Thomas C. Chen

The Unfolded Protein Response Regulator GRP78/BiP as a Novel Target for Increasing Chemosensitivity in Malignant Gliomas

Poor chemosensitivity and the development of chemoresistance remain major obstacles to successful chemotherapy of malignant gliomas. GRP78 is a key regulator of the unfolded protein response (UPR). As a Ca2+-binding molecular chaperone in the endoplasmic reticulum (ER), GRP78 maintains ER homeostasis, suppresses stress-induced apoptosis, and controls UPR signaling. We report here that GRP78 is expressed at low levels in normal adult brain, but is significantly elevated in malignant glioma specimens and human malignant glioma cell lines, correlating with their rate of proliferation. Down-regulation of GRP78 by small interfering RNA leads to a slowdown in glioma cell growth. Our studies further reveal that temozolomide, the chemotherapeutic agent of choice for treatment of malignant gliomas, leads to induction of CHOP, a major proapoptotic arm of the UPR. Knockdown of GRP78 in glioblastoma cell lines induces CHOP and activates caspase-7 in temozolomide-treated cells. Colony survival assays further establish that knockdown of GRP78 lowers resistance of glioma cells to temozolomide, and, conversely, overexpression of GRP78 confers higher resistance. Knockdown of GRP78 also sensitizes glioma cells to 5-fluorouracil and CPT-11. Treatment of glioma cells with (-)-epigallocatechin gallate, which targets the ATP-binding domain of GRP78 and blocks its protective function, sensitizes glioma cells to temozolomide. These results identify a novel chemoresistance mechanism in malignant gliomas and show that combination of drugs capable of suppressing GRP78 with conventional agents such as temozolomide might represent a novel approach to eliminate residual tumor cells after surgery and increase the effectiveness of malignant glioma chemotherapy.

Maintenance Therapy With Tumor-Treating Fields Plus Temozolomide vs Temozolomide Alone for Glioblastoma: A Randomized Clinical Trial

IMPORTANCE Glioblastoma is the most devastating primary malignancy of the central nervous system in adults. Most patients die within 1 to 2 years of diagnosis. Tumor-treating fields (TTFields) are a locoregionally delivered antimitotic treatment that interferes with cell division and organelle assembly. OBJECTIVE To evaluate the efficacy and safety of TTFields used in combination with temozolomide maintenance treatment after chemoradiation therapy for patients with glioblastoma. DESIGN, SETTING, AND PARTICIPANTS After completion of chemoradiotherapy, patients with glioblastoma were randomized (2:1) to receive maintenance treatment with either TTFields plus temozolomide (n = 466) or temozolomide alone (n = 229) (median time from diagnosis to randomization, 3.8 months in both groups). The study enrolled 695 of the planned 700 patients between July 2009 and November 2014 at 83 centers in the United States, Canada, Europe, Israel, and South Korea. The trial was terminated based on the results of this planned interim analysis. INTERVENTIONS Treatment with TTFields was delivered continuously (>18 hours/day) via 4 transducer arrays placed on the shaved scalp and connected to a portable medical device. Temozolomide (150-200 mg/m2/d) was given for 5 days of each 28-day cycle. MAIN OUTCOMES AND MEASURES The primary end point was progression-free survival in the intent-to-treat population (significance threshold of .01) with overall survival in the per-protocol population (n = 280) as a powered secondary end point (significance threshold of .006). This prespecified interim analysis was to be conducted on the first 315 patients after at least 18 months of follow-up. RESULTS The interim analysis included 210 patients randomized to TTFields plus temozolomide and 105 randomized to temozolomide alone, and was conducted at a median follow-up of 38 months (range, 18-60 months). Median progression-free survival in the intent-to-treat population was 7.1 months (95% CI, 5.9-8.2 months) in the TTFields plus temozolomide group and 4.0 months (95% CI, 3.3-5.2 months) in the temozolomide alone group (hazard ratio [HR], 0.62 [98.7% CI, 0.43-0.89]; P = .001). Median overall survival in the per-protocol population was 20.5 months (95% CI, 16.7-25.0 months) in the TTFields plus temozolomide group (n = 196) and 15.6 months (95% CI, 13.3-19.1 months) in the temozolomide alone group (n = 84) (HR, 0.64 [99.4% CI, 0.42-0.98]; P = .004). CONCLUSIONS AND RELEVANCE In this interim analysis of 315 patients with glioblastoma who had completed standard chemoradiation therapy, adding TTFields to maintenance temozolomide chemotherapy significantly prolonged progression-free and overall survival. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00916409.

Nanoparticle biointerfacing by platelet membrane cloaking

Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

PROTEIN KINASE C INHIBITORS INDUCE APOPTOSIS IN HUMAN MALIGNANT GLIOMA CELL LINES

Previous work has demonstrated the importance of the protein kinase C (PKC) system in regulating glioma growth, and has led to clinical trials utilizing PKC inhibitors as adjuncts in the therapy of patients harboring malignant gliomas. This study was performed to explore the possibility that inhibition of PKC in gliomas was triggering an apoptosis signal. Glioma cell lines were treated with PKC inhibitors staurosporine (10 nM), and tamoxifen (10 μM). DNA from cells treated with each of these drugs exhibited a ‘ladder’ pattern of oligonucleosome‐sized fragments characteristic of apoptosis, thus suggesting that in glioma cells, these drugs may be cytocidal in action.

Poor drug distribution as a possible explanation for the results of the PRECISE trial

OBJECT Convection-enhanced delivery (CED) is a novel intracerebral drug delivery technique with considerable promise for delivering therapeutic agents throughout the CNS. Despite this promise, Phase III clinical trials employing CED have failed to meet clinical end points. Although this may be due to inactive agents or a failure to rigorously validate drug targets, the authors have previously demonstrated that catheter positioning plays a major role in drug distribution using this technique. The purpose of the present work was to retrospectively analyze the expected drug distribution based on catheter positioning data available from the CED arm of the PRECISE trial. METHODS Data on catheter positioning from all patients randomized to the CED arm of the PRECISE trial were available for analyses. BrainLAB iPlan Flow software was used to estimate the expected drug distribution. RESULTS Only 49.8% of catheters met all positioning criteria. Still, catheter positioning score (hazard ratio 0.93, p = 0.043) and the number of optimally positioned catheters (hazard ratio 0.72, p = 0.038) had a significant effect on progression-free survival. Estimated coverage of relevant target volumes was low, however, with only 20.1% of the 2-cm penumbra surrounding the resection cavity covered on average. Although tumor location and resection cavity volume had no effect on coverage volume, estimations of drug delivery to relevant target volumes did correlate well with catheter score (p < 0.003), and optimally positioned catheters had larger coverage volumes (p < 0.002). Only overall survival (p = 0.006) was higher for investigators considered experienced after adjusting for patient age and Karnofsky Performance Scale score. CONCLUSIONS The potential efficacy of drugs delivered by CED may be severely constrained by ineffective delivery in many patients. Routine use of software algorithms and alternative catheter designs and infusion parameters may improve the efficacy of drugs delivered by CED.

Vagus Nerve Stimulation Activates Central Nervous System Structures in Epileptic Patients During PET H215O Blood Flow Imaging

OBJECTIVE To determine the central areas of activation by vagal nerve stimulation (VNS) in epilepsy. VNS is a promising neurosurgical method for treating patients with partial and secondary generalized epilepsy. The anti-epileptic mechanism of action from VNS is not well understood. METHODS We performed H2(15)O PET blood flow functional imaging on three patients with epilepsy in a vagal nerve stimulation study (E04 Protocol with Cyberonics). The three patients included two that had previous epilepsy surgery but continued to have frequent seizures. Seizure onset was frontal in two patients and bitemporal in the third patient. Twelve PET scans per subject were acquired every 10 minutes with a Siemens 953/A scanner. In 6 stimulus scans, VNS was activated for 60 seconds (2 mA, 30 Hz) commensurate with isotope injection. In 6 control scans no VNS was administered. No clinical seizures were present during any scan. Three way ANOVA with linear contrasts subject, task, repetition) of coregistered images identified significant treatment effects. RESULTS The difference between PET with VNS and without revealed that left VNS activated right thalamus (P < 0.0006), right posterior temporal cortex (P < 0.0003), left putamen (P < 0.0002), and left inferior cerebellum (P < 0.0009). CONCLUSIONS VNS causes activation of several central areas including contralateral thalamus. Localization to the thalamus suggests a possible mechanism to explain the therapeutic benefit, consistent with the role of the thalamus as a generator and modulator of cerebral activity.

HIV-1 Protease Inhibitors Nelfinavir and Atazanavir Induce Malignant Glioma Death by Triggering Endoplasmic Reticulum Stress

HIV type 1 (HIV-1) protease inhibitors (PI) have been shown to have anticancer activity in non-HIV-associated human cancer cells. The underlying mechanism of this effect is unclear. Here, we show that the PIs nelfinavir and atazanavir cause cell death in various malignant glioma cell lines in vitro. The underlying mechanism of this antitumor effect involves the potent stimulation of the endoplasmic reticulum (ER) stress response (ESR), as indicated by increased expression of two ESR markers, GRP78 and CHOP, and activation of ESR-associated caspase-4. Induction of ESR seems to play a central role in PI-induced cell death because small interfering RNA-mediated knockdown of the protective ER chaperone GRP78 sensitizes cells; whereas knockdown of proapoptotic caspase-4 protects cells from PI-induced cell death. Furthermore, the treatment of cells with PIs leads to aggresome formation and accumulation of polyubiquitinated proteins, implying proteasome inhibition. Thus, our results support a model whereby PIs cause tumor cell death via triggering of the ESR, inhibition of proteasome activity, and subsequent accumulation of misfolded proteins. Inhibition of glioma growth via ESR takes place in the in vivo setting as well, as nelfinavir inhibits the growth of xenografted human malignant glioma, with concomitant induction of the proapoptotic ER stress marker CHOP. Because ER stress has also been reported as the mechanism for insulin resistance and diabetes, our ER stress model of PI function may also explain why these drugs may induce insulin resistance as one of their most common side effects.

Stress Chaperone GRP78/BiP Confers Chemoresistance to Tumor-Associated Endothelial Cells

The tumor vasculature is essential for tumor growth and survival and is a key target for anticancer therapy. Glioblastoma multiforme, the most malignant form of brain tumor, is highly vascular and contains abnormal vessels, unlike blood vessels in normal brain. Previously, we showed that primary cultures of human brain endothelial cells, derived from blood vessels of malignant glioma tissues (TuBEC), are physiologically and functionally different from endothelial cells derived from nonmalignant brain tissues (BEC) and are substantially more resistant to apoptosis. Resistance of TuBEC to a wide range of current anticancer drugs has significant clinical consequences as it represents a major obstacle toward eradication of residual brain tumor. We report here that the endoplasmic reticulum chaperone GRP78/BiP is generally highly elevated in the vasculature derived from human glioma specimens, both in situ in tissue and in vitro in primary cell cultures, compared with minimal GRP78 expression in normal brain tissues and blood vessels. Interestingly, TuBEC constitutively overexpress GRP78 without concomitant induction of other major unfolded protein response targets. Resistance of TuBEC to chemotherapeutic agents such as CPT-11, etoposide, and temozolomide can be overcome by knockdown of GRP78 using small interfering RNA or chemical inhibition of its catalytic site. Conversely, overexpression of GRP78 in BEC rendered these cells resistant to drug treatments. Our findings provide the proof of principle that targeting GRP78 will sensitize the tumor vasculature to chemotherapeutic drugs, thus enhancing the efficacy of these drugs in combination therapy for glioma treatment. (Mol Cancer Res 2008;6(8):1268–75)

Aggravated Endoplasmic Reticulum Stress as a Basis for Enhanced Glioblastoma Cell Killing by Bortezomib in Combination with Celecoxib or Its Non-Coxib Analogue, 2,5-Dimethyl-Celecoxib

The proteasome inhibitor bortezomib (Velcade) is known to trigger endoplasmic reticulum (ER) stress via the accumulation of obsolete and damaged proteins. The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) causes ER stress through a different mechanism (i.e., by causing leakage of calcium from the ER into the cytosol). Each of these two mechanisms has been implicated in the anticancer effects of the respective drug. We therefore investigated whether the combination of these two drugs would lead to further increased ER stress and would enhance their antitumor efficacy. With the use of human glioblastoma cell lines, we show that this is indeed the case. When combined, bortezomib and celecoxib triggered elevated expression of the ER stress markers GRP78/BiP and CHOP/GADD153, caused activation of c-Jun NH(2)-terminal kinase and ER stress-associated caspase-4, and greatly increased apoptotic cell death. Small interfering RNA-mediated knockdown of the protective ER chaperone GRP78/BiP further sensitized the tumor cells to killing by the drug combination. The contribution of celecoxib was independent of the inhibition of COX-2 because a non-coxib analogue of this drug, 2,5-dimethyl-celecoxib (DMC), faithfully and more potently mimicked these combination effects in vitro and in vivo. Taken together, our results show that combining bortezomib with celecoxib or DMC very potently triggers the ER stress response and results in greatly increased glioblastoma cytotoxicity. We propose that this novel drug combination should receive further evaluation as a potentially effective anticancer therapy.

Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib

A drawback of extensive coxib use for antitumor purposes is the risk of life-threatening side effects that are thought to be a class effect and probably due to the resulting imbalance of eicosanoid levels. 2,5-Dimethyl-celecoxib (DMC) is a close structural analogue of the selective cyclooxygenase-2 inhibitor celecoxib that lacks cyclooxygenase-2–inhibitory function but that nonetheless is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. To further establish the potential usefulness of DMC as an anticancer agent, we compared DMC and various coxibs and nonsteroidal anti-inflammatory drugs with regard to their ability to stimulate the endoplasmic reticulum (ER) stress response (ESR) and subsequent apoptotic cell death. We show that DMC increases intracellular free calcium levels and potently triggers the ESR in various tumor cell lines, as indicated by transient inhibition of protein synthesis, activation of ER stress–associated proteins GRP78/BiP, CHOP/GADD153, and caspase-4, and subsequent tumor cell death. Small interfering RNA–mediated knockdown of the protective chaperone GRP78 further sensitizes tumor cells to killing by DMC, whereas inhibition of caspase-4 prevents drug-induced apoptosis. In comparison, celecoxib less potently replicates these effects of DMC, whereas none of the other tested coxibs (rofecoxib and valdecoxib) or traditional nonsteroidal anti-inflammatory drugs (flurbiprofen, indomethacin, and sulindac) trigger the ESR or cause apoptosis at comparable concentrations. The effects of DMC are not restricted to in vitro conditions, as this drug also generates ER stress in xenografted tumor cells in vivo, concomitant with increased apoptosis and reduced tumor growth. We propose that it might be worthwhile to further evaluate the potential of DMC as a non-coxib alternative to celecoxib for anticancer purposes. [Mol Cancer Ther 2007;6(4):1262–75]