Bortezomib (PS-341), a specific proteasome inhibitor, exhibits antitumor activity against a wide range of malignancies. However, the molecular mechanisms by which bortezomib causes apoptosis selectively in cancer cells still remain unclear. Ras signaling is involved in multiple cellular processes, including cell cycle progression, differentiation, and apoptosis, and can either promote or inhibit apoptosis depending on the type of apoptotic stimuli and the cell model. Here, we investigated the role of K-ras signaling in bortezomib-induced apoptosis. We found that K-ras-transformed cells were more susceptible to bortezomib-induced apoptosis than were nontransformed cells and that bortezomib-induced apoptosis was mainly caspase dependent in K-ras-transformed cells. We also found that mammalian sterile20-like kinase 1 (MST1) was activated by bortezomib in K-ras-transformed cells and K-ras-mutated cancer cells. Treatment of K-ras-transformed cells with bortezomib resulted in translocation of MST1 from cytoplasm into the nucleus and an increase of phosphorylated histone H2B and histone H2AX. Moreover, pretreatment with leptomycin B, an inhibitor of the nuclear export signal receptor, dramatically enhanced bortezomib-mediated MST1 activation, phosphorylation of histones H2B and H2AX, and apoptosis induction in K-ras-transformed cells. Knockdown of MST1 expression by small interfering RNA diminished bortezomib-induced apoptosis or caspase-3 activation. Our data suggested that bortezomib may be useful for treatment of K-ras-mutated cancer cells, and MST1 is one of the mediators for bortezomib-induced apoptosis in K-ras-transformed cells. (Cancer Res 2006; 66(12): 6072-9)
The ubiquitin-proteasome pathway is responsible for proteolysis of eukaryotic cellular proteins related to cell cycle regulation, cell survival, and apoptosis (1–3). Inhibition of proteasome activity is a novel therapeutic strategy against cancer cells. Bortezomib (formerly known as PS-341), a cell-permeable boronic acid dipeptide, is a specific inhibitor of the proteasome pathway (4–6). Preclinical studies have shown that bortezomib treatment has a cytotoxic effect against both hematologic malignancies and various solid tumors, including myeloma, breast, ovarian, prostate, colon, pancreatic, and lung cancers (7, 8). In addition, several clinical trials are currently ongoing in the treatment of various hematologic malignancies and solid tumors (9). Bortezomib has been reported to stabilize various proteins, including p21, p27, and p53; various transcriptional factors (e.g., c-myc, c-fos, and c-jun); IκBα; cyclins; and some Bcl-2 family members (e.g., Bak, Bax, and Bik) by inhibiting their degradation (8, 9). However, the kind of general mediator involved in bortezomib-induced apoptosis in cancer cells remains unclear. Understanding and clarifying this may lead to better therapeutic strategies with bortezomib against various cancer cells.
The role of Ras signaling has been investigated due to the consequence of different functions, such as proliferation, differentiation, development, and apoptosis (10–12). Mutation of the K-ras proto-oncogene has been found in numerous cancers, including pancreatic, colon, and ovarian cancers (13–15). Recent reports showed that K-ras mutation increases the sensitivity of human cancer cells to genotoxic stresses, such as 5-fluorouracil, tumor necrosis factor (TNF)–related apoptosis-inducing ligand, and irradiation (16–18). In addition, it was also reported that endometrial cancer cells carrying K-ras12V are more sensitive to apoptosis (19). Recently, the Ras-NORE1-mammalian sterile20-like kinase 1 (MST1) complex has been reported to be involved in mediating Ras-induced apoptosis in NIH3T3 and HEK293 cells treated with tamoxifen (20). MST1 has been identified as a caspase substrate, and MST1 activation during apoptosis requires both phosphorylation and caspase-mediated cleavage (21, 22). It has been reported that various apoptotic stimuli and cellular stresses, including UV irradiation (23), TNF-α (23), staurosporine (24), anti-Fas antibody (24, 25), and anticancer drugs such as camptothecin, doxorubicin, paclitaxel, and 5-fluorouracil (26), commonly induce MST1 cleavage by caspases, producing a 36 kDa NH2-terminal hyperactive fragment of MST1 (22, 25, 27). However, the cellular function of MST1 related to apoptotic induction still remains unclear.
The goal of this study was to investigate whether K-ras signaling affects bortezomib-induced apoptosis and to clarify its mechanisms. To avoid the influences of many genetic backgrounds on the effect of bortezomib, we used nontransformed human ovarian epithelial T29 cells and two clones generated from T29 cells with the activated K-ras or H-ras alleles, T29Kt1 and T29Ht1 cells, respectively (28). We also used colon cancer HKe-3 cell lines with homologous K-ras gene deletion (29) and the HCT116 parental cell line to investigate the effect of bortezomib. We found that K-ras-transformed or K-ras-mutated cells are more susceptible to bortezomib-induced apoptosis than are nontransformed cells. We also found that bortezomib causes rapid caspase activation and the MST1 is activated and translocated into the nucleus in both K-ras-transformed epithelial cells and K-ras-mutated cancer cell lines. These results suggest that bortezomib may have potent activity against K-ras-mutated cancer cells and that MST1 may be a crucial mediator for bortezomib-induced apoptosis in K-ras-mutated cancer cells.
Materials and Methods
Cells and culture conditions. The immortalized nontumorigenic cell line T29 was generated from mortal human ovarian epithelial cells (IOSE-29) by infecting the retrovirus expressing a full-length hTERT cDNA (30). The T29Kt1 and T29Ht1 cell lines were generated from xenograft tumors derived from T29K or T29H cells, which themselves were generated through infecting T29 cells with retrovirus expressing either K-ras12V or H-ras12V as described previously (28). The K-ras-mutated pancreatic cancer cell line ASPC-1 and the colon cancer cell line DLD-1 were obtained from the American Type Culture Collection (Rockville, MD). The human colon cancer cell lines HCT116 and its K-ras-disrupted derivative HKe-3 cells (29) were grown in DMEM supplemented with 10% heat-inactivated FCS, 25 mmol/L HEPES, 100 units/mL penicillin, and 100 mg/mL streptomycin. All cells were maintained in the presence of 5% CO2 at 37°C.
Chemicals and antibodies. Bortezomib was obtained from the pharmacy of The University of Texas M.D. Anderson Cancer Center and dissolved in PBS to a concentration of 5 μmol/L. Leptomycin B was purchased from Sigma Chemical (St. Louis, MO), dissolved in DMSO, and stored at −20°C. The general caspase inhibitor z-VAD-fmk was obtained from R&D Systems (Minneapolis, MN). Antibodies to the following proteins were used for Western blot analysis: anti-K-ras, H-ras, caspase-3, Bax, Bak, Bcl-2, and Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA); anti-caspase-8 (MBL International, Woburn, MA); anti-GFP and MST1 (BD Biosciences, San Diego, CA); anti-MST1 and caspase-9 (Cell Signaling, Beverly, MA); anti-NORE1 (Abcam, Cambridge, MA); and anti-β-actin (Sigma).
Cell proliferation assay. The antiproliferative effects of bortezomib on T29, T29Kt1, and T29Ht1 cells were determined by the sulforhodamine B assay as described previously (31).
Apoptosis assay. For detection of the cell cycle and apoptosis, cells were harvested in 0.125% trypsin, washed twice in PBS, and fixed in 70% methanol at −20°C overnight. Cells were then resuspended in PBS containing 10 μg/mL propidium iodide (Roche Diagnostics, Indianapolis, IN) and 10 μg/mL RNase A (Sigma-Aldrich, St. Louis, MO) at 37°C for 30 minutes. The cell cycle distribution and the population of apoptosis (cells with a sub-G1 DNA content) were determined as described previously (32). All experiments were conducted at least twice.
Preparation of membrane fractions and whole-cell lysates and Western blot analysis. For preparation of cytosol and membrane fractions, harvested cells were suspended in fractionation buffer [10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride] containing 1× proteinase-inhibitor cocktail (Roche Diagnostics) and lysed by sonication. The lysate was spun at 14,000 × g in a microcentrifuge at 4°C for 1 hour to obtain the cytosol fraction (supernatant). The pellet representing particulate proteins was resuspended in the above buffer containing 0.1% Triton X-100 and lysed by sonication. The lysate was spun at 14,000 × g at 4°C for 1 hour to obtain the membrane fraction (supernatant). The preparation of whole-cell extracts and Western blot analysis were as described previously (32).
Plasmid and small interference RNA transfection. The plasmid GFP-K-Ras (12V; ref. 33) was kindly provided by Dr. Y. Kloog (Tel Aviv University, Tel Aviv, Israel). Plasmid transfection was done using the FuGENE6 reagent (Roche Diagnostics) according to the protocols of the manufacturer for 30 hours, and then whole-cell lysates were analyzed by Western blotting or GFP-K-Ras-transfected cells were treated with bortezomib for an additional 36 hours. MST1 expression was silenced using a pool of four small interfering RNAs (siRNA) directed against the coding region of MST1 (Dharmacon, Lafayette, CO). T29Kt1 cells were transiently transfected with 100 nmol/L MST1 siRNA using Oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the protocols of the manufacturer for 24 to 48 hours, and then whole-cell lysates were analyzed by Western blotting or MST1 siRNA-transfected cells were treated with bortezomib for an additional 24 hours. Luciferase siRNA (Dharmacon) was used as a control.
Immunofluorescence microscopy. To investigate the localization of MST1, MST1 in bortezomib-treated T29Kt1 cells was observed by immunofluorescence microscopy. Cells were grown on a glass chamber slide (Becton Dickinson, Franklin Lakes, NJ) and treated with 50 nmol/L bortezomib for 24 hours. Cells were then washed with PBS and fixed in 3.7% formaldehyde in PBS containing 0.1% Triton X-100 for 10 minutes at room temperature. After blocking in 1% bovine serum albumin in PBS for 30 minutes, cells were incubated with an anti-MST1 polyclonal antibody (Cell Signaling) for 1 hour at ambient temperature and then incubated with a FITC-conjugated anti-rabbit secondary antibody (BD Biosciences). After extensive washing, cells were rinsed once in water, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and examined under a Nikon (Melville, NY) BX61 microscope.
Caspase-3 activity assay. Caspase-3 activity was evaluated by using the Caspase-3 Assay kit (BD PharMingen) according to the protocol of the manufacturer. T29Kt1 cells in six-well plates were transfected with control or MST1 siRNA for 36 hours and treated with 50 nmol/L bortezomib for an additional 24 hours. Cells were then harvested in cell lysis buffer, and caspase-3 activity was measured by the cleavage of the fluorometric substrate Ac-DEVD-AMC according to the instructions of the manufacturer.
Statistical analysis. Differences among the treatment groups were assessed by ANOVA using StatSoft statistical software (Tulsa, OK). P < 0.05 was considered significant.
K-ras transformation promotes bortezomib-induced cell death in human ovarian epithelial cells. Human ovarian epithelial T29 cell line and its tumorigenic derivatives expressing oncogenic K-ras12V gene (T29Kt1) or H-ras12V gene (T29Ht1) were used to investigate the effects of bortezomib. The expression of mutant forms of K-ras or H-ras protein in T29Kt1 or T29Ht1, respectively, was confirmed by Western blot analysis (Fig. 1A). We then determined the growth-inhibitory effect of bortezomib on T29, T29Kt1, and T29Ht1 cells. Cells were treated with various doses of bortezomib for 30 hours, and cell viability was then determined by the sulforhodamine B assay (Fig. 1B). Interestingly, K-ras-transformed T29Kt1 cells were much more sensitive to bortezomib than were nontransformed T29 cells and H-ras-transformed T29Ht1 cells when the cells were treated with 50 and 100 nmol/L bortezomib. By 30 hours, 50 nmol/L bortezomib–treated T29Kt1 cells exhibited apoptotic morphology, including cell shrinkage and cytoplasmic blebbing (Fig. 1C). In contrast, there was little difference in cell growth inhibition in between T29Kt1 and T29 cells treated with cisplatin or paclitaxel for 24 to 48 hours, and T29Kt1 cells were more resistant to gemcitabine than were T29 cells (data not shown). These results suggest that K-ras transformation may specifically promote bortezomib-induced cell death in our cell systems. To further confirm whether mutated K-ras expression influenced bortezomib-induced apoptosis, we investigated the effect of bortezomib in T29 cells transiently transfected with a GFP-K-ras12V plasmid. T29 cells were transfected with a GFP-K-ras plasmid for 30 hours and then treated with 50 nmol/L bortezomib for an additional 36 hours. The extent of apoptosis was then determined by flow cytometry (Fig. 1D). Transfection with GFP-K-ras12V plasmid promoted bortezomib-induced apoptosis, with peak values of 19.6% after 36 hours of treatment, whereas bortezomib induced a 12.2% sub-G1 population in control vector–transfected T29 cells.
K-ras transformation and mutation promote bortezomib-induced apoptosis through caspase activation. We next determined the proportion of apoptotic cells in cells treated with bortezomib by flow cytometry with propidium iodide staining (Fig. 2A). Bortezomib induced a marked, time-dependent increase in the sub-G1 population of T29Kt1 cells, with peak values of 63.9% after 48 hours of treatment, whereas bortezomib induced a 31.9% sub-G1 population in T29 cells. To confirm the effect of bortezomib in K-ras-mutated cancer cells, we compared the apoptotic induction by bortezomib in the isogenic colon cancer cell lines (HCT116 and HKe-3) that vary only by the presence of the mutant K-ras allele. Cells were treated with 10, 20, or 50 nmol/L bortezomib for 24 or 48 hours, and then the extent of apoptosis was determined by flow cytometry. As shown in Fig. 2B, HCT116 cells were more susceptible to bortezomib than were HKe-3 cells: In the HCT116 and HKe-3 cells treated with 20 nmol/L bortezomib, the proportion of apoptotic cells were 10.4% and 5.1%, respectively, at 24 hours, and 36.7% and 17.2%, respectively, at 48 hours. These results indicate that K-ras signaling may promote bortezomib-induced apoptosis in cancer cells.
Moreover, to confirm bortezomib-induced apoptosis in the T29 cell series, we evaluated the caspase activation in bortezomib-treated T29, T29Kt1, and T29Ht1 cells by Western blotting (Fig. 2C). Treatment of the T29Kt1 cells with 50 nmol/L bortezomib caused marked cleavages of caspase-3, caspase-8, and caspase-9 after 24 to 48 hours. Those changes were also observed in the T29 and T29Ht1 cells; however, they occurred in later phase than in the T29Kt1 cells. To further examine the consequence of caspase activation in bortezomib-treated T29Kt1 cells, we blocked the caspase activation with the general caspase inhibitor z-VAD-fmk. Cells were pretreated with z-VAD-fmk at 20 or 100 μmol/L for 30 minutes and then treated with 50 nmol/L bortezomib for another 30 hours. Cells were then analyzed for apoptosis by flow cytometry. Pretreatment with z-VAD-fmk markedly diminished the population of cells in sub-G1 phase by bortezomib treatment in T29Kt1 cells in a dose-dependent manner. The most significant difference in the apoptotic ratio was observed between cells treated with bortezomib alone (27.9 ± 1.3%) and those pretreated with 100 μmol/L z-VAD-fmk (2.3 ± 0.8%; P < 0.01, Fig. 2D). These results indicate that the bortezomib-induced apoptosis in T29Kt1 cells is mainly caspase dependent.
Bortezomib had little effect on the protein expression related to the Bcl-2 family in K-ras-transformed cells. We next investigated what kind of mediator is involved in bortezomib-induced apoptosis in K-ras-transformed cells. Our recent work showed that Bik/NBK accumulation correlates with apoptosis induction by bortezomib (34). We examined the protein expression related to the Bcl-2 family (such as Bcl-2, Bax, Bak, and Bik) in T29 cell series treated with 50 nmol/L bortezomib for 12 to 36 hours. Bcl-2 was slightly lower in T29Kt1 cells than in the T29 and T29Ht1 cells, and Bak was obviously increased in T29 and T29Kt1 cells after bortezomib treatment in a time-dependent manner (Fig. 3). However, the level of Bik did not obviously change in T29Kt1 cells, whereas bortezomib had a little effect on the Bik level in T29 cells after 36 hours. These results suggest that the regulation of these proteins has little influence on apoptotic induction in T29Kt1 cells treated with bortezomib.
Bortezomib caused activation of MST1 in K-ras-transformed cells and K-ras-mutated cancer cells. Recent reports showed that MST1 plays a key role in apoptosis induction by various apoptotic stimuli and cellular stresses in the ras signaling pathway (20, 35, 36). We next examined whether bortezomib treatment affects the expression of MST1 in the T29 cell series and in K-ras-mutated cancer cells. Cells were treated with 50 nmol/L bortezomib for the indicated time periods, and whole-cell lysates were analyzed by Western blotting. Interestingly, treatment of cells with bortezomib increased the 36 kDa fragment of MST1, indicating MST1 activation (22, 25, 27) in T29Kt1 cells but not in T29 and T29Ht1 cells (Fig. 4A). In addition, we also examined the expression of NORE1 protein, which is reported to be involved in the Ras-NORE1-MST1 complex. A slight up-regulation of NORE1 expression was observed in T29 and T29Kt1 cells treated with bortezomib, suggesting that levels of NORE1 expression is not related to bortezomib susceptibility.
We also investigated the expression of MST1 in bortezomib-treated HCT116 and HKe-3 cells. Similarly, bortezomib treatment caused MST1 activation in HCT116 cells after 36 to 48 hours but no activation occurred in HKe-3 cells (Fig. 4B). To further test whether MST1 activation is a general molecular action caused by bortezomib treatment, we checked the MST1 expression in K-ras-mutated pancreatic cancer ASPC-1 and colon cancer DLD-1 cells treated with 50 nmol/L bortezomib. The result showed that bortezomib treatment activated MST1 in those K-ras-mutated cancer cell lines after 24 to 48 hours (Fig. 4C). These results indicated that bortezomib-induced MST1 activation might be a general effect in K-ras-mutated cancer cells.
Translocation of MST1 into the nucleus and phosphorylation of histone H2B in bortezomib-treated K-ras-transformed cells. MST1 has been reported to be exclusively located in the cytoplasm; however, activated MST1 is translocated into the nucleus, where it promotes chromatin condensation and DNA fragmentation (35, 37, 38). To investigate whether bortezomib affects the localization of MST1 in T29Kt1 cells, we evaluated the localization of MST1 in bortezomib-treated T29Kt1 cells by using immunofluorescent staining. T29Kt1 cells were treated with 50 nmol/L bortezomib for 24 hours, and then fixed cells were stained with MST1 and 4′,6-diamidino-2-phenylindole (DAPI; Fig. 5A). MST1 was mainly distributed in the cytoplasm, whereas a little MST1 was localized in the nucleus in untreated T29Kt1 cells. Interestingly, MST1 was markedly translocated into the nucleus in bortezomib-treated T29Kt1 cells, although MST1 was still detected in the cytoplasm. These results indicate that bortezomib treatment caused translocation of MST1 into the nucleus and that cleaved MST1 might have a critical role for the localization of MST1 in the bortezomib-treated T29Kt1 cells. We also examined the phosphorylation of histone H2B, which is associated with DNA fragmentation in apoptotic cells (39), and histone H2AX (Fig. 5B). Similarly, bortezomib treatment caused phosphorylation of histone H2B and histone H2AX in K-ras-mutated HCT116 cells but little phosphorylation of those molecules in K-ras allele HKe-3 cells (Fig. 5C).
We next examined MST1 expression in T29Kt1 cells treated with bortezomib in the presence of the general caspase inhibitor z-VAD-fmk. T29Kt1 cells were pretreated with 50 μmol/L z-VAD-fmk for 30 minutes and then treated with 50 nmol/L bortezomib for another 24 hours, and whole-cell lysates were analyzed by Western blotting (Fig. 5D). z-VAD-fmk pretreatment blocked bortezomib-mediated cleavage of caspase-3. In addition, the bortezomib-caused MST1 activation and histone H2B phosphorylation were substantially attenuated in the presence of z-VAD-fmk. Interestingly, a little MST1 activation was observed in T29Kt1 cells treated with bortezomib plus z-VAD-fmk. The significance of this low-level MST1 activation in the presence of z-VAD-fmk is not clear.
A recent report showed that removing the two nuclear export signals of MST1 by caspases or unmasking the nuclear localization sequence results in the translocation of MST1 into the nucleus, where it promotes chromatin condensation and nuclear fragmentation (38). To investigate whether nuclear translocation of MST1 contributes to the bortezomib-mediated phosphorylation of histone H2B, we used leptomycin B, a well-characterized inhibitor of the nuclear export machinery (40). T29Kt1 cells were treated with 50 nmol/L bortezomib in the presence or absence of 5 or 20 nmol/L leptomycin B for 16 hours, and whole-cell lysates were then analyzed by Western blotting with the indicated antibodies (Fig. 5E). Leptomycin B treatment markedly enhanced bortezomib-mediated phosphorylation of histone H2B and histone H2AX, indicating that nuclear localization of MST1 might increase its ability to promote bortezomib-induced chromatin condensation. We also determined the cell viability of T29Kt1 cells treated with 50 nmol/L bortezomib in the presence or absence of 5 or 20 nmol/L leptomycin B for 16 hours by sulforhodamine B assay (Fig. 5F). Leptomycin B enhanced bortezomib-induced cell death. Viable cells after treatment with 50 nmol/L bortezomib, 20 nmol/L leptomycin B, and both were 59%, 80%, and 26%, respectively.
Knockdown of MST1 expression diminished bortezomib-induced apoptosis and inhibits histone H2B phosphorylation and caspase-3 activation. Finally, to investigate the role of MST1 in bortezomib-induced apoptosis, we knocked down the MST1 expression by silencing its expression through siRNA. T29Kt1 cells were transfected with a pool of four RNA duplexes directed against the coding region of MST1 as described in Materials and Methods. Cells were transfected with 100 nmol/L luciferase or MST1 siRNA, and the expression of MST1 protein was determined after 24 or 48 hours of transfection. As shown in Fig. 6A, basal expression of MST1 was knocked down by MST1-specific siRNA in T29Kt1 cells, with a peak inhibitory value of 83% after 48 hours of transfection. We next examined whether knockdown of MST1 expression in T29Kt1 cells affects bortezomib-induced apoptosis. T29Kt1 cells transfected with either luciferase or MST1 siRNA were treated with 50 nmol/L bortezomib for 24 hours and the extent of apoptosis was determined by flow cytometric analysis with propidium iodide staining (Fig. 6B). Transfection with MST1 siRNA markedly diminished bortezomib-induced apoptosis after 24 hours (P < 0.01). We also used Western blotting to examine the expression level of MST1 and caspase-3 and the phosphorylation of histone H2B and histone H2AX in MST1 siRNA-transfected T29Kt1 cells treated with bortezomib for 24 hours (Fig. 6C). MST1 siRNA blocked bortezomib-mediated cleavage of MST1 and phosphorylation of histone H2B and histone H2AX. Interestingly, MST1 siRNA also slightly suppressed bortezomib-mediated caspase-3 cleavage. To further investigate whether MST1 siRNA blocked bortezomib-mediated caspase-3 activation in T29Kt1 cells, we quantified the caspase-3 activity by using a caspase-3 activity kit (Fig. 6D). MST1 siRNA in the T29Kt1 cells blocked bortezomib-mediated caspase-3 activation after 24 hours (P < 0.01) compared with the same treatment without siRNA transfection. These results indicate that MST1 activation has a crucial role in bortezomib-mediated apoptosis and may be involved in the amplification of caspase-3 activation.
In this study, we characterized the role of K-ras signaling in the responsiveness to bortezomib treatment by using T29 cell series generated from ovarian epithelial cells and HCT116 and HKe-3 colon cancer cell lines that differ only in their K-ras status. We found that K-ras-transformed T29Kt1 cells were more sensitive to bortezomib than were nontransformed T29 cells and that K-ras-mutated colon cancer HCT116 cells were more susceptible to bortezomib-induced apoptosis than were K-ras allele–deleted HKe-3 cells. Our data also showed that transient K-ras expression promotes bortezomib-induced apoptosis in T29 cells. These findings suggest that K-ras signaling may have a key role in bortezomib-induced apoptosis and its selectivity in cancer cells. Nevertheless, the bortezomib-mediated growth inhibition in 60 cancer cell lines presented the National Cancer Institute (NCI) Standard Anticancer Agent Database showed no correlation with K-ras mutations. With exception of SKOV3 and NCI/ADR-Res cells that are K-ras wild-type and relatively resistant to bortezomib, most of other cancer cells are highly sensitive to bortezomib at very narrow GI50 ranges (GI50 = a dose that causes 50% growth inhibition). It is noteworthy that in the absence of a Ras mutation, increased activation of Ras pathway is commonly detected in human cancers because of gene amplification (41), overexpression (42), and an increase in upstream signals from tyrosine kinase growth factor receptors, such as Her2 (43, 44).
Our results also provided some molecular insights about bortezomib-induced apoptosis in K-ras mutant cells. First, our results on caspase activation and the effect of caspase inhibitor showed that caspase activation played a major role in bortezomib induced apoptosis. In fact, bortezomib has been reported to cause apoptosis through caspase-8 and caspase-3 activation, and the caspase-8-mediated apoptosis pathway is independent of the caspase-9 pathway (45). Second, our results showed that MST1 activation is required, at least in part, for bortezomib-induced apoptosis in K-ras-transformed cells. The roles of MST1 in the apoptotic induction by various apoptotic stimuli and cellular stresses in the K-ras signaling pathway have been previously reported by others (20, 35). Our results showed that bortezomib induces MST1 activation in both K-ras-transformed cells and K-ras-mutated cancer cells, suggesting that MST1 activation may play a key role in the bortezomib-induced apoptosis in the K-ras signaling pathway.
MST1 has been reported to have two caspase cleavage sites to generate active enzymes of 36 kDa and 40 to 41 kDa (21, 24, 37). The 36 kDa subunit has 10-fold the activity of full-length MST1 (24). Moreover, this process is paralleled by the onset of apoptosis. Our data showed that although bortezomib-mediated MST1 activation was substantially blocked when T29Kt1 cells were pretreated with the general caspase inhibitor z-VAD-fmk, suggesting that caspase activation is a major upstream signal for MST1 activation. Nevertheless, MST1 knockdown by siRNA diminished bortezomib-induced caspase-3 activation in T29Kt1 cells, indicating that MST1 activation may resulted in a positive feedback of caspase activation. Thus, bortezomib-mediated MST1 activation may be involved in the amplification of caspase signaling as well as the mediation of caspase-induced apoptotic events. This is consistent with previous reports that MST1 may function both upstream and downstream of caspases (24, 37). It has been reported that MST1 is a novel mediator of apoptotic signaling from cytoplasm to the nucleus and that it may be one of the inducers of chromatin condensation and nuclear fragmentation through nuclear protein, such as phosphorylated histone H2B, which is associated with DNA fragmentation in apoptotic cells, during apoptosis (24). Our data showed that bortezomib treatment causes translocation of MST1 into nuclear and phosphorylation of histone H2B. Moreover, MST1 activation, phosphorylation of histone H2B, and apoptosis induction were enhanced when T29Kt1 cells were treated with bortezomib in the presence of leptomycin B. These findings indicate that bortezomib-mediated nuclear localization of MST1 may enhance MST1-induced chromatin condensation and DNA fragmentation as well as apoptosis in K-ras-transformed cells.
However, the molecular mechanisms underlying the MST1-mediated positive feedback of caspase activation in K-ras-transformed or mutant cancer cells after treatment with bortezomib is not yet clear. The Ras-NORE1-MST1 complex has been reported to be involved in mediating Ras-induced apoptosis in NIH3T3 and HEK293 cells (20). Nevertheless, our data showed that bortezomib had little effect on the expression of NORE1 in K-ras-transformed cells, indicating that the levels of NORE1 expression had little influence on MST1 activation in bortezomib-treated K-ras-transformed cells. Thus, the molecular interaction between K-ras transformation and bortezomib-induced MST1 activation remain to be characterized. Nevertheless, our data suggest that bortezomib may be useful for the treatment of K-ras-mutated cancer cell and that MST1 could be a novel mediator of bortezomib-induced apoptosis in those cells. This finding may affect for future design and assessment of treatment of K-ras-mutated cancers.
Grant support: NCI grants RO1 CA092487-01A1 and RO1 CA08582-01A1 (B. Fang), Lockton Grant-Matching Fund, Lung Specialized Programs of Research Excellence grant CA 70907, and Core Grant CA16672.
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.
We thank Dr. Y. Kloog for providing GFP-K-ras plasmid, Michael Worley for editorial review, Debbie Smith for secretarial assistance, and Karen M. Ramirez for technical assistance with the flow cytometric analysis.