Kimberly J. Bussey

Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays

Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by using p-scan and a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation.

Membrane Transporters and Channels: Role of the Transportome in Cancer Chemosensitivity and Chemoresistance

Membrane transporters and channels (collectively the transportome) govern cellular influx and efflux of ions, nutrients, and drugs. We used oligonucleotide arrays to analyze gene expression of the transportome in 60 human cancer cell lines used by the National Cancer Institute for drug screening. Correlating gene expression with the potencies of 119 standard anticancer drugs identified known drug-transporter interactions and suggested novel ones. Folate, nucleoside, and amino acid transporters positively correlated with chemosensitivity to their respective drug substrates. We validated the positive correlation between SLC29A1 (nucleoside transporter ENT1) expression and potency of nucleoside analogues, azacytidine and inosine-glycodialdehyde. Application of an inhibitor of SLC29A1, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of these two drugs, indicating that SLC29A1 plays a role in cellular uptake. Three ABC efflux transporters (ABCB1, ABCC3, and ABCB5) showed significant negative correlations with multiple drugs, suggesting a mechanism of drug resistance. ABCB1 expression correlated negatively with potencies of 19 known ABCB1 substrates and with Baker’s antifol and geldanamycin. Use of RNA interference reduced ABCB1 mRNA levels and concomitantly increased sensitivity to these two drugs, as expected for ABCB1 substrates. Similarly, specific silencing of ABCB5 by small interfering RNA increased sensitivity to several drugs in melanoma cells, implicating ABCB5 as a novel chemoresistance factor. Ion exchangers, ion channels, and subunits of proton and sodium pumps variably correlated with drug potency. This study identifies numerous potential drug-transporter relationships and supports a prominent role for membrane transport in determining chemosensitivity. Measurement of transporter gene expression may prove useful in predicting anticancer drug response.

Asparagine synthetase as a causal, predictive biomarker for l-asparaginase activity in ovarian cancer cells

l-Asparaginase (l-ASP), a bacterial enzyme used since the 1970s to treat acute lymphoblastic leukemia, selectively starves cells that cannot synthesize sufficient asparagine for their own needs. Molecular profiling of the NCI-60 cancer cell lines using five different microarray platforms showed strong negative correlations of asparagine synthetase (ASNS) expression and DNA copy number with sensitivity to l-ASP in the leukemia and ovarian cancer cell subsets. To assess whether the ovarian relationship is causal, we used RNA interference to silence ASNS in three ovarian lines and observed 4- to 5-fold potentiation of sensitivity to l-ASP with two of the lines. For OVCAR-8, the line that expresses the least ASNS, the potentiation was >500-fold. Significantly, that potentiation was >700-fold in the multidrug-resistant derivative OVCAR-8/ADR, showing that the causal relationship between ASNS expression and l-ASP activity survives development of classical multidrug resistance. Tissue microarrays confirmed low ASNS expression in a subset of clinical ovarian cancers as well as other tumor types. Overall, this pharmacogenomic/pharmacoproteomic study suggests the use of l-ASP for treatment of a subset of ovarian cancers (and perhaps other tumor types), with ASNS as a biomarker for patient selection. [Mol Cancer Ther 2006;5(11):2613–23]

Mistaken Identifiers: Gene name errors can be introduced inadvertently when using Excel in bioinformatics

BackgroundWhen processing microarray data sets, we recently noticed that some gene names were being changed inadvertently to non-gene names.ResultsA little detective work traced the problem to default date format conversions and floating-point format conversions in the very useful Excel program package. The date conversions affect at least 30 gene names; the floating-point conversions affect at least 2,000 if Riken identifiers are included. These conversions are irreversible; the original gene names cannot be recovered.ConclusionsUsers of Excel for analyses involving gene names should be aware of this problem, which can cause genes, including medically important ones, to be lost from view and which has contaminated even carefully curated public databases. We provide work-arounds and scripts for circumventing the problem.

Multifactorial Regulation of E-Cadherin Expression: An Integrative Study

E-cadherin (E-cad) is an adhesion molecule associated with tumor invasion and metastasis. Its down-regulation is associated with poor prognosis for many epithelial tumor types. We have profiled E-cad in the NCI-60 cancer cell lines at the DNA, RNA, and protein levels using six different microarray platforms plus bisulfite sequencing. Here we consider the effects on E-cad expression of eight potential regulatory factors: E-cad promoter DNA methylation, the transcript levels of six transcriptional repressors (SNAI1, SNAI2, TCF3, TCF8, TWIST1, and ZFHX1B), and E-cad DNA copy number. Combined bioinformatic and pharmacological analyses indicate the following ranking of influence on E-cad expression: (1) E-cad promoter methylation appears predominant, is strongly correlated with E-cad expression, and shows a 20% to 30% threshold above which E-cad expression is silenced; (2) TCF8 expression levels correlate with (−0.62) and predict (P < 0.00001) E-cad expression; (3) SNAI2 and ZFHX1B expression levels correlate positively with each other (+0.83) and also correlate with (−0.32 and −0.30, respectively) and predict (P = 0.03 and 0.01, respectively) E-cad expression; (4) TWIST1 correlates with (−0.34) but does not predict E-cad expression; and (5) SNAI1 expression, TCF3 expression, and E-cad DNA copy number do not correlate with or predict E-cad expression. Predictions of E-cad regulation based on the above factors were tested and verified by demethylation studies using 5-aza-2′-deoxycytidine treatment; siRNA knock-down of TCF8, SNAI2, or ZFHX1B expression; and combined treatment with 5-aza-2′-deoxycytidine and TCF8 siRNA. Finally, levels of cellular E-cad expression are associated with levels of cell-cell adhesion and response to drug treatment. Mol Cancer Ther; 9(1); 1–16

ZNF367 Inhibits Cancer Progression and Is Targeted by miR-195

Background Several members of the zinc finger protein family have been recently shown to have a role in cancer initiation and progression. Zinc finger protein 367 (ZNF367) is a member of the zinc finger protein family and is expressed in embryonic or fetal erythroid tissue but is absent in normal adult tissue. Methodology/Principal Findings We show that ZNF367 is overexpressed in adrenocortical carcinoma, malignant pheochromocytoma/paraganglioma and thyroid cancer as compared to normal tissue and benign tumors. Using both functional knockdown and ectopic overexpression in multiple cell lines, we show that ZNF367 inhibits cellular proliferation, invasion, migration, and adhesion to extracellular proteins in vitro and in vivo. Integrated gene and microRNA expression analyses showed an inverse correlation between ZNF367 and miR-195 expression. Luciferase assays demonstrated that miR-195 directly regulates ZNF367 expression and that miR-195 regulates cellular invasion. Moreover, integrin alpha 3 (ITGA3) expression was regulated by ZNF367. Conclusions/Significance Our findings taken together suggest that ZNF367 regulates cancer progression.

Genomic and expression profiling of adrenocortical carcinoma: application to diagnosis, prognosis and treatment

Adrenocortical carcinoma (ACC) is an aggressive endocrine tumor with a poor 5-year survival rate of 10-20%. Current therapy is often ineffective and may be associated with intolerable side effects. Although ACC is extremely rare, recent advances in genomic and expression profiling, coupled with knowledge gained from the study of the inherited syndromes that increase ACC risk, are beginning to bring together a picture of a tumor type dependent on p53, the G2/M cell cycle transition and IGF2 stimulation. Nevertheless, ACC remains a heterogeneous disease. Only recently have sufficient tumors been characterized and results published to permit an exploration of this diversity. Advances in treatment will depend on exploiting those pathways already implicated in ACC, along with those yet to be identified, and testing those treatments in better models of the disease than the three cell lines that currently exist and are widely available to the community.

Preclinical investigation of nanoparticle albumin-bound paclitaxel as a potential treatment for adrenocortical cancer.

Background:Traditional drug discovery methods have a limited role in rare cancers. We hypothesized that molecular technology including gene expression profiling could expose novel targets for therapy in adrenocortical carcinoma (ACC), a rare and lethal cancer. SPARC (secreted protein acidic rich in cysteine) is an albumin-binding matrix-associated protein that is proposed to act as a mechanism for the increased efficacy of a nanoparticle albumin-bound preparation of the antimicrotubular drug Paclitaxel (nab-paclitaxel). Methods:The transcriptomes of 19 ACC tumors and 4 normal adrenal glands were profiled on Affymetrix U133 Plus2 expression microarrays to identify genes representing potential therapeutic targets. Immunohistochemical analysis for target proteins was performed on 10 ACC, 6 benign adenomas, and 1 normal adrenal gland. Agents known to inhibit selected targets were tested in comparison with mitotane in the 2 ACC cell lines (H295R and SW-13) in vitro and in mouse xenografts. Results:SPARC expression is increased in ACC samples by 1.56 ± 0.44 (&mgr; ± SD) fold. Paclitaxel and nab-paclitaxel show in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 &mgr;M and 0.0078 &mgr;M for paclitaxel and 0.35 &mgr;M and 0.0087 &mgr;M for nab-paclitaxel compared with mitotane concentrations of 15.9 &mgr;M and 46.4 &mgr;M. In vivo nab-paclitaxel treatment shows a greater decrease in tumor weight in both xenograft models than mitotane. Conclusions:Biological insights garnered through expression profiling of ACC tumors suggest further investigation into the use of nab-paclitaxel for the treatment of ACC.

Multi-institutional tumor banking: lessons learned from a pancreatic cancer biospecimen repository.

Clinically annotated pancreatic cancer samples are needed for progress to be made toward developing more effective treatments for this deadly cancer. As part of a National Cancer Institute-funded program project, we established a biospecimen core to support the research efforts. This article summarizes the key hurdles encountered and solutions we found in the process of developing a successful multi-institution biospecimen repository.

A fly in the ointment: reassessing mitotane's role in the treatment of adrenocortical carcinoma

Mitotane (2-[o-chlorophenyl]-2-[p-chloro phenyl]-1,1-dichloroethane or o,p´-DDD), an isomer of the banned pesticide 1,1,1-trichloro2,2-di(4-chlorophenyl)ethane (DDT), is the current standard of care for patients presenting with advanced and metastatic adrenocortical carcinoma (ACC) [1–3]. Mitotane or combinations containing mitotane have remained the standard of care for patients with ACC for over 30 years despite a lack of understanding regarding its mechanism of action or interactions with other agents and a suboptimal effectiveness. The results from the recently reported FIRM-ACT trial [4] and evidence from a study of urine steroid profiles of patients with ACC cast further doubt on the broad use of mitotane in treating ACC patients [5]. These studies highlight a need to identify markers of mitotane resistance and put significant effort into the development of new treatments, and possibly treatment paradigms, in order to improve outcomes for patients with ACC. Why mitotane is the standard of care for the treatment of ACC ACC is an aggressive cancer of the adrenal cortex. The outcome for patients with ACC has remained unchanged in the past 25 years, with the overall 5-year survival of patients undergoing surgical resection being approximately 40% [6]. The disease may have a variable course. Certain patients generally fare better, including those with stage II disease whose 5-year survival rates approach 60%, and improve to greater than 95% if their tumor does not recur within 16 months [7]. However, patients who present with large, locally invasive tumors, have involved surgical margins or present with metastatic disease fare considerably worse with 5-year survival rates of 10–20% [6]. The rationale for using mitotane as treatment for ACC stems from early observations of the adrenolytic effect of the pesticide DDT