Peptide Aptamers Targeting Mutant p53 Induce Apoptosis in Tumor Cells

Cancer Research 68, 6550-6558, August 15, 2008. doi: 10.1158/0008-5472.CAN-08-0137

Mutations in the p53 tumor suppressor gene frequently result in expression of p53 point mutants that accumulate in cancer cells and actively collaborate with tumor progression through the acquisition of novel properties. Interfering with mutant p53 functions may represent a valid alternative for blocking tumor growth and development of aggressive phenotypes. The interactions and activities of selected proteins can be specifically modulated by the binding of peptide aptamers (PA). In the present work, we isolated PAs able to interact more efficiently with p53 conformational mutants compared with wild-type p53. The interaction between mutant p53 and PAs was further characterized using molecular modeling. Transient expression of PAs was able to reduce the transactivation activity of mutant p53 and to induce apoptosis specifically in cells expressing mutant p53. These PAs could provide a potential strategy to inhibit the oncogenic functions of mutant p53 and improve mutant p53-targeted cancer therapies. [Cancer Res 2008;68(16):6550–8]

 

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The Nucleolin Targeting Aptamer AS1411 Destabilizes Bcl-2 Messenger RNA in Human Breast Cancer Cells

Cancer Research 68, 2358-2365, April 1, 2008

 

We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stabilityin MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 µmol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 µmol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 µmol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death. [Cancer Res 2008;68(7):2358–65]

 

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Detecting Simulated Patterns of Lung Cancer Biomarkers by Random Network of Single-Walled Carbon Nanotubes Coated with Nonpolymeric Organic Materials

An array of chemiresistive random network of single-walled carbon nanotubes coated with nonpolymeric organic materials shows a high potential for diagnosis of lung cancer via breath samples. The sensors array shows excellent discrimination between the volatile organic compounds (VOCs) found in the breath of patients with lung cancer, relative to healthy controls, especially if the sensors array is preceded with either water extractor and/or preconcentrator of VOCs. The pattern compositions of the healthy and cancerous states were determined by gas-chromatography linked with mass-spectroscopy (GC-MS) analysis of real exhaled breath. 

http://pubs.acs.org/cgi-bin/asap.cgi/nalefd/asap/pdf/nl801577u.pdf?isMac=477783

Drug Delivery with Carbon Nanotubes for In vivo Cancer Treatment

Zhuang Liu, Kai Chen, Corrine Davis, Sarah Sherlock, Qizhen Cao, Xiaoyuan Chen and Hongjie Dai

Key Words: Carbon nanotubes • paclitaxel • drug delivery • cancer therapy • nanobiotechnology

Chemically functionalized single-walled carbon nanotubes (SWNT) have shown promise in tumor-targeted accumulation in mice and exhibit biocompatibility, excretion, and little toxicity. Here, we show in vivo SWNT drug delivery for tumor suppression in mice. We conjugate paclitaxel (PTX), a widely used cancer chemotherapy drug, to branched polyethylene glycol chains on SWNTs via a cleavable ester bond to obtain a water-soluble SWNT-PTX conjugate. SWNT-PTX affords higher efficacy in suppressing tumor growth than clinical Taxol in a murine 4T1 breast cancer model, owing to prolonged blood circulation and 10-fold higher tumor PTX uptake by SWNT delivery likely through enhanced permeability and retention. Drug molecules carried into the reticuloendothelial system are released from SWNTs and excreted via biliary pathway without causing obvious toxic effects to normal organs. Thus, nanotube drug delivery is promising for high treatment efficacy and minimum side effects for future cancer therapy with low drug doses.

[Cancer Res 2008;68(16):6652–60]

A Stochastic Model for Cancer Stem Cell Origin in Metastatic Colon Cancer

Christine Odoux, Helene Fohrer, Toshitaka Hoppo, Lynda Guzik, Donna Beer Stolz, Dale W. Lewis, Susanne M. Gollin, T. Clark Gamblin, David A. Geller and Eric Lagasse

Key Words: Chromosomal Instability in Cancer Stem Cells

Human cancers have been found to include transformed stem cells that may drive cancer progression to metastasis. Here, we report that metastatic colon cancer contains clonally derived tumor cells with all of the critical properties expected of stem cells, including self-renewal and the ability to differentiate into mature colon cells. Additionally, when injected into mice, these cells initiated tumors that closely resemble human cancer. Karyotype analyses of parental and clonally derived tumor cells expressed many consistent (clonal) along with unique chromosomal aberrations, suggesting the presence of chromosomal instability in the cancer stem cells. Thus, this new model for cancer origin and metastatic progression includes features of both the hierarchical model for cancerous stem cells and the stochastic model, driven by the observation of chromosomal instability. 

[Cancer Res 2008;68(17):6932–41]

Targeting V600EB-Raf and Akt3 Using Nanoliposomal-Small Interfering RNA Inhibits Cutaneous Melanocytic Lesion Development

Melissa A. Tran, Raghavendra Gowda, Arati Sharma1, Eun-Joo Park, James Adair, Mark Kester, Nadine Barrie Smith and Gavin P. Robertson

Key Words: melanoma • ultrasound • B-Raf • nanoliposomes • siRNA

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and 60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation (V600EB-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, 70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting V600EB-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound–mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting V600EB-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting V600EB-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of V600EB-Raf and Akt3 led to a cooperatively acting 65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting V600EB-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas. 

[Cancer Res 2008;68(18):7638–49]

Quantitative Molecular Magnetic Resonance Imaging of Tumor Angiogenesis Using cNGR-Labeled Paramagnetic Quantum Dots

Marlies Oostendorp, Kim Douma, Tilman M. Hackeng, Anouk Dirksen, Mark J. Post, Marc A.M.J. van Zandvoort and Walter H. Backes

 

Key Words: molecular magnetic resonance imaging • tumor angiogenesis • quantum dots • cNGR • two-photon laser scanning microscopy

 

The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor-bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and decreased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature. 

[Cancer Res 2008;68(18):7676–83]

New Insights into Tumor Microstructure Using Temporal Diffusion Spectroscopy

Cancer Research, 68, 2008, 5941

Magnetic resonance images (MRI) that depict rates of water diffusion in tissues can be used to characterize the cellularity of tumors and are valuable in assessing their early response to treatment. Water diffusion rates are sensitive to the cellular and molecular content of tissues and are affected by local microstructural changes associated with tumor development. However, conventional maps of water diffusion reflect the integrated effects of restrictionsto free diffusion at multiple scales up to a specific limiting spatial dimension, typically several micrometers. Such measurements cannot distinguish effects caused by structural variations at a smaller scale. Variations in diffusion rates then largely reflect variations in the density of cells, and no information is available about changes on a subcellular scale. We report here our experiences using a new approach based on Oscillating Gradient Spin-Echo (OGSE) MRI methods that can differentiate the influence on water diffusion of structural changes on scales much smaller than the diameter of a single cell. MRIs of glioblastomas in rat brain in vivo show an increased contrast and spatial heterogeneity when diffusion measurements are selectively sensitized to shorter distance scales. These results show the benefit of OGSE methods for revealing microscopic variations in tumors in vivo and confirm that diffusion measurements depend on factors other than cellularity.

Cell Lineage Analysis of a Mouse Tumor

Cancer Research, 68, 2008, 5924

Revealing the lineage relations among cancer cells can shed light on tumor growth patterns and metastasis formation, yet cell lineages have been difficult to come by in the absence of a suitable method. We previously developed a method for reconstructing cell lineage trees from genomic variability caused by somatic mutations. Here, we apply the method to cancer and reconstruct, for the first time, a lineage tree of neoplastic and adjacent normal cells obtained by laser microdissection from tissue sections of a mouse lymphoma. Analysis of the reconstructed tree reveals that the tumor initiated from a single founder cell, 5 months before diagnosis, that the tumor grew in a physically coherent manner, and that the average number of cell divisions accumulated in cancerous cells was almost twice than in adjacent normal lung epithelial cells but slightly less than the expected figure for normal B lymphocytes. The cells were also genotyped at the TP53 locus, and neoplastic cells were found to share a common mutation, which was most likely present in a heterozygous state. Our work shows that the ability to obtain data regarding the physical appearance, precise anatomic position, genotypic profile, and lineage position of single cells may be useful for investigating cancer development, progression, and interaction with the microenvironment. 

Monitoring Serial Changes in Circulating Human Breast Cancer Cells in Murine Xenograft Models

Cancer Research, 68, 2008, 5529

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelialcancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy.