Paul C. Park

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer

BackgroundThe epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.MethodsAgilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.ResultsMorphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects.ConclusionsThis work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Evidence of multifocality of telomere erosion in high-grade prostatic intraepithelial neoplasia (HPIN) and concurrent carcinoma.

Mechanisms underlying prostate cancer (CaP) initiation and progression are poorly understood. A chromosomal instability mechanism leading to the generation of numerical and structural chromosomal changes has been implicated in the preneoplastic and neoplastic stages of CaP. Telomere dysfunction is one potential mechanism associated with the onset of such instability. To determine whether there was alteration in telomere length and chromosome number, 15 paraffin-embedded prostatectomy specimens were investigated using quantitative peptide nucleic acid (PNA) FISH analysis of representative foci of carcinoma, putative precancerous lesions (high-grade prostatic intraepithelial neoplasia, HPIN) and nondysplastic prostate epithelium. A significant decrease in telomere length was shown in both HPIN and CaP in comparison with normal epithelium. In addition, elevated rates of aneusomy suggested that increased levels of chromosomal aberrations were associated with decreased telomere length. Moreover, multiple foci of HPIN were shown to have a heterogeneous overall reduction of telomere length. This reduction was more evident in the histologic regions of the prostate containing CaP. Such observations lend support to the hypothesis that telomere erosion may be a consistent feature of CaP oncogenesis and may also be associated with the generation of chromosomal instability that characterizes this malignancy.

The breakage–fusion–bridge (BFB) cycle as a mechanism for generating genetic heterogeneity in osteosarcoma

Osteosarcoma (OS) is characterized by chromosomal instability and high copy number gene amplification. The breakage–fusion–bridge (BFB) cycle is a well-established mechanism of genome instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. To determine whether the BFB cycle could be increasing the de novo rate of formation of cytogenetic aberrations in OS, the frequency of anaphase bridge configurations and dicentric chromosomes in four OS cell lines was quantified. An increased level of anaphase bridges and dicentrics was observed in all the OS cell lines. There was also a strong association between the frequencies of anaphase bridges, dicentrics, centrosomal anomalies, and multipolar mitotic figures in all the OS cell lines, indicating a possible link in the mechanisms that led to the structural and numerical instabilities observed in OS. In summary, this study has provided strong support for the role of the BFB cycle in generating the extensive structural chromosome aberrations, as well as cell-to-cell cytogenetic variation observed in OS, thus conferring the genetic diversity for OS tumor progression.

Array comparative genomic hybridization in osteosarcoma.

Osteosarcoma, the most frequent primary bone tumor, is a malignant mesenchymal sarcoma with a peak incidence in young children and adolescents. Left untreated, it progresses relentlessly to local and systemic disease, ultimately leading to death within months. Genomically, osteosarcomas are aneuploid with chaotic karyotypes, lacking the pathognomonic genetic rearrangements characteristic of most sarcomas. The familial genetics of osteosarcoma helped in elucidating some of the etiological molecular disruptions, such as the tumor suppressor genes RB1 in retinoblastoma and TP53 in Li-Fraumeni, and RECQL4 involved in DNA repair/replication in Rothmund-Thomson syndrome. Genomic profiling approaches such as array comparative genomic hybridization (aCGH) have provided additional insights concerning the mechanisms responsible for generating complex osteosarcoma genomes. This chapter provides a brief introduction to the clinical features of conventional osteosarcoma, the predominant subtypes, and a general overview of materials and analytical methods of osteosarcoma aCGH, followed by a more detailed literature overview of aCGH studies and a discussion of emerging genes, molecular mechanisms, and their clinical implications, as well as more recent application of integrative genomics in osteosarcoma. aCHG is helping elucidate genomic events leading to tumor development and evolution as well as identification of prognostic markers and therapeutic targets in osteosarcoma.

MOLECULAR CHARACTERIZATION OF GLEASON PATTERNS 3 AND 4 PROSTATE CANCER USING REVERSE WARBURG EFFECT-ASSOCIATED GENES

BackgroundGleason scores (GS) 3+3 and 3+4 prostate cancers (PCa) differ greatly in their clinical courses, with Gleason pattern (GP) 4 representing a major independent risk factor for cancer progression. However, Gleason grade is not reliably ascertained by diagnostic biopsy, largely due to sampling inadequacies, subjectivity in the Gleason grading procedure, and a lack of more objective biomarker assays to stratify prostate cancer aggressiveness. In most aggressive cancer types, the tumor microenvironment exhibits a reciprocal pro-tumorigenic metabolic phenotype consistent with the reverse Warburg effect (RWE). The RWE can be viewed as a physiologic response to the epithelial phenotype that is independent of both the epithelial genotype and of direct tumor sampling. We hypothesize that differential expression of RWE-associated genes can be used to classify Gleason pattern, distinguishing GP3 from GP4 PCa foci.MethodsGene expression profiling was conducted on RNA extracted from laser-capture microdissected stromal tissue surrounding 20 GP3 and 21 GP4 cancer foci from PCa patients with GS 3+3 and GS ≥4+3, respectively. Genes were probed using a 102-gene NanoString probe set targeted towards biological processes associated with the RWE. Differentially expressed genes were identified from normalized data by univariate analysis. A top-scoring pair (TSP) analysis was completed on raw gene expression values. Genes were analyzed for enriched Gene Ontology (GO) biological processes and protein-protein interactions using STRING and GeneMANIA.ResultsUnivariate analysis identified nine genes (FOXO1 (AUC: 0.884), GPD2, SPARC, HK2, COL1A2, ALDOA, MCT4, NRF2, and ATG5) that were differentially expressed between GP3 and GP4 stroma (p<0.05). However, following correction for false discovery, only FOXO1 retained statistical significance at q<0.05. The TSP analysis identified a significant gene pair, namely ATG5/GLUT1. Greater expression of ATG5 relative to GLUT1 correctly classified 77.4 % of GP3/GP4 samples. Enrichment for GO-biological processes revealed that catabolic glucose processes and oxidative stress response pathways were strongly associated with GP3 foci but not GP4. FOXO1 was identified as being a primary nodal protein.ConclusionsWe report that RWE-associated genes can be used to distinguish between GP3 and GP4 prostate cancers. Moreover, we find that the RWE response is downregulated in the stroma surrounding GP4, possibly via modulation of FOXO1.

Immunohistochemical Assessment of Expression of Centromere Protein—A (CENPA) in Human Invasive Breast Cancer

Abnormal cell division leading to the gain or loss of entire chromosomes and consequent genetic instability is a hallmark of cancer. Centromere protein –A (CENPA) is a centromere-specific histone-H3-like variant gene involved in regulating chromosome segregation during cell division. CENPA is one of the genes included in some of the commercially available RNA based prognostic assays for breast cancer (BCa)—the 70 gene signature MammaPrint® and the five gene Molecular Grade Index (MGISM). Our aim was to assess the immunohistochemical (IHC) expression of CENPA in normal and malignant breast tissue. Clinically annotated triplicate core tissue microarrays of 63 invasive BCa and 20 normal breast samples were stained with a monoclonal antibody against CENPA and scored for percentage of visibly stained nuclei. Survival analyses with Kaplan–Meier (KM) estimate and Cox proportional hazards regression models were applied to assess the associations between CENPA expression and disease free survival (DFS). Average percentage of nuclei visibly stained with CENPA antibody was significantly higher (p = 0.02) in BCa than normal tissue. The 3-year DFS in tumors over-expressing CENPA (>50% stained nuclei) was 79% compared to 85% in low expression tumors (<50% stained nuclei). On multivariate analysis, IHC expression of CENPA showed weak association with DFS (HR > 60.07; p = 0.06) within our small cohort. To the best of our knowledge, this is the first published report evaluating the implications of increased IHC expression of CENPA in paraffin embedded breast tissue samples. Our finding that increased CENPA expression may be associated with shorter DFS in BCa supports its exploration as a potential prognostic biomarker.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores

Cancer biomarker studies often require nucleic acid extraction from limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissues, such as histologic sections or needle cores. A major challenge is low quantity and quality of extracted nucleic acids, which can limit our ability to perform genetic analyses, and have a significant influence on overall study design. This study was aimed at identifying the most reliable and reproducible method of obtaining sufficient high-quality nucleic acids from FFPE tissues. We compared the yield and quality of nucleic acids from 0.6-mm FFPE prostate tissue cores across 16 DNA and RNA extraction protocols, using 14 commercially available kits. Nucleic acid yield was determined by fluorometry, and quality was determined by spectrophotometry. All protocols yielded nucleic acids in quantities that are compatible with downstream molecular applications. However, the protocols varied widely in the quality of the extracted RNA and DNA. Four RNA and five DNA extraction protocols, including protocols from two kits for dual-extraction of RNA and DNA from the same tissue source, were prioritized for further quality assessment based on the yield and purity of their products. Specifically, their compatibility with downstream reactions was assessed using both NanoString nCounter gene expression assays and reverse-transcriptase real-time PCR for RNA, and methylation-specific PCR assays for DNA. The kit deemed most suitable for FFPE tissue was the AllPrep kit by Qiagen because of its yield, quality, and ability to purify both RNA and DNA from the same sample, which would be advantageous in biomarker studies.

Abstract A53: Biomarkers of chemotherapy resistance in serous epithelial ovarian cancer identified by integrative genomic and transcriptomic analysis

Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression and copy number profiling to delineate major deregulated pathways and biomarker networks associated with the development of intrinsic chemotherapy resistance with exposure to standard first-line therapy for ovarian cancer. The study cohort comprised 28 high grade serous ovarian cancer patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. Twelve patient tumors demonstrating relative resistance to platinum based chemotherapy corresponding to shorter PFS (less than 6 months) were compared to 16 tumors from platinum-sensitive patients (PFS more than 18months). Molecular profiling was performed using Affymetrix high-resolution microarray platforms to permit global comparisons of gene expression levels and copy number profiles between tumors from the resistant group with the sensitive group. Microarray data analysis using statistical methods revealed a set of 204 discriminating genes of which expression levels may be influencing differential chemotherapy response between the two groups. Pathway analysis of the differentiating genes showed IGF1 network to be significantly altered between the two groups in addition to PI3K, NFkB, distinguishing the chemotherapy resistant with the sensitive group. Copy number analysis showed differences in the chromosomal regions, 4q31.22, 5q13.2, 9p24.3, 2p23.2, 16q21, 6q14.1, 7p22.3, 12p13 and Xq. Integrative copy number and gene expression profiling will delineate the drivers of chemotherapy resistance in patients undergoing standard platinum-based treatment of ovarian cancer. Future studies to validate these markers are necessary to apply this knowledge to biomarker-based clinical trials. Citation Format: Madhuri Koti, Robert J. Gooding, Paulo Nuin, Alexandria Haslehurst, Colleen Crane, Johanne Weberpals, Timothy Chids, Peter Bryson, Moyez Dharsee, Kenneth R. Evans, Harriet E. Feilotter, Paul C. Park, Jeremy A. Squire. Biomarkers of chemotherapy resistance in serous epithelial ovarian cancer identified by integrative genomic and transcriptomic analysis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A53.

Abstract 810: Integrative genomic and transcriptomic analysis in idenfitication of biomarkers of chemoresistance in serous epithelial ovarian cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression and copy number profiling to delineate major deregulated pathways and biomarker networks associated with the development of intrinsic chemotherapy resistance with exposure to standard first-line therapy for ovarian cancer. The study cohort comprised 28 high grade serous ovarian cancer patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. Twelve patient tumors demonstrating relative resistance to platinum based chemotherapy corresponding to shorter PFS (less than 6 months) were compared to 16 tumors from platinum-sensitive patients (PFS more than 18months). Molecular profiling was performed using Affymetrix high-resolution microarray platforms to permit global comparisons of gene expression levels and copy number profiles between tumors from the resistant group with the sensitive group. Microarray data analysis revealed a set of 227 discriminating genes of which expression levels may be influencing differential chemotherapy response between the two groups. Pathway analysis of these genes showed the,PI3K,NFkB and IGF1 networks as some of the significant networks distinguishing the chemotherapy resistant with the sensitive group. Copy number analysis performed using Nexus copy number version 6.1 revealed differences in the chromosomal regions, 4q31.22, 5q13.2, 9p24.3, 2p23.2, 16q21, 6q14.1, 7p22.3, 12p13 and Xq. Integrative copy number and gene expression profiling will delineate the drivers of chemotherapy resistance in patients undergoing standard platinum-based treatment of ovarian cancer. Future studies to validate these markers are necessary to apply this knowledge to biomarker-based clinical trials. Citation Format: Madhuri Koti, Robert J. Gooding, Paulo Nuin, Alexandria Haslehurst, Colleen Crane, Johanne Weberpals, Timothy Childs, Peter Bryson, Moyez Dharsee, Kenneth Evans, Harriet E. Feilotter, Paul C. Park, Jeremy A. Squire. Integrative genomic and transcriptomic analysis in idenfitication of biomarkers of chemoresistance in serous epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2013-810