Huong Muzik

Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas.

SummaryMatrix metalloproteinases (MMPs) have been implicated as important factors in gliomas since they may both facilitate invasion into the surrounding brain and participate in neovascularization. We have tested the hypothesis that deregulated expression of gelatinase-A or B, or an activator of gelatinase-A, MT1-MMP, may contribute directly to human gliomas by quantifying the expression of these MMPs in 46 brain tumour specimens and seven control tissues. Quantitative RT-PCR and gelatin zymography showed that gelatinase-A in glioma specimens was higher than in normal tissue; these were significantly elevated in low grade gliomas and remained elevated in GBMs. Gelatinase-B transcript and activity levels were also higher than in normal brain and more strongly correlated with tumour grade. We did not see a close relationship between the levels of expression of MT1-MMP mRNA and amounts of activated gelatinase-A. In situ hybridization localized gelatinase-A and MT1-MMP transcripts to normal neuronal and glia, malignant glioma cells and blood vessels. In contrast, gelatinase-B showed a more restricted pattern of expression; it was strongly expressed in blood vessels at proliferating margins, as well as tumour cells in some cases. These data suggest that gelatinase-A, -B and MT1-MMP are important in the pathophysiology of human gliomas. The primary role of gelatinase-B may lie in remodelling associated with neovascularization, whereas gelatinase-A and MT1-MMP may be involved in both glial invasion and angiogenesis.

Suppression of ING1 expression in sporadic breast cancer

Down regulation of the ING1 candidate tumour suppressor promotes growth in soft agar and focus formation in vitro and tumour formation in vivo. ING1 encodes a nuclear, cell cycle-regulated protein, overexpression of which efficiently blocks cell growth and is capable of inducing apoptosis in different experimental systems. Here we present the first report of ING1 mutation and expression analysis in a total of 452 cancer samples. One germline missense alteration and three germline silent alterations were detected in 377 primary breast cancers while marked (2 – 10-fold) decreases in ING1 mRNA expression were seen in 44% of primary breast cancers and in ten of ten breast cancer cell lines examined. Furthermore, the majority of breast cancers (58%) showing decreased ING1 expression had metastasized to regional lymph nodes whereas only 9% of cancers with elevated ING1 expression, compared to adjacent normal tissues, were metastatic. Thus, ING1 mutation is very rare in breast or ovarian cancers, however, repression of ING1 expression frequently accompanies tumour development of breast cancer.

Localization of gelatinase-A and gelatinase-B mRNA and protein in human gliomas

Malignant gliomas maintain a poor prognosis and survival rate due to their marked local invasive growth and neovascularization. Matrix metalloproteinases (MMPs) have been implicated in glioma invasion and angiogenesis, but it is unknown whether they are produced by the tumor cells or surrounding stroma. Using in situ hybridization and immunohistochemistry, we found expression of mRNA for both gelatinase-A (MMP2) and gelatinase-B (MMP9) localized to tumor cells and vascular structures in glioma sections. Gelatinase-A protein expression was detected most prominently in tumor cells, with very little signal seen in vasculature. Gelatinase-B protein expression was prominent in vascular structures but was also expressed in tumor cells. Our data show that these proteases are produced by glioma cells and vascular structures and suggest that synthetic MMP inhibitors might be useful in this disease.

Enhanced cytotoxicity of PARP inhibition in mantle cell lymphoma harbouring mutations in both ATM and p53

Poly‐ADP ribose polymerase (PARP) inhibitors have shown promise in the treatment of human malignancies characterized by deficiencies in the DNA damage repair proteins BRCA1 and BRCA2 and preclinical studies have demonstrated the potential effectiveness of PARP inhibitors in targeting ataxia‐telangiectasia mutated (ATM)‐deficient tumours. Here, we show that mantle cell lymphoma (MCL) cells deficient in both ATM and p53 are more sensitive to the PARP inhibitor olaparib than cells lacking ATM function alone. In ATM‐deficient MCL cells, olaparib induced DNA‐PK‐dependent phosphorylation and stabilization of p53 as well as expression of p53‐responsive cell cycle checkpoint regulators, and inhibition of DNA‐PK reduced the toxicity of olaparib in ATM‐deficient MCL cells. Thus, both DNA‐PK and p53 regulate the response of ATM‐deficient MCL cells to olaparib. In addition, small molecule inhibition of both ATM and PARP was cytotoxic in normal human fibroblasts with disruption of p53, implying that the combination of ATM and PARP inhibitors may have utility in targeting p53‐deficient malignancies.

Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas

Studies have suggested that an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may contribute to the malignant phenotype of gliomas. In this study, we have undertaken a detailed analysis of expression of the TIMP family in normal human brain and malignant gliomas at both the mRNA and protein level. Reverse transcription-PCR (RT-PCR) analyses of total RNA from surgical tumour specimens revealed unique expression patterns for the 4 members of the TIMP family, with TIMP-1 and -4 showing positive and negative correlations, respectively, with glioma malignancy. By RT-PCR, TIMP-2 and TIMP-3 expression did not change with tumour grade. In situ hybridization localized TIMP-1 to glial tumour cells and also to the surrounding tumour vasculature. TIMP-4 transcripts were predominantly localized to tumour cells, though minor expression was found in vessels. Recombinant TIMP-4 reduced invasion of U251 glioma cells through Matrigel, and U87 clones overexpressing TIMP-4 showed reduced invasive capacity in vitro. TIMP-4, but not TIMP-1, blocked Membrane Type-1-MMP-mediated progelatinase-A (MMP-2) activation in human umbilical vein endothelial cells. The differential expression and localization of individual TIMPs may contribute to the pathophysiology of human malignant gliomas, particularly with regard to tumour vascularization.

Efficacy and Safety Evaluation of Human Reovirus Type 3 in Immunocompetent Animals: Racine and Nonhuman Primates

Purpose: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. Experimental Design: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. Results: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus’ antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. Conclusions: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

CXCR4 Overexpression Is Associated with Poor Outcome in Females Diagnosed with Stage IV Non-small Cell Lung Cancer

Background: It has been proposed that the chemokine receptor, CXCR4, and its ligand, stromal cell-derived factor-1 (SDF-1), play a critical role in organ-specific tumor metastasis. High CXCR4 expression in resected non-small cell lung cancer (NSCLC) tumors is associated with poorer outcome; however, its effect on patient outcome in advanced NSCLC has not been explored. Methods: After institutional ethical approval was obtained, demographic details, clinical variables, and outcome data were collected on consecutive NSCLC patients diagnosed at the Tom Baker Cancer Centre from 2003 to 2006 (Glans-Look Lung Cancer Database). Formalin-fixed paraffin-embedded diagnostic biopsies from stage IV patients were obtained and tissue microarrays generated. CXCR4 expression within NSCLC cells was analyzed by quantitative fluorescent immunohistochemistry using the HistoRx PM-2000 platform and then correlated with clinical outcome. Results: Of 832 patients, 170 had samples suitable for tissue microarray generation and analysis. Automated immunohistochemistry for CXCR4 was successfully completed on all 170 patients. High expressors had a significantly poorer median overall survival of 2.7 months versus 5.6 months for the low expressors (p = 0.0468). This difference is driven by high-expressing females who have a median overall survival of 1.6 months versus 6.4 months for the low expressors (p = 0.006). Conclusions: CXCR4 is expressed in the majority of NSCLC tumors, and overexpression is associated with significantly poorer survival in stage IV NSCLC patients. Interestingly, this poor outcome is disproportionately represented in the female population. Our results suggest a gender-dependent difference in clinical outcome based on CXCR4 overexpression in stage IV NSCLC.

Reovirus as an experimental therapeutic for brain and leptomeningeal metastases from breast cancer.

Brain and leptomeningeal metastases are common in breast cancer patients and our current treatments are ineffective. Reovirus type 3 is a replication competent, naturally occurring virus that usurps the activated Ras-signaling pathway (or an element thereof) of tumor cells and lyses them but leaves normal cells relatively unaffected. In this study we evaluated reovirus as an experimental therapeutic in models of central nervous system (CNS) metastasis from breast cancer. We found all breast cancer cell lines tested were susceptible to reovirus, with >50% of these cells lysed within 72 h of infection. In vivo neurotoxicity studies showed only mild local inflammation at the injection site and mild communicating hydrocephalus with neither diffuse encephalitis nor behavioral abnormalities at the therapeutically effective dose of reovirus (intracranial) (ie 107 plaque-forming units) or one dose level higher. In vivo, a single intratumoral administration of reovirus significantly reduced the size of tumors established from two human breast cancer cell lines and significantly prolonged survival. Intrathecal administration of reovirus also remarkably prolonged survival in an immunocompetent racine model of leptomeningeal metastases. These data suggest that the evaluation of reovirus as an experimental therapeutic for CNS metastases from breast cancer is warranted.

Susceptibility of mantle cell lymphomas to reovirus oncolysis

Mantle cell lymphoma (MCL) an incurable B-cell, non-Hodgkin lymphoma (NHL) urgently requires new treatments. We assessed reovirus mediated oncolysis in a panel of human MCL cell lines. In vitro, we found the cytopathic effect of reovirus infection ranged from high to very limited and correlated with levels of Ras activation. In vivo, a single reovirus injection intra-tumorally resulted in complete regression of both the injected and the contra-lateral tumor in a subcutaneous bi-tumor model, in one out of three cell lines tested. Reovirus treatment of MCL seems feasible but will need to be guided by the presence of molecular determinants of reovirus susceptibility.