Carcinogenesis Advance Access originally published online on May 29, 2008
Carcinogenesis 2008 29(6):1157-1163; doi:10.1093/carcin/bgn119
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RKTG sequesters B-Raf to the Golgi apparatus and inhibits the proliferation and tumorigenicity of human malignant melanoma cells
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China
* To whom correspondence should be addressed. Tel: +86 21 54920916; Fax: +86 21 54920291; Email: ychen3{at}sibs.ac.cn
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
Raf kinase trapping to Golgi (RKTG) is a newly characterized negative regulator of the Ras–Raf–mitogen-activated and extracellular signal-regulated kinase kinase (MEK)–extracellular signal-regulated kinase (ERK)-signaling pathway via sequestrating Raf-1 to the Golgi apparatus. Among Raf kinase family members, B-Raf is the most frequently mutated gene in human cancers and an activated B-Raf mutation V600E is associated with >60% of human melanomas. Here, we show that RKTG can also bind and translocate B-Raf to the Golgi apparatus. When overexpressed in A375, a human malignant melanoma cell line with B-Raf(V600E), RKTG inhibits ERK activation, cell proliferation and transformation of A375 cells. In addition, the tumorigenicity of the RKTG-expressing A375 cells is suppressed in nude mice. Consistently, cell proliferation rate was reduced in the tumor xenografts in which RKTG was overexpressed. Collectively, our results suggest that RKTG may play a suppressive role in human melanoma that harbors an oncogenic B-Raf mutation via its antagonistic action on B-Raf.
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated and extracellular signal-regulated kinase kinase; MM, malignant melanoma; RKTG, Raf kinase trapping to Golgi
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
The Raf family members, A-Raf, B-Raf and Raf-1 (or C-Raf), relay the signals from Ras to mitogen-activated and extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) (1). This signaling pathway transduces extracellular signals from the cell membrane to the nucleus via a cascade of phosphorylation events and regulates many fundamental cellular functions including cell proliferation, apoptosis, differentiation, motility and metabolism. This pathway is also implicated in many human diseases especially in the development of carcinomas of the pancreas, ovary, thyroid, colon and skin (1,2). Malignant melanoma (MM) is a form of skin cancer that originates from melanocytes, the specialized pigment-producing cells found in the basal layer of the epidermis and also in the eye (3). MM has a poor prognosis, high metastatic potential and resistance to treatment (4). The Ras–Raf–MEK–ERK pathway is activated in virtually all melanomas, and
60% of human melanomas harbor an oncogenic mutation of B-Raf(V600E). The mutation is composed of a single-base substitution T1799A, resulting in substitution of valine by glutamate at position 600 of the protein (5–7). This change renders B-Raf constitutively active, leading to elevated phosphorylation of MEK and activation of downstream mitogenic targets. Cells that express B-Raf(V600E) are dependent on it for proliferation and can grow as tumors in nude mice (8). Many proteins are implicated in the regulation of Ras–Raf–MEK–ERK-signaling cascade and several components within this pathway have been found to be regulated by subcellular compartmentalization (9,10). Ras can be shuttled between different membrane compartments to render subcellular-specific signaling through different lipid anchors (11,12). MEK–ERK can be recruited to the Golgi apparatus by Sef and such spatial regulation blocks the Ras signaling to the nucleus but not to the cytosol (13). In addition, Raf could be negatively regulated by interaction with other proteins such as Raf kinase inhibitor protein, Sprouty and its related protein Spred (14,15). We recently identified that Raf kinase trapping to Golgi (RKTG) serves as a spatial regulator of Raf-1. RKTG blocks the signaling and function of Raf-1 by sequestering it to the Golgi apparatus (16). However, the physiological outcomes underlying the regulation of Ras–Raf–MEK–ERK-signaling pathway by RKTG remain unclear. Here, we demonstrate that RKTG also interacts with B-Raf, the most frequently mutated gene of Raf kinase family during tumor development, and translocates B-Raf into the Golgi apparatus, thus blocking ERK-signaling pathway. Consequently, overexpression of RKTG inhibits ERK activation, cell proliferation and transformation of A375 human MM cells that bear B-Raf(V600E). In addition, tumor formation by inoculation of melanoma cells into nude mice is suppressed by RKTG overexpression, associated with a reduced cell proliferation rate. These findings, therefore, have uncovered a potential tumor-suppressive function of RKTG in human MM.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
Materials and cell culture
The antibodies were purchased as follows: phospho-ERK1/2 was from Cell Signaling Technology (Danvers, MA); monoclonal anti-Flag antibody and polyclonal anti-Myc antibody were from Sigma–Aldrich (St Louis, MO); monoclonal antibodies against Myc, hemagglutinin and Raf-1 and polyclonal antibodies against hemagglutinin, total ERK1/2 and actin were from Santa Cruz Biotechnology (Santa Cruz, CA); Golgin-97 monoclonal antibody, Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-mouse, and -rabbit IgG were from Molecular Probes (Eugene, OR); Cy5-labeled goat anti-mouse IgG was from Jackson Immuno Research Laboratories (West Grove, PA) and anti-Ki67 antibody was from BD Biosciences Pharmingen (San Diego, CA). ApopTag® Peroxidase In Situ Apoptosis Detection Kit was from Chemicon (Temecula, CA). Epidermal growth factor (EGF) was from Sigma–Aldrich. HEK293T, Hela and human A375 melanoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum.
Plasmids, cell transfection and stable expression of RKTG in A375 cells
The Myc-tagged and enhanced green fluorescent protein-tagged RKTG complementary DNA expression vectors have been described previously (16). RKTG was also cloned into p3XFlag-CMV-10 (Sigma–Aldrich) to fuse with three Flag tags at the N-terminus. RKTG fused with the influenza hemagglutinin epitope was cloned into pcDNA3 (Invitrogen, Carlsbad, CA) for stable transfection experiments. The Elk-reporter system contains pSG-Gal4-Elk1 and pG5.Ef lux3 (17). The constitutively active Raf-1(BXB) was kindly provided by Susanne Weg-Remers (Institut für Toxikologie und Genetik, Karlsruhe, Germany) (17); Flag-tagged Raf-1 was kindly provided by Dong Xie (Chinese Academy of Sciences); Myc-tagged wild-type B-Raf and B-Raf(V600E) were kindly provided by Dr Richard Marais (Institute of Cancer Research, London, UK) (7,18). Transient transfection was performed with the polyethylenimine method for HEK293T and Hela cells, whereas transient and stable transfection were performed by Lipofectamine 2000 (Invitrogen) for A375 melanoma cells. Stably transfected clones of mixed population were selected in G418 (2 mg/ml) and maintained in G418 (0.5 mg/ml) (19). The expression of exogenous protein by cloned cells was confirmed by indirect immunofluorescence to detect RKTG expression.
Immunoprecipitation and immunoblotting
The cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid and 1% Nonidet P-40 with a mixture of protease inhibitors and phosphatase inhibitor (Sigma–Aldrich) before immunoprecipitation and immunoblotting assays. Methods of immunoprecipitation and immunoblotting have been described previously (16). The immunoblotting results were quantified with ScionImage software (Scion, Frederick, MD).
Luciferase assay
The luciferase assay to analyze Elk-1-mediated transcriptional response was performed as described previously (17). Twelve hours after transfection, the cells were serum starved for 16 h before harvesting. The luciferase activity was measured by a luciferase assay kit (KenReal, Shanghai, China) with a luminometer (Berthold Technologies, Bad Wildbad, Germany).
Confocal microscopy
Hela cells were grown on glass coverslips. Forty-eight hours after transfection, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 5 min and incubated with primary and secondary antibodies, sequentially. Confocal images were captured with an LSM 510 confocal microscope with a 64 x 1.4 numerical aperture apochromat objective (Zeiss, Jena, Germany). The 488 line of an argon laser was used for fluorescence excitation of enhanced green fluorescent protein and Alexa 488-conjugated antibody. A helium/neon laser (543 nm) was used for excitation of Alexa 546-conjugated antibodies, and 633 nm was used for excitation of Cy5-conjugated antibody. After data acquisition, red–green–blue images were processed by using LSM 510 software.
Cell proliferation assay
A375 cells, stably transfected with the human RKTG expression vector or with empty vector (pcDNA3), were seeded in 12-well plates at 5 x 104 per well. After 24, 48, 72 and 96 h of culture, the cells were harvested and cell numbers were counted under microscope.
Soft agar assay
A bottom layer of 1 ml DMEM per well containing 0.6% agar and 10% fetal bovine serum was prepared in six-well plates. After the bottom layer was solidified, cells (400, 1000 and 4000 per well, respectively) were added in a 2 ml top layer of 0.3% agar and 10% fetal bovine serum. All the samples were prepared in triplicate and incubated at 37°C. On day 14, colonies >50 µm in diameter were counted.
Xenograft tumor assay and immunohistochemistry
Female BALB/c nude mice (6 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and maintained in pathogen-free conditions. A375 cells at 80–90% confluence were harvested and resuspended in DMEM. The cell suspension (1.6 x 106 cells in 0.2 ml DMEM per mouse) was injected subcutaneously in the dorsal flanks of the mice (n = 8 for pcDNA3 group and n = 17 for RKTG group). Tumor size was measured every other day with a vernier caliper from day 22 to day 38 after injection. Tumor volume (V) was calculated using the values of the largest (A) and the smallest (B) diameter according to the formula V = 0.5 x AB2 (20). The tumors were excised promptly after euthanasia and placed in 10% formaldehyde solution, fixed for at least 2 h and then embedded in paraffin. Paraffin sections of 4 µm were used for immunohistochemistry. Monoclonal anti-Ki67 antibody was diluted at 1:1000. Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labelling assay was performed following the manufacturer's instruction. To quantitatively analyze Ki67 staining, the number of positive cells in five high power microscope fields (x400) of each section of each tumor sample was counted.
Reverse transcription–polymerase chain reaction
The cells were lysed in TRIzol reagent (Invitrogen). Total RNA was purified according to the manufacturer's instructions, then reverse transcribed and synthesized to complementary DNA using avian myeloblastosis virus reverse transcriptase (TaKaRa). The primers used to amplify human RKTG were 5'-ATGCATCAGAAGCTGCTGAAG-3' and 5'- GACATAGCAGCCCAGTATTC-3'. The primers for glyceraldehyde 3-phosphate dehydrogenase were 5'-GTCTTCACCACCATGGAGAAGG-3' and 5'-TCGCTGTTGAAGTCAGAGGAGA-3'.
Statistics
Statistical analysis was done using the Student's t-test.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
RKTG suppresses B-Raf-stimulated ERK signaling
Our previous studies have demonstrated that in HEK293T cells, overexpression of RKTG was able to markedly reduce the phosphorylation level of ERK induced by EGF and inhibit Elk-1 transactivation by an activated Raf-1 through sequestrating Raf-1 to the Golgi apparatus (16). Although Raf-1 can potentially be an oncogenic protein as it is overexpressed in some ovarian and pulmonary carcinomas, Raf-1 mutations are rarely found in human cancers (5,21). It has been demonstrated recently that another member of the Raf kinase family, B-Raf, plays an important role in carcinogenesis as it is predominantly mutated in a wide range of human cancers, such as in 66% of MMs and in many other carcinomas with moderate to high rates (5,7,22). To characterize the potential regulatory role of RKTG in the signaling of cancer-associated B-Raf mutants, we analyzed the function of RKTG in A375 human MM cells that bear B-Raf(V600E), which is the most common B-Raf mutation found in human MM (7).
To examine whether RKTG is able to inhibit ERK activation in A375 cells, EGF-induced ERK phosphorylation was analyzed with or without overexpression of RKTG. As shown in Figure 1A, A375 cells exhibited high basal levels of phosphorylated ERK in the absence of EGF stimulation, consistent with previous studies (8). Remarkably, RKTG overexpression reduced the basal level of ERK activation in these cells. EGF treatment was able to increase ERK phosphorylation slightly (at 5 min) that was rapidly attenuated afterward in the absence of RKTG overexpression. However, in the presence of RKTG, EGF was no longer able to induce ERK phosphorylation. This finding provided the first clue that RKTG could antagonize ERK activation in melanoma cells that bear B-Raf(V600E).
|
To provide additional evidence that RKTG is able to antagonize B-Raf signaling, we performed a reporter assay that measures the transcriptional activity of Elk-1, a nuclear target of ERK. The wild-type Raf-1, constitutively active Raf-1(BXB) that only contains the Raf-1 kinase domain (23), wild-type B-Raf and B-Raf(V600E) were transiently expressed in HEK293T cells together with an Elk-1 luciferase reporter (17). As shown in Figure 1B, wild-type Raf-1 had no obvious effect on Elk-1 transactivation. As expected, Raf-1(BXB), wild-type B-Raf and B-Raf(V600E) markedly elevated Elk-1-mediated transcriptional response. When RKTG was overexpressed in these cells, the Elk-1-mediated transcriptional activity stimulated by Raf-1(BXB), wild-type B-Raf and B-Raf(V600E) were all significantly suppressed. These findings indicate that in addition to the inhibitory effect of RKTG on Raf-1 as reported previously (16), RKTG is able to antagonize ERK activation by either wild-type B-Raf or activated B-Raf mutation (V600E).
Interaction of RKTG with B-Raf and translocation of B-Raf to the Golgi by RKTG
Taking into consideration that through binding Raf-1, RKTG traps Raf-1 to the Golgi apparatus, thus insulating the signal transduction from Ras to MEK–ERK (16), we hypothesized that RKTG may affect B-Raf-stimulated ERK signaling through interaction with B-Raf. We analyzed the interaction of B-Raf with RKTG by coimmunoprecipitation assays. When coexpressed in HEK293T cells, both wild-type B-Raf and B-Raf(V600E) were found to associate with RKTG (Figure 2). In addition, repeated coimmunoprecipitation experiments revealed that there was no significant difference between the interactions of wild-type or V600E B-Raf with RKTG.
|
We next investigated whether the interaction of B-Raf with RKTG could also translocate B-Raf into the Golgi apparatus. Hela cells were transiently transfected with enhanced green fluorescent protein–RKTG and Myc-tagged wild-type B-Raf or B-Raf(V600E). As described previously by us, RKTG protein is exclusively localized at the Golgi apparatus (16). When coexpressed with pEGFP-C1 control vector, wild-type B-Raf or B-Raf(V600E) was diffusely distributed in the cytoplasm and not colocalized with a Golgi marker Golgin-97 (Figure 3). Intriguingly, when RKTG was cotransfected, the majority of wild-type B-Raf or B-Raf(V600E) was translocated to the Golgi apparatus shown as almost complete colocalization with RKTG and Golgin-97. Taken together, these observations suggest that RKTG may inhibit ERK activation downstream of B-Raf by specifically binding and sequestering cytoplasmic wild-type B-Raf or B-Raf(V600E) to the Golgi apparatus.
|
Overexpression of RKTG inhibits cell proliferation and transformation of A375 melanoma cells
To further analyze the physiological function of RKTG in melanoma cells, we generated A375 cell lines that stably expressed RKTG (supplementary Figure 1 is available at Carcinogenesis Online). Using these cells, we found that the growth rate of RKTG-expressing A375 cells was significantly reduced (Figure 4A), indicating that RKTG had a growth-suppressive function in these cells. Consistent with our observation that transient RKTG overexpression was able to inhibit ERK phosphorylation (Figure 1A), we found that the stably expressed RKTG also reduced ERK phosphorylation in A375 cells (Figure 4B). Previously, A375 cells have been shown to possess a transforming activity due to the activating mutation of B-Raf (24). We next examined the abilities of these cells to form colonies in soft agar, a characteristic of anchorage-independent cancer cell growth and transformation. When RKTG was overexpressed, the colony-forming activity of A375 cells was significantly reduced (Figure 4C and D), consistent with the growth suppressive activity of RKTG in these cells.
|
Overexpression of RKTG suppresses tumorigenicity of A375 melanoma cells
As RKTG overexpression was able to suppress cell proliferation and transformation of A375 cell line in vitro, we used immunodeficient nude mice to determine the effect of RKTG on the tumorigenicity of A375 cells in vivo. The same numbers of RKTG-transfected or control vector-transfected A375 cells were subcutaneously injected into nude mice and the size and volume of the xenograft tumors were measured. As shown in Figure 5, control vector-transfected A375 cells formed rapidly growing tumors, reaching an average tumor weight of
0.45 g on day 36 after cell inoculation. In contrast, RKTG overexpression significantly inhibited in vivo tumor growth of A375 cells, leading to formation of small tumors with an average tumor weight of 0.15 g at the 36th day after inoculation. These results indicate that overexpression of RKTG might possess a suppressive function on mitogenesis and tumor growth of melanoma cells.
|
To investigate the potential mechanism underlying the suppressive function of RKTG in A375-induced xenograft tumors, we used immunohistochemical staining with a proliferation marker Ki67 to analyze the cell proliferative status of the tumor samples. As shown in Figure 6, the number of Ki67-positive cells was significantly reduced in RKTG-overexpressed tumors. On the other hand, we examined the profile of cell apoptosis using TUNEL assay and found no obvious induction of apoptosis by RKTG (supplementary Figure 2 is available at Carcinogenesis Online). Taken together, these results suggest that the suppressive activity of RKTG in A375 xenograft tumors is probably due to inhibition of cell proliferation but not induction of apoptosis.
|
MM is the rarest form of skin cancer but by far the most deadly one due to its high metastatic potential and resistance to treatment. There is an urgent need to understand the molecular mechanism of MM (25). The Ras–Raf–MEK–ERK pathway mediates cellular responses to growth signals and is commonly constitutively activated in the majority of human melanoma. Raf proteins play a central role in such pathway. The V600E somatic mutation of B-Raf is associated with >60% of MM, implicating that activating oncogenic mutations of B-Raf functions as a critical promoter during malignancy (7).
In our present study, we demonstrated that RKTG was able to bind and translocate B-Raf as well as its activating mutant V600E to the Golgi apparatus, resulting in the suppression of B-Raf-stimulated ERK activation. When RKTG was overexpressed in A375 cells, ERK phosphorylation, cell proliferation and colony-forming ability were reduced. Consistently, overexpression of RKTG led to reduced mitogenesis and tumor formation of A375 cells in nude mice, accompanied by a decrease in cell proliferation. Taken together, our results reveal for the first time that RKTG may play a suppressive role in human melanoma that harbors oncogenic B-Raf mutation via negative regulation of the Ras–Raf–MEK–ERK pathway. In addition, our unpublished data showed that RKTG may function as a tumor suppressor in chemical-induced skin mitogenesis and tumorigenesis (Xie and Chen, unpublished data). Considering such important function of RKTG in carcinogenesis, the next challenge will be to uncover the regulation and function of RKTG in human cancers from the patients.
|
Supplementary materials |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
Supplementary Figures 1 and 2 can be found at http://carcin.oxfordjournals.org/
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
Chinese Academy of Sciences (One Hundred Talents Program and the Knowledge Innovation Program KSCX1-YW-02); National Natural Science Foundation of China (30588002 and 30470870); Ministry of Science and Technology of China (2007CB947100) to Y.C.
|
Acknowledgments |
|---|
We thank Drs Richard Marais, Susanne Weg-Remers and Dong Xie for providing the plasmids.
Conflict of Interest Statement: None declared.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary materialsFundingReferences |
|---|
- Madhunapantula SV, et al. Is B-Raf a good therapeutic target for melanoma and other malignancies? Cancer Res. (2008) 68:5–8.
[Abstract/Free Full Text] - Rapp UR, et al. BuCy RAFs drive cells into MEK addiction. Cancer Cell (2006) 9:9–12.[CrossRef][Web of Science][Medline]
- Hurst EA, et al. Ocular melanoma: a review and the relationship to cutaneous melanoma. Arch. Dermatol. (2003) 139:1067–1073.
[Abstract/Free Full Text] - Gray-Schopfer VC, et al. The role of B-RAF in melanoma. Cancer Metastasis Rev. (2005) 24:165–183.[CrossRef][Web of Science][Medline]
- Michaloglou C, et al. BRAF(E600) in benign and malignant human tumours. Oncogene (2008) 27:877–895.[CrossRef][Web of Science][Medline]
- Panka DJ, et al. Targeting the mitogen-activated protein kinase pathway in the treatment of malignant melanoma. Clin. Cancer Res. (2006) 12:2371s–2375s.
[Abstract/Free Full Text] - Davies H, et al. Mutations of the BRAF gene in human cancer. Nature (2002) 417:949–954.[CrossRef][Medline]
- Dhomen N, et al. New insight into BRAF mutations in cancer. Curr. Opin. Genet. Dev. (2007) 17:31–39.[CrossRef][Web of Science][Medline]
- Mor A, et al. Compartmentalized Ras/MAPK signaling. Annu. Rev. Immunol. (2006) 24:771–800.[CrossRef][Web of Science][Medline]
- Harding A, et al. Subcellular localization determines MAP kinase signal output. Curr. Biol. (2005) 15:869–873.[CrossRef][Web of Science][Medline]
- Choy E, et al. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell (1999) 98:69–80.[CrossRef][Web of Science][Medline]
- Rocks O, et al. Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors. Curr. Opin. Cell Biol. (2006) 18:351–357.[CrossRef][Web of Science][Medline]
- Torii S, et al. Sef is a spatial regulator for Ras/MAP kinase signaling. Dev. Cell (2004) 7:33–44.[CrossRef][Web of Science][Medline]
- Yeung K, et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature (1999) 401:173–177.[CrossRef][Medline]
- Wakioka T, et al. Spred is a Sprouty-related suppressor of Ras signalling. Nature (2001) 412:647–651.[CrossRef][Medline]
- Feng L, et al. Spatial regulation of Raf kinase signaling by RKTG. Proc. Natl Acad. Sci. USA (2007) 104:14348–14353.
[Abstract/Free Full Text] - Weg-Remers S, et al. Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway. EMBO J. (2001) 20:4194–4203.[CrossRef][Web of Science][Medline]
- Marais R, et al. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases. J. Biol. Chem. (1997) 272:4378–4383.
[Abstract/Free Full Text] - Pricop L, et al. Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines. J. Immunol. (2001) 166:531–537.
[Abstract/Free Full Text] - Chen X, et al. A small interfering CD147-targeting RNA inhibited the proliferation, invasiveness, and metastatic activity of malignant melanoma. Cancer Res. (2006) 66:11323–11330.
[Abstract/Free Full Text] - McPhillips F, et al. Raf-1 is the predominant Raf isoform that mediates growth factor-stimulated growth in ovarian cancer cells. Carcinogenesis (2006) 27:729–739.
[Abstract/Free Full Text] - Beeram M, et al. Raf: a strategic target for therapeutic development against cancer. J. Clin. Oncol. (2005) 23:6771–6790.
[Abstract/Free Full Text] - Frost JA, et al. Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity. Mol. Cell. Biol. (1994) 14:6244–6252.
[Abstract/Free Full Text] - Panka DJ, et al. The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells. Cancer Res. (2006) 66:1611–1619.
[Abstract/Free Full Text] - Collisson EA, et al. Treatment of metastatic melanoma with an orally available inhibitor of the Ras-Raf-MAPK cascade. Cancer Res. (2003) 63:5669–5673.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Jiang, X. Xie, Y. Zhang, X. Luo, X. Wang, F. Fan, D. Zheng, Z. Wang, and Y. Chen Regulation of G-Protein Signaling by RKTG via Sequestration of the G{beta}{gamma} Subunit to the Golgi Apparatus Mol. Cell. Biol., January 1, 2010; 30(1): 78 - 90. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||