Tracy Litzi

Elevated AKAP12 in paclitaxel-resistant serous ovarian cancer cells is prognostic and predictive of poor survival in patients.

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (q < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients.

MicroRNAs in endometrial cancers from black and white patients.

OBJECTIVE Previous studies have identified differences in gene mutations among endometrial cancers from whites and blacks suggesting that differences in tumor biology may explain racial disparities in patient outcome. Micro RNAs (miRNAs) have emerged as regulators of transcript expression and their aberrant expression has been discovered in many diseases, including endometrial cancer. We performed quantitative polymerase chain reaction-based analysis in a set of endometrial cancers to identify whether there are racial differences in miRNA expression. STUDY DESIGN Tumor cells from 50 stage-I endometrioid endometrial cancer specimens from 41 white and 9 black patients were prepared by laser microdissection and miRNA extracts were analyzed using TaqMan (Life Technologies, Carlsbad, CA) low-density arrays. Statistically significant, differentially expressed miRNAs between blacks and whites were identified using multidimensional scaling, Wilcoxon testing, and analysis of variance. RESULTS There were no global differences in miRNA expression between endometrial cancers from 41 white and 9 black patients. To minimize potential bias introduced by unbalanced sample size, we performed a subset analysis with stage- and histology-matched specimens from 9 whites and 9 blacks that identified 18 differentially abundant miRNAs (>2-fold at P < .005). Quantitative polymerase chain reaction validated miRNA-337-3p in an independent set of endometrial cancer specimens from 23 white and 24 black women. There were no racial differences in hsa-miR-337-3p expression in normal endometrium. CONCLUSION These data indicate that hsa-mir-337-3p is more frequently down-regulated in endometrial cancers from whites compared to blacks. Future studies are focused on determining the phenotypic impact of miR-337-3p and whether its differential expression is associated with clinical outcome.

Transcript expression in endometrial cancers from Black and White patients

OBJECTIVE Previous studies suggest that differences in molecular features of endometrial cancers between racial groups may contribute to the poorer survival in Blacks. The objective of this investigation was to determine whether gene expression among endometrial cancers is different between Blacks and Whites. METHODS Fresh frozen tumors from 25 Black patients were matched by stage, grade, and histology to endometrial cancer specimens from 25 White patients. Each case was macrodissected to produce specimens possessing a minimum of 75% cancer cellularity. A subset of 10 matched pairs was also prepared using laser microdissection (LMD) to produce specimens possessing a minimum of 95% cancer cells. Total RNA isolated from each sample was analyzed using the Affymetrix Human Genome U133 Plus 2.0 arrays. Data were analyzed using principal component analysis and binary class comparison analyses. RESULTS Unsupervised analysis of the 50 endometrial cancers failed to identify global gene expression profiles unique to Black or White patients. In a subset analysis of 10 matched pairs from Blacks and Whites prepared using LMD and macrodissection, unsupervised analysis did not reveal a unique gene expression profile associated with race in either set, but associations were identified that relate to sample preparation technique, histology and stage. CONCLUSIONS Our microarray data revealed no global gene expression differences and identified few individual gene differences between endometrial cancers from Blacks and Whites. More comprehensive methods of transcriptome analysis could uncover RNAs that may underpin the disparity of outcome or prevalence of endometrial cancers in Blacks and Whites.

NUAK1 (ARK5) Is Associated with Poor Prognosis in Ovarian Cancer

Background and objective Nua kinase 1 (NUAK1) was identified in multigene signatures of survival and suboptimal debulking in high-grade serous ovarian cancer (HGSOC). This study investigates the individual clinical and biologic contributions of NUAK1 in HGSOC patients and cell lines. Methods Public transcript expression, clinical, and outcome data were used to interrogate the relationship between NUAK1 and clinicopathologic factors and patient outcomes including progression-free survival (PFS) and molecular subtypes using logistic and Cox modeling. Analysis of NUAK1 transcript expression was performed in primary tumors from 34 HGSOC patients with < or ≥2 years PFS. The impact of silencing NUAK1 by RNA interference (RNAi) on the migratory potential and chemosensitivity of SOC cells was assessed in vitro. Results Elevated NUAK1 transcript expression was associated with worse PFS (hazard ratio = 1.134), advanced stage (odds ratio, OR = 1.7), any residual disease (OR = 1.58), and mesenchymal disease subtype (OR = 7.79 ± 5.89). Elevated NUAK1 transcript expression was observed in HGSOC patients with < vs. ≥2 years PFS (p < 0.045). RNAi-mediated silencing of NUAK1 expression attenuated migration of OV90 and E3 HGSOC cells in vitro, but did not modulate sensitivity to cisplatin or paclitaxel. Conclusion Elevated NUAK1 was associated with poor survival as well as advanced stage, residual disease after cytoreductive surgery and mesenchymal molecular subtype. NUAK1 impacted migration, but not chemosensitivity, in vitro. Additional studies are needed to further develop the concept of NUAK1 as a clinically deployable biomarker and therapeutic target in HGSOC.

Race‐specific molecular alterations correlate with differential outcomes for black and white endometrioid endometrial cancer patients

The objective of this study was to identify molecular alterations associated with disease outcomes for white and black patients with endometrioid endometrial cancer (EEC).

Abstract 1966: Analysis of micro RNAs in the racial disparity of endometrial cancer .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In the United States endometrial cancer is recognized for having a distinct racial disparity. This disparity by race occurs for both the prevalence of disease and for survival outcome. Caucasians are about two times more likely to develop endometrial cancer than are African Americans. However, African American women are more likely to die from this disease than are Caucasians. The basis for this disparity remains unknown. Previous studies have identified differences in the types and frequencies of gene mutations among endometrial cancers from Caucasians and African-Americans suggesting that the tumors from these two groups might have differing underlying genetic defects. In addition previous studies have utilized gene expression microarray studies in an effort to identify differentially expressed transcripts between African-American and Caucasian womens endometrial cancers. MicroRNAs (miRNA) have emerged as additional regulators of cell function and their aberrant expression and function is noted in many diseases including endometrial cancers. We performed an analysis in a set of early stage endometrial cancers to identify whether differences in miRNA expression may underlie some biologic aspect of this racial disparity. We assayed 50 laser microdissected endometrial cancers using TaqMan Low Density arrays and compared the expression of miRNAs between African-American (9 patients) and Caucasian (41 patients) cancers. Similarly to previous mRNA comparisons by our group, no global differences in miRNA expression were evident between African American and Caucasian endometrial cancers. We further refined our analyses using Partial Least Squares Regression (PLSR) where race-, stage-, and grade-dependent variances were examined which indicated that the depth of invasion affects global associations. An analysis of variance (ANOVA) that considered race and stage as factors and a Wilcoxon signed rank test of these groups revealed a small number of differentially expressed miRNAs. Since the unbalanced sample sizes (41 Caucasian versus 9 African-American cases) may introduce bias, we performed a paired analysis (9 AA cancers v. 9 C cancers). Of several differentially identified miRNAs, hsa-miR-337-3p was consistently identified in class comparisons. We validated the differential expression of hsa-miR-337-3p in an independent set of endometrial cancers from African Americans (n=24) and Caucasians (n=23). Analysis of normal endometrial epithelial expression indicated that hsa-miR-337-3p expression is down-regulated in cancers as compared to normal expression and that there is no difference in expression of hsa-miR-337-3p in the normal endometrium according to race. These data indicate that hsa-mir-337-3p is specifically down-regulated in Caucasian womens endometrial cancers and may play a role in the more frequent development of uterine cancers in Caucasians. Citation Format: G. Larry Maxwell, GVR Chandramouli, Tracy Litzi, Andrew Berchuck, Thomas P. Conrads, John I. Risinger. Analysis of micro RNAs in the racial disparity of endometrial 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 1966. doi:10.1158/1538-7445.AM2013-1966

Abstract 799: Comprehensive proteomic analysis of cisplatin resistance in ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We have conducted a comprehensive proteomic analysis of an ovarian cancer cell line (A2780) and its cisplatin resistant daughter line (A2780-CP20) to identify key regulators and signaling pathways that confer cisplatin resistance. Quantitative proteomic profiling was conducted of defined biological compartments including that from global lysates, the nuclear fraction, the phosphoproteome and the secretome. Total cell lysates and nuclear fractions were harvested from A2780 and A2780-CP20 cultured in the presence or absence of cisplatin (3 µM) for 72 h. The secretome fractions were collected from A2780 and A2780-CP20 cultured in serum-free, phenol red-free media in the presence or absence of cisplatin (3 µM) for 24 h. Phosphopeptides were enriched from peptide digests of total lysates of A2780 and A2780-CP20 cultured in the presence or absence of cisplatin (3 µM) for 15 min using TiO2. Sample digests were analyzed by high-resolution LC-MS/MS that resulted in the identification of 2703, 2286, 1815, and 1362 proteins from the global, nuclear, secretome, and phosphoproteome fractions, respectively. Spectral counting was utilized to quantify the relative abundance of proteins identified. Nuclear proteins identified at elevated abundance in the cisplatin resistant A2780-CP20 including an array of chromatin remodeling proteins, such as SATB1, SATB2, coilin, AT-rich interactive domain-containing protein 3A (ARID3A), ARID3B, and ARID3Cwere validated by Western blot and qPCR in A2780/A2780-CP20 as well as other gynecologic cancer cell lines. Modulation of the abundance level of these candidates and the phenotypic impact on cisplatin resistance utilizing shRNA knockdown will be described. Differential proteins selected from the secretome analysis for western blot validation include Fras1-related extracellular matrix protein 2 (FREM2), stanniocalcin-1 (STC1), angio-associated migratory cell protein (AAMP), plasminogen activator inhibitor 1 (SERPINE1), high mobility group protein B1 (HMGB1), and chloride intracellular channel protein 2 (CLIC4), stromelysin-2 (MMP10), interstitial collagenase (MMP2), and CD166 antigen. Validated candidate protein targets from the secretome analysis will be tested by IHC on formalin fixed tissue sections as well as ELISA and multiple reaction monitoring (MRM) by mass spectrometry in serum from recurrent and non-recurrent ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 799. doi:1538-7445.AM2012-799