Pang-ning Teng

Advances in proximal fluid proteomics for disease biomarker discovery.

Although serum/plasma has been the preferred source for identification of disease biomarkers, these efforts have been met with little success, in large part due the relatively small number of highly abundant proteins that render the reliable detection of low abundant disease-related proteins challenging due to the expansive dynamic range of concentration of proteins in this sample. Proximal fluid, the fluid derived from the extracellular milieu of tissues, contains a large repertoire of shed and secreted proteins that are likely to be present at higher concentrations relative to plasma/serum. It is hypothesized that many, if not all, proximal fluid proteins exchange with peripheral circulation, which has provided significant motivation for utilizing proximal fluids as a primary sample source for protein biomarker discovery. The present review highlights recent advances in proximal fluid proteomics, including the various protocols utilized to harvest proximal fluids along with detailing the results from mass spectrometry- and antibody-based analyses.

Differential Proteomic Analysis of Renal Cell Carcinoma Tissue Interstitial Fluid

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.

Standardization of a sample preparation and analytical workflow for proteomics of archival endometrial cancer tissue.

The goal of the present study was to establish a standard operating procedure for mass spectrometry (MS)-based proteomic analysis of laser microdissected (LMD) formalin-fixed, paraffin-embedded (FFPE) uterine tissue. High resolution bioimage analysis of a large endometrial cancer tissue microarray immunostained for the breast cancer type 1 susceptibility protein enabled precise counting of cells to establish that there is an average of 600 cells/nL of endometrial cancer tissue. We sought to characterize the peptide recovery from various volumes of tissue gathered by LMD and processed/digested using the present methodology. We observed a nearly linear increase in peptide recovery amount with increasing tissue volume dissected. There was little discernible difference in the peptide recovery from stromal versus malignant epithelium, and there was no apparent difference in the day-to-day recovery. This methodology reproducibly results in 100 ng of digested peptides per nL of endometrial tissue, or ∼25 pg peptides/endometrial cancer cell. Results from liquid chromatography (LC)-MS/MS experiments to assess the impact of total peptide load on column on the total number of peptides and proteins identified from FFPE tissue digests prepared with the present methodology indicate a demonstrable increase in the total number of peptides identified up to 1000 ng, beyond which diminishing returns were observed. Furthermore, we observed no impact on the peptide identification rates from analyses of equivalent peptide amounts derived from lower volume LMD samples. These results show that this single-tube collection-to-injection proteomics (CTIP) workflow represents a straightforward, scalable, and highly reliable methodology for sample preparation to enable high throughput LMD-MS analysis of tissues derived from biopsy or surgery.

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.

Restraint stress and stress hormones significantly impact T lymphocyte migration and function through specific alterations of the actin cytoskeleton

Stress triggers complex response mechanisms designed to recognize and adapt to perturbations in homeostasis. The immune system is highly responsive to stress, although the complete mechanisms linking stress and immune mediators including T lymphocytes, are not fully understood. Stress exerts its effects on immune effectors through two primary pathways: the sympathetic-adrenal-medullary pathway, and the hypothalamic-pituitary-adrenal pathway which modulate adaptive immunity and lymphocyte migration. In this report we show that stress via release of stress hormones induces early T cell activation and greatly impacts the cytoskeleton by modulating numerous actin-regulating proteins. In particular, proteomic profiling revealed significant decreases in numerous key actin-binding proteins including moesin. Although confocal microscopy showed that moesin and actin were uniformly distributed on the surface of resting T cells, a remarkable polarization and redistribution of moesin and actin was observed following treatment with stress hormones with moesin localizing at the distal pole complex. In addition, the alteration in moesin localization and eventual decrease in expression were accompanied by a loss of CD43; a receptor involved in negatively regulating T cell activation. In conclusion, we have defined a novel molecular mechanism whereby stress hormones negatively impact T cell activation and migration through regulation of key cytoskeletal and plasma membrane factors.

Abstract 3758: Inhibition of ATR, but not ATM, sensitizes gynecologic cancer cells to cisplatin

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Resistance to platinum-based therapies remains a major hurdle in the management of gynecologic (GYN) cancer especially in ovarian cancer. Platinum such as cisplatin damages DNA by inducing DNA crosslinks that stalls DNA replication forks where significant accumulation of single-stranded DNA from persistently stalled replication forks could ultimately lead to double strand breaks and activation of apoptosis. Ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) are two main DNA damage response (DDR) protein kinases that recognize genotoxic stress and function to initiate cell cycle arrest and DNA repair mechanisms. We hypothesized by combining genotoxic stress with inhibition of DDR kinases ATR and/or ATM, therapeutic response of GYN cancer cells to platinum-based chemotherapy could be improved. To test our hypothesis, we assessed cell survival of multiple GYN cell lines including ovarian (A2780, A2780-CP20, OVCAR3), endometrial (KLE, HEC1B), and cervical (HELA, SIHA) carcinoma cells exposed to cisplatin along with ATR inhibitor (ETP-46464) and/or ATM inhibitor (KU55933). We observed inhibition of ATR significantly enhanced cisplatin induced cell death in all seven cell lines tested, resulting in 65 - 96% increased sensitivity to cisplatin. Inhibition of ATM did not sensitize GYN cancer cells to cisplatin and cells were not further sensitized by co-inhibition of ATM and ATR beyond that observed by inhibition of ATR alone. DDR signaling was assessed by immunoblotting in cells exposed to cisplatin with or without the presence of ETP-46464 and/or KU55933 where elevated levels of phospho-ATM (Ser1981), phospho-Chk2 (Thr68) and phospho-Chk1 (Ser345) observed in cisplatin treated cells were attenuated in cells that were co-treated with cisplatin and ETP-46464. In addition, increased levels of cleaved PARP1 and caspase 3 were observed in cisplatin-treated, ATR-inhibited cells, suggesting the enhancement of cisplatin induced cell death with ATR inhibition occurs through elevated apoptosis. Further, no differential effect was observed in GYN cancer cells harboring wild type (A2780, OVCAR3, HELA, SIHA) or mutant (CP20, KLE, HEC1B) TP53. These data support further investigation of pharmacologic inhibitors of ATR in combination with existing platinum based therapeutics for treating GYN cancer. Citation Format: Pang-Ning Teng, Nicholas W. Bateman, Chad A. Hamilton, G. Larry Maxwell, Christopher J. Bakkenist, Thomas P. Conrads. Inhibition of ATR, but not ATM, sensitizes gynecologic cancer cells to cisplatin. [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 3758. doi:10.1158/1538-7445.AM2014-3758

Abstract 3377: Integrated molecular analysis of ovarian cancer cells identifies the ATR signaling axis central to cisplatin resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer is the most lethal gynecological malignancy due primarily to the high percentage of women diagnosed at advanced stage. Although most patients initially respond to treatment, recurrence is common and second-line treatment is less effective, particularly in platinum-resistant disease. An integrated molecular analysis of an ovarian cancer cell line, A2780, and its isogenic cisplatin resistant line, A2780-CP20, was conducted utilizing next generation sequencing of the exome and the transcriptome (RNA-seq), along with a comprehensive analysis of the proteome and the phosphoproteome. Integration of these data revealed a number of differentially expressed candidates at the level of the proteome, the mechanistic nature of which could be explained by mutational status or differential splicing. We focused our attention on the ataxia telangiectasia mutated and Rad3-related (ATR) kinase. ATR activity is induced by DNA damage when single strand DNA results from persistently stalled replication forks. The gene and transcript for ATR were observed as mutated, alternatively spliced or altered at the transcript level in A2780-CP20 versus A2780 cells. The gene product was identified and validated to be ∼50-fold elevated in A2780-CP20 relative to A2780 cells. We hypothesized that inhibition of ATR would sensitize ovarian cancer cells to cisplatin-induced DNA lesions. To test this hypothesis undifferentiated (A2780, A2780-CP20) and papillary serous ovarian cancer cells (OV90 and OVCAR3) were challenged with cisplatin in the presence and absence of a selective ATR small molecule inhibitor (5 μM ATRi, [ETP46464][1]). The results demonstrate significantly increased cisplatin sensitivity in all cell lines tested by 54-99%. We further evaluated the ATR signaling axis, and repeated the cisplatin challenge experiments with a selective small molecule inhibitor against the serine/threonine kinase Chk1 (LY2603618), a downstream target that is phosphorylated by activated ATR. Similarly to what we observed with inhibition of ATR, these data reveal significant sensitization of ovarian cancer cells to cisplatin when co-cultured with the Chk1 inhibitor. To evaluate the selectivity of this sensitization response, the ovarian cancer cells were co-cultured with a selective inhibitor to the serine/threonine kinase Chk2 (2-(4-(4-Chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide), which revealed no change in cisplatin sensitivity. We conclude that selective inhibition of ATR or Chk1, but not Chk2, significantly sensitizes ovarian carcinoma cell lines to cisplatin. Furthermore, we suggest that ATR and Chk1 represent key signaling nodes that are centrally involved in cisplatin resistance in ovarian cancer, and therefore represent attractive candidates for molecularly targeted therapy in the setting of resistant/recurrent ovarian cancer. Citation Format: Pang-Ning Teng, Guisong Wang, Tracy J. Litzi, Brian L. Hood, Brian Blanton, Kelly A. Conrads, Kathleen M. Darcy, Chad A. Hamilton, William P. Mcguire, G. Larry Maxwell, Chris J. Bakkenist, Thomas P. Conrads. Integrated molecular analysis of ovarian cancer cells identifies the ATR signaling axis central to cisplatin resistance. [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 3377. doi:10.1158/1538-7445.AM2013-3377 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=ETP46464&atom=%2Fcanres%2F73%2F8_Supplement%2F3377.atom