Summary Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nrf2/Nfe2l2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine EGFR signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

A novel approach to pancreatic cancer biomarker discovery has been developed, which employs a stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS). Secreted proteins from CAPAN-2 human pancreatic cancer derived cells were collected after conducting stable isotope labeling by amino acids in cell culture (SILAC). The resulting SILAP standard contained <0.5% of individual unlabeled proteins. Pooled sera from patients with early stage pancreatic cancer or controls were prepared, and an equal amount of the SILAP standard was added to each sample. Proteins were separated by isoelectric focusing (IEF) prior to two-dimensional liquid chromatography (2D-LC)–MS/MS analysis. A total of 1065 proteins were identified of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. ELISA validation of these findings was successfully performed for two proteins, ICAM-1 and BCAM. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC-MS/MS for the identification and relative quantitation of potential biomarkers of pancreatic cancer. This technique allows for the detection of low-abundance proteins, and focuses only on biologically relevant proteins derived from pancreatic cancer cells.

Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain < 0.5 % of the endogenous unlabeled forms. Identified proteins were screened to provide a set of fifteen candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the fifteen candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.

Objective Pancreatic stellate cells (PSCs) are important players in pancreatic fibrosis and are major contributors to the extracellular matrix proteins observed with the stromal response characteristic of pancreatic ductal adenocarcinoma (PDAC). PSCs are also believed to secrete soluble factors that promote tumor progression, however no comprehensive analysis of the PSC proteome in either the quiescent or activated state has been reported. Methods Using two-dimensional tandem mass spectrometry and the RLT-PSC cell line, we present the first comprehensive study describing and comparing the quiescent and activated human PSC secreted proteomes. Results Very few proteins are secreted in the quiescent state. In stark contrast, activated PSCs secreted a vast array of proteins. Many of these proteins differed from those secreted by PDAC derived cell lines. Proteins associated with wound healing, proliferation, apoptosis, fibrosis and invasion were characterized. Selected proteins were verified in human tissue samples from PDAC, dysplastic pancreas, and normal pancreas using Western blot analysis and immunohistochemical staining. Conclusions Our study represents the first comprehensive analysis of proteins secreted by PSCs. These findings lay the foundation for characterizing PSC derived proteins involved in stroma-tumor interactions and the promotion of pancreatitis and PDAC.

Background:The relationship between divergence of amino-acid sequence and divergence of function among homologous proteins is complex. The assumption that homologs share functionthe basis of transfer of annotations in databases -must therefore be regarded with caution. Here, we present a quantitative study of sequence and function divergence, based on the Gene Ontology classification of function. We determined the relationship between sequence divergence and function divergence in 6828 protein families from the PFAM database. Within families there is a broad range of sequence similarity from very closely related proteins -for instance, orthologs in different mammals -to very distantly-related proteins at the limit of reliable recognition of homology.