Mohammad T. Malik

Regulation of angiogenesis and invasion by human Pituitary tumor transforming gene (PTTG) through increased expression and secretion of matrix metalloproteinase-2 (MMP-2)

BackgroundPituitary tumor transforming gene (PTTG) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Existence of a relationship between PTTG levels and tumor angiogenesis and metastasis has been reported. However, the mechanisms by which PTTG achieve these functions remain unknown. In the present study, we investigated the effect of overexpression of PTTG on secretion and expression of metastasis-related metalloproteinase-2 (MMP-2) in HEK293 cells, cell migration, invasion and tubule formation.ResultsTransient or stable transfection of HEK293 cells with PTTG cDNA showed a significant increase in secretion and expression of MMP-2 measured by zymography, reverse transcriptase (RT/PCR), ELISA, and MMP-2 gene promoter activity. Furthermore, in our studies, we showed that tumor developed in nude mice on injection of HEK293 cells that constitutively express PTTG expressed high levels of both MMP-2 mRNA and protein, and MMP-2 activity. Conditioned medium collected from the HEK293 cells overexpressing PTTG showed a significant increase in cell migration, invasion and tubule formation of human umbilical vein endothelial cells (HUVEC). Pretreatment of conditioned medium with MMP-2-specific antibody significantly decreased these effects, suggesting that PTTG may contribute to tumor angiogenesis and metastasis via activation of proteolysis and increase in invasion through modulation of MMP-2 activity and expression.ConclusionOur results provide novel information that PTTG contributes to cell migration, invasion and angiogenesis by induction of MMP-2 secretion and expression. Furthermore, we showed that tumors developed in nude mice on injection of HEK293 cells that constitutively express PTTG induce expression of MMP-2 and significantly increase its functional activity, suggesting a relationship between PTTG levels and MMP-2 which may play a critical role in regulation of tumor growth, angiogenesis and metastasis. Blocking of function of PTTG or down regulation of its expression in tumors may result in suppression of tumor growth and metastasis, through the down regulation of MMP-2 expression and activity. To our knowledge, this study is the first study demonstrating the modulation of MMP-2 expression and biological activity by PTTG.

Plasma membrane Ca2+-ATPase associates with CLP36, α-actinin and actin in human platelets

The plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca2+ in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts via its C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the last ten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pump to the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitation assays coupled with liquid chromatography/tandem mass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing protein associated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extract using a fusion protein containing the C-terminal PDZ domain binding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized platelets demonstrated the existence of a 1,000-kDa complex containing PMCA and CLP36, and in addition, α-actinin and actin. Immunoflourescence microscopy confirmed the co-localization of PMCA with CLP36 in resting and activated platelets.Taken together these results suggest that PMCA is localized in non-filamentous actin complexes in resting platelets by means of PDZ domain interactions and then associates with the actin cytoskeleton during cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine and tyrosine phosphorylation events previously described in human platelets, PMCA function may be regulated by interactions with anchoring and cytoskeletal proteins.

Gold Nanoplates as Cancer-Targeted Photothermal Actuators for Drug Delivery and Triggered Release

The selective exposure of cancerous tissue to systemically delivered chemotherapeutic agents remains a major challenge facing cancer therapy. To address this question, a near infrared responsive oligonucleotide-coated AS1411, hairpin, or both gold nanoplate loaded with doxorubicin is demonstrated to be nontoxic to cells without triggered release, while being acutely toxic to cells after 5 minutes of laser exposure to trigger DOX release. Conjugation of oligonucleotides to the nanoplates is confirmed by an average increase in hydrodynamic diameter of 30.6 nm, an average blue shift of the plasmon resonance peak by 36 nm, and an average −10 mV shift in zeta potential of the particles. DOX loading through intercalation into the hairpin DNA structure is confirmed through fluorescence measurements. For both GNP-Hairpin and GNP-Hairpin-AS1411, ~60% of loaded DOX is released after the first 5 minutes of laser exposure λ=817 nm, with complete release after two more 5-minute exposures. Preliminary proof of concept is demonstrated in vitro using A549 and MDA-MB-231 cell lines as models for breast and lung cancer, respectively. Exposure of cells to untriggered DOX-loaded conjugate with no laser exposure results in little to no toxicity, while laser-triggered release of DOX causes significant cell death.

Development of cystic glandular hyperplasia of the endometrium in Mullerian inhibitory substance type II receptor–pituitary tumor transforming gene transgenic mice

The pituitary tumor transforming gene (PTTG)/securin is an oncogene that is involved in cell cycle regulation and sister chromatid separation. PTTG is highly expressed in various tumors including ovarian tumors, suggesting that PTTG may play a role in ovarian tumorigenesis. Overexpression of PTTG resulted in induction of cellular transformation in vitro and tumor formation in nude mice. To ascertain PTTG function in ovarian tumorigenesis, we generated a transgenic mouse model of PTTG by cloning PTTG cDNA downstream of Mullerian inhibitory substance type II receptor gene promoter (MISIIR) in order to target the ovarian surface epithelium. By screening of transgenic animals, we identified five founders (four males and one female). Using the four male founders, we developed four transgenic lines. PTTG expression was increased in ovarian surface epithelium, ovarian granulosa cells, as well as in the pituitary gland. Transgenic females did not develop any visible ovarian tumors at 8-10 months of age; however, there was an overall increase in the corpus luteum mass in transgenic ovary, suggesting increased luteinization. These changes were associated with an increase in serum LH and testosterone levels. In addition, there was a generalized hypertrophy of the myometrium of MISIIR-PTTG transgenic uteri with cystic glandular and hyperplasia of the endometrium. Based on these results, we conclude that the overexpression of PTTG may be required to initiate precancerous conditions but is not sufficient to induce ovarian tumorigenesis and may require another partner to initiate cellular transformation.

Effects of plasma membrane Ca2+‐ATPase tyrosine phosphorylation on human platelet function

Summary.  Background: The plasma membrane Ca2+‐ATPase (PMCA) plays an essential role in maintaining low intracellular Ca2+ ([Ca2+]i) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity. Objectives: The objective was to determine the functional consequences of PMCA tyrosine phosphorylation. Methods: A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura‐2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation. Results: Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin‐permeabilized, thrombin‐treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca2+]i during thrombin‐mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca2+]i was a significant delay in the onset of thrombin‐mediated platelet aggregation. Conclusions: The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca2+]i during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca2+]i accompanying platelet activation.

Abstract 5688: Multifunctional gold nanoparticles linked with aptamers and fluorophores for breast cancer imaging and therapy

Breast cancer is a leading cause of mortality among women worldwide and there is a need for improved methods for early detection, new therapies that are more effective with fewer side effects, and personalized treatment options. Nucleic acid aptamers are synthetic oligonucleotides that bind to specific target proteins and have potential for both imaging and therapy. They have a targeting mechanism similar to monoclonal antibodies (i.e. shape-specific recognition), but may have substantial advantages, including easier synthesis and storage, better tumor penetration, and non-immunogenicity. Previously, we developed AS1411 (now renamed ACT-GRO-777), a nucleolin-binding DNA aptamer that has antiproliferative activity against cancer cells with little effect on non-malignant cells. AS1411 was the first anticancer aptamer to be tested in human clinical trials, which indicated promising activity with no evidence of serious side effects. The molecular target for AS1411 is nucleolin, a protein highly expressed on the surface of breast cancer cells and breast tumor-associated endothelial cells, suggesting the potential utility of AS1411 in this disease. Here we report on new research in which we conjugated gold nanoparticles (GNPs) to AS1411 and assessed their potential for breast cancer imaging and therapy. The antiproliferative activity of AS1411 linked to 5 nm gold nanoparticles (GNP) was determined by MTT and clonogenic assays. The GI50 value for AS1411-GNPs against breast cancer cell lines was less than 100 nM (aptamer concentration), which is at least 20-fold lower than unconjugated AS1411. Moreover, AS1411-GNPs retained the cancer-selectivity of AS1411 and had no effect on non-malignant breast cells. Confocal microscopy revealed increased uptake in breast cancer cells for GNP-AS1411 compared to GNP alone or GNP conjugated to a control oligonucleotide. In addition, GNP-AS1411 potently induced breast cancer cell vacuolization and death, similar to that seen at higher concentrations of AS1411. Furthermore, in vivo studies in nude mice with established MDA-MB-231 xenografts have shown that systemic administration of AS1411-GNPs for 12 days could completely inhibit tumor growth in a specific manner. We further examined if the GNP-AS1411 conjugated to fluorophore Cy5 has a potential for in-vivo optical imaging to specifically detect the tumor in established breast cancer xenograft model. Preliminary data from biodistribution studies suggest that GNP-AS1411 can very efficiently detect tumors using in vivo imaging modalities. Altogether, our data suggest that this strategy could be used to develop multifunctional tumor-targeting nanoparticles that can serve as imaging agents to detect breast cancers or monitor clinical response, as well as to specifically deliver therapeutic agents to the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5688. doi:1538-7445.AM2012-5688

Abstract 5447: A novel inhibitor of DNA methylation

We previously reported the discovery of XB05, a synthetic small molecule with antiproliferative activity. XB05 was found to be a strong inhibitor of DNMT1 activity in cell-free and cell-based assays. However, screening in the NCI 60 tumor cell lines and analysis using COMPARE indicated a novel mechanism of action for XB05, different from standard demethylating agents such as 5-azacytidine (azaC) and decitabine. Here, we report on new research to further characterize XB05 activity. We have examined the effects of XB05 on clonogenicity, promoter methylation, gene expression, and DNMT1 protein. XB05 was found to inhibit colony formation of HCT116 colon carcinoma cells in a dose-dependent manner with a sub-micromolar IC 50 . Western blots of nuclear extracts from HCT116 cells treated for 72 h with 100 nM XB05 or 10 µM azaC (as positive control) showed greatly decreased levels of DNMT1, suggesting that binding to XB05 can induce degradation of DNMT1 (previous research indicates direct drug-enzyme binding). Analysis of CDKN2A promoter methylation by bisulfite modification and sequencing demonstrated heavy methylation in untreated HCT116 cells. This was reversed by treatment of with 100 nM XB05 or 10 µM azaC, resulting in re-expression of the gene (which encodes tumor suppressor, p16), as shown by qRT-PCR. Experiments to assess promoter methylation using multiplex arrays and to examine gene expression changes using DNA microarrays were recently completed and data analysis is ongoing. To evaluate in vivo activity, we treated mice bearing subcutaneous HCT116 xenografts by IP injection of XB05 with multiple doses of up to 10 mg/kg. There was no evidence of toxicity as judged by body weights and gross necropsies. The tumors of mice treated with XB05, although not significantly smaller than in control animals, were characterized by extensive necrosis in the center of the tumor, leading to a “hollow” tumor in the majority (12/20) of mice. No similar necrosis was observed in vehicle-treated or azaC-treated mice, suggesting that this effect is treatment related. Because induced necrosis is seen with vascular disrupting agents, we also examined the effect of XB05 on endothelial cells in an in vitro assay, and observed reproducible inhibition of endothelial tube formation at 400 nM or higher. In summary, our new data confirm that XB05 is a potent inhibitor of DNA methylation in cultured cancer cells, and provide the first evidence of in vivo activity, which may involve effects on the tumor vasculature, as well as the tumor itself. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5447.