Natsuko Nomura

Prostate specific membrane antigen (PSMA) expression in primary gliomas and breast cancer brain metastases

BackgroundPrimary and secondary brain cancers are highly treatment resistant, and their marked angiogenesis attracts interest as a potential therapeutic target. Recent observations reveal that the microvascular endothelium of primary high-grade gliomas expresses prostate specific membrane antigen (PSMA). Breast cancers express PSMA and they frequently form secondary brain tumors. Hence we report here our pilot study addressing the feasibility of PSMA targeting in brain and metastatic breast tumors, by examining PSMA levels in all glioma grades (19 patients) and in breast cancer brain metastases (5 patients).MethodsTumor specimens were acquired from archival material and normal brain tissues from autopsies. Tissue were stained and probed for PSMA, and the expression levels imaged and quantified using automated hardware and software. PSMA staining intensities of glioma subtypes, breast tumors, and breast tumor brain metastases were compared statistically versus normals.ResultsNormal brain microvessels (4 autopsies) did not stain for PSMA, while a small proportion (<5%) of healthy neurons stained, and were surrounded by an intact blood brain barrier. Tumor microvessels of the highly angiogenic grade IV gliomas showed intense PSMA staining which varied between patients and was significantly higher (p < 0.05) than normal brain. Grade I gliomas showed moderate vessel staining, while grade II and III gliomas had no vessel staining, but a few (<2%) of the tumor cells stained. Both primary breast cancer tissues and the associated brain metastases exhibited vascular PSMA staining, although the intensity of staining was generally less for the metastatic lesions.ConclusionsOur results align with and extend previous data showing PSMA expression in blood vessels of gliomas and breast cancer brain metastases. These results provide a rationale for more comprehensive studies to explore PSMA targeted agents for treating secondary brain tumors with PSMA expressing vasculature. Moreover, given that PSMA participates in angiogenesis, cell signaling, tumor survival, and invasion, characterizing its expression may help guide later investigations of the poorly understood process of low grade glioma progression to glioblastoma.

Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs

BackgroundGlioblastoma (GBM) is a therapeutic challenge, associated with high mortality. More effective GBM therapeutic options are urgently needed. Hence, we screened a large multi-class drug panel comprising the NIH clinical collection (NCC) that includes 446 FDA-approved drugs, with the goal of identifying new GBM therapeutics for rapid entry into clinical trials for GBM.MethodsScreens using human GBM cell lines revealed 22 drugs with potent anti-GBM activity, including serotonergic blockers, cholesterol-lowering agents (statins), antineoplastics, anti-infective, anti-inflammatories, and hormonal modulators. We tested the 8 most potent drugs using patient-derived GBM cancer stem cell-like lines. Notably, the statins were active in vitro; they inhibited GBM cell proliferation and induced cellular autophagy. Moreover, the statins enhanced, by 40-70 fold, the pro-apoptotic activity of irinotecan, a topoisomerase 1 inhibitor currently used to treat a variety of cancers including GBM. Our data suggest that the mechanism of action of statins was prevention of multi-drug resistance protein MDR-1 glycosylation. This drug combination was synergistic in inhibiting tumor growth in vivo. Compared to animals treated with high dose irinotecan, the drug combination showed significantly less toxicity.ResultsOur data identifies a novel combination from among FDA-approved drugs. In addition, this combination is safer and well tolerated compared to single agent irinotecan.ConclusionsOur study newly identifies several FDA-approved compounds that may potentially be useful in GBM treatment. Our findings provide the basis for the rational combination of statins and topoisomerase inhibitors in GBM.

High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile

Staurosporine (STS) is a potent pan-kinase inhibitor with marked activity against several chemotherapy-resistant tumor types in vitro. The translational progress of this compound has been hindered by poor pharmacokinetics and toxicity. We sought to determine whether liposomal encapsulation of STS would enhance antitumor efficacy and reduce toxicity, thereby supporting the feasibility of further preclinical development. We developed a novel reverse pH gradient liposomal loading method for STS, with an optimal buffer type and drug-to-lipid ratio. Our approach produced 70% loading efficiency with good retention, and we provide, for the first time, an assessment of the in vivo antitumor activity of STS. A low intravenous dose (0.8 mg/kg) inhibited U87 tumors in a murine flank model. Biodistribution showed preferential tumor accumulation, and body weight data, a sensitive index of STS toxicity, was unaffected by liposomal STS, but did decline with the free compound. In vitro experiments revealed that liposomal STS blocked Akt phosphorylation, induced poly(ADP-ribose) polymerase cleavage, and produced cell death via apoptosis. This study provides a basis to explore further the feasibility of liposomally encapsulated STS, and potentially related compounds for the management of resistant solid tumors.

In silico modeling predicts drug sensitivity of patient-derived cancer cells

BackgroundGlioblastoma (GBM) is an aggressive disease associated with poor survival. It is essential to account for the complexity of GBM biology to improve diagnostic and therapeutic strategies. This complexity is best represented by the increasing amounts of profiling (“omics”) data available due to advances in biotechnology. The challenge of integrating these vast genomic and proteomic data can be addressed by a comprehensive systems modeling approach.MethodsHere, we present an in silico model, where we simulate GBM tumor cells using genomic profiling data. We use this in silico tumor model to predict responses of cancer cells to targeted drugs. Initially, we probed the results from a recent hypothesis-independent, empirical study by Garnett and co-workers that analyzed the sensitivity of hundreds of profiled cancer cell lines to 130 different anticancer agents. We then used the tumor model to predict sensitivity of patient-derived GBM cell lines to different targeted therapeutic agents.ResultsAmong the drug-mutation associations reported in the Garnett study, our in silico model accurately predicted ~85% of the associations. While testing the model in a prospective manner using simulations of patient-derived GBM cell lines, we compared our simulation predictions with experimental data using the same cells in vitro. This analysis yielded a ~75% agreement of in silico drug sensitivity with in vitro experimental findings.ConclusionsThese results demonstrate a strong predictability of our simulation approach using the in silico tumor model presented here. Our ultimate goal is to use this model to stratify patients for clinical trials. By accurately predicting responses of cancer cells to targeted agents a priori, this in silico tumor model provides an innovative approach to personalizing therapy and promises to improve clinical management of cancer.

Demonstration of Non-Gaussian Restricted Diffusion in Tumor Cells Using Diffusion Time-Dependent Diffusion-Weighted Magnetic Resonance Imaging Contrast

The diffusion-weighted magnetic resonance imaging (DWI) technique enables quantification of water mobility for probing microstructural properties of biological tissue and has become an effective tool for collecting information about the underlying pathology of cancerous tissue. Measurements using multiple b-values have indicated biexponential signal attenuation, ascribed to “fast” (high ADC) and “slow” (low ADC) diffusion components. In this empirical study, we investigate the properties of the diffusion time (Δ)-dependent components of the diffusion-weighted (DW) signal in a constant b-value experiment. A xenograft gliobastoma mouse was imaged using Δ = 11 ms, 20 ms, 40 ms, 60 ms, and b = 500–4000 s/mm2 in intervals of 500 s/mm2. Data were corrected for EPI distortions, and the Δ-dependence on the DW-signal was measured within three regions of interest [intermediate- and high-density tumor regions and normal-appearing brain (NAB) tissue regions]. In this study, we verify the assumption that the slow decaying component of the DW-signal is non-Gaussian and dependent on Δ, consistent with restricted diffusion of the intracellular space. As the DW-signal is a function of Δ and is specific to restricted diffusion, manipulating Δ at constant b-value (cb) provides a complementary and direct approach for separating the restricted from the hindered diffusion component. We found that Δ-dependence is specific to the tumor tissue signal. Based on an extended biexponential model, we verified the interpretation of the diffusion time-dependent contrast and successfully estimated the intracellular restricted ADC, signal volume fraction, and cell size within each ROI.

Hyaluronan expression in primary and secondary brain tumors

BACKGROUND Collectively, primary and secondary brain tumors represent a major public health challenge. Glioblastoma (GBM) is the most common primary brain tumor in adults and is associated with a dismal 5-year survival of only 10%. Breast cancer causes secondary tumors; it occurs in 200,000 patients yearly and 30% of these individuals develop brain metastases which also lead to a very poor prognosis. GBM and primary breast tumors are known to express hyaluronan (HA) which may serve as a therapeutic target. METHODS For the present study we had two aims: (I) to identify suitable preclinical models for HA in GBM by examining HA expression in human GBM cell lines implanted orthotopically in mice; and (II) to determine whether breast cancer brain metastases in human patients express HA similar to the primary tumor. Forty human surgical samples of primary breast tumors and breast cancer brain metastases were processed and stained for HA. Athymic nu/nu mice were orthotopically implanted with one of 15 GBM lines and after tumors were established, quantitative immunohistochemistry determined whether. RESULTS HA was expressed. All GBM cell lines and patient-derived orthotopic tumors did express HA, with 3 primary human lines expressing the highest staining intensity, above that of normal brain. All 40 human primary breast tumors and brain metastases examined also contained HA, though staining intensity was highly variable. CONCLUSIONS Our data support the use of specific patient-derived GBM cell lines in nu/nu mice for preclinical studies on HA-targeting therapies. Additionally, our research provides a basis for the assessment of HA expression and HA-targeting therapeutic agents for the treatment of breast cancer brain metastases.

Anti-cancer effects of nitrogen-containing bisphosphonates on human cancer cells

Zoledronic acid, a potent nitrogen-containing bisphosphonate (NBP), has been extensively used to limit bone turnover in a various diseases including tumors. Recent clinical studies have demonstrated direct anti-cancer effects of zoledronic acid, in addition to its clinical benefits for skeletal-related events. Here we investigated the effects of 4 clinically available NBPs on human tumor cell proliferation. Our data demonstrate a potent anti-proliferative effect of zoledronic acid against glioblastoma (GBM) cell lines, breast cancer cells and GBM patient-derived lines. Zoledronic acid also effectively inhibited GBM tumor growth in xenograft mouse models. Zoledronic acid strongly stimulated autophagy but not apoptotic signals in all tested cells. Only one intermediate product of cholesterols synthesis pathway, geranylgeranyl diphosphate (GGPP) rescued cells from the cytotoxic effects of zoledronic acid. To further investigate the effect of GGPP, we knocked down RABGGTA, which encodes a subunit of the Rabgeranylgeranyltransferase protein. This knockdown induced an effect similar to zoledronic acid in cancer cell lines. These data are promising and suggested a potential for zoledronic acid as an anti-cancer agent, through its ablation of the function of Rab proteins.

Pritumumab, the first therapeutic antibody for glioma patients

Immunotherapy is now at the forefront of cancer therapeutic development. Gliomas are a particularly aggressive form of brain cancer for which immunotherapy may hold promise. Pritumumab (also known in the literature as CLNH11, CLN-IgG, and ACA-11) was the first monoclonal antibody tested in cancer patients. Pritumumab is a natural human monoclonal antibody developed from a B lymphocyte isolated from a regional draining lymph node of a patient with cervical carcinoma. The antibody binds ecto-domain vimentin on the surface of cancer cells. Pritumumab was originally tested in clinical trials with brain cancer patients in Japan where it demonstrated therapeutic benefit. It was reported to be a safe and effective therapy for brain cancer patients at doses 5-10 fold less than currently approved antibodies. Phase I dose escalation clinical trials are now being planned with pritumumab for the near future. Here we review data on the development and characterization of pritumumab, and review clinical trails data assessing immunotherapeutic effects of pritumumab for glioma patients.

A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma

BackgroundThe mitochondrial protein p32 is a validated therapeutic target of cancer overexpressed in glioma. Therapeutic targeting of p32 with monoclonal antibody or p32-binding LyP-1 tumor-homing peptide can limit tumor growth. However, these agents do not specifically target mitochondrial-localized p32 and would not readily cross the blood–brain barrier to target p32-overexpressing gliomas. Identifying small molecule inhibitors of p32 overexpressed in cancer is a more rational therapeutic strategy. Thus, in this study we employed a pharmacophore modeling strategy to identify small molecules that could bind and inhibit mitochondrial p32.MethodsA pharmacophore model of C1q and LyP-1 peptide association with p32 was used to screen a virtual compound library. A primary screening assay for inhibitors of p32 was developed to identify compounds that could rescue p32-dependent glutamine-addicted glioma cells from glutamine withdrawal. Inhibitors from this screen were analyzed for direct binding to p32 by fluorescence polarization assay and protein thermal shift. Affect of the p32 inhibitor on glioma cell proliferation was assessed by Alamar Blue assay, and affect on metabolism was examined by measuring lactate secretion.ResultsIdentification of a hit compound (M36) validates the pharmacophore model. M36 binds directly to p32 and inhibits LyP-1 tumor homing peptide association with p32 in vitro. M36 effectively inhibits the growth of p32 overexpressing glioma cells, and sensitizes the cells to glucose depletion.ConclusionsThis study demonstrates a novel screening strategy to identify potential inhibitors of mitochondrial p32 protein overexpressed in glioma. High throughput screening employing this strategy has potential to identify highly selective, potent, brain-penetrant small molecules amenable for further drug development.

Abstract 5611: Use of ecto-domain vimentin to target brain cancers

Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. For brain cancers, immunohistological analysis showed the binding of pritumumab to EDV to be restricted to tumor cells and not normal cells and tissues suggesting EDV may be a useful biomarker. A recombinant version of the mAb was made using the GPEx® system in CHO cells that showed comparable binding activity by flow cytometry, immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC; EC50 of 39.6ng/ml) activity. Further studies include establishing xenograft models with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth of both brain cancer stem cells as well as pancreatic cancer in nude mice but not in SCID mice suggesting an active immune response is necessary for optimal treatment. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest that EDV is a suitable target and biomarker for brain cancers. Citation Format: Ivan Babic, Rajesh Mukthavaram, Pengfei Jiang, Eric Glassy, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Use of ecto-domain vimentin to target brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5611. doi:10.1158/1538-7445.AM2017-5611