signaling cascades important for the growth and survival of cancer cells has led to the development of targeted cancer therapeutics aimed at blocking these signals. The mitogen-activated protein kinase (MAPK) pathway has a well-defined role in cancer biology and has been an important target in the development of targeted therapies. Recently, several small-molecule inhibitors of MAPK/extracellular signal-regulated kinase kinase (MEK), a key intermediary of MAPK signaling, have been developed and are currently being tested in clinical trials. Herein, we review the MAPK pathway, the development of small-molecule MEK inhibitors, and the results obtained to date with MEK inhibitors in human cancer trials.     

Interleukin-6, osteopontin and Raf/MEK/ERK signaling modulate the sensitivity of human myeloma cells to alkylphosphocholines

Alkylphosphocholines are highly active against multiple myeloma (MM) cells in vitro and are devoid of myelotoxicity. Little is known about the determinants of MM cell responsiveness or resistance to these drugs. In this study we investigated the effects of disease-relevant cytokines, such as interleukin-6 (IL-6) and osteopontin (OPN), on the in vitro antimyeloma activity of erufosine and perifosine. The role of the Raf/MEK/ERK pathway was also studied. Exogenous IL-6 reduced the cytotoxicity of erufosine against OPM-2 cells and, to a smaller extent, against U-266 cells. This was accompanied by inhibition of apoptosis in OPM-2 cells. The efficacy of perifosine was similarly affected, but to a greater extent. IL-6 slightly enhanced the sensitivity of RPMI-8226 cells to erufosine, thus emphasizing the heterogeneity of MM. Induced overexpression of OPN isoforms made OPM-2 cells less sensitive to erufosine. In all cases of IL-6- or OPN-induced resistance, the effective concentrations of erufosine were still within the clinically achievable range. Like other alkylphosphocholines, erufosine enhanced Raf/MEK/ERK signaling in MM cells but in some cases this contributed to cytotoxicity.     

Conferring specificity on the ubiquitous Raf/MEK signalling pathway

The Raf-MEK-ERK signalling pathway controls fundamental cellular processes including proliferation, differentiation and survival. It remains enigmatic how this pathway can reliably convert a myriad of extracellular stimuli in specific biological responses. Recent results have shown that the Raf family isoforms A-Raf, B-Raf and Raf-1 have different physiological functions. Here we review how Raf isozyme diversity contributes to the specification of functional diversity, in particular regarding the role of Raf isozymes in cancer.     

Raf kinase inhibitors in oncology

The importance of the MAP kinase pathway, which includes the kinases Raf, MEK1/2, and ERK1/2, for the proliferation and survival of tumor cells recently increased with the discovery of activating BRAF mutations in human tumors. Therefore, in addition to a role in controlling tumors with Ras mutations and activated growth factor receptors, inhibitors of Raf kinase may harbor therapeutic potential in tumors carrying a BRAF oncogene. A variety of agents have been discovered to interfere with Raf kinase, including antisense oligonucleotides and small molecules. These inhibitors prevent the expression of Raf protein, block Ras/Raf interaction, or obstruct its kinase activity. Raf inhibitors that are currently undergoing clinical evaluation show promising signs of anti-cancer efficacy with a very tolerable safety profile. Clinically most advanced is the Raf inhibitor BAY 43-9006, which recently entered phase III clinical testing. This review addresses the rationale for targeting Raf kinase and the current status of various pharmacological approaches.     

Targeting Raf/MEK/ERK pathway in pituitary adenomas

Pituitary adenomas are the common neoplasms that cause mass effect and/or endocrine dysfunction. Studies in the pathogenesis and functional regulation of pituitary adenomas are mainly focused on the following two topics: (a) the origin of pituitary adenomas and abnormal physical adjustment due to the activation of oncogenes and loss of function for tumour-suppressor genes; and (b) the mechanistic anomalies of the intracellular signal transduction. Among which, the Raf/MEK/ERK signalling has been considered to be one of the major and central pathways in disease aetiology. Raf/MEK/ERK signalling is evolutionarily conserved that controls cellular growth, differentiation and survival. Altered functionality of this signalling pathway has been found to be involved in the development of several types of cancers in humans including pituitary adenomas. This review summarises the roles of Raf/MEK/ERK signalling pathway in pituitary tumourigenesis and highlights the clinical potential of this signalling pathways to be a therapeutic target for intervention and treatment of pituitary adenomas.     

Modulation of Raf/MEK/ERK kinase activity does not affect the chemoresistance profile of advanced prostate cancer cells

The Raf/MEK/ERK signaling cascade has been extensively studied for its roles in growth and differentiation of a variety of cell types. Confliciting evidence exists regarding the function of classical MAPK signaling with regards to the development of chemotherapeutic drug resistance; some reports describe an pro-survival role, whereas others have suggested that activation of Raf/MEK/ERK is essential for drug-induced death. To elucidate the importance of MAPK signaling in the development of advanced prostate cancer drug resistance, DU145 and PC3 prostate cells were stably-infected/transfected with constitutively-activated mutants of both Raf-1 and B-Raf. Results from MTT analyses suggested that activation of either Raf-1 or B-Raf is inconsequential in prostate cancer chemoresistance. To confirm these findings, the MAPK signal transduction cascade was activated with EGF and response to doxorubicin or paclitaxel was measured in the presence/absence of the MEK-specific inhibitor, U0126. These results showed that inhibition of signals transduced by the MAPK pathway are insufficient to affect the chemoresistance profile of advanced prostate cancer cells. Together, these data demonstrate that the response of prostatic tumors to the chemotherapeutic compounds doxorubicin and paclitaxel is independent of Raf/MEK/ERK signaling.     

Raf激酶抑制蛋白与恶性肿瘤

Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的成员。RKIP在Raf、核转录因子-κB(NF-κB)及G蛋白偶联受体(GPCR)等蛋白激酶信号传导通路中起重要调节作用。RKIP参与神经发育、心脏功能、精子发生、细胞迁移及巨噬细胞分化,同时还具有丝氨酸蛋白酶活性。近年来众多资料表明,RKIP在多种恶性肿瘤中表达减弱或丢失,其在恶性肿瘤中作用机制已经成为研究的热点,该类研究将为今后肿瘤治疗提供新的靶点。     

Raf激酶抑制蛋白与恶性肿瘤

Raf激酶抑制蛋白（RKIP）是磷脂酰乙醇胺结合蛋白家族的成员。RKIP在Raf、核转录因子-κB（NF-κB）及G蛋白偶联受体（GPCR）等蛋白激酶信号传导通路中起重要调节作用。RKIP参与神经发育、心脏功能、精子发生、细胞迁移及巨噬细胞分化，同时还具有丝氨酸蛋白酶活性。近年来众多资料表明，RKIP在多种恶性肿瘤中表达减弱或丢失，其在恶性肿瘤中作用机制已经成为研究的热点，该类研究将为今后肿瘤治疗提供新的靶点。     

Leukemia inhibitory factor can mediate Ras/Raf/MEK/ERK-induced growth inhibitory signaling in medullary thyroid cancer cells

Medullary thyroid carcinoma (MTC) is a multiple endocrine neoplasia type 2 syndrome caused by mutations in extracellular receptor or intracellular kinase domains of the RET proto-oncogene. Activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest by secreting leukemia inhibitory factor (LIF) in MTC cells harboring a RET receptor domain mutation. Here, we report that Ras/Raf/MEK/ERK can also mediate, via LIF, growth inhibition in MTC cells harboring a RET kinase domain mutation. Ras/Raf/MEK/ERK activation was sufficient to induce growth inhibition and LIF expression in the human MTC line MZ-CRC-1. Presence of LIF-mediated signaling was determined by blocking the activity of culture medium conditioned by Raf-activated cells using anti-LIF neutralizing antibody. In addition, recombinant LIF effectively suppressed cell proliferation via cell cycle arrest in G0/G1 phase. Expression of dominant negative STAT3 abrogated LIF effects, indicating that LIF mediates its signaling through the JAK/STAT3 pathway. These results suggest that growth inhibition and activation of the autocrine/paracrine signaling through LIF/JAK/STAT may be a common response to Ras/Raf activation in different MTC types, and justify further evaluation of LIF as a potential anticancer agent for MTC.     

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.     

Empiric Therapy with BRAF and MEK Inhibitors in Metastatic Melanoma

BRAF and MEK inhibitors are highly active in the setting of BRAF^V600 mutant melanoma. Rarely, patients without previous testing present with fulminant progression necessitating emergent treatment prior to BRAF testing results. The safety and efficacy of empiric treatment in this setting is unclear. Herein, we present two patients treated with empiric BRAF and MEK inhibitors, resulting in dramatic clinical improvement in one patient later found to have a BRAF mutation, and lack of improvement (but no accelerated progression) in a patient lacking this mutation. Empiric BRAF and MEK inhibitor treatment should not be routinely pursued but may be given safely in rare, emergent situations.     

Raf蛋白激酶与肿瘤治疗相关研究的现状

目的:讨论Raf蛋白激酶在肿瘤治疗中的作用。方法:使用PubMed和万方数据库检索,以Raf激酶、抗肿瘤为关键词检索出82篇文章,以与ERK通路、BAY43-9006、ISIS5132、LE-rafAON和MCP1具有相关性的和2006、2007年内发表的为限制筛选25篇文章,以这些文章为基础并扩展文章的参考文献进行讨论。结果:多种拮抗Raf激酶的生物分子的临床研究已深入到不同的阶段,并且得到了较大的进展,其中包括Raf激酶抑制剂-BAY43-9006,反义寡核苷酸ISIS5132、LErafAON、Raf去稳定剂-格尔德霉素以及Ras-Raf的衔接抑制物-MCP1。结论:对于不同部位的肿瘤治疗,Raf激酶可作为单独的攻击靶点或者与传统的化疗方案相结合,而且这两种方式都产生了较有效的作用。     

Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf

The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.     

Targeting mitogen-activated protein kinase kinase (MEK) in solid tumors

The Raf-mitogen activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) protein kinase signaling cascade is an important intracellular pathway whose activation influences many fundamental cellular processes and whose aberrancy is associated with cancer cell growth. In addition to activation from within by, for example, Raf mutations, this pathway is frequently activated from above by mutated Ras or epidermal growth factor receptor (EGFR). Given the near ubiquity of derangements affecting at least part of this network in cancer, there is a strong and clear rationale for interrupting it. In recent times, in colorectal and lung cancer, Ras and EGFR mutant status have been shown to be critically important and mutually exclusive predictors of response to anti-EGFR therapies. These developments underline the importance of targeting downstream effectors, and MEK inhibition has been the subject of intense scientific and clinical research for some time now. This article reviews the current status of MEK inhibitors with regard to their clinical development.     

Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma

The Ras/Raf/MEK/ERK signalling cascade is frequently deregulated in tumourigenic diseases and known to be involved in proliferation and transformation of cells. Also in hepatocellular carcinoma (HCC) increased ERK levels are observed and known to correlate with tumour progression, but the underlying molecular mechanism are unknown. We analyzed expression of Raf-1 kinase inhibitory protein (RKIP) in HCC. Expression of RKIP mRNA and protein was downregulated in HCC cell lines and tissue as compared to primary human hepatocytes (PHH) or non-tumorous liver tissue, respectively. Transfection of an HCC cell line with an RKIP expression construct blocked the Raf kinase pathway resulting in decreased activity of ERK1/2 and AP-1. In contrast, downregulation of RKIP by transfection with an antisense RKIP construct led to increased ERK1/2 and AP-1 activity. Since HCC develop in the majority of cases in cirrhotic liver tissue and cirrhosis is the main risk factor for HCC development, we analyzed RKIP expression also in non-cancerous cirrhotic liver tissues by immunohistochemistry. In contrast to normal liver tissue, where the staining was equally distributed within the cytoplasm, hepatocytes in cirrhotic liver revealed an intense RKIP staining of the membrane. It can be speculated that this changed RKIP expression pattern parallels impaired protein function in PHH in cirrhotic livers that may predispose PHH to malignant transformation. In addition, our study demonstrates functional relevance of downregulation of RKIP in HCC that may play an important role in HCC development and progression.     

Raf kinase inhibitor protein (RKIP) in cancer

Raf kinase inhibitory protein (RKIP) was initially identified as phosphatidylethanolamine binding protein in bovine brain. It was later identified as a protein that inhibits Raf kinase activation of MEK. Further exploration has revealed that RKIP modulates several other signaling pathways including NF–κB and G-protein signaling. A gene array screen revealed that RKIP expression was low in a metastatic compared with non-metastatic prostate cancer cell line. Further experiments revealed that RKIP fits the criteria for a metastasis suppressor gene. RKIP expression has been shown to be downregulated in metastatic tissues, compared with non-metastatic tissue in multiple cancers, suggesting that loss of RKIP metastasis suppressor activity is a broad mechanism leading to metastasis. Additionally, loss of RKIP has been shown to impact therapy through conferring radioresistance and chemoresistance. Taken together, these data indicate understanding RKIP’s contributions to cancer may lead to important therapeutic strategies to prevent metastasis and promote therapeutic efficacy.     

Raf激酶抑制蛋白增强卵巢癌细胞的化疗敏感性

摘 要　目的：探讨Raf激酶抑制蛋白（Raf kinase inhibitor protein,RKIP）对卵巢癌SKOV-3细胞化疗敏感性的影响。方法：以脂质体法将含有人全长RKIP基因的真核表达质粒pcDNA3.1-ssRKIP转染入SKOV-3细胞中,Western blotting检测SKOV-3细胞中RKIP蛋白的表达。不同浓度顺铂作用转染后的SKOV-3细胞,MTS法观察RKIP基因转染对顺铂处理后SKOV-3细胞增殖的影响,流式细胞仪检测RKIP基因转染对顺铂诱导SKOV-3细胞凋亡及细胞周期的影响。结果：pcDNA3.1-ssRKIP转染的SKOV-3细胞RKIP表达明显升高。不同浓度顺铂处理细胞24、48、72 h后,RKIP基因转染细胞增殖抑制率显著高于对照细胞（P<0.05）。用2.5 μg/ml顺铂作用SKOV-3细胞24 h后,RKIP转染细胞的凋亡率为（10.86±0.73）%,明显高于未转染细胞的（4.27±0.67）%和空质粒转染细胞的（4.02±0.43）%（P<0.01);在无顺铂作用情况下,RKIP转染细胞的凋亡率为（3.11±0.78）%,仍然高于未转染细胞的（1.51±0.13）%和转染空质粒细胞的（1.97±0.46）%（P<0.01)。细胞周期检测结果显示,RKIP转染细胞G0/G1期的比例下降,S期的比例增加,转染的SKOV-3细胞发生S期阻滞。结论：RKIP基因的转染可以增加卵巢癌SKOV-3细胞对化疗药物顺铂的敏感性。