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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Division of Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School and 2 Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts
Requests for reprints: James W. Mier, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail: jmier{at}bidmc.harvard.edu.
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Introduction |
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80% of benign nevi (7), indicating that it is only one of several factors contributing to melanoma development. The mitogen-activated protein kinase (MAPK) pathway is activated in virtually all melanomas regardless of the status of the B-raf gene (8), suggesting the existence of several mechanisms independent of the B-rafV599E mutation that contribute to the constitutive extracellular signal-regulated kinase (ERK) phosphorylation observed in melanoma cells. Satyamoorthy et al., for example, have shown that Ras is in a GTP-loaded state and c-raf is phosphorylated in melanoma cells (9). They have shown that neutralizing antibodies against hepatocyte and basic fibroblast growth factors partially inhibit ERK phosphorylation, suggesting the existence of an autocrine loop that regulates MAPK activity in these cells (9). Activating N-ras mutations have been reported in a minority of melanomas, primarily those without B-raf mutations (10). Melanoma cells frequently lack endogenous raf inhibitors, and their absence contributes to MAPK activation. For example, Raf-1 kinase inhibitory protein is down-regulated in melanoma cells, and the restored expression of this inhibitor reduces ERK phosphorylation (11). The down-modulation of various members of the Sprouty (SPRY) family may also contribute to the activation of the MAPK pathway (12, 13). The COOH' termini of SPRY2 and SPRY4 bind to Raf-1 and wild-type (WT) B-raf but not B-rafV599E and inhibit their kinase activity (14, 15). A recent microarray analysis comparing the gene expression profiles of melanomas with WT B-raf with those harboring the B-rafV599E mutation identified SPRY2 as a gene selectively down-modulated in melanomas with WT B-raf (15), suggesting that the loss of SPRY2 may contribute to the constitutive ERK phosphorylation observed in melanomas with WT B-raf. Collectively, these data attest to the diversity of biochemical pathways used by melanomas to ensure the activation of the MAPK pathway.
In melanoma cells with the B-rafV599E mutation, the depletion of this oncogene with a specific small interefering RNA (siRNA) resulted in growth arrest, the loss of anchorage-independent growth, and the induction of apoptosis (6). Although the mechanism of cell death in this circumstance is unclear, the inactivation of MAP/ERK kinase (MEK) and ERK presumably plays a role because pharmacologic inhibition of MEK has similar effects (16). Studies by Eisenmann et al. suggest that the critical event downstream of ERK dephosphorylation responsible for the apoptosis induced by the MEK inhibitor PD184352 may be the inactivation of the ERK substrate pp90rsk and the ensuing dephosphorylation of the propapoptotic BH3-only Bcl-2 family member Bad (16). When phosphorylated, Bad remains in the cytosol as an inert complex with 14-3-3, but when dephosphorylated, it translocates to the mitochondria, where it binds to Bcl-2 and Bcl-XL, preventing them from forming inhibitory heterodimers with the proapoptotic bcl-2 family members Bak and Bax (17–22). Eisenmann et al. have shown that a constitutively activated p90rsk mutant was able to block the lethal effects of MEK inhibition (16). These findings suggest that melanoma cell death resulting from raf inhibition (with a specific siRNA or with the pharmacologic inhibitor BAY 43-9006) might similarly depend on the inactivation of p90rsk and the dephosphorylation of Bad.
Other studies, however, suggest that the proapoptotic effects of raf inhibition may be more complex. For example, Erhardt et al. have shown that cytochrome c does not activate caspases when added to lysates of cells overexpressing B-raf (23). This finding suggests that B-raf may function downstream of the mitochondria to prevent apoptosis. Although raf-1 knockout mice die during early embrogenesis, mice homozygous for a dual raf-1 mutation (Y340F/Y341F) that has no in vitro kinase activity toward MEK are phenotypically normal (24). These and other studies lend support to the notion that the predominant antiapoptotic effects of the various raf isoforms may be independent of MEK/ERK signaling (24–27), and the apoptosis induced by raf inhibition may not be due to the loss of MEK/ERK activity.
Regardless of the mechanism by which raf inhibition induces apoptosis, the data cited above underline the potential value of MAPK inhibitors in the treatment of melanoma. Indeed, several such agents have demonstrable activity in animal models and have recently entered clinical trials (28, 29). Perhaps the best characterized of these new agents is BAY 43-9006, a potent inhibitor of c-raf and B-raf as well as other potentially important kinase targets, such as the platelet-derived growth factor (PDGF) receptor and c-kit (29). This agent has substantial activity in human tumor xenograft models and in phase II clinical trials, especially when administered in conjunction with carboplatinum and paclitaxel (30). These data lend credence to the notion that the MAPK pathway may be a clinically exploitable Achilles' heel for an otherwise untreatable malignancy.
The studies reported in this article were undertaken to determine the mechanism by which BAY 43-9006 induces apoptosis in melanoma cells and in particular, the extent to which the suppression of ERK and Bad phosphorylation contributes to the process. Our results suggest that biochemical events independent of MAPK inhibition may be the more important determinants of the lethality of this novel agent for melanoma cells.
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Materials and Methods |
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Western blots. Cells were treated as described in Results and then lysed in Lysis Solution (Cell Signaling) supplemented with sodium fluoride (10 µmol/L, Fisher Scientific, Hampton, NH) and phenylmethylsulfonyl fluoride (100 µg/mL, Sigma-Aldrich, St. Louis, MO). Lysates were fractionated in either 8% or 12% SDS-polyacrylamide gels as indicated, and the separated proteins were transferred to nitrocellulose. The blots were probed for the proteins of interest with specific antibodies followed by a second antibody-horseradish peroxidase conjugate and then incubated with SuperSignal chemiluminescence substrate (Pierce, Rockford, IL). The blots were then exposed to Kodak X-Omat Blue XB-1 film. The ERK and apoptosis-inducing factor (AIF) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); the phospho-ERK (p-ERK), MEK, p-MEK, bcl-2, bad, p-bad (both Ser75 and Ser99), and poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling. The antibody used to detect p-Ser75 in Bad was generated against a murine Bad phosphopeptide spanning Ser112, the equivalent site in the murine molecule. Likewise, the antibody used to detect Bad phosphorylation at Ser99 was generated against a murine Bad phosphopeptide spanning Ser136, the equivalent serine in the murine molecule. The vinculin antibody was obtained from Sigma; the Cox4 antibody was obtained from BD Clontech (Palo Alto, CA); the SMAC/Diablo antibody was purchased from eBioscience (San Diego, CA), and the Bcl-XL antibody was from Upstate Biotechnology (Lake Placid, NY).
Apoptosis and mitochondrial potential assays. In each of these assays, the adherent cells were detached from the underlying plastic by treatment with 20 mmol/L EDTA in PBS for 5 minutes and then combined with the floating, nonadherent cells. In assays in which staining with FITC-Annexin V with or without propidium iodide was used as an apoptotic index, FITC-Annexin V (5 µL, BD PharMingen) with or without propidium iodide (5 ng/mL, Sigma) were added to the cell pool, and after 20 minutes at room temperature, the cells were analyzed by flow cytometry with a BD Biosciences FACScan or Coulter FC 500 cytometer. In each of these assays except those involving Ds-Red–transfected cells (see below), the percentage of cells dually staining with Annexin V and propidium iodide was taken to represent the extent of apoptosis induced by the experimental condition being tested. In those assays involving Ds-Red–transfected cells, staining with Annexin V (without propidium iodide) was taken to represent the extent of apoptosis induced in the cells.
Assays assessing the mitochondrial transmembrane potential were carried out using the BD Mitosensor reagent (BD Clontech; ref. 31). In these studies, Mitosensor (250 µL of a 1:1,000 dilution) was added to the drug-treated cells, and after 20 minutes at 37°C, the cells were analyzed by dual color flow cytometry as described above for the FITC-Annexin V/propidium iodide studies.
Bak and Bax activation assay. Cells were fixed in paraformaldehyde (0.25%) for 5 minutes and then permeabilized with digitonin (100 µg/mL). The primary murine anti-Bak (Calbiochem) or anti-Bax (BD PharMingen) antibody (5 µg/mL) and a FITC-conjugated goat anti-mouse antibody (1:100 dilution; Caltag, Burlingame, CA) were sequentially added and the cells then analyzed with a Coulter FC 500 cytometer.
Preparation and transfection of siRNAs for Bad and AIF. siRNAs for Bad and AIF were obtained from Santa Cruz Biotechnology. Cells were transfected with the Ds-Red Express construct (BD Clontech) alone or jointly with one of the siRNAs with the siRNA present in a 10:1 molar excess. The transfections were carried out using the TransIT-TKO Transfection reagent (Mirus Corp., Madison, WI) following the manufacturer's protocol. After 48 hours, a portion of the cells were sorted for the Ds-Red–positive fraction using a MoFLO cell sorter (DakoCytomation, Ft. Collins, CO). The sorted red-fluorescent cells were then lysed and analyzed by Western blot to confirm that the siRNA had the desired effect on the level of the protein of interest. The remaining cells were treated with Bay 43-9006 (20 µmol/L for 20 hours), stained with FITC-Annexin V, and then analyzed for red (FL2) and green (FL1) fluorescence using a Coulter FC 500 Flow cytometer as described above. Data were reported as the percentage of red-fluorescent cells staining with FITC-Annexin V.
Detection of cytochrome c, SMAC, and AIF release from the mitochondria. Mitochondria-enriched and cytosolic fractions were isolated from Dounce-homogenized cells using the ApoAlert Cell Fractionation Kit (BD Clontech). The quality of the mitochondria-enriched fractions was validated by Western blots using an antibody for the mitochondrial protein cytochrome c oxidase subunit IV (Cox4, BD Clontech; ref. 32). Cytosolic fractions were obtained during the isolation of the mitochondria. Lysates from the mitochondrial and cytosolic fractions were analyzed for cytochrome c (BD Clontech) and SMAC/Diablo by Western blot as described above. Nuclei were isolated according to a standard protocol (33), lysed, and analyzed along with the corresponding cytosolic fractions for AIF by Western blot as described above.
To determine if AIF translocation to the nucleus could be detected in intact cells by immunohistochemistry, A2058 cells were plated onto eight-well Tissue Culture Treated slides (BD Falcon, Bedford, MA). The cells were treated with BAY 43-9006 (20 µmol/L), PD98059 (50 µmol/L), or U0126 (20 µmol/L) for 5 hours and fixed with 0.5 % zinc chloride and 0.5 % zinc acetate for 15 minutes. The cells were then treated with rabbit anti-AIF (1:100, Santa Cruz Biotechnology) followed by goat-anti-rabbit coupled to Alexa Fluor 488 (1:100; Molecular Probes, Eugene, OR). Nuclei were detected with Bisbenzimide H33342 (Alexis Biochemicals, San Diego, CA) as previously described (34). The slides were mounted and analyzed using a Zeiss Axioplan fluorescence microscope. The anti-AIF (green colored) and nuclear (blue colored) stains were photographed and analyzed using Adobe Photoshop.
Statistical analysis. As previously stated, the apoptotic effects of drug exposure and the modulatory effects of transfection with any of several siRNAs on drug-induced apoptosis were quantified by measuring the percentage of cells staining with Annexin V alone or with both Annexin V and propidium iodide. In each of these studies, >1.0 x 105 events were analyzed. Studies comparing the percentage of Annexin or Annexin/propidium iodide staining cells within two disparately treated populations were analyzed with the two-sample Z test. Studies comparing the mean fluorescence intensities of two populations were analyzed with the two-sample t test.
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Results |
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To assess the effects of BAY 43-9006 on mitochondrial polarity, cells were incubated with the drug (20 µmol/L for 20 hours). The Mitosensor reagent was added, and the cells were then analyzed by flow cytometry for polymeric (intramitochondrial) and monomeric (cytosolic) forms of the Mitosensor. As shown in Fig. 2A, exposure to BAY 43-9006, markedly increased the amount of monomeric Mitosensor within the cells, indicating a diminution in mitochondrial uptake and transmembrane potential. The mean fluorescence intensity (MFI) for the monomer was increased 4-fold in A375 cells, 5-fold in A2058 cells, and 2-fold in SKMEL5 cells as a result of exposure to BAY 43-9006. The two MEK inhibitors PD98059 and U0126 had no effect on the subcellular distribution of the Mitosensor in A375 and A2058 cells and only a modest effect in SKMEL5 cells at concentrations that inhibited ERK phosphorylation (50 and 20 µmol/L, respectively). The BAY 43-9006–induced accumulation of the Mitosensor in the cytosol was highly significant (P < 0.001 for each cell line).
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2-fold in all cell lines as a result of drug exposure and that of Bax increased from 1.4-fold (in A2058 cells) to 2.9-fold (in A375 cells). This increase in Bak and Bax staining was not due to an increase in protein levels as no increase in either protein could be detected by Western blot (Fig. 2C). These increases in MFI were highly significant by t test (P < 0.0001 for both Bak and Bax in each cell line). Effects of BAY 43-9006 on proapoptotic and antiapoptotic members of the Bcl-2 family. The accumulation of dephosphorylated Bad in the mitochondria has been proposed as a mechanism by which MEK inhibitors induce programmed cell death in melanoma cells (16). To determine if the raf inhibitor BAY 43-9006 similarly inhibits Bad phosphorylation, melanoma cells were exposed to the drug (20 µmol/L for 20 hours) and lysed, and the lysates were then analyzed by Western blot using 12% SDS-PAGE. Cells treated with the MEK inhibitors PD98059 (50 µmol/L) or U0126 (20 µmol/L) were similarly analyzed. For these studies, we used a phospho-specific antibody that recognizes p-Ser75, the site phosphorylated by the ERK substrate p90rsk, and an antibody specific for p-Ser99, the site phosphorylated by Akt and the mammalian target of rapamycin target p70rsk (16, 36). As shown in Fig. 3A, BAY 43-9006 as well as the two MEK inhibitors each suppressed Bad phosphorylation on Ser75 in all three cell lines. Ser99 was also dephosphorylated by BAY 43-9006, although this effect was delayed relative to the dephosphorylation of Ser75 and not observed in cells treated with either of the MEK inhibitors, suggesting that BAY 43-9006–induced Ser99 dephosphorylation cannot be attributed to MAPK inhibition.
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We also examined the effects of BAY 43-9006 on the expression of the antiapoptotic proteins Bcl-2 and Bcl-XL. As shown in Fig. 4, the drug had little effect on Bcl-2 and Bcl-XL levels in A375 cells. However, it nearly eliminated both proteins from the more sensitive A2058 and SKMEL5 cells. The down-modulation of Bcl-2 was evident within an hour of adding the drug, whereas that of Bcl-XL was more gradual. ERK phosphorylation was equally inhibited in all three cell lines in this study. Of note is the observation that neither of the two MEK inhibitors tested (PD98059 and U0126) induced the down-modulation of Bcl-2 or Bcl-XL, suggesting that this effect of BAY 43-9006 may not be attributable to the inhibition of the MAPK pathway. The data shown in Figs. 3 and 4 suggest that the down-modulation of Bcl-2 and Bcl-XL may obviate the need for Bad dephosphorylation in the induction of apoptosis and that the dephosphorylation of Bad may play an essential role in BAY 43-9006–induced cell death only in those cell lines that maintain high Bcl-2 and Bcl-XL levels in the presence of the drug.
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To assess the contribution of caspase activation to the lethality of BAY 43-9006, the pan-caspase inhibitor Z-VAD-fmk (20 µmol/L) was added to the melanoma cells. Thirty minutes later, BAY 43-9006 (20 µmol/L) was added, and the cells were subsequently analyzed by flow cytometry for staining with Annexin V/propidium iodide. As shown in Fig. 5D, the caspase inhibitor had only a modest protective effect on the cells. In the case of the SKMEL5 cells, for example, Annexin V/propidium iodide staining after 24 hours of exposure to BAY 43-9006 was only slightly reduced (95-84%) by the inclusion of Z-VAD at a concentration that reduced the extent of PARP cleavage from nearly complete to scarcely detectable. For the purpose of statistical analysis, we took the percentage of Annexin/propidium iodide (+) Z-VAD alone–treated cells to represent the Annexin/propidium iodide staining that would be observed in cells treated with both BAY 43-9006 and Z-VAD in the event that Z-VAD completely inhibited BAY 43-9006–induced apoptosis (i.e., 100% inhibition) and that of BAY 43-9006 alone–treated cells, respectively, to represent that of cells treated with both drugs in the event Z-VAD had no inhibitory effect (0% inhibition). Using this criterion to quantify drug-induced apoptosis, the probability that Z-VAD inhibited BAY 43-9006–induced apoptosis (i.e., Annexin/propidium iodide staining) by even 50% was <0.001 for all three cell lines at both the 6- and 24-hour time points. These data indicate that Z-VAD at concentrations that block PARP cleavage failed to prevent BAY 43-9006–induced apoptosis in melanoma cells.
BAY 43-9006 induces mitochondrial release and nuclear translocation of AIF. To determine if AIF is translocated from the mitochondria to the nucleus in response to BAY 43-9006, melanoma cells were treated with the drug (20 µmol/L for 20 hours), Dounce homogenized, and fractionated into subcellular components. The mitochondrial and nuclear fractions were then detergent solubilized, fractionated by 12% SDS-PAGE, and analyzed for AIF by Western blot. As shown in Fig. 6A, BAY 43-9006–induced nuclear translocation of AIF followed a pattern similar to that of the SMAC release depicted in Fig. 5B in that it was undetectable in the A375 cells but nearly quantitative in the A2058 and SKMEL5 cells. The accumulation of AIF in the nucleus was also readily demonstrable by immunohistochemistry, as shown in Fig. 6B. In this study, the cells were examined after only 5 hours of drug exposure. In the untreated cells, AIF seems to be scattered throughout the cytosol, but within 5 hours, nuclear staining is clearly evident, indicating that the nuclear translocation of AIF is an early event in the apoptosis induced by BAY 43-9006. Of note is the observation that neither of the two MEK inhibitors PD98059 or U0126 at concentrations sufficient to suppress ERK phosphorylation (i.e., 50 and 20 µmol/L, respectively) induced appreciable AIF translocation, suggesting that this particular effect of BAY 43-9006 is not the result of MAPK inhibition.
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Discussion |
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Exposure to BAY 43-9006 activates Bak and Bax. In healthy cells, Bax and Bak are in an inactive conformation in which their NH2 and COOH termini are folded into a hydrophobic pocket (18–20, 35). In response to apoptotic stimuli, the proteins unfold and form multimers in the mitochondrial membrane. The lethality of this multimerization is blocked through the formation of heterodimers with Bcl-2 or Bcl-XL (22) and favored by signaling events that render Bcl-2 and Bcl-XL unavailable to partner with Bak and Bax.
In A2058 and SKMEL5 cells, BAY 43-9006 down-modulates Bcl-2 and Bcl-XL, an effect predicted to favor Bak and Bax homodimerization and apoptosis induction. This down-modulation of Bcl-2 and Bcl-XL could not be duplicated with either of the MEK inhibitors PD98059 or U0126 and is therefore presumably not mediated through the inhibition of MAPK. In cells in which Bcl-2 and Bcl-XL levels are maintained (e.g., A375), heterodimerization with Bak and Bax is presumably prevented by competition with Bad. When present in the mitochondria, Bad binds Bcl-2 and Bcl-XL, rendering them unavailable for heterodimerization with Bax and Bak (17). The dephosphorylation and mitochondrial translocation of Bad induced by MAPK inhibiton would therefore be expected to have the same consequences for cell viability as the down-modulation of Bcl-2 and Bcl-XL. The results of our experiments with the Bad siRNA are entirely consistent with this notion as the effect of Bad depletion on the susceptibility to BAY 43-9006–induced apoptosis correlated with Bad levels and the effects of the drug on Bcl-2 and Bcl-XL. A2058 and SKMEL5 cells, both of which down-modulate Bcl-2 and Bcl-XL in response to BAY 43-9006 and have relatively low levels of Bad, were not protected by the Bad knockdown, whereas the A375 cells, which maintain high Bcl-2 and Bcl-XL levels in the presence of the drug and have high levels of Bad expression, were protected. These data suggest the existence of at least two distinctive patterns of response to BAY 43-9006. In some melanoma cell lines, the drug down-modulates Bcl-2 and Bcl-XL through a process independent of MAPK inhibition. Other cell lines respond to BAY 43-9006 as they might to a MEK inhibitor, in which case the dephosphorylation of Bad serves as the primary means of Bak and Bax activation and apoptosis induction.
Exposure to BAY 43-9006 resulted in cytochrome c release from the mitochondria and caspase activation (PARP cleavage) in all three cell lines examined. These events, however, played only a minor role in BAY 43-9006–induced apoptosis because the pan-caspase inhibitor Z-VAD-fmk had little effect on drug-induced Annexin V staining at concentrations sufficient to inhibit PARP cleavage. These data suggest that BAY 43-9006–induced apoptosis is largely caspase independent. BAY 43-9006 also induced the mitochondrial release of the IAP inactivator SMAC and AIF. The release of these proapoptotic proteins was highly variable from one cell line to another, scarcely detectable in the A375 cells but nearly complete in SKMEL5 and A2058 cells. As with the down-modulation of Bcl-2 and Bcl-XL, AIF release could not be induced with either of the MEK inhibitors, suggesting that this effect of BAY 43-9006 was not due to MAPK inhibition.
AIF is a flavoprotein with both oxidoreductase and DNA-binding domains but no intrinsic DNase activity (37–40). Depending on the circumstances, AIF can function to promote cell survival or induce apoptosis. In the mitochondria, AIF is involved in cellular respiration (37, 38) and is essential for cell survival, especially in retinal and cerebellar neurons (40). When translocated to the nucleus, AIF binds the DNase Endo G, another mitochondrial constituent released in response to apoptotic stimuli (39). This recruitment of EndoG results in DNA fragmentation and cell death (41, 42).
The release of AIF from the mitochondria and its subsequent translocation to the nucleus is one of the earliest events observed in melanoma cells after exposure to BAY 43-9006. These events were demonstrable by Western blot and immunohistochemistry within a few hours of drug exposure. To determine if AIF might be involved in the apoptosis induced by BAY 43-9006, we introduced an siRNA for AIF into melanoma cells and compared the responses of control and AIF-deficient cells to the drug. In each cell line, BAY 43-9006–induced Annexin V staining was reduced by the siRNA, suggesting that the AIF knockdown protected the cells from the drug. This protective effect was, however, highly variable depending on the cell line. The A2058 and SKMEL5 cells, which release AIF in response to the drug, were clearly protected by AIF depletion, whereas the A375 cells (in which the knockdown was most successful) were the least protected by the AIF siRNA. This result is consistent with the fact that AIF release was nearly undetectable in the A375 cells.
The apoptosis induced in sensitive melanoma cell lines (e.g., A2058 and SKMEL5) by BAY 43-9006 has several features not shared with MEK inhibitors, including rapid onset, depolarization of the mitochondria, the down-modulation of Bcl-2 and Bcl-XL, the nuclear translocation of AIF, and the fact that the process is independent of Bad dephosphorylation. In the case of the more resistant A375 cells, BAY 43-9006–induced apoptosis is dependent on Bad dephosphorylation and is unaccompanied by either Bcl-2/Bcl-XL down-modulation or AIF translocation and, in this respect, resembles that induced by MEK inhibitors. It is conceivable that the distinctions between the effects of raf and MEK inhibitors might be due to the ability of raf to promote cell survival through a mechanism independent of the MAPK pathway. Indeed, several non-MEK raf-binding proteins have been identified that clearly play a role in cell survival (26, 27). However, none of these alternative substrates are known to require phosphorylation by raf to exert their biological effects, and it is therefore unclear how their function might be affected by a raf kinase inhibitor, such as BAY 43-9006. It is of course possible that some of the apoptotic effects of BAY 43-9006 may be due to the inhibition of non-raf targets, such as c-kit or the PDGF receptor-ß (29), and studies are currently under way to determine the extent to which the inhibition of these membrane tyrosine kinases contributes to BAY 43-9006–induced apoptosis and tumor regression.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3/16/05. Revised 10/20/05. Accepted 11/11/05.
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 M. J. Edick, L. Tesfay, L. E. Lamb, B. S. Knudsen, and C. K. Miranti Inhibition of Integrin-mediated Crosstalk with Epidermal Growth Factor Receptor/Erk or Src Signaling Pathways in Autophagic Prostate Epithelial Cells Induces Caspase-independent Death Mol. Biol. Cell, July 1, 2007; 18(7): 2481 - 2490. [Abstract] [Full Text] [PDF]
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 S. N. Markovic, L. A. Erickson, R. D. Rao, R. H. Weenig, B. A. Pockaj, A. Bardia, C. M. Vachon, S. E. Schild, R. R. McWilliams, J. L. Hand, et al. Malignant Melanoma in the 21st Century, Part 2: Staging, Prognosis, and Treatment Mayo Clin. Proc., April 1, 2007; 82(4): 490 - 513. [Abstract] [Full Text] [PDF]
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 J. Szwaya, C. Bruseo, E. Nakuci, D. McSweeney, X. Xiang, D. Senator, D. France, and C.-R. Chen A Novel Platform for Accelerated Pharmacodynamic Profiling for Lead Optimization of Anticancer Drug Candidates J Biomol Screen, March 1, 2007; 12(2): 159 - 166. [Abstract] [PDF]
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A. Glading, J. A. Koziol, J. Krueger, and M. H. Ginsberg PEA-15 Inhibits Tumor Cell Invasion by Binding to Extracellular Signal-Regulated Kinase 1/2 Cancer Res., February 15, 2007; 67(4): 1536 - 1544. [Abstract] [Full Text] [PDF]
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L. Liu, Y. Cao, C. Chen, X. Zhang, A. McNabola, D. Wilkie, S. Wilhelm, M. Lynch, and C. Carter Sorafenib Blocks the RAF/MEK/ERK Pathway, Inhibits Tumor Angiogenesis, and Induces Tumor Cell Apoptosis in Hepatocellular Carcinoma Model PLC/PRF/5 Cancer Res., December 15, 2006; 66(24): 11851 - 11858. [Abstract] [Full Text] [PDF]
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 A. P. Mitra, R. H. Datar, and R. J. Cote Molecular Pathways in Invasive Bladder Cancer: New Insights Into Mechanisms, Progression, and Target Identification J. Clin. Oncol., December 10, 2006; 24(35): 5552 - 5564. [Abstract] [Full Text] [PDF]
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 L. Chin, L. A. Garraway, and D. E. Fisher Malignant melanoma: genetics and therapeutics in the genomic era. Genes & Dev., August 15, 2006; 20(16): 2149 - 2182. [Abstract] [Full Text] [PDF]
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