Daniel E. Spratt

Targeting the Mechanisms of Resistance to Chemotherapy and Radiotherapy with the Cancer Stem Cell Hypothesis

Despite advances in treatment, cancer remains the 2nd most common cause of death in the United States. Poor cure rates may result from the ability of cancer to recur and spread after initial therapies have seemingly eliminated detectable signs of disease. A growing body of evidence supports a role for cancer stem cells (CSCs) in tumor regrowth and spread after initial treatment. Thus, targeting CSCs in combination with traditional induction therapies may improve treatment outcomes and survival rates. Unfortunately, CSCs tend to be resistant to chemo- and radiation therapy, and a better understanding of the mechanisms underlying CSC resistance to treatment is necessary. This paper provides an update on evidence that supports a fundamental role for CSCs in cancer progression, summarizes potential mechanisms of CSC resistance to treatment, and discusses classes of drugs currently in preclinical or clinical testing that show promise at targeting CSCs.

Targeted Nanoparticles That Deliver a Sustained, Specific Release of Paclitaxel to Irradiated Tumors

To capitalize on the response of tumor cells to XRT, we developed a controlled-release nanoparticle drug delivery system using a targeting peptide that recognizes a radiation-induced cell surface receptor. Phage display biopanning identified Gly-Ile-Arg-Leu-Arg-Gly (GIRLRG) as a peptide that selectively recognizes tumors responding to XRT. Membrane protein extracts of irradiated glioma cells identified glucose-regulated protein GRP78 as the receptor target for GIRLRG. Antibodies to GRP78 blocked the binding of GIRLRG in vitro and in vivo. Conjugation of GIRLRG to a sustained-release nanoparticle drug delivery system yielded increased paclitaxel concentration and apoptosis in irradiated breast carcinomas for up to 3 weeks. Compared with controls, a single administration of the GIRLRG-targeted nanoparticle drug delivery system to irradiated tumors delayed the in vivo tumor tripling time by 55 days (P = 0.0001) in MDA-MB-231 and 12 days in GL261 (P < 0.005). This targeting agent combines a novel recombinant peptide with a paclitaxel-encapsulating nanoparticle that specifically targets irradiated tumors, increasing apoptosis and tumor growth delay in a manner superior to known chemotherapy approaches.

Dose to the inferior pharyngeal constrictor predicts prolonged gastrostomy tube dependence with concurrent intensity-modulated radiation therapy and chemotherapy for locally-advanced head and neck cancer.

BACKGROUND AND PURPOSE To determine if dose and/or dose-volume parameters to anatomic swallowing structures are predictive of gastrostomy tube (PEG) dependence from chemotherapy-intensity modulated radiotherapy (IMRT) in locally advanced head and neck cancer (LAHNC). METHODS AND MATERIALS A retrospective study was performed on 141 consecutive patients with LAHNC (squamous cell) treated with definitive chemoIMRT with weekly concurrent carboplatin and paclitaxel. Late dysphagia was assessed by length of PEG requirement. Analysis of IMRT dose was retrospectively performed for critical swallowing structures. RESULTS Approximately 62% of patients required PEG, the majority placed during treatment. Mean and median time for PEG was 7.7 and 4.4 months respectively (range 1.4-43.8). Only IMRT dose to the inferior constrictor was significantly associated with length of PEG. Mean dose (of individual mean doses) was 47 Gy for prolonged PEG use versus 41 Gy for PEG ⩽ 12 months. V40 to the inferior constrictor also correlated with PEG >12 months (p = 0.02) with a mean V40 of 48% versus 41% for PEG ⩽ 12 months. CONCLUSIONS IMRT dose to the inferior constrictor correlated with persistent dysphagia requiring prolonged PEG use. Maintaining mean inferior constrictor dose to ⩽ 41 Gy and V40 to ⩽ 41% may help minimize gastrostomy tube dependence.

An automated algorithm to improve the precision of basilar artery diameter measurements before and after subarachnoid hemorrhage-induced vasospasm in an animal model.

OBJECTIVEQuantifying vasospasm has traditionally been performed manually, a method prone to imprecision and user bias. An alternative approach is to use computerized image analysis techniques to define and quantify the diameter of a vessel. The goal of this article is to demonstrate a novel automated vessel measurement algorithm specific to the needs of vasospasm studies and to compare it with traditional manual measurements in an animal model of vasospasm. METHODSA total of 576 arterial diameter measurements were collected by 4 independent, blinded examiners from 24 angiograms in a rabbit subarachnoid hemorrhage (SAH) model. Measurements were taken from 3 segments of the basilar artery in anteroposterior and lateral projections, both before SAH and after SAH-induced vasospasm. Means and standard deviations of 288 manual measurements were compared with 288 automated measurements. RESULTSThe precision of automated measurements was significantly improved compared with standardized manual measurements (85.7% decrease in variation; P < .001). When using automated measurements, the precision was not affected by vessel size, but when using manual measurements, smaller arteries were less precise (P = .04). There was no significant difference in precision between 2 different contrast concentrations (P = .32). CONCLUSIONAutomated measurements of basilar artery diameters are more precise than manual measurements, both before and after SAH-induced vasospasm. The variability in the manual group worsens when the artery is smaller secondary to vasospasm, indicating a need for the use of this segmentation method.

Abstract 1398: Aurora kinase A inhibition with MLN8054 radiosensitizes prostate cancer by inducing cell cycle arrest leading to increased prolonged DNA damage and apoptosis

The purpose of this study is to investigate a potential new radioenhancer for the treatment of prostate cancer. Despite multiple promising treatment modalities, prostate cancer remains a significant cause of diminished quality of life and cancer death, indicating the need for more aggressive, targeted therapy. Aurora kinase A (AURKA) is a promising treatment target, but its inhibition has never been investigated in combination with radiotherapy in prostate cancer. Using the selective AURKA inhibitor, MLN8054, we performed clonogenic assay to investigate in vitro radiosensitization, cell cycle analysis to observe changes induced by AURKA inhibition, tumor growth delay to study in vivo effects, and histologic active caspase staining to measure apoptosis. We demonstrated radiosensitizing effects in vitro in PC3 and DU145 prostate cancer cell lines, which was confirmed in vivo by tumor growth delay in PC3 xenografts in nude mice. In addition, we investigated a potential mechanism for radiosensitization. We report that MLN8054 induces cell cycle arrest at G2/M and polyploidy in both prostate cancer cell lines, which is associated with sustained DNA damage from radiation as seen by increased immunofluorescence for γ-H2AX, a measure of DNA double-strand breaks. Staining for caspase-3 in xenograft samples showed increased apoptosis in irradiated samples pre-treated with MLN8054 compared to irradiation or inhibitor alone. Our findings support the potential use of AURKA inhibition with MLN8054 to enhance the effectiveness of radiation therapy for the treatment of prostate cancer, and suggest a potential mechanism underlying its radiosensitization effects. 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 1398.

Abstract 5548: Targeted nanoparticle drug delivery to radiation inducible GRP78 in tumor vasculature

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose/Objectives: GRP78 is a major endoplasmic reticulum chaperone that suppresses stress-induced apoptosis. High GRP78 levels have been shown to correlate with a worse pathologic grade and poor patient survival in various cancer types. GRP78 is also known to confer chemoresistance to tumors and tumor-associated endothelial cells. The goal of this study is to elucidate the mechanism responsible for radiation-mediated neoantigen expression in tumor microvasculature. GIRLRG, a peptide already shown to bind specifically to radiation (XRT) treated, responding tumors, was used in tumor protein extraction to identify its possible cellular targets. GRP78 was identified from the protein extraction as a possible receptor for GIRLRG. We tested the hypothesis that GRP78 is upregulated in response to radiation and can be used as a radiation-inducible drug delivery target. Methods: Proteins from untreated and XRT treated GL261 gliomas were extracted and incubated with agarose beads coated with GIRLRG and then analyzed by gel electrophoresis and mass spectrometry. A fluorescently labeled antibody to GRP78 was used to assess location of GRP78 expression post-XRT in vitro. WB analysis, immunohistochemistry (IHC), and in vivo imaging of GRP78 expression levels was performed in XRT treated and untreated GL261 and MDA-MB-231 breast tumors 48 hours post-XRT. In vivo blocking experiments were performed to assess GRP78 as the radiation inducible receptor for the GIRLRG peptide. Finally, GIRLRG was conjugated to a slow release nanoparticle drug delivery system encapsulating paclitaxel to target GRP78 in vivo. Results: Mass spectrometry analysis revealed that GIRLRG specifically extracted a 78k-dalton protein, GRP78 from XRT treated tumor samples. Similarly, WB, IHC, and in vivo imaging analysis showed an upregulation of GRP78 expression in XRT treated GL261 and MDA-MB-231 samples. In vitro imaging showed GRP78 to be upregulated at the cell surface in response to XRT. A blocking antibody to GRP78 was able to significantly abrogate GIRLRG binding post-XRT (p < 0.05). The GIRLRG-nanoparticle drug delivery system produced a significant tumor growth delay relative to all control groups (p < 0.05). Conclusions: GRP78 has been identified as GIRLRGs binding partner. We found that GRP78 is induced by XRT. This finding correlates with GRP78s known stress induced anti-apoptotic function, as XRT is a known cellular stressor. Furthermore, we utilized upregulation of GRP78 post-XRT by combining chemotherapeutic agents to a peptide ligand, GIRLRG, that recognizes the XRT-induced receptor, providing a targeting system that increases chemotherapeutic efficacy for irradiated tumors in vivo. In the future we plan to investigate if blocking GRP78 could potentially radiosensitize tumors by promoting an apoptotic state. Overall the regulation of GRP78 has the potential to provide exciting new targets for anti-cancer strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5548.