Alexander Baras

A 20-gene model for molecular nodal staging of bladder cancer: development and prospective assessment

BACKGROUND Neoadjuvant chemotherapy before cystectomy confers a survival benefit in bladder cancer, but it has not been widely adopted since most patients do not benefit and we are at present unable to predict those that do. Since the most important predictor of recurrence after cystectomy is pathologically positive nodes, our aim was to assess techniques that define this stage for the selection of patients for neoadjuvant chemotherapy. METHODS We developed a gene expression model (GEM) to predict the pathological node status in primary tumour tissue from three independent cohorts of patients who were clinically node negative. From a subset of transcripts detected faithfully by microarrays from both paired frozen and formalin-fixed tissues (32 pairs), we developed both the GEM and cutoffs that identified patient strata with raised risk of nodal involvement by use of two separate training cohorts (90 and 66 patients). We then assessed the GEM and cutoffs to predict node-positive disease in tissues from a phase 3 trial cohort (AUO-AB-05/95; 185 patients). FINDINGS We developed a 20-gene GEM with an area under the curve of 0·67 (95% CI 0·60-0·75) for prediction of nodal disease at cystectomy in AUO-AB-05/95. The cutoff system identified patients with high relative risk (1·74, 95% CI 1·03-2·93) and low relative risk (0·70, 95% CI 0·51-0·96) of node-positive disease. Multivariate logistic regression showed the GEM predictor was independent of age, sex, pathological stage, and lymphovascular space invasion (coefficient 9·81, 95% CI 1·64-18·00; p=0·019). INTERPRETATION Selecting patients for neoadjuvant chemotherapy on the basis of risk of node-positive disease has the potential to benefit high-risk patients while sparing other patients toxic effects and delay to cystectomy. FUNDING US National Cancer Institute (R01CA143971).

Knockdown of Sox4 expression by RNAi induces apoptosis in ACC3 cells

Microarray RNA gene expression profiling analysis has shown that Sox4 (Sry-related high mobility group (HMG) box 4) is one of the most upregulated genes in adenoid cystic carcinoma (ACC), relative to non-neoplastic tissue of origin. Here, we show that Sox4 protein is similarly upregulated in ACC by immunohistochemistry of 28 primary cancers and 20 normal tissues. To elucidate the functional significance of these findings, RNA interference (RNAi)-mediated RNA silencing was used to downregulate Sox4 expression in the ACC-derived cell line, ACC3. With confirmed knockdown of Sox4 protein, cell viability was reduced by 51%, with a corresponding increase of apoptosis to 85% as compared to 12% in controls. Apoptosis was confirmed by cell morphology, DNA fragmentation and flow cytometry. Cells could be rescued from the proapoptotic effects of Sox4 RNAi by co-transfection with a construct expressing functional Sox4. Microarray gene expression profiling of RNAi knockdown experiments shows that downregulation of Sox4-modulated expression of critical genes involved in apoptosis and cell cycle control. Overall, our findings suggest that Sox4 contributes to the malignant phenotype of ACC cells by promoting cell survival.

The COXEN Principle: Translating Signatures of In vitro Chemosensitivity into Tools for Clinical Outcome Prediction and Drug Discovery in Cancer

Substantial effort has been devoted to in vitro testing of candidate chemotherapeutic agents. In particular, the United States National Cancer Institute Developmental Therapeutics Program (NCI-DTP) Human Tumor Cell Line Screen has screened hundreds of thousands of compounds and extracts, for which data on more than 40,000 compounds tested on a panel of 60 cancer cell lines (NCI-60) are publically available. In tandem, gene expression profiling has brought about a sea change in our understanding of cancer biology, allowing discovery of biomarkers or signatures able to characterize, classify, and prognosticate clinical behavior of human tumors. Recent studies have used tumor profiling matched to clinical trial outcome data to derive gene expression models predicting therapeutic outcomes, though such efforts are costly, time-consuming, tumor type-specific, and not amenable to rare diseases. Furthermore, addition of new or established drugs to multidrug combinations in which such models are already available requires the entire model to be re-derived. Can the aforementioned in vitro testing platform, coupled to the universal language of genomics, be used to develop, a priori, gene expression models predictive of clinical outcomes? Recent advances, including the CO-eXpression ExtrapolatioN (COXEN) algorithm, suggest that development of these models may be possible and raise important implications for future trial design and drug discovery.

Expression of Ral GTPases, Their Effectors, and Activators in Human Bladder Cancer

Purpose: The Ral family of small G proteins has been implicated in tumorigenesis, invasion, and metastasis in in vitro and animal model systems; however, a systematic evaluation of the state of activation, mutation, or expression of these GTPases has not been reported in any tumor type. Experimental Design: We determined the activation state of the RalA and RalB paralogs in 10 bladder cancer cell lines with varying Ras mutation status. We sequenced RalA and RalB cDNAs from 20 bladder cancer cell lines and functionally evaluated the mutations found. We determined the expression of Ral, Ral activators, and Ral effectors on the level of mRNA or protein in human bladder cancer cell lines and tissues. Results: We uncovered one E97Q substitution mutation of RalA in 1 of 20 cell lines tested and higher Ral activation in cells harboring mutant HRAS. We found overexpression of mRNAs for RalA and Aurora-A, a mitotic kinase that activates RalA, in bladder cancer (both P < 0.001), and in association with tumors of higher stage and grade. RalBP1, a canonical Ral effector, mRNA and protein was overexpressed in bladder cancer (P < 0.001), whereas Filamin A was underexpressed (P = 0.004). We determined that RalA mRNA levels correlated significantly with protein levels (P < 0.001) and found protein overexpression of both GTPases in homogenized invasive cancers. Available data sets suggest that RalA mRNA is also overexpressed in seminoma, glioblastoma, and carcinomas of the liver, pancreas, and prostate. Conclusion: These findings of activation and differential expression of RalA and RalB anchor prior work in model systems to human disease and suggest therapeutic strategies targeting both GTPases in this pathway may be beneficial.

Development and characterization of xenograft model systems for adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common malignancies to arise in human salivary glands, and it also arises in the glandular tissue of other organ systems. To address the paucity of experimental model systems for this tumor type, we have undertaken a program of transplanting tissue samples of human ACC into immunodeficient nu/nu mice to create xenograft model systems. In 17 of 23 attempts (74%), xenograft tumors were successfully grown. In all cases, the histologic appearance of the donating tumor was recapitulated in the subsequent xenograft. Characterization of a subset of xenograft models by immunohistochemical biomarkers and by RNA transcript microarray analysis showed good fidelity in the recapitulation of gene expression patterns in the xenograft tumors compared with the human donor tumors. As ACC is known to frequently contain a t(6;9) translocation that fuses the MYB and NFIB genes, fluorescence in situ hybridization (FISH) of 12 ACC xenograft models was performed that assayed MYB locus break-apart and MYB–NFIB locus fusion. Of 12 xenograft models, 11 (92%) revealed MYB locus rearrangement and 10 (83%) showed evidence of fusion of the MYB and NFIB loci. The two related xenograft models (derived from primary and metastatic tumors, respectively, of the same human subject) were karyotyped, showing a t(1;6) translocation, suggesting MYB translocation to a novel fusion partner gene. Overall, our results indicate that ACC is amenable to xenografting and that ACC xenograft models recapitulate the molecular and morphologic characteristics of human tumors, suggesting utility as valid experimental and preclinical model systems for this disease.

Transcriptional signatures of Ral GTPase are associated with aggressive clinicopathologic characteristics in human cancer

RalA and RalB are small GTPases that support malignant development and progression in experimental models of bladder, prostate, and squamous cancer. However, demonstration of their clinical relevance in human tumors remains lacking. Here, we developed tools to evaluate Ral protein expression, activation, and transcriptional output and evaluated their association with clinicopathologic parameters in common human tumor types. To evaluate the relevance of Ral activation and transcriptional output, we correlated RalA and RalB activation with the mutational status of key human bladder cancer genes. We also identified and evaluated a transcriptional signature of genes that correlates with depletion of RalA and RalB in vivo. The Ral transcriptional signature score, but not protein expression as evaluated by immunohistochemistry, predicted disease stage, progression to muscle invasion, and survival in human bladder cancers and metastatic and stem cell phenotypes in bladder cancer models. In prostate cancer, the Ral transcriptional signature score was associated with seminal vesicle invasion, androgen-independent progression, and reduced survival. In squamous cell carcinoma, this score was decreased in cancer tissues compared with normal mucosa, validating the experimental findings that Ral acts as a tumor suppressor in this tumor type. Together, our findings show the clinical relevance of Ral in human cancer and provide a rationale for the development of Ral-directed therapies.

Concurrent loss of heterozygosity and copy number analysis in adenoid cystic carcinoma by SNP genotyping arrays

Adenoid cystic carcinoma (ACC) is one of the most common malignancies to arise in the salivary glands, yet very little is known of the genetic alterations that are involved in the pathogenesis of this disease. To further examine the genetic changes that underlie ACC, we analyzed genomic DNA obtained from 22 primary ACC and two ACC-derived cell lines by high-density oligonucleotide single-nucleotide polymorphism genotyping arrays (Affymetrix GeneChip® Human Mapping 100K Set). Allelotype calls were analyzed by the Haplotype Correction version of the Linkage Disequilibrium Hidden Markov Model to determine loss of heterozygosity using information derived only from tumor samples. Comparison of data obtained from matched tumor-normal samples suggested that only deletion calls of >3 Mb were reliable. Within these parameters, ACC samples revealed a mean of three deletions per tumor, and no consensus areas of deletion were observed across the majority of tumors. Similarly, copy number analysis of primary hybridization data revealed no consensus areas of gene amplification. This is in contrast to a much higher rate of genomic alterations detected in a cohort of squamous carcinomas analyzed by the same methods. Our data show that most ACC have predominantly stable genomes, which is consistent with the theory that telomere crisis does not play a significant role in early stages of ACC tumor progression. Our data suggest that gene mutation and/or epigenetic events that cannot be detected by assay of gross alteration of chromosomal structure are likely to underlie the malignant transformation events of this tumor type.

Combined genomic and gene expression microarray profiling identifies ECOP as an upregulated gene in squamous cell carcinomas independent of DNA amplification

To identify dysregulated genes that may play a role in the pathogenesis of tobacco-related human squamous cell carcinoma (SCC), a cohort of SCCs from smokers (29 SCC of the head and neck, 3 SCC of the esophagus and 46 SCC of the lungs) were concomitantly analyzed for gene expression using Affymetrix U133A 2.0 arrays and for genomic variation using Affymetrix Human Mapping 100 K set. Gene expression profiling clearly separated benign squamous mucosa (BSM) from SCC and identified several candidate genes relevant to the biology of SCC. The single-nucleotide polymorphism array data adapted for copy number analysis identified two discrete areas of high-level genomic amplification, including 7p11.2 (EGFR (epidermal growth factor receptor)) and 11q13.3 (CCND1 (cyclin D1)). When gene expression measures were correlated with amplification status at 7p11.2 locus, EGFR overexpression in relation to benign tissue was dependent on amplification and occurred in only 9% of cases. However, an adjacent gene (∼0.4 Mb), EGFR-co-amplified and overexpressed protein (ECOP), showed strong over-expression in the majority (90%) of SCCs regardless of gene amplification status. This finding was corroborated with quantitative real-time PCR assays and protein immunoblots. Interestingly, small interfering RNA-mediated knockdown of ECOP gene products in a SCC cell line (SCC-9) resulted in increased cell death. The results of these studies identify ECOP as a protein relevant to the biology of SCC.

Intracellular localization of GASP/ECOP/VOPP1

Vesicular Over-expressed in cancer Prosurvival Protein 1 (VOPP1), also known as Glioblastoma Amplified and Secreted Protein and EGFR-Coamplified and Over-expressed Protein has been previously shown to be over-expressed in human glioblastoma multiforme and squamous cell carcinoma. Additionally, previous experimental work suggests that it confers a prosurvival cellular phenotype. A query of a public database of gene expression profiling data (Oncomine™) shows that the VOPP1 transcript is also highly expressed in several other common human cancers, including breast carcinoma, pancreatic carcinoma, and lymphoma. Analysis of VOPP1 sequence structure shows both a signal sequence and a transmembrane domain, and examination of a public microarray dataset for endoplasmic reticulum (ER)-bound mRNA transcripts is consistent with the VOPP1 protein product being synthesized into the ER. Immunoblot analysis of cell culture and conditioned media confirms that the protein product is not secreted and is retained intracellularly. VOPP1 protein tagged with a fluorescence reporter, as well as antibody-mediated visualization of recombinant and native forms of the protein reveals an intracellular vesicular pattern of localization. Co-localization experiments reveal that VOPP1 vesicles do not co-localize with mitochondria or peroxisomes, but show partial co-localization with perinuclear lysosomes. Additionally, markers of endocytosis and autophagy show partial perinuclear co-localization, suggesting that VOPP1-containing vesicles enter final common pathways of the lysosomal system. These findings throw into doubt the hypothesis that VOPP1 interacts directly with cytoplasmic mediators of the NF kappa B pathway, and suggest that the prosurvival phenotype conferred by this gene product is mediated by other mechanisms.

Loss of VOPP1 overexpression in squamous carcinoma cells induces apoptosis through oxidative cellular injury.

The vesicular overexpressed in cancer prosurvival protein 1 (VOPP1) gene product (previously known as GASP and ECOP) has a poorly characterized functional role in cancer cells, although its expression levels are known to be elevated in many cancer types. To determine the role that VOPP1 has in human squamous cell carcinoma (SCC), a series of siRNA-mediated expression knockdown experiments were performed in carcinoma-derived model systems with confirmed endogenous VOPP1 overexpression (three SCC-derived cell lines: SCC-9, FaDu, and H2170, as well as the cervical adenocarcinoma HeLa cell line, which has been examined in relevant previous reports). The data indicate that VOPP1 knockdown induces cell death at 72 h post-transfection and this is caused by the induction of apoptosis via the intrinsic pathway. Analysis of microarray gene expression profiling showed that genes whose expression was affected by VOPP1 knockdown exhibited enrichment in annotations of oxidative stress and mitochondrial dysfunction. Reporters of reactive oxygen species (ROS) and mitochondrial membrane potential show that ROS levels become elevated and mitochondrial dysfunction occurs with VOPP1 knockdown at time points before the activation of effector caspases and cell death seen at later time points. Furthermore, the introduction of the antioxidant N-acetyl cysteine was able to abrogate the induction of apoptosis observed with VOPP1 knockdown in a dose-responsive manner. Reporter constructs for NF-κB-mediated transcription are not affected in SCC cell lines by VOPP1 knockdown. Taken together, these data support the hypothesis that VOPP1 overexpression in cancer participates in the control of the intracellular redox state, and that its loss leads to oxidative cellular injury leading to cell death by the intrinsic apoptotic pathway.