Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease with a 5-year survival rate of 4%. A key hallmark of PDAC is extensive stromal involvement, which makes capturing precise ...tumor-specific molecular information difficult. Here we have overcome this problem by applying blind source separation to a diverse collection of PDAC gene expression microarray data, including data from primary tumor, metastatic and normal samples. By digitally separating tumor, stromal and normal gene expression, we have identified and validated two tumor subtypes, including a 'basal-like' subtype that has worse outcome and is molecularly similar to basal tumors in bladder and breast cancers. Furthermore, we define 'normal' and 'activated' stromal subtypes, which are independently prognostic. Our results provide new insights into the molecular composition of PDAC, which may be used to tailor therapies or provide decision support in a clinical setting where the choice and timing of therapies are critical.
Protein kinase activity forms the backbone of cellular information transfer, acting both individually and as part of a broader network, the kinome. Their central role in signaling leads to kinome ...dysfunction being a common driver of disease, and in particular cancer, where numerous kinases have been identified as having a causal or modulating role in tumor development and progression. As a result, the development of therapies targeting kinases has rapidly grown, with over 70 kinase inhibitors approved for use in the clinic and over double this number currently in clinical trials. Understanding the relationship between kinase inhibitor treatment and their effects on downstream cellular phenotype is thus of clear importance for understanding treatment mechanisms and streamlining compound screening in therapy development. In this work, we combine two large-scale kinome profiling data sets and use them to link inhibitor-kinome interactions with cell line treatment responses (AUC/IC
). We then built computational models on this data set that achieve a high degree of prediction accuracy (R
of 0.7 and RMSE of 0.9) and were able to identify a set of well-characterized and understudied kinases that significantly affect cell responses. We further validated these models experimentally by testing predicted effects in breast cancer cell lines and extended the model scope by performing additional validation in patient-derived pancreatic cancer cell lines. Overall, these results demonstrate that broad quantification of kinome inhibition state is highly predictive of downstream cellular phenotypes.
An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to ...separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg–1 s–1, for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.
Numerous aspects of cellular signaling are regulated by the kinome—the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by ...both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation driving many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. In this work, we attempt to link the state of the human kinome, or kinotype, with cell viability in treated, patient-derived PDAC tumor and cancer-associated fibroblast cell lines. We applied classification models to independent kinome perturbation and kinase inhibitor cell screen data, and found that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines, including the understudied kinases CSNK2A1/3, CAMKK2, and PIP4K2C. We next utilized these models to predict the response of new, clinical kinase inhibitors that were not present in the initial dataset for model devlopment and conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the perturbed state of the human protein kinome provides significant opportunity for better understanding of signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapeutic strategies for PDAC.
Abstract
Genetic alterations in signaling pathways and processes, including mutations, deletions, and amplifications, have been found in many pancreatic cancers. Prominent among these pathways is the ...phosphoinositide 3-kinase (PI3-K) pathway, which regulates multiple functions, including: proliferation; stress response; and apoptosis. Akt (also known as protein kinase B or PKB) is a serine/threonine kinase in the PI3-K pathway whose activity has been linked to providing cancer cells with anti-apoptotic properties. In pancreatic cancer, constitutive PI3-K/Akt activity appears to be an indicator of tumor aggressiveness, with high levels of active Akt associated with decreased patient survival. Currently, the gold standard for assessment of Akt activity is Western blot, where cellular homogenates are probed with anti-Akt antibodies to measure active Akt. However, this technology lacks single-cell resolution and yields an average result for a population of cells. Heterogeneity of Akt activity in single cells is thought to be an important mechanism in chemotherapy resistance, and analysis of multiple cell types suggests that Akt activity may be bimodal. A population average-based measurement would fail to capture the existence of high and low states of activity, whereas single-cell interrogation should reveal this behavior.
We describe the single-cell measurement of Akt activity in single human pancreatic cancer cells. An optimized peptide substrate incorporating non-native residues for peptidase resistance was used to measure Akt activity within cells. Briefly, the peptide was microinjected into a single cell, where it was available for phosphorylation by Akt. Single cells were lysed and their contents loaded into an overlying fused-silica capillary for electrophoretic separation of the non-phosphorylated and phosphorylated peptide. We first validated the system in 3 model pancreatic cancer cell lines before applying it to primary cells from human pancreatic cancer xenografts. Some tumor cell lines exhibited statistically significant bimodal behavior in the level of Akt activity. In the primary cells, Akt exhibited highly variable activity at the single-cell level, with some cells showing little to no activity while others demonstrated very high levels of activity as indicated by nearly complete phosphorylation of the substrate peptide. Single-cell Akt activity was blocked in the presence of wortmannin, a PI3-K inhibitor. This system also enabled characterization of peptidase action on the substrate with primary cells demonstrating a 10-fold lower level of peptidase activity. Future work focuses on utilizing this technology for higher-throughput analysis of Akt activity in primary patient tissue from multiple cancer types.
Citation Format: Angela Proctor, S. Gabriela Herrera-Loeza, Jen Jen Yeh, Nancy L. Allbritton. Measurement of Akt activity in single primary human pancreatic cancer cells using chemical cytometry. 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 3345. doi:10.1158/1538-7445.AM2014-3345
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