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B-Raf is the most mutated gene in melanoma; however, mechanism through which it promotes early melanomas remains uncertain. Most nevi contain activated V600EB-Raf but few develop into melanoma and expression in melanocytes is inhibitory with low protein levels present in surviving cells, suggesting unknown cooperative oncogenic events are necessary for melanoma development. Since many melanomas have V600EB-Raf and active Akt3, it is possible these proteins cooperatively facilitate melanocyte transformation. In this study, Akt3 is shown to phosphorylate V600EB-Raf to lower its activity as well as that of the downstream MAP kinase pathway to levels promoting early melanoma development. Expression of active Akt3 in early melanoma cells containing V600EB-Raf reduced MAP kinase signaling and promoted anchorage independent growth. Furthermore, expression of both V600EB-Raf and active Akt3 in melanocytes promoted a transformed phenotype. Mechanistically, aberrant Akt3 activity in early melanomas serves to phosphorylate serines 364 and 428 on V600EB-Raf in order to reduce activity of V600EB-Raf to levels that promote rather than inhibit proliferation, which aids melanocytic transformation. Inhibition of V600EB-Raf or Akt3 in advanced melanoma cells in which both pathways were active reduced anchorage independent growth and tumor development in a cooperatively acting manner. Inhibition of Akt3 alone in these cells led to increased MAP kinase signaling. In summary, these results suggest that activating B-Raf mutations initially promote nevi development but the resulting high, intense activation of the MAP kinase pathway inhibits further tumor progression requiring Akt3 activation to bypass this barrier and aid melanoma development.
Despite being the least prevalent type of skin cancer, malignant melanoma is by far the deadliest due to its highly metastatic nature (1). PI3 and MAP kinase pathways play prominent roles in melanoma development by regulating diverse cellular processes including cellular proliferation, survival, invasion and angiogenesis (2-4). The mechanism through which these pathways interact or cooperate to promote melanoma development remains to be fully elucidated (2).
Increased activity of the PI3 kinase pathway occurs in ~70% of sporadic melanomas due to loss of PTEN and/or increased expression of Akt3 resulting from increased gene copy number (5). Highest level of Akt3 activity occur in metastatic melanomas, deregulating apoptotic signaling and promoting chemoresistance, making it attractive therapeutic target for metastatic disease (6, 7), (8). Targeting Akt3 decreases tumorigenesis by increasing cellular apoptosis in advanced stage melanomas but it is not known whether it plays a second independent role in early melanoma development (7, 9).
The MAP kinase pathway is activated through Ras mutations in 10-15% and B-Raf mutations in ~60% of melanomas (10-16). A single-base mutation converting T to A at nucleotide 1799 of B-Raf, substituting a valine for a glutamic acid at codon 600 (V600E) in exon 15 is the most prominent mutation (12) and is acquired but not inherited during development of sporadic melanomas (17-19). Since B-Raf is the most mutated gene in melanomas, it is an attractive therapeutic target for melanoma patients (20).
While V600EB-Raf regulates melanoma cellular proliferation (21-23), angiogenesis (21), survival (22, 23), invasion and metastasis (24, 25), it alone is not sufficient for melanoma development. Ectopic expression of Ras or Raf in primary or cancer cell lines causes cell cycle arrest or senescence (26), likely due to duration or level of MAP kinase pathway activation (27), which induces cyclin dependent kinase inhibitors (28, 29). Thus, other factors are necessary to facilitate melanoma tumor progression, likely involving loss of tumor suppressor genes or acquisition of cooperating oncogene activation (30-33). Possibly, deregulation of p53 (31, 33) and/or Rb pathway through p16INK4A loss (30, 31) might be necessary. Another unexplored possibility might involve cooperation between V600EB-Raf and Akt3 signaling cascades, which are frequently activated in the same cells (3, 4). In support of this possibility, Akt has been shown to phosphorylate B-Raf to decrease its activity in normal cells; however, it is unknown whether this process could play any role in melanoma development (34-36).
In this study, V600EB-Raf and Akt3 are shown to cooperatively promote melanoma development. Akt3 is demonstrated to phosphorylate mutant V600EB-Raf in order to reduce its and MAP kinase pathway activity to levels promoting rather than retarding melanocytic cell growth and transformation. Thus, these results suggest that activating V600EB-Raf mutation initially promotes nevi development but the resulting high, intense activation of the MAP kinase pathway inhibits further tumor progression requiring Akt3 activation to phosphorylate V600EB-Raf in order to bypass this barrier and promote melanoma development.
Normal human melanocytes and melanoma cell lines; WM35, UACC 903, and SK-MEL-24 cells were cultured as previously described (7, 37-39). Mouse melan-a melanocytes were grown in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and 200 nM TPA.
The PI3 kinase inhibitor LY-294002 (Qbiogene, Carlsbad, CA) was used to pharmacologically inhibit Akt activity. Briefly, 1×106 NHEM, WM35, SK-MEL-24 or 1.5×105 UACC 903 cells were grown in complete media supplemented with FBS or growth factors Two days later, cells were fed with serum/growth factor free media. 24-hours later, cells were treated with 50 mM LY-294002 or DMSO for 20 minutes followed by stimulation using serum or growth factors upto180 minutes. At each time point protein lysates were collected for Western blot analysis.
Protein lysates were collected, quantitated, and analyzed by Western blotting as detailed previously (21). Antibodies to pErk and pAkt serine-473 and caspase-3 from Cell Signaling Technologies (Danvers, MA); cyclin D1, p27 Kip1, α-enolase, anti-hemagglutinin (HA), and B-Raf from Santa Cruz Biotechnology (Santa Cruz, CA); and Akt3 from Upstate Biotechnology (Lake Placid, NY) were used.
Hemagglutinin tagged wild type and kinase-dead Akt3 (40) were subcloned (Eco RI and Xba I) into pcDNA3.1puro. A myristoylation tag was added by PCR to the N terminus of Akt3 and subcloned into the Eco RI and Xba I. Site directed mutagenesis (Quick Change, Stratagene, La Jolla, CA) was used to create E40K Akt3 mutant with primers TGGCTCATTCATAGGATATAAAAAGAAACCTCAAGATGTGGATTT and AAATCCACATCTTGAGGTTTCTTTTTATATCCTATGAATGAGCCA. For B-Raf constructs, HA-tagged wild type B-Raf (provided by Ann Vojtek, University of Michigan) (41) was excised (Hind III/Eco RV) and subcloned into pcDNA6/V5-HisA (Invitrogen, Carlsbad, CA). Site directed mutagenesis with primers GATTTTGGTCTAGCTACAGAGAAATCTCGATGGAGTGGG and CCCACTCCATCGAGATTTCTCTGTAGCTAGACCAAAATC generated V600EB-Raf. Similarly, primers CGAGACCGATCCTCAGCAGCTCCCAATGTGCAT and ATGCACATTGGGAGCTGCTGAGGATCGGTCTCG generated S364A V600EB-Raf, primers GCGAGAAAGGAAGTCAGCTTCATCCTCAGAAGAC and GTCTTCTGAGGATGAAGCTGACTTCCTTTCTCGC generated S428A V600EB-Raf. Construct inserts were sequence verified. Plasmids were introduced into cell lines with the Amaxa Nucleofector (Koeln, Germany). Reagent R, K17 program was used for UACC 903 cells while NHEM reagent, U20 program was used for human melanocytes, WM35 and melan-a cells. Transfection efficiencies were determined to be ~60% using pMaxGFP as a GFP expression plasmid control.
Plates were coated with PolyHEMA (12.0 mg/ml) (Sigma, St Louis, MI) in 95% ethanol to assess anchorage independent growth.
Duplexed “Stealth” siRNA from Invitrogen (Carlsbad, CA) was used for inhibiting protein expression as described previously (7, 9, 25, 39). siRNA (100 or 200 pmoles) was nucleofected into 2×106 human melanocytes, UACC 903 or WM35 cells allowed to recover for 1.5-2 days. Cells were then trypsinized and 2×104 seeded into PolyHEMA coated 96 well plates. Protein lysates for Western blot analysis was harvested. For UACC 903, 1×104 cells/well were seeded into DMEM supplemented with 10% FBS or 2×104 cells/well in serum free conditions. Cell proliferation was quantified 3 days later by MTS assay (Promega, Madison, WI).
HA-tagged Akt3 constructs (1.45 pmoles pcDNA3.1puro HA wt-Akt3, E40K-Akt3, myr-Akt3, and dead-Akt3) were nucleofected alone or in combination with siRNA to PRAS40 (100 pmoles) or a scrambled siRNA into 2×106 cells, 5×104 cells/50 μl plated into 96-well PolyHEMA coated plates. Twenty-four hours later, cells were selected using 1.6 μg/ml puromycin containing media. Following 7-days of selection, anchorage independent growth was assessed by MTS assay.
2×106 cells were nucleofected with 1.45 pmoles of pcDNA6 V600EB-Raf, pcDNA6 AA-V600EB-Raf alone or in combination with pcDNA3.1puro myr-Akt3 or E40K-Akt3. 4×104 cells/50 μl were added to 96-well PolyHEMA coated plates. After overnight recovery, 50 μl DMEM supplemented 10% FBS, 6 μg/ml puromycin and/or 6 μg/ml blasticidin was added to give a final concentration of 3 μg/ml. After 3 days selection, cell viability was quantified by MTS assay.
1.45 pmoles of HA-tagged pcDNA6 V600EB-Raf, S364A V600EB-Raf, S428A V600EB-Raf, AA-V600EB-Raf (S364A, S428A) or Dead B-Raf were nucleofected together with 1.45 pmoles pcDNA 3.1myc-His myr-Akt3 into melan-a cells to phosphorylate ectopically expressed B-Raf at serine residues 364 and 428. Cell lysates were harvested 3 days later using lysis buffer containing 20 mM Tris pH 8.0, 2 mM EDTA, 50 mM beta-glycerophosphate, 1mM activated sodium orthovanadate, 1% Triton X-100, 10% glycerol, 1 mM PMSF, and Protease Inhibitor Cocktail (Sigma, St Louis, MI). Various HA-tagged B-Raf proteins were immunoprecipitated from 150 μg of total lysates using 0.2 μg of antibody against HA, which was pre-coupled to Gamma Bind G sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). Activity of immunoprecipitated B-Raf was measured as previously reported (42) using full length Mek protein substrate (Santa Cruz Biotechnology, Santa Cruz, CA). Samples were separated on duplicate NuPAGE gels (Invitrogen, Carlsbad, CA), with protein on one gel transferred onto PVDF membrane (Western blot analysis) and other gel fixed in 10% acetic acid/50% methanol, dried, and then exposed to HyBlot CL autoradiography film (Denville, Metuchen, NJ) for 32P Mek detection.
HA-tagged B-Raf expressing plasmids (pcDNA6 WT B-Raf, V600EB-Raf, S364A V600EB-Raf, and AA-V600EB-Raf (S364A, S428A)) were nucleofected separately or in combination with pcDNA 3.1myc-His myr-Akt3 into melan-a cells, seeded into PolyHEMA coated plates and cell lysates harvested 3 days later. Various HA-tagged B-Raf proteins were immunoprecipitated from 20-40 μg of total lysates using 0.1 μg of antibody against HA using Gamma Bind G sepharose. Samples were separated on NuPAGE gels for Western blot analysis and probed with anti-Hemagglutinin and anti-phosphorylated-Akt substrate monoclonal antibody (Cell Signaling, Danvers, MA). Western blot images were scanned and quantified using the Image Gauge 4.0 software (Fuji Photo Film Co., Tokyo, Japan).
siRNA (112.5 pmoles for Akt3, C-Raf and scrambled; 12.5 pmoles for V600EB-Raf together with 100 pmoles of scrambled siRNA; or 12.5 pmoles for V600EB-Raf together with 100 pmoles of Akt3 siRNA) was nucleofected into 1×106 UACC 903 cells. 36-hours later, 1×106 UACC 903 cells in 0.2 ml of DMEM solution were injected subcutaneously into the left and right flanks of 4-6 week old nude mice. Dimensions of developing tumors were measured on alternate days up to day 17.5.
One-way or Two-way Analysis of Variance (ANOVA) was used for groupwise comparisons, followed by the Tukey’s or Bonferroni post hoc test. Results were considered significant at a p-value of < 0.05. Chou-Talalay method (Calcusyn software) was used for determining the combination Index (Biosoft, Cambridge, United Kingdom) (43).
In melanomas, the PI3k pathway is activated through increased Akt3 expression and/or PTEN loss (7, 39). The MAP kinase pathway is primarily activated through mutation of B-Raf to a constitutively active V600E form (7, 21, 22, 24, 39, 44). Whether these pathways cooperate in early melanoma development remains an unanswered question. To investigate this possibility, activation of each pathway in melanocytes, having normal levels of Akt3 activity and wild type B-Raf, was compared to UACC 903 melanoma cells having increased Akt3 activity as well as constitutively active V600EB-Raf, following growth factor starvation and stimulation. Densitometric analysis of Western blots quantified pErk and pAkt levels, which estimates MAP and PI3 kinase pathway activity respectively, and were plotted as a continuum from 0 to 180 minutes following serum stimulation (Figs. 1A & 1B). Note, profiles represent best-fit lines showing minor perturbations due to graphical software. In starved melanocytes, activity levels of both pathways were initially low (Fig. 1A), however, upon growth factor stimulation, activity of both pathways increases transiently and declined over time to basal levels due to receptor desensitization, as reported for other model systems (34). In contrast, UACC 903 cells, which had significantly higher basal activity than melanocytes showed in Fig. 1B an increase in pAkt accompanied by a corresponding decrease in MAP kinase pathway activity determined by measuring pErk (7, 14, 21). Following serum stimulation, activity of both pathways in UACC 903 cells increased; however, as observed with other cell systems, growth factor stimulation led to transient increases in activity of both pathways which declined over time (34). This decrease was not observed following Akt inhibition using LY-294002; rather, pErk plateaued at high levels. Representative Western blots showing pErk and pAkt levels 30 and 60 minutes after growth factor or serum stimulation are shown in Fig. 1C. While melanocytes showed no increase in pErk levels following inhibition of Akt using LY-294002, inhibition increased pErk levels for both UACC 903 and SK-MEL-24 cells lines (Fig. 1C). Different serum stimulation times that corresponded to the peak pAkt/Erk levels for each cell line were selected in order to easily observe changes in phosphorylation following agent manipulation (34). Thus, inhibition of PI3k pathway increased MAP kinase activity suggesting inhibitory cross-regulation between these pathways in melanoma cells but not melanocytes.
To establish whether Akt3 in the PI3k signaling cascade downregulated MAP kinase pathway activity, siRNA targeting Akt3 was introduced into UACC 903 cells to inhibit protein expression and cells starved, then stimulated with serum followed by measurement of pAkt/Erk levels. Reducing Akt3 protein expression (activity) in UACC 903 cells caused a corresponding decrease in pAkt levels (Fig. 1D). In contrast, no change was observed in levels of pErk in melanocytes following siRNA-mediated knockdown of Akt3, similar to results observed following pharmacological inhibition of Akt signaling in melanocytes (Fig. 1C). Significantly, decreased Akt3 activity caused a corresponding increase in MAP kinase activity observed by measuring pErk1/2 levels. Thus, Akt3 regulated activity of the MAP kinase pathway.
The interactive roles of Akt3 and mutant V600EB-Raf in melanoma development were evaluated measuring in vitro anchorage independent growth of WM35 cells. Cells were derived from an early stage melanocytic lesion in the radial growth phase containing the (T1799A) B-Raf mutation (19). However, unlike UACC 903 cells, WM35 cells have low endogenous Akt3 activity due to presence of functional PTEN (7). Thus, WM35 cells are similar to ~90% of human nevi containing V600EB-Raf and having low Akt3 activity (6, 7, 14, 15, 44), making it a suitable model for evaluating the role played by Akt3 in melanocytic tumor progression. Densitometric analysis of Western blots showed pErk/Akt levels in WM35 cells following stimulation with growth factors after 24 hours starvation (Fig. 2A). Upon growth factor stimulation, activity of both pathways increased transiently in WM35 cells and, over time, pAkt declined to basal levels due to receptor desensitization similar to that observed for melanocytes since both cell types contain PTEN (19, 34). In contrast, levels of pErk did not decline to near baseline levels, rather pErk levels remained at near maximal amounts for duration of the experiment due to V600EB-Raf constitutively activating the downstream MAP kinase pathway (Figs. (Figs.1A1A and and2A).2A). Thus, presence of V600EB-Raf and absence of constitutively activated Akt3 led to high MAP kinase activity, with potential to retard tumor progression.
Regulation of V600EB-Raf activity by Akt3 was examined using pharmacological inhibition of Akt or expression of active myristoylated Akt3 in WM35 cells to examine effect on pErk/Akt levels. While WM35 cells have low levels of pAkt compared to UACC 903 cells, inhibition of Akt using LY-294002 decreased levels of pAkt causing a corresponding increase in pErk (Fig. 2B), similar to that observed for advanced melanoma cell lines (Fig. 1C). Since WM35 cells had levels of pAkt similar to those observed in normal melanocytes, constructs ectopically expressing constitutively active Akt3 were used to produce pAkt levels similar to those observed in more aggressive melanomas to confirm Akt3-mediated regulation of MAP kinase pathway activity. Western blot analysis showed increased pAkt accompanied by a corresponding decrease in pErk following ectopic expression of active myristoylated Akt3 but not dead Akt3 (control producing inert protein) expression (Fig. 2B). Thus, cross regulation between Akt3 and MAP kinase pathway activity occur with active Akt3.
Effect of V600EB-Raf and Akt3 expression on melanocytic transformation was measured by regulating expression of each protein and quantifying anchorage independent growth (a phenotypic measurement of cell transformation). WM35 cells were nucleofected with siRNA against B-Raf and seeded onto PolyHEMA coated 96-well plates. A significant decrease of viable WM35 cells was observed following nucleofection with B-Raf siRNA compared to a scrambled control (Fig. 2C, p<0.05; One-way ANOVA). Western blot analysis confirmed B-Raf protein knockdown and corresponding decrease in downstream pErk (Fig. 2C). Next, myristoylated-Akt3 having high activity or the E40K-Akt3 mutant having activity in the normal physiological range was expressed in WM35 cells containing endogenous V600EB-Raf to determine whether it could enhance anchorage independent growth (45). Akt3 expression significantly enhanced anchorage independent growth compared to vector controls, which was directly proportional to level of pAkt (Fig. 2D, p<0.001; One-way ANOVA). Expression of wild type protein had lowest pAkt and least effect on anchorage independent growth. To demonstrate that Akt3 mediated anchorage independent growth of WM35 cells was not due to decreased apoptosis reported in advanced melanoma (7, 9), the downstream target of Akt3 regulating apoptosis, called PRAS40, was knocked down using siRNA while active Akt3 was expressed (9). Similar anchorage independent growth was observed irrespective of levels of PRAS40 expression (Fig. 2D). Thus, deregulated apoptosis mediated by active Akt3 was not leading to enhanced anchorage independent growth. This raised the possibility that Akt3 could be interacting with V600EB-Raf to regulate anchorage independent growth since Akt has been reported to downregulate B-Raf activity through direct phosphorylation (34-36). Thus, V600EB-Raf and Akt3 mediate anchorage independent growth in the WM35 cell model and potentially cooperate in melanocyte transformation.
Since Akt3 and V600EB-Raf independently regulated anchorage independent growth of WM35 cells, effect of ectopic co-expression on melanocyte transformation was examined using melan-a mouse melanocytes to assess transformation by anchorage independent growth. This model was chosen since melan-a cells undergo transformation following low level V600EB-Raf expression (46, 47). Ectopically expressed V600EB-Raf or active-Akt3 alone, promoted anchorage independent growth compared to empty vector (p<0.001, Fig. 3A). Numerous groups have reported the oncogenic properties of myr-Akt3 (48). However, a limitation is that the activity resulting from myr-Akt3 is very high, which could be an explanation for cell transformation. Therefore, we incorporated the E40K Akt3 mutant that leads to more moderate physiological pAkt levels similar to those present in more aggressive melanoma cells. Co-expression of V600EB-Raf with active E40K- or myr-Akt3 led to statistically significant enhanced anchorage independent growth compared to single genes (Lanes 5 vs 2 or 3, p<0.05; Lanes 6 vs. 2 or 4, p<0.001; Fig. 3A). Western blot analysis of cell lysates showed that active myr-Akt3 reduced levels of pErk accompanying V600EB-Raf expression (Fig. 3A). A similar but less significant effect was seen following E40K Akt3 coexpression with V600EB-Raf (Fig. 3A). These results suggest that active Akt3 and V600EB-Raf cooperate in melanocyte transformation, promoting anchorage independent growth.
To determine whether phosphorylation mediated by Akt3 regulated V600EB-Raf activity, serine residues 364 and/or 428 in V600EB-Raf were converted to alanine. Plasmids expressing these B-Raf constructs were co-nucleofected with myr-Akt3 into melan-a cells to promote phosphorylation of ectopically expressed B-Raf proteins on serines 364 and 428. V600EB-Raf constructs containing alanine at residues 364 and/or 428 could not be phosphorylated by Akt3. Various HA-tagged B-Raf proteins were then immunoprecipitated and activity measured using an in vitro kinase assay with Mek as the substrate (Fig. 3B). AA-V600EB-Raf (S364A, S428A) exhibited highest level of activity compared to V600EB-Raf, V600EB-Raf (S364A) or V600EB-Raf (S428A) (p<0.05; Fig. 3B) demonstrating that direct phosphorylation of V600EB-Raf protein by Akt3 decreased its activity. Thus, phosphorylation of V600EB-Raf has significant potential to decrease activity of the mutant protein.
To demonstrate that Akt3 phosphorylates mutant V600EB-Raf in melanocytic cells, melan-a melanocytes were transfected with HA-tagged V600EB-Raf, V600EB-Raf (S364A), or AA-V600EB-Raf (S364A, S428A) alone or with myr-Akt3. Western blot analysis using an antibody recognizing consensus sequences typically phosphorylated by Akt was used to quantify levels of phosphorylation of immunoprecipitated HA tagged B-Raf from cells (Fig. 3C). V600EB-Raf (S428A) was not used since the antibody does not recognize this site. Increased phosphorylation of V600EB-Raf occurred when coexpressed with active Akt3 (p<0.01; Fig. 3C). Mutation of serine 364 to alanine (V600EB-Raf (S364A)) decreased pB-Raf levels (p<0.01; Fig.3C). Furthermore, mutation of both serine 364 and serine 428 to alanines (AA-V600EB-Raf (S364A, S428A)) completely blocked phosphorylation by Akt3 (p<0.01; Fig. 3C). Thus, active Akt3 is capable of phosphorylating V600EB-Raf in melanocytic cells.
Effect of phosphorylated versus unphosphorylated V600EB-Raf on melanocytic transformation was examined by expressing V600EB-Raf or AA-V600EB-Raf (S364A, S428A) alone or with active Akt3 in melan-a cells and measuring anchorage independent growth. Compared to V600EB-Raf expression alone, AA-V600EB-Raf (S364A, S428A) protein significantly decreased cell proliferation (p<0.001; Fig. 4A). Similarly, co-expression of AA-V600EB-Raf (S364A, S428A) with myristoylated or E40K Akt3 also significantly reduced proliferation when compared to cells expressing V600EB-Raf or Akt3 (Fig. 4A, P<0.001). Western blot analysis of cell lysates showed AA-V600EB-Raf (S364A, S428A) expression accompanied by higher MAP kinase pathway activity than occurred with V600EB-Raf (Fig. 4B). Furthermore, co-expression of AA-V600EB-Raf (S364A, S428A) with myristoylated-Akt3 did not lower pErk levels as observed when V600EB-Raf and myristoylated Akt3 were co-expressed (Fig. 4B). Induction of cyclin D1 was also impaired following AA-V600EB-Raf (S364A, S428A) expression (Fig. 4B). Thus, phosphorylation of V600EB-Raf by Akt3 decreased MAP kinase signaling overcoming growth inhibitory effects associated with high pathway activity.
To evaluate the therapeutic potential of simultaneously inhibiting Akt3 and mutant V600EB-Raf in melanoma tumorigenesis, a siRNA was used to inhibit protein expression. Effects on tumor development was studied by nucleofecting siRNA against each target into UACC 903 cells, allowing 1.5 days of recovery in culture followed by subcutaneous injection of 1 million cells into nude mice. Reduced expression of each protein was observed for as long as 9.5 days after nucleofection in cultured cells and in tumors of nude mice (data not shown) (25). Size of developing tumors was measured using calipers every other day until day 17. Initially, siRNA against Akt3 and B-Raf was titered to establish optimal concentration inhibiting tumorigenesis to a similar extent. One hundred pmoles of Akt3 siRNA was found to inhibit tumorigenesis similar to that occurring with 12.5 pmoles of siRNA against B-Raf (Fig. 5A). In comparison to controls (buffer, scrambled siRNA and C-Raf siRNA), siRNA against Akt3 or V600EB-Raf significantly decreased the tumorigenic potential of the UACC 903 cells (Fig. 5A). An even more dramatic reduction in tumorigenesis was observed following simultaneous inhibition of both Akt3 and B-Raf. Under these conditions cooperatively acting tumor inhibition was observed (Fig. 5A, p<0.001; Two-way ANOVA). Tumors removed from mice 9.5 days after siRNA nucleofection were found to have reduced Akt3 (Fig. 5B) or V600EB-Raf (Fig. 5C) protein expression demonstrating effective knockdown of these proteins. Thus, targeting both Akt3 and B-Raf led to cooperatively acting tumor inhibition.
Since targeting Akt3 and V600EB-Raf led to cooperative tumor inhibition, mechanistic basis for inhibition was examined next. siRNA targeting Akt3 and/or V600EB-Raf was nucleofected into UACC 903 cells and effect on in vitro anchorage independent growth examined. Simultaneous siRNA-mediated inhibition of Akt3 and B-Raf dramatically inhibited growth of cells compared to either Akt3 or B-Raf (Fig 6A, Lanes 2, 4, and 7, and Lanes 2, 5 and 8; ANOVA, p<0.001) (49). To determine whether inhibition of anchorage independent growth was additive or synergistic following siRNA-mediated inhibition of Akt3 and V600EB-Raf the Chou-Talalay method for determining the combination index (CI) using Calcusyn software was undertaken (43). Using this approach, CI values < 0.9 are synergistic, > 1.1 are antagonistic, and values 0.9-1.1 are nearly additive. Experimentally, Akt3 siRNA was kept at a constant dose of 200 pmoles while the concentration of B-Raf siRNA was titrated at doses of 3, 6, and 12 pmoles. The resulting CI for each combination was calculated to be 0.909, 1.023, and 1.107, for 3, 6, and 12 pmoles of B-Raf siRNA respectively combined with 200 pmoles of Akt3 siRNA, which suggest nearly additive cooperation. Thus, simultaneous targeting V600EB-Raf and Akt3 inhibits anchorage independent growth in a cooperatively additive manner.
Mechanistic basis for inhibition of anchorage independent growth following V600EB-Raf and Akt3 targeting was examined using Western blotting to measure levels of protein expression involved in proliferation (cyclin D1) and apoptosis (cleaved caspase-3). Control UACC 903 cells had high cyclin D1 and low cleaved caspase-3 levels (Lane 1, Fig 6B). In contrast, siRNA-mediated inhibition of Akt3 increased cleaved caspase-3 levels indicating increased cellular apoptosis (Lane 2, Fig 6B). B-Raf siRNA did not significantly alter levels of cleaved caspase 3 but did significantly decreased cyclin D1 levels with a corresponding increase in p27 (Fig 6B, Lanes 3 to 5). Significantly, inhibition of both Akt3 and mutant V600EB-Raf dramatically increased levels of cleaved caspase 3 indicative of high apoptosis compared to cells in which Akt3 alone had been inhibited (Fig. 6B; Lanes 6, 7, and 8 versus lane 2). Furthermore, the combination approach led to more dramatic decrease in cyclin D1 than observed when V600EB-Raf was inhibited alone. Thus, simultaneously targeting both Akt3 and V600EB-Raf has a more dramatic effect on the signaling cascades of these pathways than inhibiting each individually.
Nevi or moles are benign proliferations of melanocytes that initially proliferated and then stopped growing (30). Approximately 90% harbor activating B-Raf mutations, but never progress into melanomas (12, 14, 15). This is caused by cellular senescence associated with high MAP kinase pathway activity due to presence of V600EB-Raf, which is linked to induction of cyclin dependent kinase inhibitors, such as p21Cip1, p16Ink4a, and p27Kip1, functioning as a defense mechanism of normal cells to overcome oncogene activation (26, 28, 29). In contrast, moderate levels of MAP kinase pathway activation promote cell cycle progression and cancer development (27). Therefore cells containing V600EB-Raf require additional genetic changes for senescent melanocytic cells to reenter the cell cycle (31).
Identification of proteins cooperating with V600EB-Raf in melanocytic transformation are being characterized by studying nevi development using transgenic zebrafish (30). Prolonged hyperactivation of MAP kinase signaling in zebrafish melanocytes mediated by V600EB-Raf initially cause promote cellular growth and development of structures resembling nevi followed by a senescent phase; a situation comparable to that occurring with most human moles (30). Zebrafish nevi did not develop into melanomas until p53 was deleted, suggesting deregulation of multiple pathways for melanocytic tumor progression (30). Thus, genes such as Akt3 with prominent roles development ~70% of melanomas likely cooperate with V600EB-Raf in melanocytic transformation (7, 9, 39, 44, 50).
Akt had been shown to regulate B-Raf activity (34, 35); however, it was unknown whether Akt3 would phosphorylate mutant V600EB-Raf, and if so, whether this process played any role in melanoma development. In this report, using a combination of pharmacological and genetic approaches, Akt3 was shown to downregulate V600EB-Raf mediated activation of MAP kinase pathway signaling in order to promote tumor progression. Ectopic expression of active Akt3 in early melanoma cells, phenotypically resembling nevi cells by containing V600EB-Raf with low endogenous Akt3 activity, increased anchorage independent growth. Furthermore, expression of both proteins in melanocytes promoted anchorage independent growth, demonstrating that V600EB-Raf and active Akt3 act cooperatively together to promote melanocyte transformation; thereby, confirming the importance of these proteins in early melanoma development. Mechanistically, Akt3 was shown to directly phosphorylate B-Raf on serine residues 364 and 428 to regulate activity of mutant V600EB-Raf protein in order to promote melanocyte transformation (41). This second role for Akt in melanoma development was confirmed by inhibiting the downstream target of Akt3 regulating apoptosis, called PRAS40 (9). Constitutively active Akt3 was expressed to activate the signaling cascade, while siRNA-mediated knockdown was used to inhibit PRAS40 expression/activity. Since similar anchorage independent growth was observed irrespective of levels of PRAS40 expression, deregulated apoptosis mediated by active Akt3 was not leading to enhanced anchorage independent growth. Thus, a second important role has been identified for Akt3 in melanoma development.
Melan-a mouse melanocytes used as a model of transformation are considered to be a normal mouse melanocyte cell line and is an accepted model for studying V600EB-Raf mediated transformation (47). However, it lacks p16INK4a as well as ARF protein expression. Data presented in this study show that melan-a transformation mediated by V600EB-Raf expression in a p16INK4a /ARF null background requires expression of active Akt3 to promote anchorage independent growth. This suggests that not only was loss of p16INK4a /ARF important in bypassing V600EB-Raf induced senescence, but Akt3 activation was required to phosphorylate V600EB-Raf in order to modulate the level of MAP kinase activity.
The therapeutic potential of simultaneously targeting Akt3 and V600EB-Raf is also demonstrated in this study. Tumor inhibition was most significant in animals containing cells in which both proteins had been targeted. In vitro studies confirmed the effects of the combination approach demonstrating that targeting both led to cooperatively acting inhibition significantly increasing cellular apoptosis and decreasing proliferation. Thus, therapeutics targeting both pathways in human patients is predicted to be more effective than targeting each individually.
In conclusion, Akt3 has been shown to cooperate with V600EB-Raf in promoting melanoma development by phosphorylating the mutant protein in order to decrease its activity to levels that promote rather than inhibit melanocytic cell growth. Furthermore, simultaneously targeting both Akt3 and V600EB-Raf was more effective at inhibiting melanomas that targeting each individually, which suggest that therapies for human patients should simultaneously target these signaling pathways for maximal clinical efficacy.
Grant support: The Melanoma Research Foundation, The American Cancer Society (RSG-04-053-01-GMC), The Foreman Foundation for Melanoma Research, NIH grant CA-127892-01A1, and Elsa U. Pardee Foundation