Proteomic and PROTEOMEX profiling of mammary cancer progression in a HER‐2/neu oncogene‐driven animal model system

Abstract

The prevention of mammary carcinoma by immunological strategies targeting the HER-2/neu receptor has proved to be effective in preclinical models. Thus, a well-characterized HER-2/neu oncogene-driven mammary carcinogenesis model was analysed by various profiling strategies following "triplex" vaccination to identify new candidate targets for breast cancer immunoprevention. 2-DE-based proteomic profiling of preneoplastic and tumour lesions versus normal and aged mammary tissue demonstrated that tumour progression was associated with an up-regulation of molecular chaperones including glucose-regulated protein (GRP)78 and of proteins favouring cell motility, which was in line with the corresponding transcriptomic profiling data. Furthermore, PROTEOMEX analyses suggested that naturally induced autoantibody responses occur during early phases of mammary cancer progression. Most of the cancer progression-induced antibodies targeted proteins of normal and preneoplastic mammary glands. However, three proteins were only recognized by sera obtained from vaccinated mice, including 2 isoforms of annexin A6. The distinct expression patterns for annexin A6 and GRP78 during tumour progression were further verified by western blot and/or immunoprecipitation. In addition, an inhibitor-mediated blockade of GRP78 expression in a model cell line caused a reduced cell growth. Thus, the proteome-based approaches applied in the murine BALB-NeuT model might indeed provide candidates for immunoprevention strategies in breast cancer.

A Proteomic View at T Cell Costimulation

“…Preparative gels were loaded with 750 µg total protein of cell lysates, the spots of interest were subsequently digested in the respective gel in situ and subjected to mass spectrometry using the matrix-assisted laser-desorption/ionization time of flight (MALDI-TOF) instrument Voyager DE™ PRO (Applied Biosystems, Forster City, CA, USA) as previously described [57]. However, due to the very limited sample material available, preparative gels were run with samples representing unfractionated Ficoll-isolated T cells obtained from healthy donors.…”

Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

“…In addition, the biological variances within the respective expression profiles were assessed by performing further comparative expression profilings applying either minimal or saturating labeling difference gel electrophoresis (DIGE) strategies according to the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany) as recently described [43], [44].…”

Impact of the mitogen‐activated protein kinase pathway on the subproteome of detergent‐resistant microdomains of colon carcinoma cells

“…Equal protein amounts of total cellular lysate or aliquots of sucrose gradient fractions were separated by SDS‐PAGE according to the method of Laemmli and transferred onto PVDF (Roche Diagnostics, Mannheim, Germany) or nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) as previously described . Membranes were blocked for 1 h at room temperature in TBS containing 0.1% Tween 20 (Applichem, Darmstadt, Germany and 5% w/v skim milk (Difco, Becton Dickinson (BD), Heidelberg, Germany) membranes were incubated overnight at 4°C with the respective primary antibodies (Ab) directed against CAV1 (# 611339, BD), CD71 (sc‐7087), Ezrin (sc‐32759), FLOT 2 (sc‐28320), FYN (sc‐434), LYN (sc‐7274) phospho‐ERK (sc‐7383; all from Santa Cruz Biotechnology, Heidelberg, Germany), GAPDH (# 2118S), ERK 1/2 (# 4695 and # 9102), phospho‐ERK 1/2 (# 9101), SRC (# 2108S) and YES (# 3201S, all from Cell Signaling Technology (CST)/ New England Biolabs, Frankfurt, Germany) or β‐actin (A2228, Sigma‐Aldrich, Munich, Germany), the latter serving next to GAPDH as loading controls.…”

“…Equal protein amounts of total cellular lysate or aliquots of sucrose gradient fractions were separated by SDS-PAGE according to the method of Laemmli [43] and transferred onto PVDF (Roche Diagnostics, Mannheim, Germany) or nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) as previously described [45]. After several washing steps in TBS supplemented with 0.1% Tween 20 (TBST) the membranes were incubated with respective secondary antibodies (# 7074, CST; P0260 and P0160, DAKO, Hamburg, Germany) for 2 h at room temperature, rinsed several times in TBST and then developed using the ECL method or Lumi-Light Western Blotting Substrate (Roche) as recently described [46].…”