Stephen Schleicher

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.

Progress in the unraveling of the endoplasmic reticulum stress/autophagy pathway and cancer: Implications for future therapeutic approaches

Given the inherent resistance to apoptosis that characterizes cancer, the targeting of alternative pathways is an attractive strategy to improve anti-tumor therapy. Endoplasmic reticulum (ER) stress, which is basally activated in many cancers, and the subsequent activation of autophagy represent novel cancer treatment targets. While these associated pathways are often protective and promote cell survival, when excessive, ER stress results in autophagic cell death. Therefore, depending on the circumstances, either inhibition or activation of ER stress and autophagy can improve cancer therapy. This review provides an update on how ER stress relates to autophagy, and how these associated pathways can serve dual functions to promote survival or cell death in cancer. Furthermore, it lays out a spectrum of potential pharmacological agents and combinatorial approaches that target these pathways to enhance tumor cell kill.

Autotaxin and LPA Receptors Represent Potential Molecular Targets for the Radiosensitization of Murine Glioma through Effects on Tumor Vasculature

Despite wide margins and high dose irradiation, unresectable malignant glioma (MG) is less responsive to radiation and is uniformly fatal. We previously found that cytosolic phospholipase A2 (cPLA2) is a molecular target for radiosensitizing cancer through the vascular endothelium. Autotaxin (ATX) and lysophosphatidic acid (LPA) receptors are downstream from cPLA2 and highly expressed in MG. Using the ATX and LPA receptor inhibitor, α-bromomethylene phosphonate LPA (BrP-LPA), we studied ATX and LPA receptors as potential molecular targets for the radiosensitization of tumor vasculature in MG. Treatment of Human Umbilical Endothelial cells (HUVEC) and mouse brain microvascular cells bEND.3 with 5 µmol/L BrP-LPA and 3 Gy irradiation showed decreased clonogenic survival, tubule formation, and migration. Exogenous addition of LPA showed radioprotection that was abrogated in the presence of BrP-LPA. In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells. Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation. However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect. Using heterotopic tumor models of GL-261, mice treated with BrP-LPA and irradiation showed a tumor growth delay of 6.8 days compared to mice treated with irradiation alone indicating that inhibition of ATX and LPA receptors may significantly improve malignant glioma response to radiation therapy. These findings identify ATX and LPA receptors as molecular targets for the development of radiosensitizers for MG.

Inhibition of JAK2 signaling by TG101209 enhances radiotherapy in lung cancer models.

Introduction: Persistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. Methods: We tested whether TG101209, a small-molecule inhibitor of JAK2 (a STAT3-activating tyrosine kinase), affected survivin expression and sensitized lung cancer to radiation. We investigated whether inhibition of JAK2 signaling with TG101209 can be used to reduce survivin expression and enhance radiosensitivity of lung cancer cells in vitro and tumor growth delay in vivo. JAK2 downstream signaling, including PI3-K/Akt and Ras/MAPK/ERK pathways, was also explored. Results: TG101209 inhibited STAT3 activation and survivin expression and sensitized HCC2429 (dose enhancement ratio = 1.34, p = 0.002) and H460 (dose enhancement ratio = 1.09, p = 0.006) cells to radiation in clonogenic assays. Radiation promoted phospho-Akt and phospho-ERK in H460 cells, while their levels were unchanged in HCC2429. After treatment with TG101209, phospho-ERK protein levels were reduced in both HCC2429 and H460 cells. HCC2429 cells transfected with KRAS-12V mutant were more resistant to radiation- and TG101209-induced apoptosis than wild-type control cells. In vivo, addition of TG101209 to radiation in lung xenografts produced a significant tumor growth delay (>10 days) compared with radiation alone and was well tolerated. Immunohistochemistry staining of tumor sections showed that TG101209 increased apoptosis and decreased cell proliferation and vascular density, suggesting that TG101209 also has antiangiogenic effects. Conclusions: TG101209 enhanced the effects of radiation in lung cancer in vitro and in vivo. This study suggests the potential utility of selecting lung cancer patients according to KRAS mutation status for future clinical trials testing combination of TG101209 and radiotherapy.

Role of Insulin-Like Growth Factor-1 Signaling Pathway in Cisplatin-Resistant Lung Cancer Cells

PURPOSE The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. METHODS AND MATERIALS H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. RESULTS Cisplatin-R cells illustrated greater expression of the markers CD133 and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. CONCLUSIONS The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.

Enhanced Radiosensitivity of Androgen-Resistant Prostate Cancer: AZD1152-Mediated Aurora Kinase B Inhibition

Aurora kinase B (AURKB) is critical to the process of mitosis, aiding in chromosome condensation by phosphorylating histone H3. We investigated the effects of AZD1152, an AURKB inhibitor, on radiosensitivity of androgen-insensitive prostate cancer cells. The goal of this study was to test whether AZD1152 increases the susceptibility of hormone-refractory prostate cancer cells to radiation-induced DNA damage and to determine the conditions of AZD1152 treatment that maximize radiosensitization. PC3 and DU145 cells were treated with various AZD1152 doses for various durations to elucidate the conditions that yielded maximal increases in G2/M-phase and polyploid cells. To assess DNA damage, &ggr;-H2AX phosphorylation was quantified for cells grown under radiosensitizing conditions and subjected to either no radiation or 5 Gy radiation. Radiosensitivity was determined by clonogenic assays. Cell cycle effects in both cell lines were maximized by treatment with 60 nM AZD1152 for 48 h. AZD1152-treated cells exhibited significantly increased DNA damage 30 min postirradiation (PC3: 100% compared to 68%, P  =  0.035; DU145: 100% compared to 69%, P  =  0.034), with additional DNA damage 6 h postirradiation (PC3: 85% compared to 15%, P  =  0.002; DU145: 67% compared to 21%, P  =  0.012). Radiosensitivity was increased in both cell lines, with dose enhancement ratios of 1.53 for PC3 cells (P  =  0.017) and 1.71 for DU145 cells (P  =  0.02). This study identifies the optimal AZD1152 treatment conditions to maximize the radiosensitization of PC3 and DU145 cells. These results suggest a major role for DNA damage and impairment of DNA repair mechanisms in AZD1152-induced radiosensitization of prostate cancer cells.

GSK-3β inhibition promotes cell death, apoptosis, and in vivo tumor growth delay in neuroblastoma Neuro-2A cell line

Neuroblastoma is the most common extracranial solid tumor of childhood. While survival rates are high for localized disease, treatment response remains poor for a subset of patients with large tumors or disseminated disease. Thus, there remains much room for improvement in treatment strategies for this disease. Using in vitro and in vivo systems, we present glycogen synthase kinase-3β (GSK-3β) inhibition as a potential mechanism to treat neuroblastoma. Using the specific GSK-3β inhibitor SB415286, we demonstrate that GSK-3β inhibition decreases the viability of Neuro-2A cells, as determined by cell proliferation assay and clonogenic survival. Moreover, we show that GSK-3β inhibition induces apoptosis in neuroblastoma cells, as determined by Annexin V staining and confirmed with DAPI staining. Using flow cytometry, we are able to demonstrate that SB415286 induces the accumulation of cells in the G2/M phase of the cell cycle. Finally, we show that these in vitro results translate into delayed tumor growth in vivo using a heterotopic tumor model in nude mice treated with SB415286. These findings suggest that GSK-3β is a potential molecular target for the treatment of neuroblastoma.

The EGFR Polymorphism rs884419 is Associated With Freedom From Recurrence in Patients With Resected Prostate Cancer

PURPOSE Prognostic biomarkers are needed to optimize treatment decisions for prostate cancer. Single nucleotide polymorphisms participate in the individual genetic background modulating risk and clinical outcomes of cancer. We tested whether EGFR polymorphisms are associated with prostate cancer clinical outcomes. MATERIALS AND METHODS The study population consisted of 212 patients with clinically localized prostate cancer treated with radical prostatectomy from 1997 to 1999. Resected prostatic tissues were genotyped with allele specific probes for 9 haplotype tagging single nucleotide polymorphisms, which were located in intronic, exonic and flanking regions of linkage disequilibrium in the EGFR gene. Correlations between alleles, and recurrence and survival data were investigated using univariate and multivariate genetic analysis models. RESULTS There was a statistically significant association between the single nucleotide polymorphism rs884419 and prostate cancer recurrence, as defined in the study by at least prostate specific antigen biochemical recurrence (log rank test p <0.001). The incidence of the recurrence risk enhancing genotype A/A was 3.1% vs 17.4% and 80% for the risk decreasing genotypes A/G G/G, respectively. Based on Cox proportional hazard regression modeling patients carrying G/G and A/G genotypes were associated with a decreased risk of prostate cancer recurrence compared to those with the A/A genotype (HR 0.10, 95% CI 0.02-0.41 and 0.13, 95% CI 0.04-0.46, respectively, p <0.002). CONCLUSIONS These data suggest that a polymorphism flanking the EGFR gene is an independent prognostic genetic biomarker that predicts prostate cancer biochemical recurrence after radical prostatectomy.

MLN8054, A SMALL MOLECULE INHIBITOR OF AURORA KINASE A, SENSITIZES ANDROGEN-RESISTANT PROSTATE CANCER TO RADIATION

PURPOSE To determine whether MLN8054, an Aurora kinase A (Aurora-A) inhibitor causes radiosensitization in androgen-insensitive prostate cancer cells in vitro and in vivo. METHODS AND MATERIALS In vitro studies consisted of culturing PC3 and DU145 prostate cancer cells and then immunoblotting Aurora A and phospho-Aurora A after radiation and/or nocodazole with MLN8054. Phases of the cell cycle were measured with flow cytometry. PC3 and DU145 cell lines were measured for survival after treatment with MLN8054 and radiation. Immunofluorescence measured γ-H2AX in the PC3 and DU145 cells after treatment. In vivo studies looked at growth delay of PC3 tumor cells in athymic nude mice. PC3 cells grew for 6 to 8 days in mice treated with radiation, MLN8054, or combined for 7 more days. Tumors were resected and fixed on paraffin and stained for von Willebrand factor, Ki67, and caspase-3. RESULTS In vitro inhibition of Aurora-A by MLN8054 sensitized prostate cancer cells, as determined by dose enhancement ratios in clonogenic assays. These effects were associated with sustained DNA double-strand breaks, as evidenced by increased immunofluorescence for γ-H2AX and significant G2/M accumulation and polyploidy. In vivo, the addition of MLN8054 (30 mg/kg/day) to radiation in mouse prostate cancer xenografts (PC3 cells) significantly increased tumor growth delay and apoptosis (caspase-3 staining), with reduction in cell proliferation (Ki67 staining) and vascular density (von Willebrand factor staining). CONCLUSION MLN8054, a novel small molecule Aurora-A inhibitor showed radiation sensitization in androgen-insensitive prostate cancer in vitro and in vivo. This warrants the clinical development of MLN8054 with radiation for prostate cancer patients.

A Novel Bioluminescence Orthotopic Mouse Model for Advanced Lung Cancer

Lung cancer is the leading cause of cancer-related death in the United States despite recent advances in our understanding of this challenging disease. An animal model for high-throughput screening of therapeutic agents for advanced lung cancer could help promote the development of more successful treatment interventions. To develop our orthotopic lung cancer model, luciferase-expressing A549 cancer cells were injected into the mediastinum of athymic nude mice. To determine whether the model would allow easy monitoring of response to therapeutic interventions, tumors were treated with 30 mg/kg Paclitaxel or were irradiated with 5 fractions of 2 Gy, and tumor burden was monitored using bioluminescence imaging. Evidence of radiation-induced lung injury was assessed using immunohistochemical staining for phospho-Smad2/3 and cleaved caspase-3. We found that tumor implantation recapitulated advanced human lung cancer as evidenced by tumor establishment and proliferation within the mediastinum. The tumor responded to Paclitaxel or radiation as shown by decreased tumor bioluminescence and improved overall survival. Immunohistochemistry revealed increased phospho-Smad2/3 and cleaved caspase-3 in irradiated lungs, consistent with radiation-induced lung injury. This orthotopic lung cancer model may help provide a method to assess therapeutic interventions in a preclinical setting that recapitulates locally advanced lung cancer.