Nicole L. Simone

Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.

Protein arrays are described for screening of molecular markers and pathway targets in patient matched human tissue during disease progression. In contrast to previous protein arrays that immobilize the probe, our reverse phase protein array immobilizes the whole repertoire of patient proteins that represent the state of individual tissue cell populations undergoing disease transitions. A high degree of sensitivity, precision and linearity was achieved, making it possible to quantify the phosphorylated status of signal proteins in human tissue cell subpopulations. Using this novel protein microarray we have longitudinally analysed the state of pro-survival checkpoint proteins at the microscopic transition stage from patient matched histologically normal prostate epithelium to prostate intraepithelial neoplasia (PIN) and then to invasive prostate cancer. Cancer progression was associated with increased phosphorylation of Akt (P<0.04), suppression of apoptosis pathways (P<0.03), as well as decreased phosphorylation of ERK (P<0.01). At the transition from histologically normal epithelium to PIN we observed a statistically significant surge in phosphorylated Akt (P<0.03) and a concomitant suppression of downstream apoptosis pathways which proceeds the transition into invasive carcinoma.

Laser-capture microdissection: opening the microscopic frontier to molecular analysis.

As the list of expressed human genes expands, a major scientific challenge is to understand the molecular events that drive normal tissue morphogenesis and the evolution of pathological lesions in actual tissue. Laser capture microdissection (LCM) has been developed to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections, in one step, under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA and proteins) is retained and held on the transfer film. With the advent of LCM, cDNA libraries can be developed from pure cells obtained directly from stained tissue, and microhybridization arrays of thousands of genes can now be used to examine gene expression in microdissected human tissue biopsies. The fluctuation of expressed genes or alterations in the cellular DNA that correlate with a particular disease stage can ultimately be compared within or between individual patients. Such a fingerprint of gene-expression patterns can provide crucial clues for etiology and might, ultimately, contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with a defined pathological lesion might serve as imaging ot therapeutic targets.

Ionizing Radiation-Induced Oxidative Stress Alters miRNA Expression

Background MicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H2O2). Methodology/Principal Findings Normal human fibroblasts were exposed to radiation, H2O2, or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H2O2 treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation. Conclusions These results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H2O2, and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.

Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip

The complicated, changing pattern of protein expression should contain important information about the pathologic process taking place in the cells of actual tissue. Utilization of this information for the selection of druggable targets could be possible if a means existed to rapidly analyze and display changes in protein expression in defined microscopic cellular subpopulations. As a demonstration of feasibility, we show the generation of sensitive, rapid, and reproducible molecular weight protein profiles of patient‐matched normal, premalignant, malignant, and metastatic microdissected cell populations from stained human esophageal, prostate, breast, ovary, colon, and hepatic tissue sections through the application of an affinity‐based biochip. Reproducible, discriminatory protein biomarker profiles can be obtained from as few as 25 cells in less than 5 min from dissection to the generation of the protein fingerprint. Furthermore, these protein pattern profiles reveal reproducible changes in expression as cells undergo malignant transformation, and are discriminatory for different tumor types. Consistent protein changes were identified in the microdissected cells from patient‐matched tumor and normal epithelium from eight out of eight different malignant esophageal tissue sets and three out of three malignant prostate tissue sets. A means to rapidly generate a display of expressed proteins from microscopic cellular populations sampled from tissue could be an important enabling technology for pharmacoproteomics, molecular pathology, drug intervention strategies, therapeutic assessment of drug entities, disease diagnosis, toxicity, and gene therapy monitoring. Drug Dev. Res. 49:34–42, 2000. Published 2000 Wiley‐Liss, Inc.

Sensitive Immunoassay of Tissue Cell Proteins Procured by Laser Capture Microdissection

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Laser Capture Microdissection: Beyond Functional Genomics to Proteomics

Proteomics will drive biology and medicine beyond genomics, and can have a profound impact on molecular diagnostics. The posttranslational modifications of cellular proteins that govern physiology and become deranged in disease cannot be accurately portrayed by gene expression alone. Consequently, new technology is being developed to discover, and quantitatively monitor, proteomic changes that are associated with disease etiology and progression. In the past, proteomic technologies were restricted to tumor cell lines or homogenized bulk tissue specimens. This source material may not accurately reflect molecular events taking place in the specific cells of the tissue itself. This article describes a completely new class of proteomic-based approaches aimed at the identification and investigation of protein markers in the actual histologically defined cell populations that are immersed in heterogeneous diseased tissue. It is envisioned that these investigations will eventually lead to novel diagnostic, prognostic, or therapeutic markers that can be applied to monitor therapeutic toxicity or efficacy.

Comparison of intensity-modulated radiotherapy, adaptive radiotherapy, proton radiotherapy, and adaptive proton radiotherapy for treatment of locally advanced head and neck cancer

BACKGROUND AND PURPOSE Various radiotherapy planning methods for locally advanced squamous cell carcinoma of the head and neck (SCCHN) have been proposed to decrease normal tissue toxicity. We compare IMRT, adaptive IMRT, proton therapy (IMPT), and adaptive IMPT for SCCHN. MATERIALS AND METHODS Initial and re-simulation CT images from 10 consecutive patients with SCCHN were used to quantify dosimetric differences between photon and proton therapy. Contouring was performed on both CTs, and plans (n=40 plans) and dose-volume histograms were generated. RESULTS The mean GTV volume decreased 53.4% with re-simulation. All plans provided comparable PTV coverage. Compared with IMRT, adaptive IMRT significantly reduced the maximum dose to the mandible (p=0.020) and mean doses to the contralateral parotid gland (p=0.049) and larynx (p=0.049). Compared with IMRT and adaptive IMRT, IMPT significantly lowered the maximum doses to the spinal cord (p<0.002 for both) and brainstem (p<0.002 for both) and mean doses to the larynx (p<0.002 for both) and ipsilateral (p=0.004 IMRT, p=0.050 adaptive) and contralateral (p<0.002 IMRT, p=0.010 adaptive) parotid glands. Adaptive IMPT significantly reduced doses to all critical structures compared with IMRT and adaptive IMRT and several critical structures compared with non-adaptive IMPT. CONCLUSIONS Although adaptive IMRT reduced dose to several normal structures compared with standard IMRT, non-adaptive proton therapy had a more favorable dosimetric profile than IMRT or adaptive IMRT and may obviate the need for adaptive planning. Protons allowed significant sparing of the spinal cord, parotid glands, larynx, and brainstem and should be considered for SCCHN to decrease normal tissue toxicity while still providing optimal tumor coverage.

Cellular Stress Induced Alterations in MicroRNA let-7a and let-7b Expression Are Dependent on p53

Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53−/− colon cancer cell line (HCT116) or ATM−/− human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53+/+ cells but not HCT116 p53−/− cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.

Early Tumor Progression Associated with Enhanced EGFR Signaling with Bortezomib, Cetuximab, and Radiotherapy for Head and Neck Cancer

Purpose: A phase I clinical trial and molecular correlative studies were conducted to evaluate preclinical evidence for combinatorial activity of the proteasome inhibitor bortezomib, the epidermal growth factor receptor (EGFR) inhibitor cetuximab, and radiation therapy. Experimental Design: Patients with radiotherapy-naive stage IV or recurrent squamous cell carcinoma of the head and neck (SCCHN) were studied. Escalating doses of bortezomib (0.7, 1.0, and 1.3 mg/m2) were given intravenously twice weekly on days 1, 4, 8, and 11, every 21 days, with weekly cetuximab beginning 1 week prior and concurrently with intensity-modulated radiotherapy, delivered in 2 Gy fractions to 70 to 74 Gy. Molecular effects were examined in serial serum and SCCHN tumor specimens and the cell line UMSCC-1. Results: Seven patients were accrued before the study was terminated when five of six previously untreated patients with favorable prognosis oropharyngeal SCCHN progressed within 1 year (progression-free survival = 4.8 months; 95% CI, 2.6–6.9). Three patients each received bortezomib 0.7 or 1.0 mg/m2, without dose-limiting toxicities; one patient treated at 1.3 mg/m2 was taken off study due to recurring cetuximab infusion reaction and progressive disease (PD). Expected grade 3 toxicities included radiation mucositis (n = 4), dermatitis (n = 4), and rash (n = 1). SCCHN-related cytokines increased in serial serum specimens of patients developing PD (P = 0.029). Bortezomib antagonized cetuximab- and radiation-induced cytotoxicity, degradation of EGFR, and enhanced prosurvival signal pathway activation in SCCHN tumor biopsies and UMSCC-1. Conclusions: Combining bortezomib with cetuximab and radiation therapy showed unexpected early progression, evidence for EGFR stabilization, increased prosurvival signaling, and SCCHN cytokine expression, warranting avoidance of this combination. Clin Cancer Res; 17(17); 5755–64. ©2011 AACR.

Caloric restriction augments radiation efficacy in breast cancer

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.