Guisong Wang

Mitochondrial proteomic analysis of cisplatin resistance in ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death among women with gynecologic malignancies and accounts for approximately 6% of cancer deaths among women. Cisplatin and its analogues form the backbone of the most active chemotherapy regimens in advanced EOC; however, development of platinum resistance is common and typically marks a transition in which curing the patient is no longer possible. An emerging theme in many cancers is that mitochondrial dysfunction contributes to an aggressive carcinogenic phenotype. We hypothesized that changes in the mitochondrial proteome are required to support development of cisplatin resistance in human EOC. To investigate this hypothesis, an organellar proteomics approach was utilized to quantify alterations in protein abundance in mitochondria enriched from isogenic cisplatin-sensitive (A2780) and -resistant (A2780-CP20) human EOC cells. Protein isolates from mitochondria-enriched fractions were analyzed by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), and relative abundance of identified proteins was quantified by spectral counting. Pathway analyses revealed significant increases in notch signaling pathways, cell survival, and alternate apoptotic pathways in the A2780-CP20 subtype. Among the alterations identified in the mitochondrial proteomic composition in cisplatin-resistant EOC cells, activated leukocyte cell adhesion molecule (AKAP12) and A kinase anchoring protein 12 (AKAP12) were elevated, while nestin was diminished in the mitochondrial fraction of A2780-CP20 relative to A2780. This was verified by immunoblot analysis. These results confirm that important changes in the mitochondrial proteome, many of which promote evasion of apoptosis and tumor invasiveness and metastasis, are present in cisplatin-resistant EOC.

Progesterone enhances calcitriol antitumor activity by upregulating vitamin D receptor expression and promoting apoptosis in endometrial cancer cells.

Human studies suggest that progesterone and calcitriol may prove beneficial in preventing or inhibiting oncogenesis, but the underlying mechanism is not fully understood. The current study investigates the effects of progesterone, calcitriol, and their combination on immortalized human endometrial epithelial cells and endometrial cancer cells and identifies their targets of action. Combination treatment with both agents enhanced vitamin D receptor expression and inhibited cell proliferation through caspase-3 activation and induction of G0–G1 cell-cycle arrest with associated downregulation of cyclins D1 and D3 and p27 induction. We used mass spectrometry–based proteomics to measure protein abundance differences between calcitriol-, progesterone-, or combination-exposed endometrial cells. A total of 117 proteins showed differential expression among these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), IFN-induced, double-stranded RNA-activated protein kinase (EIF2AK2), and Bcl-2–associated X protein (BAX). Abundance levels of selected candidates were low in endometrial cancer cell lines versus the immortalized endometrial epithelial cell line. All four proteins displayed elevated expression in cancer cells upon exposure to calcitriol, progesterone, or the combination. Further BAX analysis through gain- or loss-of-function experiments revealed that upregulation of BAX decreased cell proliferation by changing the BAX:BCL-2 ratio. Knockdown of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol upregulate the expression of BAX along with other apoptosis-related proteins, which induce inhibition of endometrial cancer cell growth by apoptosis and cell-cycle arrest. Cancer Prev Res; 6(7); 731–43. ©2013 AACR.

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.

Proteomics of the Human Endometrial Glandular Epithelium and Stroma from the Proliferative and Secretory Phases of the Menstrual Cycle

ABSTRACT Despite its importance in reproductive biology and womens health, a detailed molecular-level understanding of the human endometrium is lacking. Indeed, no comprehensive studies have been undertaken to elucidate the important protein expression differences between the endometrial glandular epithelium and surrounding stroma during the proliferative and midsecretory phases of the menstrual cycle. We utilized laser microdissection to harvest epithelial cells and stromal compartments from proliferative and secretory premenopausal endometrial tissue and performed a global, quantitative mass spectrometry-based proteomics analysis. This analysis identified 1224 total proteins from epithelial cells, among which 318 were differentially abundant between the proliferative and secretory phases (q < 0.05), and 1005 proteins from the stromal compartments, 19 of which were differentially abundant between the phases (q < 0.05). Several proteins were chosen for validation by immunohistochemistry in an independent set of uterine tissues, including carboxypeptidase M, tenascin C, neprilysin, and ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3). ENPP3, which was elevated in epithelial glandular cells in the secretory phase, was confirmed to be elevated in midsecretory-phase baboon uterine lavage samples and also observed to have an N-linked glycosylated form that was not observed in the proliferative phase. This study provides a detailed view into the global proteomic alterations of the epithelial cells and stromal compartments of the cycling premenopausal endometrium. These proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins and their role in reproductive biology and endometrial pathologies.

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.

Nestin: A biomarker of aggressive uterine cancers

OBJECTIVE Evidence of potential prognostic and predictive value for nestin was investigated in well-annotated uterine cancers (UCs). METHODS Nestin expression and previously-published biomarkers were evaluated by immunohistochemistry (IHC) in UC tissue microarrays. Biomarkers were categorized as low vs. high, and nestin was cut at 10% positive staining. Relationship between nestin and clinicopathologic factors, biomarkers and outcome were evaluated using exact/log-rank testing or logistic/Cox modeling. RESULTS There were 323 eligible cases, 34% had advanced stage disease, 37% had type II disease, and 5% were carcinosarcomas. High nestin, observed in 19% of cases, was more common in advanced vs. early stage disease, type II cancers or uterine carcinosarcoma vs. type I cancers, grade 3 disease, positive lymphovascular space invasion (LVSI) and tumors >6cm (p<0.05). Nestin was inversely correlated with ER, PR and TFF3, and correlated with p53 and IMP3. Women with high vs. low nestin had worse progression-free survival (PFS) and cancer-specific survival overall, and worse PFS in the subset who received no adjuvant therapy or radiation, or had early stage, type I disease or tumors with both low and high ER, PR, TFF3, PTEN, p53 or IMP3. The relationship between nestin and PFS was independent of stage, LVSI and risk categorization but not type of UC. CONCLUSIONS High nestin was more common in UCs with aggressive features and poor outcome. Nestin may represent a predictive biomarker for treatment selection for patients previously considered to be lower risk and a candidate for no or radiation-based adjuvant therapy, and compliment ER/PR testing.

Abstract 5277: Proteome and transcriptome alterations in black endometrial cancer patients correlate with poor disease outcome

Objective: Black endometrial cancer patients are more than twice as likely to die from their disease as White patients. This study sought to identify alterations in the proteome and transcriptome of primary tumor tissues from White and Black endometrioid endometrial cancer (EEC) patients associated with differential outcome. Methods: An integrated proteomic and transcriptomic analysis (LC-MS/MS and RNA-seq) was performed on White (n=13) and Black (n=17) EEC patient tissues. Significant and concordantly altered protein and transcript candidates were validated against publicly available RNA-seq data (TCGA UCEC) from White (n=216) and Black (n=49) EEC patients. Validated candidates were further correlated with overall (OS, n=356 White and Black patients) and progression-free survival (PFS, n=331 White and Black patients) to identify candidates significantly associated with differential disease outcome. Alterations of outcome-associated candidates were validated in an independent cohort of White (n=115) and Black (n=17) EEC patient transcript expression data. Results: We identified and validated 89 proteins and transcripts significantly altered between White vs Black EEC patients. Pathway analyses revealed candidates elevated in White EEC patients correlated with marked activation of molecular signaling pathways regulating viral infection, but inhibition of those regulating cell death and necrosis. Candidates elevated in Black EEC patients largely correlated with activation of cell viability and nucleic acid metabolism, but inhibition of cell death, glucose metabolism disorder and inflammatory signaling. Correlation with patient outcome measures revealed 11 candidates significantly associated with differential OS and 8 candidates with differential PFS in EEC patients. All outcome-associated candidates elevated in White patients significantly correlated with a low risk of poor OS and poor PFS (Hazard Ratio (HR) 1, Wald p-value Conclusions: Our analyses identified and confirmed molecular alterations between White and Black EEC patients, including outcome-associated candidates largely supportive of better outcome in White patients, but poor outcome in Black patients. These findings define molecular alterations in White and Black EEC patients consistent with the historic disparity of poor outcome for Black patients warranting further investigation of these candidates in Black EEC disease pathology. Citation Format: Nicholas W. Bateman, Elizabeth Dubil, Guisong Wang, Brian L. Hood, Tracy Litzi, Julie Oliver, Kathleen M. Darcy, Chad A. Hamilton, Thomas P. Conrads, George L. Maxwell. Proteome and transcriptome alterations in black endometrial cancer patients correlate with poor disease outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5277. doi:10.1158/1538-7445.AM2017-5277

Abstract LB-111: Elevated CXXC5 is associated with recurrence, poor overall survival and cell viability in endometrial cancer

Endometrial cancer is the fourth most common cancer in women and the most prevalent gynecologic malignancy in the United States, however, there has been little improvement in treatment strategies. This problem will become more eminent in the future as the incidence and prevalence of this disease is increasing overall. As it did decades ago, determination of therapeutic modality still relies heavily on two subjective measures: surgical staging and pathological classification. Thus, new markers are needed to more objectively assess disease state and predict potential progression. We have used two independent datasets (Internal and TCGA) containing quantified measures of the transcriptome derived from both serous and endometrioid endometrial cancers to identify upregulated transcripts that are predictive of overall survival (OS) and/or disease-free survival (DFS). In initial analysis of our internal dataset we identified 114 transcripts at p In this study we focused on the most impressive of the transcripts we have identified, CXXC5, which is an understudied gene. Elevated levels of CXXC5 demonstrated an impressive association with recurrence (p = 0.000009 in TCGA). We next determined that those TCGA cancers that are high CXXC5 expressors (upper quartile) also had worse OS and DFS as compared to those in the low expressor group. Finally, we verified CXXC5 overexpression as being predictive of recurrence by quantitative RT-PCR (p≤0.03) in an independent set of 80 endometrial cancers. We examined several oncogenic phenotypes in two uterine serous cancer cell line models following shRNA mediated CXXC5 knockdown. CXXC5 expression was effectively knocked down in 4 of 5 lentiviral shRNAs targeting CXXC5 as compared to non-targeting controls and was confirmed for both the protein and RNA level. CXXC5 knockdown cells displayed a slower metabolic rate as measured by MTS assay, showed a dramatic (>70%) reduction in colony formation and also had increased levels of caspase 3/7 activity. Overall, our results suggest that elevated CXXC5 levels promote cell viability in endometrial cancers overexpressing CXXC5. Thus, CXXC5 could serve not only as a predictive clinical marker for poor outcome disease, but also as a potential novel therapeutic target. Citation Format: Alyssa Fedorko, Gadisetti V.R. Chandramouli, Hong Im Kim, Yutaka Shoji, Rusheeswar Challa, Kathleen M. Darcy, Guisong Wang, Julian Schink, Thomas P. Conrads, John Risinger. Elevated CXXC5 is associated with recurrence, poor overall survival and cell viability in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-111.

Abstract 4632: Poor survival associated with NUAK1 overexpression in serous ovarian cancer may be explained by chemotherapy resistance

Objectives: The purpose of this study was to identify and validate novel gene transcripts associated with poor prognosis ovarian cancer. Methods: Clinical, pathologic, outcomes and transcriptomics data for 22,277 probesets were downloaded for each of 1,183 eligible patients representing six datasets. Cohorts 1 (n = 638) and 2 (n = 545) were used for discovery and validation, respectively. Associations with survival were evaluated using univariate Cox modeling with Wald test and Kaplan-Meier method with log rank test. Odds ratios were evaluated using the Mantel-Haenszel test. NUAK1 expression in ovarian cancer cell lines was examined using immunoblotting. Results: There were 293 (1.3%) probesets whose expression was associated with survival in serous ovarian cancer patients in cohort 1 that passed false discovery correction, and 19 of these, including NUAK1, validated in cohort 2 (q Conclusions: Overexpression of NUAK1, also known as AMPK-related protein kinase 5 (ARK5) and a survival factor in nutrient starved cells with low oxygen tension, was identified and validated to predict poor prognosis in a cohort of 1,183 serous ovarian cancer patients, and to be associated with chemoresistance using in vitro models of platinum and taxane resistance in human ovarian cancer. Citation Format: Neil T. Phippen, Nicholas W. Bateman, Guisong Wang, Chad A. Hamilton, George L. Maxwell, Kathleen M. Darcy, Thomas P. Conrads. Poor survival associated with NUAK1 overexpression in serous ovarian cancer may be explained by chemotherapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4632. doi:10.1158/1538-7445.AM2015-4632

Abstract 1570: ARID1A regulation of ATAD2 in gynecologic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA ARID1A the DNA binding component of the SWI/SNF nucleosome and chromatin remodeling complex has recently been identified as a tumor suppressor and inactivating mutations have been reported in several tumor types including uterine and ovarian cancer. Approximately 30% of ovarian endometrioid cancers (OEC), 50% of clear cell ovarian cancers (OCCC) and 40% of uterine endometrial cancers harbor ARID1A inactivating mutations. Little is known regarding the subunit composition of SWI/SNF complexes that contain ARID1A in ovarian and uterine cells and less is known regarding the transcriptional and functional consequences of ARID1A loss. In order to further explore the function of ARID1A in gynecologic cancers we restored ARID1A in ACI-98 (undifferentiated uterine cancer) an ARID1A negative and TP53 positive cell line using an inducible Tet-on system. Restoration of ARID1A induced apoptosis in ACI-98 cells after 48h doxycycline (Dox) treatment. We examined the proteome of these cells and catalogued the protein changes following ARID1A complementation using LC MS/MS. Specifically we identified 523 highly significantly differentially expressed proteins (z-score <0.01) from this analysis. Among these was the AAA domain containing 2 (ATAD2) that was dramatically down-regulated in ARID1A restored cells. ATAD2 is a highly conserved protein normally expressed in germ cells but is also over-expressed in some cancers. Affymetrix gene expression results also showed that ATAD2 is over-expressed in some ovarian and uterine cancers compared to normal controls. We further examined the clinical consequence of ATAD2 expression in ovarian cancers using the Kaplan-Meier plots website (http://kmplot.com) and found that the high-expression group has a worse overall survival than those in the low-expression group. ATAD2 associates via its bromodomain with histone H3 and it is known to act as a co-factor for E2Fs, MYC, androgen and estrogen receptors and whose over-expression drives the expression of target genes that induce cell proliferation and resistance to apoptosis. qRT-PCR results indicated that transcription of ATAD2 was not changed by restoration of ARID1A, moreover, anti-ARID1A ChIP-sequence revealed that SWI/SNF-ARID1A is not binding to the promoter of ATAD2. From these results, we suspect that the down regulation of ATAD2 is through a non-transcriptional mechanism. In addition, ARID1A-IP-MS result suggested that ARID1A is itself making complexes with ATAD2. We performed ATAD2 and ARID1A immunohistochemistry on a set of OCCC and OEC primary cancers and found that all cases with ARID1A negative staining preferentially over-express ATAD2. In summary our data indicate that ARID1A decreases levels of ATAD2, that ARID1A-negative OCCC and OEC are a subset of ovary cancer that preferentially overexpress ATAD2, and that ATAD2 over-expressing cancers have worse survival than weak expressers. Citation Format: Yutaka Shoji, Kelly A. Conrads, Rusheeswar Challa, Brian L. Hood, Guisong Wang, Kathleen M. Darcy, Chad A. Hamilton, George Larry Maxwell, Thomas P. Conrads, John I. Risinger. ARID1A regulation of ATAD2 in gynecologic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1570. doi:10.1158/1538-7445.AM2014-1570