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The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Global analysis of biological systems is becoming increasingly feasible as technologies that facilitate genome-wide analyses of gene expression are developed. Proteomics is the global analysis of expressed proteins (including posttranslational modifications) and seeks to establish the relationship between genome sequence, expressed proteins, protein-protein interactions, and cell and tissue phenotype. While the relative abundance of transcripts can be quantified using gene expression microarrays, the identification and quantitation of expressed proteins is more challenging. Nevertheless, the potential payoff for global protein analyses is immense because identification of distinctive protein signatures associated with cell function may provide novel therapeutic targets, molecular markers of disease, and increased understanding of determinants of cell phenotype. The challenges and promises of applications of established and emerging proteome strategies to detect and quantify differentially expressed proteins in culture cells are discussed.

The success of the various genome sequencing projects (1) and the ability to measure differences in gene expression routinely at the transcription level (2), has shifted a considerable amount of focus towards proteomics—the characterization of gene expression at the protein level (3). Much of this focus has concentrated on methods to identify and measure effectively the relative abundance of a large number of proteins in a rapid fashion (4). Major developments in the areas of protein and peptide fractionation and instrument technology have been required to accomplish these goals. In the vast majority of global proteomic projects most proteins are identified by mass spectrometry (MS)-based methods. The proteins are identified either by matching a set of peptide masses measured by MS to a list of predicted peptide masses or by generating sequence-related information of a single peptide by tandem MS and searching this MS/MS spectrum against an appropriate database. This identification strategy usually provides the identity of the analyzed proteins and sequences of their degraded peptides.

CD82/KAI1 was one of the first metastasis suppressors identified and is capable of inhibiting the metastatic process in orthotopic tumor models. The expression of the CD82/KAI1 is down-regulated during tumor progression in many tumor types including prostate, breast, lung, bladder, and pancreatic cancers, and this down regulation appears to be at the level of transcription or post-transcription. The gene is not mutated, deleted or silenced by promoter methylation. CD82/KAI1 is also down regulated in advanced gynecologic cancers where CD82/KAI1 loss is associated with the poor overall survival in ovarian cancer. The mechanisms controlling the level of CD82/KAI1 expression remains unclear particularly the widespread loss in advanced cancers. The p53 tumor suppressor (TP53) can directly activate the CD82/KAI1 gene by interacting with the 5-prime upstream region of the CD82/KAI1 gene. Mutant TP53 is unable to bind this promoter site and fails to support CD82/KAI1 transcription. Taken together these data support a direct relationship between TP53 and CD82/KAI1 genes and suggested that the loss of TP53 function, which is common in many types of advanced cancer can lead to downregulation of CD82/KAI1, which then may result in metastases. However many cancers without TP53 mutation also exhibit loss of CD82/KAI1 suggesting that the mechanism for CD82/KAI1 loss is not completely explained by mutant TP53. Furthermore the mutant TP53 state and CD82/KAI1 loss is not strongly correlated in ovarian cancer. In some sarcomas the mechanism of CD82/KAI1 loss is related to the increase of the gp-78 E3 ligase and increased proteasomal degradation. Therefore the regulation of CD82/KAI1 in cells is likely complex. The AT Rich DNA binding protein ARID1A gene is frequently mutated in endometrial and ovarian cancers. In order to explore the functions of ARID1A in these cancers we complemented mutant ARID1A in the ACI-98 endometrial cancer cell line. We utilized a doxycycline inducible system and catalogued the protein changes following ARID1A complementation using LC MS/MS. Among the most differentially expressed proteins was CD82/KAI1. Levels of CD82 were dramatically increased following ARID1A induction. ACI-98 cells were found to be TP53 wild-type and CD82 expression null. QRT-PCR assays confirmed the dramatic increase in CD82/KAI1 mRNA levels and western blots also confirmed restoration of CD82/KAI1 protein following Doxycycline induced ARID1A restoration. We performed ChIP and confirmed that ARID1A was bound to the CD82/KAI1 promoter region close to the TP53 interaction area. Interestingly ACI-98 cells are mutant in both ARID1 homologs A and B. Ectopic expression of ARID1B was also able to restore expression of CD82/KAI1. These data suggest that the SWI/SNFARID1A/B complex is necessary for chromatin remodeling and expression of the CD82/KAI1 gene. Citation Format: Rusheeswar Challa, Yutaka Shoji, Kelly A. Conrads, Brian L. Hood, Guisong Wang, Kathleen M. Darcy, Chad A. Hamilton, George Larry Maxwell, Thomas P. Conrads, John I. Risinger. Metastasis suppressor CD82/KAI1 expression is dependent on functional SWI/SNF ARID1A/B. [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 990. doi:10.1158/1538-7445.AM2014-990

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

Objectives: The management of gynecological malignancies includes exposure of tumor cells to genotoxic insults, such as ionizing radiation (IR), and focuses on damaging cellular DNA and inducing subsequent cell death. As cells possess innate mechanisms to repair DNA damage, this study focused on combining IR with pharmacological inhibition of key mediators of DNA repair, ATR (Ataxia telangiectasia and Rad3 related) and ATM (Ataxia telangiectasia mutated), to further sensitize gynecological cancer cells to this mode of genotoxic stress. Methods: Clonogenic survival assays: A panel of human cell line models of ovarian, endometrial and cervical cancer were treated with drug vehicle, 5.0 µM ATR inhibitor (ETP-46464), 10.0 µM ATM inhibitor (KU55933) or a combination of ATRi and ATMi, prior to exposure to IR doses up to 6.0 Gy. Drug was removed four hours later and resulting colonies were counted when mean colony size was ≥ 50 cells. Western blot: ATM, ATR and downstream canonical signaling effectors were assessed in cell lysates harvested from representative cell line models of ovarian (A2780), endometrial (HEC1B) and cervical (HELA) cancers, one hour following treatment with inhibitors and IR exposure (2.0 Gy only) as described above. Results: Clonogenic survival assays revealed that inhibition of ATR and ATM increased sensitization to IR across all cell line models of gynecological cancer assessed. This effect was further increased with combined ATR and ATM inhibitor treatments. Western blot analyses revealed activation of ATM and ATR signaling in response to IR via increases in phospho-ATM (Ser1981), p-Chk1 (Ser345) and p-Chk2 (T68) levels. Activation of Chk1, a downstream effector of ATR, was attenuated in ATRi-treated conditions and activation of Chk2, a downstream effector of ATM, were reduced in ATMi-treated conditions. Conclusions: These studies revealed that inhibition of the DNA damage response kinases, ATR and ATM, markedly sensitizes diverse gynecological cancer subtypes to IR. They provide evidence to support further consideration of therapies pairing modulation of DNA damage signaling with IR in the treatment of gynecological malignancies. Citation Format: Nicholas Bateman, Pang-ing Teng, Kelly Conrads, Chad Hamilton, George Maxwell, Christopher Bakkenist, Thomas Conrads. Pharmacological inhibition of the DNA damage response kinases, ATR (Ataxia telangiectasia and Rad3 related) and ATM (Ataxia telangiectasia mutated), broadly sensitizes diverse subtypes of gynecological cancer cells to ionizing radiation. [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 2747. doi:10.1158/1538-7445.AM2014-2747

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 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

5559 Background: Folate Receptor Alpha (FRa) (aka Folate Binding protein (FBP)) is an immunogenic protein that is over-expressed in breast, lung, endometrial (EC) and ovarian cancers (OC). In fact,...

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.

Approximately 1 million people in the United States suffer from interstitial cystitis, a chronic painful urinary bladder disorder characterized by thinning or ulceration of the bladder epithelial lining; its etiology is unknown. We have identified a glycosylated frizzled-related peptide inhibitor of cell proliferation that is secreted specifically by bladder epithelial cells from patients with this disorder. This antiproliferative factor (APF) profoundly inhibits bladder cell proliferation by means of regulation of cell adhesion protein and growth factor production. The structure of APF was deduced by using ion trap mass spectrometry (MS), enzymatic digestion, lectin affinity chromatography, and total synthesis, and confirmed by coelution of native and synthetic APF derivatives on microcapillary reversed-phase liquid chromatography (μRPLC)/MS. APF was determined to be an acidic, heat-stable sialoglycopeptide whose peptide chain has 100% homology to the putative sixth transmembrane domain of frizzled 8. Both synthetic and native APF had identical biological activity in normal bladder epithelial cells and T24 bladder cancer cells. Northern blot analysis indicated binding of a probe containing the sequence for the frizzled 8 segment with mRNA extracted from cells of patients with interstitial cystitis but not controls. APF is therefore a frizzled-related peptide growth inhibitor shown to contain exclusively a transmembrane segment of a frizzled protein and is a potential biomarker for interstitial cystitis.

Immunoaffinity isolation enables the identification of phosphotyrosine peptides on a global level.

The t(5;17)(q35;q21) variant of acute promyelocytic leukemia fuses nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Expression of the NPM-RAR fusion protein leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. Using a proteomic approach to identify proteins that bind NPM-RAR, we previously demonstrated that TRADD, amongst other proteins, is a distinct binding partner for NPM-RAR. TRADD serves as an adapter protein that mediates TNF-receptor-mediated activation of the caspase, NFkB, and JNK pathways. We reported that NPM-RAR inhibits TNF-activation of caspase 8 and caspase 3, with subsequent impairment of TNF-induced apoptosis. Extending these findings, we assayed activation of JNK kinase in NPM-RAR-expressing subclones of HELA and U937 cells. We find that TNF activated JNK kinase in NPM-RAR HELA and U937 cells to a similar extent as nontransfected parental controls. This indicates that NPM-RAR binding to TRADD selectively inhibits TNF’s ability to activate caspases, without impairing its ability to activate JNK. In this regard, NPM-RAR effects on TRADD is similar to the EBV oncogenic viral protein LMP1, which binds to the death domain of TRADD, to selectively inhibit caspase activity, while still allowing TNF-mediated activation of survival pathways. Our studies identify a novel mechanism through which NPM-RAR may impact leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

6051 Background: The epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) pathways are upregulated in HNSCC. Crosstalk potentiates growth, proliferation and invasion. Preclinical models show synergistic anti-tumor effects from dual blockade. We evaluated biomarker modulation in paired tumor specimens from a 3-arm phase 0 trial of erlotinib, a small molecule EGFR inhibitor; erlotinib + sulindac, a non-selective COX inhibitor; vs. placebo. Methods: Patients (pts) with untreated stage II-IVb HNSCC were randomized 5:5:3 to neoadjuvant erlotinib 150 mg daily (Arm 1, n = 19), erlotinib + sulindac 150 mg twice daily (Arm 2, n = 16), or placebo (n = 12). Pts were treated for 7-14 days. Tumor specimens were collected pre- and post-treatment. The primary endpoint was change in Ki-67 proliferative index. We hypothesized an ordering in Ki-67 down-modulation, with Arm 2 > Arm 1 > placebo. A Jonckheere-Terpstra test evaluated trend; a Wilcoxon test was applied to pairwise contrasts. We prepared tissue microarrays for IHC, to explore pharmacodynamic modulation of 21 EGFR and COX-2 pathway constituents including EGFR, Src, STAT3, Akt, EP2 and EP4. Results: From Dec 2005 – Dec 2008, 48 pts enrolled; 28 tumor pairs were evaluable for biomarker modulation. Class toxicities of EGFR inhibition, including acneiform rash and diarrhea, were observed on Arms 1 and 2. Compared to placebo, Ki-67 was reduced by erlotinib (p = 0.0005) or by erlotinib-sulindac (p = 0.0001). There was a trend in ordering of Ki-67 down-modulation, with greater effect from the addition of sulindac (exact Jonckeheere-Terpstra, Arm 1 vs. 2, p = 0.003). Among 21 protein candidates tested for association with Ki-67, only low baseline p-Src correlated with greater Ki-67 reduction in both erlotinib groups (R2 = 0.312, p = 0.024). Conclusions: Relative to placebo, brief neoadjuvant treatment with erlotinib or erlotinib-sulindac significantly decreased the proliferative index in operable HNSCC. Sulindac enhanced Ki-67 down-modulation. Efficacy studies of dual EGFR-COX inhibition are justified. Baseline p-Src warrants further study as a resistance biomarker for anti-EGFR therapy. Clinical trial information: NCT01515137.

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

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. The combined cell treatment enhanced vitamin D receptor expression and synergistically inhibited cell proliferation through caspase-3 activation and induction of G0/G1 cell cycle arrest with associated down-regulation of cyclin D1, 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 amongst these three treatments. Four proteins were then selected for validation studies: histone H1.4 (HIST1H1E), histidine triad nucleotide-binding protein 2 (HINT2), interferon-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:Bcl2 ratio. Knock down of BAX attenuated progesterone- and calcitriol-induced cell growth inhibition. Our results showed that progesterone and calcitriol up-regulate the expression of BAX along with other apoptosis-related proteins, which induced inhibition of endometrial cancer cell growth by apoptosis and cell cycle arrest. Citation Format: Laura R. Lee, Brian L. Hood, Huyen Nguyen, Leyla Kavandi, Jane M. Turbov, Larry G. Thaete, Chad A. Hamilton, Gustavo C. Rodriguez, George L. Maxwell, Thomas P. Conrads, Viqar Syed. Calcitriol and progesterone synergistically inhibit growth in endometrial cancer cells. [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 1716. doi:10.1158/1538-7445.AM2013-1716

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 women's 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 women's 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

Global proteomics has been witness to the solutionbased revolution in which mixtures of proteins are digested into peptides prior to fractionation and analysis by mass spectrometry (MS). In stark contrast to gelbased methods, the complexity of the protein mixture is increased rather than decreased prior to MS analysis. This “bottom up” approach makes peptide mapping as a protein identification tool obsolete as a typical MS scan will detect peptides originating from many different proteins. Instead, solution-based global proteomic studies rely on tandem MS to identify peptides, which are then extrapolated as originating from intact proteins. The results as far as the number of proteins identified using solution-based methods are quite impressive, with more than 1000 proteins being routinely identified in such studies [1].

Abstract 5126 The t(5;17)(q35;q21) variant of Acute Promyelocytic Leukemia fuses nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). The resultant NPM-RAR fusion protein blocks myeloid differentiation, and leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. The contribution of the N-terminal 117 amino acids of NPM contained within NPM-RAR has not been well studied. NPM is a molecular chaperone, and binds to a variety of proteins implicated in leukemogenesis. We performed a proteomic analysis to identify NPM-RAR interacting proteins. Vectors encoding NPM-RAR or RARA (as control) fused in frame to calmodulin binding peptide and Protein A were expressed in HEK293 cells, and interacting proteins subjected to tryptic digest. Peptides were analyzed by nanoflow reversed-phase liquid chromatography-mass spectrometry. Amongst other targets, we identified tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD) as a distinct binding partner for NPM-RAR. TRADD did not bind to wild-type RARA or NPM, suggesting that the interaction was unique to the fusion protein. The NPM-RAR/TRADD interaction was verified by reciprocal co-precipitation. Though NPM-RAR localizes primarily in the nucleoplasm, we also found a low level of NPM-RAR/TRADD dimer in the cytoplasm utilizing confocal microscopy. Expression of NPM-RAR in U937 cells impaired TNF activation of caspase 8 and caspase 3. TNF-induced acquisition of Annexin V, generation of sub-G0/G1 nuclear content, and cleavage of PARP were all blunted, indicating that NPM-RAR blocks TNF-induced apoptosis. Our studies identify a novel mechanism through which NPM-RAR impacts leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

e15560 Background: Optimal surgical cytoreduction (1cm) debulking as primary treatment for advanced stage epithelial ovarian cancer (EOC). Molecular descriptions of optimally and suboptimally debulked ovarian tumors are lacking. We have performed a global, quantitative proteomic analysis of optimally and suboptimally debulked EOC tumor samples. Methods: A cohort of 16 optimally (8) and suboptimally (8) debulked patients with archival formalin-fixed, paraffin-embedded tumor specimens was identified. Homogenous tumor cell populations were collected and protein digests were prepared and analyzed by high resolution mass spectrometry for identification and quantification by spectral counting. Immunohistochemistry (IHC) was performed and slides were scored using a semi-quantitative method (H-score). Results: An average of 1,110 proteins were identified within each cohort, of which 21 possessed significant (p

Epithelial ovarian cancer (EOC) is the leading cause of death among women with gynecologic malignancies and the fifth leading cause of cancer death in women in the United States. Cisplatin is one of the most effective and active agents in the treatment of advanced EOC, however, development of chemoresistance is a major obstacle. A recurrent theme is emerging that mitochondria and mitochondrial dysfunction are centerpiece in contributing to an aggressive carcinogenic phenotype. Similarly, we hypothesized that changes in the mitochondrial proteome are required to support development of cisplatin resistance in human EOC. We applied an organellar proteomics approach to elucidate quantitative alterations in protein abundance in mitochondria enriched from an isogenic pair of cisplatin sensitive (A2780) and resistant (A2780-CP20) human EOC cells. Protein isolates from mitochondria were analyzed by high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantitative determination of changes in the relative abundance level of identified proteins was accomplished by spectral counting. This analysis confirmed that there are substantial alterations in the mitochondrial proteomic composition in cisplatin resistant EOC cells. Differential proteins selected for western blot validation include hypoxia up-regulated protein 1 (HYOU1), activated leukocyte cell adhesion molecule (ALCAM), isoform 1 of dynamin-like 120kDa protein (OPA1), A kinase anchoring protein 12 (AKAP12), and ferredoxin reductase (FDXR). Our proteomic analysis demonstrated overexpression of HYOU1, ALCAM, AKAP12, and OPA1 in the resistant EOC cells. This panel of protein candidates have been implicated in other tumor types and are involved in relevant cell mechanisms to include evasion of apoptosis, increased tumor invasiveness, tumor hypoxia, cellular respiration, and mitochondrial fragmentation. 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 842. doi:1538-7445.AM2012-842

Arsenite oxidase, a member of the DMSO reductase family of molybdenum enzymes, has two molecules of guanosine dinucleotide molybdenum cofactor coordinating the molybdenum at the active site. X-ray absorption spectroscopy indicates that the Mo-S bonds shorten from 2.47 to 2.37 A upon reduction with the physiological substrate. It also indicates the presence of an oxo ligand at 1.70 A in both oxidized and reduced forms of the enzyme, together with a short, 1.83 A, Mo-O bond in the oxidized form that is lost upon reduction. Resonance Raman spectroscopy indicates that the two pterin dithiolene moieties have different aromaticities, with one, the Q-pterin, having a more discrete dithiolate structure while the other, the P-pterin, has considerable pi-delocalization. Our results indicate that the structure of arsenite oxidase is intermediate between that seen in other molybdenum enzymes, in which one ligand to the metal is provided by the polypeptide (serine, cysteine, or selenocysteine), and tungsten enzymes that lack a peptide ligand.

Mitochondria are important gatekeepers of cell survival in melanoma cells and their role as oxygen sensors, ATP producers, or inducers of reactive oxygen species are established. However, the role(s) of metabolic processes other than glycolysis are not well characterized in melanoma development and progression. Here we show that melanoma cells exhibit higher levels of oxidative phosphorylation (Oxphos) compared to human epidermal melanocytes. Furthermore, we present several independent lines of evidence (bioenergetics analysis using Seahorse XF24, stable isotope labeling of melanoma cells in culture, immunoblot analysis of isolated mitochondria, electron microscopy, and rho zero cells) that elesclomol, an inducer of oxidative stress, suppresses Oxphos in melanoma cells without a significant effect on glycolysis in vitro. Melanoma cells with intrinsically high levels of glycolysis are less sensitive to elesclomol. In addition, following chronic continuous drug exposure, resistant cells manifest a high glycolytic phenotype. Finally hypoxia inducible factor 1 alpha, a master metabolic switch of cell metabolism towards glycolysis, is upregulated following elesclomol treatment in a number of melanoma cell lines examined. Our results suggest that Oxphos is an important therapeutic target and that elesclomol specifically impinges on this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B144.

Isothiocyanates (ITCs), such as phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), are effective cancer chemopreventive compounds. Induction of cell cycle arrest and apoptosis is believed to be a major mechanism for their cancer preventive activity. Our previous studies indicate that covalent binding to target proteins is an important upstream event. However, the identity of ITC binding targets remains largely unknown. To identify proteins that are covalently modified by ITCs, human non-small cell lung cancer A549 cells were treated with 14C-PEITC and 14C-SFN and the cell lysates were extracted for analysis by 2-D gel electrophoresis and mass spectrometry. After superimposing the colloidal Coomassie blue protein staining pattern with the pattern of radioactivity obtained from X-ray films, it was clear that only a small fraction of cellular proteins contained radioactivity, presumably resulting from selective binding with PEITC or SFN via thiocarbamation. In this study, more than 30 proteins with a variety of biological functions, including cytoskeleton, redox regulation, protein quality control, stress response, mitochondria, and signaling transduction, were identified with high confidence. Here we report the identities of these potential ITC target proteins and discuss their biological relevance. The discovery of the protein targets may facilitate studies of the mechanisms by which ITCs exert their cancer preventive activity and provide molecular basis for designing more efficacious ITC compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C12.

e15527 Background: Identification of platinum resistance is key to improving ovarian cancer treatment and patient outcomes. Although DNA excision repair proteins function to remove platinum-induced...

5094 Background: Black women have higher mortality rates of uterine cancer than white women in the U.S. This disparity has often been attributed to Blacks having later stage of cancer at diagnosis ...

Background Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit. Results We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed. Conclusions The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging.

Objective: To develop a workflow for collecting specimens for proteomic analysis from the peritoneum, fallopian tubes (FT), and ovarian surface epithelium (OSE) at the time of risk-reducing surgery in women at increased risk for developing malignancies due to a germline BRCA mutation. Methods: A pilot set of 6 subjects with BRCA2 mutations were consented for an IRB-approved protocol for surgical specimen collection. During laparoscopic removal of the FT and ovaries, peritoneal washings, OSE brushings, and FT lavage specimens were collected without interfering with standard pathological analysis of the tissues. After immunodepletion, samples were loaded in a stacking gel and digested in-gel with trypsin. Digests were analyzed in duplicate by nanoflow reversed-phase liquid chromatography coupled online to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database from the European Bioinformatics Institute using SEQUEST. Differences in protein abundance between samples were derived by spectral counting. Immunohistochemical (IHC) staining for p53 and loss of heterozygosity (LOH) testing for p53 at the D17S-1844, D17S-516, and D17S-786 loci were performed on all paraffin-embedded FT specimens. Results: A total of 265, 328, and 241 different proteins were identified by 2 or more unique peptides each from the any of the OSE, FT and peritoneal samples, respectively. The number of proteins identified on both the right and left side in individual subjects ranged from 16-33% for OSE and 31-36% for FT. 37 common proteins involved in cell signaling, cell growth, cellular assembly, acute phase response, and DNA replication and repair were found in at least 1 specimen from all 6 subjects. 23 of the common proteins are involved in cancer pathways. All surgical pathology was negative both for malignancy and possible precursor lesions such as tubal intraepithelial carcinoma. MUC16 (CA-125) was elevated by proteomic analysis in the right FT of 1 subject; the left FT lavage in this subject could not be collected due to tubal scarring. Although IHC for p53 was normal in both of these FT, LOH for p53 was present in both. IHC was also negative for all remaining FT specimens. LOH was absent in both FT in 3 subjects, present in 1 FT but not the other in 2 subjects, and present bilaterally in 1 subject mentioned above. Conclusions: This workflow allows analysis of the proteomes of peritoneal, FT, and OSE specimens collected at the time of laparoscopic risk-reducing surgery, a technique which has not been described previously. It presents a unique opportunity for closer examination of the tissues at risk for malignant transformation in women with BRCA mutations. Harvesting proteins from the environment where ovarian and related cancers arise will inform current attempts to use the “p53 signature” to evaluate the FT as the potential source of many pelvic serous tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2011-4876

Triple negative breast cancer (TNBC) is clinically characterized as an aggressive form of cancer which is unresponsive to hormone and trastuzumab based therapies and associated with poorer overall patient prognosis. TNBC can be treated with chemotherapeutic agents but unfortunately typically half of patients do not respond to treatment because their breast cancer cells become resistant. Recent evidence suggests that psychological distress in cancer patients equates to poor chemotherapeutic response, indeed the National Cancer Institute reports that 50% of cancer patients report anxiety due to the cancer diagnosis, the fear of dying and chemotherapy. Historically, single markers depicting breast cancer prognosis have failed to readily translate to the clinic therefore the expression pattern of multiple molecules may prove better predictors of drug resistance to chemotherapeutic agents. We hypothesize that drug resistance in breast cancer may arise in part due to stress hormone-induced alterations in intrinsic cellular proteins/genes in cancer cells. To examine the effects of stress, via stress hormones on drug resistance MDA-MB-231 breast cancer cells were incubated with paclitaxel in the presence of physiological concentrations of cortisol, norepinephrine and epinephrine for 48 h. We utilized a quantitative proteomic strategy, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), and LC-MS/MS to compare differences in protein abundance between paclitaxel-treated and stress hormone/paclitaxel-treated cells. For proteomic profiling, paclitaxel-treated cells cultured in unlabeled (no stress hormones) and labeled (stress hormones) SILAC media were mixed, lysed, and proteins were resolved by 1D-SDS PAGE, digested in-gel with trypsin and analyzed by LC-MS/MS. For genomic analyses, total RNA was isolated using TRIzol, cRNA was generated and hybridized to Affymetrix GeneChip HGU133 plus 2.0 arrays. We identified 220 proteins and 1201 genes whose expression levels were significantly altered by stress hormones. We further identified a unique molecular profile of predictive candidate biomarkers related to stress hormone- induced drug resistance including a significant decrease in the mitotic checkpoint serine/threonine-protein kinase BUB1 and the DNA damage response enzyme PARP-1, and increases in the immunophillin, FKBP-5 and the cell proliferation/apoptosis regulator C-MYC. In conclusion, our results demonstrate that stress hormones promote resistance to paclitaxel through modulation of several markers of drug resistance in TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2572.

Dietary inorganic phosphate represents a novel chemoprevention target. Recent results from two different mouse models of tumorigenesis; the two-stage skin carcinogenesis and KrasLA1 lung tumor models suggest that a high phosphate diet increases tumor/papilloma number by approximately 50% relative to a low phosphate diet. Additionally, a low phosphate diet resulted in delayed incidence and growth of papillomas. These results suggest that increased consumption of dietary inorganic phosphate may have long-term consequences on overall health and particularly to tumorigenesis and consequently reducing dietary phosphate intake may be a novel chemoprevention strategy. Our in vitro studies have revealed an active role of inorganic phosphate in altering cell function in various cell types and suggest that the amount of available inorganic phosphate may have significant cell autonomous consequences on cell behavior including activation of ras and ERK1/2 signaling, and increased expression of transformation and metastasis associated genes such as osteopontin (spp1). However, the mechanism by which cells sense and respond to changes in extracellular phosphate resulting in increased proliferation and transformation is not fully understood. We have performed an analysis of the temporal changes in gene expression in response to elevated phosphate. Results identified the strong stimulation of immediate early genes within 15 minutes of exposure to elevated phosphate. Reporter and DNA binding assays revealed the requirement of serum response and AP-1 transcriptional elements and taken together characterize phosphate as a novel mitogen. Furthermore, we have identified the requirement of FGF receptor signaling as one of the earliest events in the phosphate signaling cascade. This finding provides a novel link between growth factor signaling, nutrient sensing and cell growth and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3789.

The incidence rate of esophageal adenocarcinoma (EAC) has risen by 350% since 1970, outpacing all other cancers (www.nci.gov). There are approximately 15,000 new cases of esophageal cancer per year in the United States, half of which are EAC. The incidence of EAC is 4 cases per 100,000 person years. This cancer is associated with a dismal prognosis, with an overall five-year survival of less than 15%. Although the survival rate depends on the stage of disease, more than 50% of patients present with dysphagia (difficulty swallowing) secondary to obstruction from the tumor and with incurable disease. Upon review of the Surveillance Epidemiology and End Results database for the last 27 years, it is ominous that the incidence rate for esophageal cancer is mirrored precisely by the age-adjusted mortality for a given year. Currently, risk for esophageal cancer is determined solely by the histologic appearance of Barrett's esophagus (BE), however there is a large degree of intra- and inter-observer variability among pathologists. The ability to identify and validate protein biomarkers of disease progression from Barrett's metaplasia to esophageal adenocarcinoma, when treatment outcome is still favorable, would result in a reduction in mortality by aiding in the identification of new diagnostic and treatment options for this disease. We hypothesized that signature protein biomarkers predictive of BE progression could be identified in tissue biopsies that correlate with the metaplasia-dysplasia-carcinoma pathway, which may provide a more reliable set of diagnostic tools, prognostic markers, and therapeutic targets for EAC. Cells from formalin-fixed paraffin embedded (FFPE) tissue sections from defined histological regions of metaplasia (n=9), dysplasia (n=10) and adenocarcinoma (n=12) of the esophagus were procured by laser capture microdissection. A mass spectrometry (MS)-friendly heat-induced/enzyme-mediated antigen retrieval method developed in our laboratory was utilized to produce global tryptic digests from the LCM-procured cells. The digests were randomized and analyzed by high-resolution liquid chromatography-tandem mass spectrometry with an Orbitrap MS, resulting in the identification of an average of 2015 peptides and 652 proteins per sample, with an average relative standard deviation of 15%. Spectral counting and hierarchical cluster analysis enabled the identification of over 80 proteins whose abundances significantly change as a function of neoplastic progression of BE. Several of these putative BE progression biomarkers were validated by quantitative multiple reaction monitoring MS and/or immunohistochemistry, including mucin 5 A/C and 2, cytokeratin 20 and galectin 3. This study is the largest and most comprehensive discovery-driven proteomic analysis of Barrett's esophagus tissue where multiple candidates of progression have been validated by orthogonal techniques. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4565.

MicroRNAs (miRNAs) represent an endogenous, post-transcriptional gene expression regulatory mechanism that is mediated by small, non-coding double-stranded RNAs (dsRNA) which target discrete messenger RNAs (mRNAs) resulting in their degradation or translational repression. Current computational methodologies predict that an average of 200 unique genes may be targeted by a single miRNA with the vast majority remaining, as yet, experimentally unvalidated. Furthermore, as miRNAs regulate their gene targets post-transcriptionally, the application of global, quantitative proteomic analysis to miRNA target discovery will yield a more comprehensive assessment of miRNA activity than gene expression analysis alone. Several miRNAs have been shown to be robustly downregulated in breast cancer, including microRNA-145 (miR-145). Recent investigations of miR-145 in breast cancer have revealed tumor suppressor activities for this miRNA and that loss of miR-145 expression may be associated with early stages of breast cancer pathogenesis. To elucidate targets of miR-145 and its role in breast cancer, we restored expression in the human breast cancer cell lines MDA-MB-231 and SKBR3 and conducted both phenotypic and global, quantitative protein expression analysis utilizing mass spectrometry-based proteomics. Phenotypic analyses indicated that MDA-MB-231 and SKBR3 cells stably expressing miR-145 exhibit a growth advantage relative to control cells. In support of this evidence, GO enrichment analysis of significantly differentially expressed proteins in miR-145-expressing MDA-MB-231 (MDA-145) cells revealed modulation of factors associated with cell cycle regulation, such as increased expression of the G2/M-phase regulatory factors anaphase promoting complex subunit 1 (ANAPC1) and cell division cycle 20 homolog (CDC20) expression and decreased expression of DnaJ (Hsp40) homolog (DNAJA3). Investigation of miR-145-specific effects via sequence analysis of 3’ untranslated regions derived from genes corresponding to proteins which were decreased in abundance in MDA-145 cells revealed 33 target candidates that contained a miR-145 “seed” motif. Of these 33 candidates, 21 have been previously predicted as miR-145 targets (miRNAMap). Target candidates are associated with cell cycle regulation and mitochondrial function and include integrin linked kinase (ILK), the cytokine growth factor chromosome 19 open reading frame 10 (C19ORF10) and microsomal glutathione S-transferase 1 (MGST1). Validation of target candidates and exploration of their role in the miR-145-mediated growth advantage phenotype are currently underway. In conclusion, these results indicate that restoration of stable miR-145 expression in breast cancer cells confers a growth advantage which is underscored by modulation of proteins associated with cell cycle regulation and mitochondrial activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2062.

Background Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src.

By combining chromatographic retention with MS, SELDI-TOF MS can generate protein profiles from as little as 1 μL of serum or as few as 25–50 cells.