Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.

We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.

Please cite this article in press as: Antberg L, et al. Pathway-centric analysis of the DNA damage response to chemotherapeutic agents in two breast cell lines. EuPA Open Proteomics (2015), http://dx.Available online xxx Keywords: DNA repair DNA damage Chemotherapy Breast Cancer Label-free Quantification Pathway-centric analysis Selected reaction monitoring Shotgun proteomics a b s t r a c tThe response to DNA damage by alkylation and DNA topoisomerase inhibition was studied in two breast cancer cells lines. We present data from both a shotgun and a targeted, pathway-centric approach to highlight the different DNA repair pathway modulation in the cell lines and the correlation with viability and DNA damage assays. This type of focussed profiling may be of utility in rapidly defining non-responders undergoing systemic neoadjuvant therapy.

Estrogen receptor is a key driver in breast cancer and is expressed in about 75% of breast tumors. ER positive tumors are susceptible to endocrine therapies; however, the major obstacle for curative treatment is recurrence due to resistance to anti-estrogens. Endocrine therapies may induce a selective pressure promoting growth of estrogen independent cell subclones. Our aim was to reveal molecular changes occurring in tumors in response to anti-estrogen treatment, and to identify subpopulations of cells able to withstand anti-estrogen treatment. A luminal-like estrogen-dependent orthotopically growing xenograft model was treated with fulvestrant, or exposed to estrogen deprivation. The effect of ER-signaling inhibition was analyzed using quantitative mass spectrometry (MS) -based proteomic analysis and high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). Cell surface marker expression (CD24 and SSEA-4) was monitored by flow cytometry, allowing detailed comparison of protein expression between intratumor cell subpopulations. We found that both modes of anti-estrogen therapy restrained tumor growth and induced expression of enzymes involved in TCA cycle, oxidative phosphorylation and fatty acid beta-oxidation. This was accompanied by changes in levels of specific metabolites indicative of a possible reprogramming of cell metabolism and utilization of oxidative phosphorylation in preference to aerobic glycolysis (decrease in Warburg effect). Furthermore, anti-estrogen treatment seemed to have selective effects on intratumor cell subpopulations, specified by expression of the markers CD24 and SSEA-4. More specifically, highly tumorigenic CD24low/SSEA-4low (dbl. low) cells were eliminated and the seemingly more benign CD24high/SSEA-4high (dbl. high) cells were enriched in the residual tumor. When comparing the proteome in dbl. low verus dbl. high cells sorted from untreated tumors, metabolism was one of the most differentially enriched processes. Enzymes involved in glycolysis, TCA cycle, respiratory electron transport chain and fatty acid were more abundant in the dbl. high subpopulation. These results suggest that cancer cells may reprogram their metabolism in response to anti-estrogen therapy to support a less estrogen-dependent phenotype. Moreover, subpopulations of cells with different metabolism may exist within the growing tumor, and these may respond differently to anti-estrogen treatment. Citation Format: Nirma Skrbo, Ufuk Kirik, Alexandr Kristian, Paolo Cifani, Linn Antberg, Siver A. Moestue, Olav Engebraaten, Gunhild M. Mælandsmo, Kristin Andersen, Peter James, Therese Sørlie. Protein expression analysis of intratumor heterogeneity in a luminal-like breast cancer xenograft. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A36.

High degree of intratumor cellular diversity represent a major challenge when attempting to cure cancer. The presence of tumor cell subpopulations with enhanced in vivo tumorigenic capacity (tumor initiating cells, TICs), and high resistance to conventional cancer therapy, compared with the “bulk” tumor cell populations, has for the last decade been the focus of many breast cancer research groups. The cellular heterogeneity of an orthotopic luminal-like breast cancer xenograft model was investigated using IHC, flow cytometry, whole genome expression profiling and mass spectrometry-based proteomics combined with in vivo tumorigenicity and targeted therapy assays. Epithelial cell adhesion molecule, EpCAM, highly specific for the human tumor epithelial cells, was used to separate human tumor cells from the mouse stromal compartment. Further flow analysis of the EpCAM positive tumor cell population revealed diverse expression of several cell surface markers, including CD24 and SSEA-4 (stage specific embryonic antigen 4). SSEA-4-/CD24-, SSEA-4+/CD24- and SSEA-4-/CD24+ populations were capable of initiating tumors in NOD SCID mice while SSEA-4+/CD24+ cells were non-tumorigenic. Tumors resulting from the SSEA-4+/CD24- subpopulation did not express CD24, while tumors arising from the SSEA-4-/CD24- and SSEA-4-/CD24+ populations, contained all four subpopulations. As measured by whole genome expression analysis and mass spectrometry-based proteomics, the molecular differences were most pronounced between tumorigenic subpopulations and the non-tumorigenic subpopulation. While the mRNA expression data revealed a high degree of similarity among the four subpopulations, the proteomics data suggested that several signaling pathways might have distinct and different activity across the populations. The effects of targeted therapy against ER and WNT signaling on heterogeneity and tumorigenicity have been evaluated by in vivo experiments. Citation Format: Nirma Skrbo, Kristin Andersen, Alexandr Kristian, Linn Antberg, Geir Olav Hjortland, Olav Egenbråten, Peter James, Gunhild Mari Mælandsmo, Therese Sørlie. Phenotypically diverse cancer cell subpopulations in a luminal-like breast cancer xenograft model are associated with different signaling pathways. [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 245. doi:10.1158/1538-7445.AM2013-245