Jun Ying Zhou

Mitogen-Activated Protein Kinase Phosphatase-1 Is Required for Cisplatin Resistance

Mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 is a member of the MKP family that negatively regulates MAPK signaling. MKP-1 has been implicated in cell survival in response to stressful stimuli, including anticancer treatment, but its role in cisplatin resistance is not fully understood. Here, we show that cisplatin induces MKP-1 in several human cancer cell lines. Induction of MKP-1 by cisplatin was through the transcriptional mechanism regulated by extracellular signal-regulated kinase (ERK). Overexpression of MKP-1 rendered human lung cancer cells resistant to cisplatin. Conversely, down-regulation of MKP-1 by small interfering RNA silencing sensitized human lung cancer cells to cisplatin-induced cell death. Using primary mouse embryonic fibroblasts (MEF) from MKP-1 knockout mice, we show that induction of MKP-1 by cisplatin correlates with inactivation of c-Jun NH(2)-terminal kinase (JNK) but not ERK and p38. Furthermore, apoptosis induced by cisplatin was significant in MKP-1(-/-) MEFs, whereas such change was minimal in MKP-1(+/+) MEFs. More importantly, cisplatin-induced cell death is inhibited by blocking JNK but not ERK and p38 activities. Collectively, our results establish a critical role of JNK in cisplatin-induced apoptosis and suggest that MKP-1 is required for cisplatin resistance.

The Phosphatase MKP1 Is a Transcriptional Target of p53 Involved in Cell Cycle Regulation

The tumor suppressor p53 protein suppresses cell growth by inducing cell cycle arrest or apoptosis. Despite the fact that p53-dependent p21-mediated G1 arrest induced by DNA damage is well defined, the role of p53 in the cell cycle in response to the MAKP signaling remains to be determined. Here we show that MKP1, a member of the dual specificity protein phosphatase family capable of inactivating MAPKs, is a transcriptional target of p53. MKP1 mRNA and protein levels were increased upon p53 activation in several well defined p53-regulated cell systems. p53 bound to a consensus p53 binding site located in the second intron of the MKP1 gene and transactivated MKP1 in reporter gene assays. Inhibition of phosphatase activity impaired p53-mediated G1 arrest in arrested human glioblastoma GM cells in response to growth factor stimuli. Importantly conditional expression of MKP1 prevented arrested human cancer cells from entering into the cell cycle. Thus, these results provide a novel mechanism by which p53 controls the cell cycle in response to the MAPK signaling in the absence of DNA damage and suggest that p53 may negatively control the MAKP pathway via MKP1.

The Role of Mitogen-Activated Protein Kinase Phosphatase-1 in Oxidative Damage–Induced Cell Death

Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a member of the MAPK phosphatase family that functions as a negative regulator of MAPK signaling. MKP-1 is induced by oxidative stress, but the role of its induction in cell death is not fully understood. Here, we show that hydrogen peroxide (H(2)O(2)) induces MKP-1 and activates MAPKs. Induction of MKP-1 by H(2)O(2) correlated with inactivation of p38 and c-Jun-NH(2)-kinase (JNK). Overexpression of MKP-1 increased cell resistance to H(2)O(2)-induced death. Furthermore, we show by small interfering RNA silencing that down-regulation of MKP-1 increases phosphorylated p38 and JNK and subsequent cell death induced by H(2)O(2). More importantly, primary embryonic fibroblasts from mice lacking MKP-1 had a higher level of phosphorylated p38 and JNK and were more sensitive to H(2)O(2)-induced cell death compared with corresponding cells with MKP-1, indicating that p38 and JNK pathways may play important roles in H(2)O(2)-mediated cell death. Thus, these results suggest that activation of MKP-1 is a survival mechanism against oxidative damage.

ERK-Dependent MKP-1–Mediated Cisplatin Resistance in Human Ovarian Cancer Cells

Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is the MAPK phosphatase family member that negatively regulates MAPK signaling. Our previous study showed that MKP-1 is involved in cisplatin resistance, but the mechanism underlying its resistance is not understood. Here, we show that ERK2-mediated MKP-1 expression is critical for cisplatin resistance. Specifically, we showed that in the human ovarian cancer cell lines, cisplatin induces MKP-1 through phosphorylation. We also showed that inhibition of ERK2 activity by the MEK1/2 inhibitor U0126 or by small interfering RNA silencing decreases MKP-1 induction, leading to an increase in cisplatin-induced cell death, which mimicked the results obtained with cells in which MKP-1 is down-regulated. Importantly, down-regulation of ERK2 decreased cisplatin-induced MKP-1 phosphorylation, suggesting that MKP-1 phosphorylation depends on ERK2 activity. Furthermore, down-regulation of ERK2 or MKP-1 enhanced cisplatin-induced apoptosis. In addition, we showed that down-regulation of ERK2 or MKP-1 decreases the basal level of Bcl-2 protein and that inhibition of Bcl-2 activity sensitizes ovarian cancer cells to cisplatin. Collectively, our results indicate that induction of MKP-1 by cisplatin is through phosphorylation involving ERK signaling and that MKP-1 plays a critical role in ERK-mediated cisplatin resistance. Thus, our results suggest that targeting ERK-MKP-1 signaling could overcome cisplatin resistance in human ovarian cancer.

Dual inactivation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects.

TIC10 is a small molecule that activates Foxo3a through dual inactivation of Akt and ERK, up-regulates the expression of the TRAIL gene, an endogenous tumor suppressor, and effectively improves the therapeutic properties and utility of TRAIL as an anticancer therapy. TIC’ing Up the TRAIL TRAIL is a naturally occurring tumor suppressor: It stimulates cell death pathways in a variety of human cancers and thus has been a popular target for the development of anticancer drugs. Previous TRAIL-targeting strategies include synthesis of the recombinant protein and stimulatory antibodies. All of these agents exhibit some of the typical drawbacks of protein-based therapeutics, such as short half-lives and a need to administer the drugs directly into the bloodstream or even into the tumor. Now, Allen and colleagues have discovered a drug, TIC10, which can stimulate production of TRAIL while avoiding the shortcomings of protein-based therapies. The authors demonstrated that TIC10 can increase TRAIL and stimulate the death of multiple types of human cancer cells both in culture and in mice. The drug was equally effective when given orally or intravenously and effectively penetrated the blood-brain barrier to target glioblastoma, a difficult-to-treat brain tumor. Whereas recombinant TRAIL displayed a short half-life of ~30 min, TIC10 activity persisted in the mice for days, allowing for once-a-week dosing. Toxicity analysis in mice showed no detectable adverse effects from treatment with TIC10. The authors also showed that TIC10 boosts TRAIL function through inactivation of the Akt and MEK signaling proteins, which results in translocation of the transcription factor Foxo3a into the cell nucleus, where it stimulates TRAIL gene expression. Before TIC10 can be used to treat patients, the drug will need to be tested in clinical trials to confirm safety and efficacy results from mouse studies. In addition, further work is needed to determine the mechanism by which TIC10 causes the dephosphorylation and resulting inactivation of Akt and MEK. However, the discovery of TIC10 clears a path to versatile TRAIL-based cancer therapies. Recombinant tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal–regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL.

Role of the Akt/mTOR survival pathway in cisplatin resistance in ovarian cancer cells.

The mechanism of cisplatin resistance in cancer cells is not fully understood. Here, we show that the Akt/mTOR survival pathway plays an important role in cisplatin resistance in human ovarian cancer cells. Specifically, we found that cisplatin treatment activates the Akt/mTOR survival pathway and that inhibition of this pathway by the PI3K inhibitor LY294002 or knockdown of Akt sensitizes ovarian cancer cells to cisplatin. Furthermore, we generated cisplatin-resistant cells and found that resistant cells express a higher level of activated Akt as compared to their cisplatin sensitive counterparts. Importantly, inhibition of Akt or mTOR sensitized resistant cells to cisplatin-induced apoptosis. Taken together, our data indicate that activation of the Akt/mTOR pathway prevents cisplatin-induced apoptosis, leading to cisplatin resistance. Therefore, our study suggests that cisplatin resistance can be overcome by targeting the Akt/mTOR survival pathway in human ovarian cancer cells.

Activation of the Akt Survival Pathway Contributes to TRAIL Resistance in Cancer Cells

The mechanism of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in cancer cells is not fully understood. Here, we show that the Akt survival pathway plays an important role in TRAIL resistance in human cancer cells. Specifically, we found that TRAIL treatment activates the Akt survival pathway and that inhibition of this pathway by the PI3K inhibitor LY294002 or knockdown of Akt sensitizes resistant cancer cells to TRAIL. Since Akt is negatively regulated by the tumor suppressor PTEN, we examined the TRAIL sensitivity in PTEN knockdown mouse prostate epithelial cells and found that PTEN−/− cells are more resistant than PTEN+/+ cells while the sensitivity of PTEN+/− cells fell in between. Further, we showed that overexpression of a mutant PTEN confers TRAIL resistance in PTEN+/+ cells, supporting a role of PTEN in TRAIL sensitivity. In TRAIL resistant breast T47D cells, overexpression of the mutant PTEN further increased their resistance to TRAIL. Taken together, our data indicate that inactivation of functional PTEN and the consequent activation of the Akt pathway prevents TRAIL-induced apoptosis, leading to TRAIL resistance. Therefore, our results suggest that TRAIL resistance can be overcome by targeting PTEN or the Akt survival pathway in cancer cells.

Involvement of MKP-1 and Bcl-2 in acquired cisplatin resistance in ovarian cancer cells

Although cisplatin is a very effective anticancer agent against several types of cancer including ovarian cancer, the mechanisms of acquired resistance are not fully understood. By chronically exposing cisplatin to ovarian cancer cell lines, we established two cisplatin-resistant cell lines OV433 and TOV112D. Our results indicate that the mechanisms underlying their cisplatin resistance are distinct. In OV433 cells, cisplatin resistance is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). By knocking down MKP-1 expression by siRNA or inhibiting MKP-1 expression by its pharmacological inhibitor triptolide, cisplatin-resistant OV433 cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. In TOV112D cells, on the other hand, acquired cisplatin resistance is associated with increased levels of Bcl-2 protein. By inhibiting the activity of Bcl-2 protein with its pharmacological inhibitor gossypol or knocking down Bcl-2 expression by siRNA, cisplatin-resistant TOV112D cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. Therefore, our data suggest that the mechanisms of acquired cisplatin resistance vary among ovarian cancer cells, which involve up-regulation of molecules associated with the cell survival pathways.

Bim Protein Degradation Contributes to Cisplatin Resistance

Cisplatin is the first-line chemotherapy for the treatment of several cancers. However, the development of cisplatin resistance represents a major clinical problem, and the mechanisms of acquired resistance are not fully understood. Here we show that degradation of the Bcl-2 homology 3-only proapoptotic protein Bim plays an important role in cisplatin resistance in ovarian cancer. Specifically, we show that treatment of ovarian cancer cells with cisplatin caused Bim phosphorylation and subsequent degradation and that its degradation is associated with cisplatin resistance. We also show that cisplatin treatment caused the activation of ERK, which correlated with Bim phosphorylation and degradation. By inhibiting ERK phosphorylation with the MEK inhibitor and knocking down ERK expression with siRNA, we show that Bim phosphorylation and degradation were blocked, which suggests that Bim is phosphorylated by ERK and that such phosphorylation is responsible for cisplatin-induced Bim degradation. We show that ERK was activated in cisplatin-resistant OV433 cells as compared with their counterpart parental OV433 cells. We also show that Bim was phosphorylated and degraded in cisplatin-resistant OV433 cells but not in the parental OV433 cells. Importantly, we show that inhibition of Bim degradation by the proteasome inhibitor MG132 sensitized resistant OV433 cells to cisplatin-induced death. Taken together, our data indicate that degradation of Bim via ERK-mediated phosphorylation can lead to cisplatin resistance. Therefore, these findings suggest that cisplatin resistance can be overcome by the combination of cisplatin and the proteasome inhibitors in ovarian cancer cells.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Is Required for Tumor Necrosis Factor α–Mediated Sensitization of Human Breast Cancer Cells to Chemotherapy

Tumor necrosis factor alpha (TNFalpha) induces apoptosis and sensitizes cancer cells to chemotherapy, but the mechanism underlying its sensitization is not fully understood. Here, we report that TNFalpha-mediated sensitization of cancer cells to chemotherapy involves activation of the TRAIL pathway. We show that the combined treatment of breast cancer cells with TNFalpha and Adriamycin significantly increases cell death compared with the treatment with either agent alone. The combined treatment activated both death receptor and mitochondrial apoptotic pathways, whereas Adriamycin alone activated only the mitochondrial pathway, and TNFalpha failed to activate either. Furthermore, we show that TNFalpha induces TRAIL through a transcriptional mechanism. Using reporter gene assays in conjunction with chromatin immunoprecipitation assays, we show that TRAIL induction by TNFalpha is regulated via both nuclear factor-kappaB and Sp1 binding sites. Importantly, down-regulation of TRAIL by small interfering RNA silencing decreased TNFalpha-mediated Adriamycin-induced caspase activation and apoptosis, and thus enhanced breast cancer cell resistance to Adriamycin. Collectively, our results suggest that induction of TRAIL by TNFalpha is critical for sensitization of breast cancer cells to chemotherapy.