Jay F. Dorsey

Cell Cycle–Dependent and Schedule-Dependent Antitumor Effects of Sorafenib Combined with Radiation

The antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G2-M. In synchronized cells, sorafenib causes a reversible G1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies.

Selective targeting of brain tumors with gold nanoparticle-induced radiosensitization.

Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ∼1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature.

Gold-Loaded Polymeric Micelles for Computed Tomography-Guided Radiation Therapy Treatment and Radiosensitization

Gold nanoparticles (AuNPs) have generated interest as both imaging and therapeutic agents. AuNPs are attractive for imaging applications since they are nontoxic and provide nearly three times greater X-ray attenuation per unit weight than iodine. As therapeutic agents, AuNPs can sensitize tumor cells to ionizing radiation. To create a nanoplatform that could simultaneously exhibit long circulation times, achieve appreciable tumor accumulation, generate computed tomography (CT) image contrast, and serve as a radiosensitizer, gold-loaded polymeric micelles (GPMs) were prepared. Specifically, 1.9 nm AuNPs were encapsulated within the hydrophobic core of micelles formed with the amphiphilic diblock copolymer poly(ethylene glycol)-b-poly(ε-capralactone). GPMs were produced with low polydispersity and mean hydrodynamic diameters ranging from 25 to 150 nm. Following intravenous injection, GPMs provided blood pool contrast for up to 24 h and improved the delineation of tumor margins via CT. Thus, GPM-enhanced CT imaging was used to guide radiation therapy delivered via a small animal radiation research platform. In combination with the radiosensitizing capabilities of gold, tumor-bearing mice exhibited a 1.7-fold improvement in the median survival time, compared with mice receiving radiation alone. It is envisioned that translation of these capabilities to human cancer patients could guide and enhance the efficacy of radiation therapy.

Detection of brain tumor cells in the peripheral blood by a telomerase promoter-based assay.

Blood tests to detect circulating tumor cells (CTC) offer great potential to monitor disease status, gauge prognosis, and guide treatment decisions for patients with cancer. For patients with brain tumors, such as aggressive glioblastoma multiforme, CTC assays are needed that do not rely on expression of cancer cell surface biomarkers like epithelial cell adhesion molecules that brain tumors tend to lack. Here, we describe a strategy to detect CTC based on telomerase activity, which is elevated in nearly all tumor cells but not normal cells. This strategy uses an adenoviral detection system that is shown to successfully detect CTC in patients with brain tumors. Clinical data suggest that this assay might assist interpretation of treatment response in patients receiving radiotherapy, for example, to differentiate pseudoprogression from true tumor progression. These results support further development of this assay as a generalized method to detect CTC in patients with cancer.

Modulation of CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 expression by nelfinavir sensitizes glioblastoma multiforme cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Human glioblastoma multiforme cells demonstrate varying levels of sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Endoplasmic reticulum (ER) stress has been shown to trigger cell death through apoptosis. We therefore pursued a strategy of integrating clinically relevant investigational agents that cooperate mechanistically through the regulation of ER stress and apoptosis pathways. Nelfinavir belongs to the protease inhibitor class of drugs currently used to treat patients with HIV and is in clinical trials as an anti-tumor agent. We found that Nelfinavir treatment led to ER stress-induced up-regulation of the DR5 receptor. This transactivation was mediated by the transcription factor CCAAT/enhancer binding protein homologous protein (CHOP). We also determined that ER stress-induced ATF4 up-regulation was responsible for modulation of CHOP. In contrast, DR4 receptor expression was unchanged by Nelfinavir treatment. Combining Nelfinavir with TRAIL led to a significantly enhanced level of apoptosis that was abrogated by siRNA silencing of DR5. We provide evidence that Nelfinavir-induced ER stress modulates DR5 expression in human glioblastoma multiforme cells and can enhance TRAIL efficacy. These studies provide a potential mechanistic rationale for the use of the Food and Drug Administration-approved agent Nelfinavir in combination with DR5 agonists to induce apoptosis in human malignancies.

Replication stress, defective S-phase checkpoint and increased death in Plk2-deficient human cancer cells.

We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis. Our goals have been to better understand why Plk2 is regulated by p53 and how Plk2 signals protection from cell death through checkpoint activation. We found that following knock-down of Plk2 in wild-type p53 expressing H460 human non-small cell lung cancer cells there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block. The highest levels of cell death were observed when Plk2-deficient cells were released from both aphidicolin and etoposide treatment. These results suggested that a defective S-phase checkpoint may contribute to enhanced sensitivity of Plk2-deficient cells to replication stress. Consistent with this hypothesis, we observed higher levels of Serine 139 H2AX phosphorylation in Plk2-deficient as compared to control cells before and after aphidicolin treatment indicating that there is more DNA damage when Plk2 is depleted. We also observed higher levels of Chk1 protein in Plk2-deficient cells that were associated with reduced levels of Serine 317-phosphorylated Chk1. In aphidicolin-treated cells, there were lower levels of Serine 317-phosphorylated Chk1 when Plk2 was knocked-down. Plk2 was demonstrated to interact with Chk2, Chk1, Serine 317-phoshorylated Chk1 and p53. Thus, increased cell death observed after aphidicolin treatment and release in Plk2-deficient cells may result from both higher levels of replication stress-induced DNA damage and a dysfunctional S-phase checkpoint.

Stereotactic radiosurgery to the resection bed for intracranial metastases and risk of leptomeningeal carcinomatosis

OBJECT Following resection of a brain metastasis, stereotactic radiosurgery (SRS) to the cavity is an emerging alternative to postoperative whole-brain radiation therapy (WBRT). This approach attempts to achieve local control without the neurocognitive risks associated with WBRT. The authors aimed to report the outcomes of a large patient cohort treated with this strategy. METHODS A retrospective review identified 91 patients without a history of WBRT who received Gamma Knife (GK) SRS to 96 metastasis resection cavities between 2007 and 2013. Patterns of intracranial control were examined in the 86 cases with post-GK imaging. Survival, local failure, and distant failure were estimated by the Kaplan-Meier method. Prognostic factors were tested by univariate (log-rank test) and multivariate (Cox proportional hazards model) analyses. RESULTS Common primary tumors were non-small cell lung (43%), melanoma (14%), and breast (13%). The cases were predominantly recursive partitioning analysis Class I (25%) or II (70%). Median preoperative metastasis diameter was 2.8 cm, and 82% of patients underwent gross-total resection. A median dose of 16 Gy was delivered to the 50% isodose line, encompassing a median treatment volume of 9.2 cm(3). Synchronous intact metastases were treated in addition to the resection bed in 43% of cases. Patients survived a median of 22.3 months from the time of GK. Local failure developed in 16 cavities, for a crude rate of 18% and 1-year actuarial local control of 81%. Preoperative metastasis diameter ≥ 3 cm and residual or recurrent tumor at the time of GK were associated with local failure (p = 0.04 and 0.008, respectively). Distant intracranial failure occurred in 55 cases (64%) at a median of 7.3 months from GK. Salvage therapies included WBRT and additional SRS in 33% and 31% of patients, respectively. Leptomeningeal carcinomatosis developed in 12 cases (14%) and was associated with breast histology and infratentorial cavities (p = 0.024 and 0.012, respectively). CONCLUSIONS This study bolsters the existing evidence for SRS to the resection bed. Local control rates are high, but patients with larger preoperative metastases or residual/recurrent tumor at the time of SRS are more likely to fail at the cavity. While most patients develop distant intracranial failure, an SRS approach spared or delayed WBRT in the majority of cases. The risk of leptomeningeal carcinomatosis does not appear to be elevated with this strategy.

A multifunctional nanoplatform for imaging, radiotherapy, and the prediction of therapeutic response.

Gold nanoparticles have garnered interest as both radiosensitzers and computed tomography (CT) contrast agents. However, the extremely high concentrations of gold required to generate CT contrast is far beyond that needed for meaningful radiosensitization, which limits their use as combined therapeutic-diagnostic (theranostic) agents. To establish a theranostic nanoplatform with well-aligned radiotherapeutic and diagnostic properties for better integration into standard radiation therapy practice, a gold- and superparamagnetic iron oxide nanoparticle (SPION)-loaded micelle (GSM) is developed. Intravenous injection of GSMs into tumor-bearing mice led to selective tumoral accumulation, enabling magnetic resonance (MR) imaging of tumor margins. Subsequent irradiation leads to a 90-day survival of 71% in GSM-treated mice, compared with 25% for irradiation-only mice. Furthermore, measurements of the GSM-enhanced MR contrast are highly predictive of tumor response. Therefore, GSMs may not only guide and enhance the efficacy of radiation therapy, but may allow patients to be managed more effectively.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and paclitaxel have cooperative in vivo effects against glioblastoma multiforme cells

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in conjunction with microtubule-targeting agents may be a promising novel anticancer treatment strategy. In vitro studies have suggested that relatively low concentrations of TRAIL enhance the lethality of paclitaxel (Taxol) against human cancer cells. The increased efficacy may be due to the triggering of caspase activation, resulting in mitotic checkpoint abrogation and catastrophe. We show here that wild-type p53 protects cells from caspase-dependent death induced by this therapeutic combination in vitro. We have now also developed an imaging-based model system to test the in vivo efficacy of combined TRAIL and Taxol, in which tumor growth and treatment response can be monitored noninvasively and in real-time. We further utilize bioluminescence, F18-fluorodeoxyglucose-positron emission tomography, and microscale computed tomography imaging to confirm the effects of combined treatment on tumors. These studies together provide the first in vivo confirmation that combined TRAIL plus paclitaxel results in better tumor control compared with either TRAIL or paclitaxel alone, and with no discernable increased normal tissue toxicity in the mouse. Interestingly, the in vivo antitumor response elicited by combined treatment was not affected by the p53 status of the tumor cells. These preclinical observations together suggest the therapeutic potential of combining TRAIL plus paclitaxel in cancer treatment, and support further preclinical and future clinical testing. [Mol Cancer Ther 2009;8(12):3285–95]

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.