Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells.

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.

Serine phosphorylation of glutathione S-transferase P1 (GSTP1) by PKCα enhances GSTP1-dependent cisplatin metabolism and resistance in human glioma cells.

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 13.63µM to 36.49µM in MGR1 and from 20.75µM to 38.45µM 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.

Treatment of HER2-positive breast carcinomatous meningitis with intrathecal administration of alpha-particle-emitting (211)At-labeled trastuzumab.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Intrathecal (211)At-labeled trastuzumab shows promise as a treatment for patients with HER2-positive breast CM.

Tyrosine phosphorylation of the human glutathione S-transferase P1 by epidermal growth factor receptor.

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.

Treatment of intracerebral neoplasia and neoplastic meningitis with regional delivery of oncolytic recombinant poliovirus.

PURPOSE: 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). EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: Our findings suggest oncolytic PV recombinants as a viable treatment option for CNS complications of breast cancer.

Good clinical course in infants with desmoplastic cerebral neuroepithelial tumor treated by surgery alone.

We investigated why surgery alone provides for a benign clinical course in patients with desmoplastic infantile ganglioglioma and astrocytoma (DIG/A). The clinical course of 4, less than six-month-old girls, surgically treated at our institutions, was evaluated retrospectively. All presented with the clinical symptom of increasing head circumference. CT and MRI scans revealed a solid tumor attached to the dura that was surrounded by large, multiple cysts, in fronto-temporo-parietal lobe. Gross total removal succeeded in all 4 cases because the solid components of the tumor were very firm in contrast to the soft adjacent brain tissue. Microscopically, the surgical specimens consisted almost entirely of dense fibrous connective tissue containing generally elongate cells with inconspicuous cytoplasm. Most of these cells were immunopositive for GFAP. There was no evidence of tumor cells in the cyst wall. In 3 cases, some small neurons were positive for neurofilament immunostain. A high proportion of undifferentiated small cells in a less demoplastic area far from the dura were immunopositive for MIB-1. All of the 4 patients have been free of recurrence for more than five years. In patients with DIG/A, there are 5 reasons for a good clinical course. [1] At surgery, the tumor margin is clearly discernible because of the difference between the solid tumor and the soft adjacent brain tissue. [2] The tumor is located in the superficial cerebral hemisphere. [3] Large, multiple cysts surround the tumor. [4] The growth point appears to be adjacent to the cysts. [5] The cyst walls are free of invading tumor cells.

Myelodysplastic syndrome following therapy for brain tumor--two case reports.

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.

O(6)-methylguanine-DNA methyltransferase (MGMT) as a determinant of resistance to camptothecin derivatives.

The precise mechanisms of resistance to camptothecin (CPT)-derived DNA topoisomerase (topo I) inhibitors and the determinants remain unclear. We found that a DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), participated in resistance to irinotecan hydrochloride (CPT-11), its active metabolite SN-38, and a novel CPT derivative, DX-8951f. In 17 human cancer cell lines, MGMT gene expression level closely correlated with sensitivity to the CPT derivatives, and inhibition of MGMT activity by nontoxic 5 microM O(6)-benzylguanine augmented the drug activity in relation to the MGMT expression levels in 8 cell lines examined. Transfection of pCR / MGMT-sense into U-251MG and pCR / MGMT-antisense into T98G and HEC-46 cells revealed that increased MGMT expression decreased the sensitivity to CPT-11, SN-38, and DX-8951f, whereas repressed MGMT expression sensitized cells to the drugs. Western analysis revealed that treatment of MGMT-expressing T98G cells with the drugs caused a decrease of both MGMT and topo I in a dose-dependent manner. Although, in the transfectants, MGMT expression did not so closely correlate with the sensitivity to drugs as to nimustine hydrochloride (ACNU), MGMT is probably an important resistance determinant to CPT derivatives, and may play some role in the topo I-mediated DNA damage and / or the repair process.