Ziqiang Yuan

Erlotinib, an effective epidermal growth factor receptor tyrosine kinase inhibitor, induces p27KIP1 up-regulation and nuclear translocation in association with cell growth inhibition and G1/S phase arrest in human non-small-cell lung cancer cell lines.

Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been shown to have potent antitumor effects against human non-small-cell lung cancer (NSCLC) cell growth; however, the mechanism of such an effect is not elucidated. Here, we demonstrate that erlotinib-induced cell growth inhibition in EGFR high-expressing human H322 NSCLC cells was accompanied by G1/S phase arrest, which was largely caused by a decrease in expression of G1/S-related cyclins, suppression of activities of cyclin-dependent kinase (CDK) 2 and CDK4, induction of CDK inhibitor p27KIP1, and retinoblastoma hypophosphorylation. To further understand the role of p27KIP1 in G1/S arrest and cell growth inhibition by erlotinib, we determined its effect on the expression of p27KIP1 at transcriptional and posttranscriptional levels. Studies using real-time reverse transcription-polymerase chain reaction analysis and p27 promoter-driven luciferase reporter showed that erlotinib treatment resulted in the promotion of p27 gene transcription. In addition, erlotinib treatment led to an increase in p27KIP1 half-life by inhibiting p27KIP1 phosphorylation at Thr187 and by down-regulating Skp2 expression. Furthermore, immunofluorescence staining and cell fractionation showed that erlotinib treatment led to p27KIP1 translocation to the nucleus. Knockdown of p27KIP1 expression with p27KIP1 small interfering RNA significantly abrogated erlotinib-induced G1 phase arrest and cell growth inhibition, suggesting that induction of p27KIP1 is required for G1 arrest and cell growth inhibition by erlotinib. It is noteworthy that we found that G1 arrest and p27KIP1 up-regulation by erlotinib occurred in the tested sensitive cell lines but to a lesser extent in the resistant cell lines. Taken together, these results suggest that erlotinib inhibits human NSCLC cell growth predominantly by inducing p27KIP1 expression and by suppressing cell-cycle events involved in the G1/S transition.

PM02734 (Elisidepsin) Induces Caspase-Independent Cell Death Associated with Features of Autophagy, Inhibition of the Akt/mTOR Signaling Pathway, and Activation of Death-Associated Protein Kinase

Purpose: PM02734 (elisidepsin) is a synthetic marine-derived cyclic peptide of the kahalalide family currently in phase II clinical development. The mechanisms of cell death induced by PM02734 remain unknown. Experimental Design: Human non–small-cell lung cancer (NSCLC) cell lines H322 and A549 were used to evaluate PM02734-induced cytotoxicity, apoptosis, and autophagy, as well as effects on cell death–related signaling pathways. Results: PM02734 at clinically achievable concentrations (0.5–1 μmol/L) was cytotoxic to H322 and A549 cells but did not cause nuclear fragmentation, PARP cleavage, or caspase activation, suggesting that classical apoptosis is not its main mechanism of cell death. In contrast, PM02734-induced cell death was associated with several characteristics of autophagy, including an increase in acidic vesicular organelle content, levels of GFP-LC3–positive puncta, elevation of the levels of Atg-5/12 and LC3-II, and an associated compromise of the autophagic flux resulting in increased number of autophagosomes and/or autolysosomes. Cotreatment with 3-methyladenine (3-MA) and downregulation of Atg-5 gene expression by siRNA partially inhibited PM02734-induced cell death. PM02734 caused inhibition of Akt/mTOR signaling pathways and cotreatment with the Akt inhibitor wortmannin or with the mTOR inhibitor rapamycin led to a significant increase in PM02734-induced cell death. Furthermore, PM02734 caused the activation of death-associated protein kinase (DAPK) by dephosphorylation at Ser308, and downregulation of DAPK expression with siRNA caused also a partial but significant reduction of PM02734-induced cell death. In vivo, PM02734 significantly inhibited subcutaneous A549 tumor growth in nude mice (P < 0.05) in association with induction of autophagy. Conclusions: Our data indicate that PM02734 causes cell death by a complex mechanism that involves increased autophagosome content, due for the most part to impairment of autophagic flux, inhibition of the Akt/mTOR pathway, and activation of DAPK. This unique mechanism of action justifies the continued development of this agent for the treatment of NSCLC. Clin Cancer Res; 17(16); 5353–66. ©2011 AACR.

Differential expression of DOC-1 in microsatellite-unstable human colorectal cancer

The precise genetic mechanism of malignant transformation in DNA mismatch repair deficient, microsatellite-unstable colorectal cancer (CRC) has yet to be elucidated. We employed cDNA microarray to identify patterns of gene expression among CRC cell lines and to compare directly lines with and without microsatellite instability. This study was undertaken to test the hypothesis that microsatellite-unstable CRC cell lines demonstrate specific patterns of gene expression that differ significantly from those observed among microsatellite-stable CRC. Multiple differential expression patterns were identified. Genes demonstrating differential expression included deleted-in-oral-cancer-1 (DOC-1), a highly conserved growth suppressor. DOC-1 expression correlated with microsatellite status, with significantly decreased expression in microsatellite-unstable cell lines and constitutive expression in microsatellite-stable cell lines. We also observed alterations in the biologic behavior of p12DOC-1-deficient cell lines, with increased S phase and decreased apoptosis compared to microsatellite-stable (DOC-1+) cell lines. Transfection of p12DOC-1 into SW48, which lacks p12DOC-1 expression, resulted in cell cycle and apoptosis profiles similar to other p12DOC-1+ cell lines. These results support the hypothesis that microsatellite-unstable CRC is characterized by novel patterns of gene expression different from those associated with microsatellite-stable CRC, and demonstrate that p12DOC-1 has tumor suppressor potential in colon epithelial cells.

Loss of MEN1 activates DNMT1 implicating DNA hypermethylation as a driver of MEN1 tumorigenesis

Multiple endocrine neoplasia type 1 (MEN1) syndrome results from mutations in the MEN1 gene and causes tumor formation via largely unknown mechanisms. Using a novel genome-wide methylation analysis, we studied tissues from MEN1-parathyroid tumors, Men1 knockout (KO) mice, and Men1 null mouse embryonic fibroblast (MEF) cell lines. We demonstrated that inactivation of menin (the protein product of MEN1) increases activity of DNA (cytosine-5)-methyltransferase 1 (DNMT1) by activating retinoblastoma-binding protein 5 (Rbbp5). The increased activity of DNMT1 mediates global DNA hypermethylation, which results in aberrant activation of the Wnt/β-catenin signaling pathway through inactivation of Sox regulatory genes. Our study provides important insights into the role of menin in DNA methylation and its impact on the pathogenesis of MEN1 tumor development.

Deleted in oral cancer-1 expression upregulates proapoptosis elements in microsatellite-unstable human colorectal cancer.

AbstractBackground: We previously reported differential expression of the growth suppressor, deleted in oral cancer-1 (DOC-1), in microsatellite-unstable (MSI+) versus microsatellite-stable colorectal cancer (CRC) cell lines. MSI+ CRC cell lines demonstrated decreased DOC-1 expression and decreased apoptosis. Transfection of wild-type DOC-1 into an MSI+ cell line (SW48) resulted in increased apoptosis. We undertook our current experiment to identify specific elements modulated by DOC-1 expression that result in increased apoptosis. Methods: SW48 is an MSI+ CRC cell line that does not constitutively express DOC-1. SW48 was suspended in culture medium and incubated to 60% confluence. Half the plates were transfected with cytomegalovirus (CMV)-DOC-1. At 30 hours, RNA and protein were isolated with Trizol. Complementary DNA microarray was performed to compare SW48CMV-DOC-1 with SW48, which lacks DOC-1. Signal intensity was analyzed by GenePix Pro 3.0 software. Expression ratios ≤.67 and ≥1.5 were considered significant. Poor-quality spots were flagged and excluded from analysis. Real-time polymerase chain reaction was performed to determine DOC-1 levels in both cell lines. Results: Successful transfection of DOC-1 was confirmed by real-time polymerase chain reaction and by Western blot. Microarray revealed significant differential expression of DOC-1, as expected. Increased DOC-1 expression in SW48CMV-DOC-1 was associated with significantly increased expression of proapoptosis components of the caspase cascade (CASP7, CASP9) and bcl2/bax pathway (BNIP3, BNIP3L, BID). Conclusions: DOC-1 expression promotes apoptosis by upregulation of specific elements of the caspase cascade and bcl2/bax pathways. DOC-1 therefore deserves further study as a candidate for the therapeutic modulation of apoptosis in MSI+ CRC.

Development of a method to implement whole-genome bisulfite sequencing of cfDNA from cancer patients and a mouse tumor model

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Targeting Tumor Vasculature Using Adeno-Associated Virus Phage Vectors Coding Tumor Necrosis Factor-α

An advantage of targeted gene therapy is the potential for effectively delivering anti-vascular and antitumor therapy directly to the site of the tumor, thus maximizing therapeutic efficacy and minimizing toxicity. The use of a vector capable of directed cell transduction can result in the sustained expression of the therapeutic agent. This chapter reviews the current status of vector strategies, targeted gene therapy with tumor-specific promoter and antibody and peptide conjugation, and outlines the challenges faced for the next-generation of gene transfer technology. Furthermore, it summarizes the results of preclinical anti-angiogenic gene therapy investigations, which utilize novel targeted tumor necrosis factor-α treatment, and discusses potential clinical applications of this treatment strategy.

Novel Dual-Action Targeted Nanomedicine in Mice With Metastatic Thyroid Cancer and Pancreatic Neuroendocrine Tumors

Background The advantages of nanomedicines include preferential delivery of the payload directly to tumor tissues. CYT-21625 is the novel, first-in-class gold nanomedicine designed to target tumor vasculature and cancer cells by specifically delivering recombinant human tumor necrosis factor alpha (rhTNF) and a paclitaxel prodrug. Methods We analyzed TNF receptor expression in publicly available gene expression profiling data and in thyroid tissue samples. Mice with metastatic FTC-133 and 8505C xenografts and the MEN1 conditional knock-out mice were treated weekly with CYT-21625 and gold nanoparticles with rhTNF only (CYT-6091); controls included mice treated with either paclitaxel or saline. In vivo luciferase activity was used to assess the effects on tumor growth. Computed tomography, magnetic resonance imaging, and 18F-Fludeoxyglucose positron emission tomography were used to study tumor selectivity in mice with insulin-secreting pancreatic neuroendocrine tumors (PNETs). All statistical tests were two-sided. Results Anaplastic thyroid cancer (ATC) expressed statistically significantly higher levels of TNF receptor superfamily 1A and 1B messenger RNA (n = 11) and protein (n = 6) than control samples (n = 45 and 13, respectively). Mice (n = 5-7 per group) with metastatic ATC (P < .009) and FTC-133 xenografts (P = .03 at week 3, but not statistically significant in week 4 owing to reduced sample size from death in non-CYT-21625 groups) treated with CYT-21625 had a statistically significantly lower tumor burden. Treatment with CYT-21625 resulted in loss of CD34 expression in intratumoral vasculature, decreased proliferating cell nuclear antigen, and increased cleaved caspase-3. Intratumoral vascular leakage occurred only in mice with PNET and ATC treated with CYT-6091 and CYT-21625. CYT-6091 and CYT-21625 preferentially deposited in PNETs and statistically significantly decreased serum insulin levels (n = 3 per group, P < .001). There were no toxicities observed in mice treated with CYT-21625. Conclusions CYT-21625 is effective in mice with PNETs and metastatic human thyroid cancer with no toxicities. Thus, CYT-21625 should be studied in patients with advanced PNETs and thyroid cancer.

Abstract 3911: Bacteriophage associated silicon particles (BASP) for targeting pancreatic neuroendocrine tumors

The purpose of this project is to design and develop a multifunctional nanosystem for the diagnosis and treatment of pancreatic neuroendocrine tumors (PNETs). PNETs also known as Islet Cell Tumors are rare and slow-growing highly vascularized malignancies with an incidence rate of a thousand new cases per year. Their occurrence has increased five-fold within the past few decades and with surgery being the primary treatment modality, there is an immediate need to devise efficient diagnostic and therapeutic procedures. For this purpose, the synergistic tissue targeting capabilities of phage, optical and physical properties of gold nanoparticles (AuNP)/phage scaffold and the drug carrying potential of the hydrodynamically engineered silicon-based multistage nanovectors were employed to create the Bacteriophage Associated Silicon Particles (BASP). The BASP were characterized in vitro, optimized for in vivo imaging and are currently being investigated for therapeutic efficiency in vivo when loaded with Abraxane. CRKL is evaluated as a potential target expressed in the PNET microenvironment. CRKL targeting phage is incorporated into the BASP and the targeting capability is explored. Our data show that bacteriophage, displaying CRKL-recognizing peptide sequences, enhanced cellular association of BASP in human pancreatic cancer and murine microvascular endothelial cell lines. There is a significant increase in association of CRKL targeted BASP with endothelial cells as opposed to non-targeted assemblies mainly under the flow conditions with shear stresses similar these in the tumor vessels. Further, intravital microscopy studies in highly vascularized tumors demonstrated preferential accumulation of BASP in the tumor vasculature, based on their hydrodynamic characteristics and biological targeting. Overexpression of CRKL was confirmed in histological samples from PNET patients and mice with homozygous deletion of the Men1 gene in the pancreas (PNET model). MRI and CT imaging using CRKL targeted BASP loaded with SPION demonstrated that the systems can aid in detecting very small tumors using various imaging modalities. 3D CT images showed increasing density in the pancreatic tumor region in the mice for CRKL targeted BASP loaded with SPION treatment. Furthermore, the mice were imaged and validated the biodistribution and concentration of nanoparticles using a 9.4T MRI scanner. MRI images reveal nanoparticles accumulation in the region of tumor growth in the pancreas of mice at post-injection with CRKL targeted BASP loaded with SPION. This innovative multifunctional nanosystem has the potential to deliver a novel platform for both imaging and therapy for patients with PNETs as well as other cancers. Citation Format: Ziqiang Yuan, S Srinivasab, Jenolyn Francisca Alexander, X Liu, Wadih Arap, Renata Pasqualini, Mauro Ferrari, Steven K. Libutti, Biana Godin Vilentchouk. Bacteriophage associated silicon particles (BASP) for targeting pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3911.

In Vivo Proton Magnetic Resonance Spectroscopy of Pancreatic Neuroendocrine Tumors (PNETs) in a Multiple Endocrine Neoplasia Type 1 (MEN1) Conditional Knockout Mouse Model

MR spectroscopy (MRS) can improve diagnosis and follow treatment in cancer. However, no study has yet reported application of in vivo 1H‐MRS in malignant pancreatic lesions. This study quantitatively determined whether in vivo 1H‐MRS on multiple endocrine neoplasia type 1 (Men1) conditional knockout (KO) mice and their wild type (WT) littermates could detect differences in total choline (tCho) levels between tumor and control pancreas.