Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with ...an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR.
Spread to the central nervous system (CNS) and the leptomeninges is a frequent complication of systemic cancers that is associated with serious morbidity and high mortality. We have evaluated a novel ...therapeutic approach against CNS complications of breast cancer based on the human neuropathogen poliovirus (PV).
Susceptibility to PV infection and ensuing rapid cell lysis is mediated by the cellular receptor of PV, CD155. We evaluated CD155 expression in several human breast tumor tissue specimens and cultured breast cancer cell lines. In addition, we tested an oncolytic PV recombinant for efficacy in xenotransplantation models of neoplastic meningitis and cerebral metastasis secondary to breast cancer.
We observed that breast cancer tissues and cell lines derived thereof express CD155 at levels mediating exquisite sensitivity toward PV-induced oncolysis in the latter. An association with the immunoglobulin superfamily molecule CD155 renders breast cancer a likely target for oncolytic PV recombinants. This assumption was confirmed in xenotransplantation models for neoplastic meningitis or solitary cerebral metastasis, where local virus treatment dramatically improved survival.
Our findings suggest oncolytic PV recombinants as a viable treatment option for CNS complications of breast cancer.
Recently, we reported that the human GSTP1 is phosphorylated and functionally activated by the PKC class of serine/threonine kinases. In this study, we investigated the contribution of this ...post-translational modification of GSTP1 to tumor cisplatin resistance. Using two malignant glioma cell lines, MGR1 and MGR3, the ability of PKCα-phosphorylated GSTP1 to catalyze the conjugation of cisplatin to glutathione was assessed and correlated with cisplatin sensitivity and cisplatin-induced DNA interstrand cross-links and apoptosis of the cells. The results showed PKCα activation and associated phosphorylation of GSTP1 to correlate significantly with increased glutathionylplatinum formation, decreased DNA interstrand cross-link formation and increased cisplatin resistance. Following PKC activation, the IC
50 of cisplatin increased from 13.63
μM to 36.49
μM in MGR1 and from 20.75
μM to 38.45
μM in MGR3. In both cell lines, siRNA-mediated GSTP1 or PKCα transcriptional suppression similarly decreased cisplatin IC
50 and was associated with decreased intracellular levels of glutathionylplatinum metabolite. Combined inhibition/transcriptional suppression of both PKCα and GSTP1 was synergistic in enhancing cisplatin sensitivity. Although, cisplatin-induced apoptosis was associated with the translocation of Bax to mitochondria, release of cytochrome
c and caspase-3/7 activation, the levels of relocalized Bax and cytochrome
c were significantly greater following GSTP1 knockdown. These results support a mechanism of cisplatin resistance mediated by the PKCα-dependent serine phosphorylation of GSTP1 and its associated increased cisplatin metabolism, and suggest the potential of simultaneous targeting of GSTP1 and PKCα to improve the efficacy of cisplatin therapy.
Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, ...GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.
Background: GSTP1 is a downstream EGFR phosphorylation target.
Results: EGFR-dependent C-terminal Tyr-198 phosphorylation shifts GSTP1 to the monomeric state, facilitates JNK binding and inhibition, and suppresses apoptosis in brain tumor cells.
Conclusion: Enhanced suppression of JNK signaling by EGFR-phosphorylated GSTP1 provides a survival advantage for tumors.
Significance: The GSTP1-EGFR cross-talk is a mechanism of tumor cell survival and drug resistance.
Recently, we reported that the human GSTP1 is phosphorylated and functionally activated by the PKC class of serine/threonine kinases. In this study, we investigated the contribution of this ...post-translational modification of GSTP1 to tumor cisplatin resistance. Using two malignant glioma cell lines, MGR1 and MGR3, the ability of PKCa-phosphorylated GSTP1 to catalyze the conjugation of cisplatin to glutathione was assessed and correlated with cisplatin sensitivity and cisplatin-induced DNA interstrand cross-links and apoptosis of the cells. The results showed PKCa activation and associated phosphorylation of GSTP1 to correlate significantly with increased glutathionylplatinum formation, decreased DNA interstrand cross-link formation and increased cisplatin resistance. Following PKC activation, the IC sub(50) of cisplatin increased from 13.63 kM to 36.49 kM in MGR1 and from 20.75 kM to 38.45 kM in MGR3. In both cell lines, siRNA-mediated GSTP1 or PKCa transcriptional suppression similarly decreased cisplatin IC sub(50) and was associated with decreased intracellular levels of glutathionylplatinum metabolite. Combined inhibition/transcriptional suppression of both PKCa and GSTP1 was synergistic in enhancing cisplatin sensitivity. Although, cisplatin-induced apoptosis was associated with the translocation of Bax to mitochondria, release of cytochrome c and caspase-3/7 activation, the levels of relocalized Bax and cytochrome c were significantly greater following GSTP1 knockdown. These results support a mechanism of cisplatin resistance mediated by the PKCa-dependent serine phosphorylation of GSTP1 and its associated increased cisplatin metabolism, and suggest the potential of simultaneous targeting of GSTP1 and PKCa to improve the efficacy of cisplatin therapy.
Carcinomatous meningitis (CM) is a devastating disease characterized by the dissemination of malignant tumor cells into the subarachnoid space along the brain and spine. Systemic treatment with ...monoclonal antibody (mAb) trastuzumab can be effective against HER2-positive systemic breast carcinoma but, like other therapies, is ineffective against CM. The goal of this study was to evaluate the therapeutic effect of alpha-particle emitting (211)At-labeled trastuzumab following intrathecal administration in a rat model of breast carcinoma CM.
Athymic rats were injected intrathecally with MCF-7/HER2-18 breast carcinoma cells through a surgically implanted indwelling intrathecal catheter. In Experiment 1, animals received 33 or 66 muCi (211)At-labeled trastuzumab, cold trastuzumab or saline. In Experiment 2, animals were inoculated with a lower tumor burden and received 46 or 92 muCi (211)At-labeled trastuzumab or saline. In Experiment 3, animals received 28 muCi (211)At-labeled trastuzumab, 30 muCi (211)At-labeled TPS3.2 control mAb or saline. Histopathological analysis of the neuroaxis was performed at the end of the study.
In Experiment 1, median survival increased from 21 days for the saline and cold trastuzumab groups to 45 and 48 days for 33 and 66 muCi (211)At-labeled trastuzumab, respectively. In Experiment 2, median survival increased from 23 days for saline controls to 68 and 92 days for 46 and 92 muCi (211)At-labeled trastuzumab, respectively. In Experiment 3, median survival increased from 20 days to 29 and 36 days for animals treated with (211)At-labeled TPS3.2 and (211)At-labeled trastuzumab, respectively. Long-term survivors were observed exclusively in the (211)At-trastuzumab-treated groups.
Intrathecal (211)At-labeled trastuzumab shows promise as a treatment for patients with HER2-positive breast CM.
A 3-month-old boy and a 29-year-old woman presented with myelodysplastic syndrome (MDS) following therapy for primary malignant brain tumor. Both received intensive alkylating agent doses for ...induction and maintenance chemotherapy combined with craniospinal or cranial radiation for medulloblastoma and anaplastic astrocytoma, respectively. They developed refractory anemia and pancytopenia. Approximately 9 years after the completion of induction chemoradiotherapy, chromosomal analysis of bone marrow cells resulted in the diagnosis of MDS. The boy died of leukemic evolution 15 months later, the woman died of hematopoietic failure 3 months later. The most common symptom of MDS is refractory anemia, either alone or as part of bi- or pancytopenia. Clonal proliferation with chromosomal analysis of bone marrow cells establishes the diagnosis of MDS. Patients with malignant brain tumors are at risk of the development of MDS as a late complication of chemotherapy based on high cumulative doses of alkylating agents.
A network composed of activation and inactivation pathways to regulate mitomycin C (MMC) action is suggested to exist in human cancer cells. COLO201 colon cancer cells were stably transfected with ...human NQO1 cDNA that encodes NAD(P)H: quinone oxidoreductase (DT‐diaphorase, DTD), and a clonal cell line with about 57‐fold elevated DTD activity was obtained. Northern analysis revealed that expression of the NADPH:cytochrome P450 reductase (P450 reductase) gene was decreased in the transfectant, COLO201/NQO1, associated with the increase of NQO1 expression. Biochemical characterization of the cells showed a significant increase of the glutathione (GSH) content concomitantly with the decrease of the P450 reductase activity. As a result of these coordinated modulations, sensitivity of COLO201/NQO1 to MMC was not increased as compared to the parent cells. Analyses of inhibition by specific inhibitors of DTD, P450 reductase and glutathione S‐transferase (GST) in 5 human colon cancer cell lines including the transfectant showed that DTD and P450 reductase play significant roles in MMC activation in cells with sufficiently high DTD activity and with marginal DTD activity, respectively. In contrast, GST appeared to participate in MMC inactivation in cells with a high level of GST activity. These results indicated that DTD, P450 reductase, GSH and GST may act together compensatively or competitively, depending on their levels in cells, to determine the cellular sensitivity to MMC.
Carcinomatous meningitis (CM) is a devastating disease characterized by the dissemination of malignant tumor cells into the subarachnoid space along the brain and spine. Systemic treatment with ...monoclonal antibody (mAb) trastuzumab can be effective against HER2-positive systemic breast carcinoma but, like other therapies, is ineffective against CM. The goal of this study was to evaluate the therapeutic effect of α-particle emitting
211At-labeled trastuzumab following intrathecal administration in a rat model of breast carcinoma CM.
Athymic rats were injected intrathecally with MCF-7/HER2-18 breast carcinoma cells through a surgically implanted indwelling intrathecal catheter. In Experiment 1, animals received 33 or 66 μCi
211At-labeled trastuzumab, cold trastuzumab or saline. In Experiment 2, animals were inoculated with a lower tumor burden and received 46 or 92 μCi
211At-labeled trastuzumab or saline. In Experiment 3, animals received 28 μCi
211At-labeled trastuzumab, 30 μCi
211At-labeled TPS3.2 control mAb or saline. Histopathological analysis of the neuroaxis was performed at the end of the study.
In Experiment 1, median survival increased from 21 days for the saline and cold trastuzumab groups to 45 and 48 days for 33 and 66 μCi
211At-labeled trastuzumab, respectively. In Experiment 2, median survival increased from 23 days for saline controls to 68 and 92 days for 46 and 92 μCi
211At-labeled trastuzumab, respectively. In Experiment 3, median survival increased from 20 days to 29 and 36 days for animals treated with
211At-labeled TPS3.2 and
211At-labeled trastuzumab, respectively. Long-term survivors were observed exclusively in the
211At-trastuzumab-treated groups.
Intrathecal
211At-labeled trastuzumab shows promise as a treatment for patients with HER2-positive breast CM.
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