BAG-1 haplo-insufficiency impairs lung tumorigenesis

Abstract

Background: BAG-1 is a multifunctional co-chaperone of heat shock proteins (Hsc70/Hsp70) that is expressed in most cells. It interacts with Bcl-2 and Raf indicating that it might connect protein folding with other signaling pathways. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial.

Methods: We have evaluated the effect of BAG-1 heterozygosity in mice in a model of non-small-cell lung tumorigenesis with histological and molecular methods. We have generated mice heterozygous for BAG-1, carrying a BAG-1 null allele, that in addition express oncogenic, constitutively active C-Raf kinase (SP-C C-Raf BxB) in type II pneumocytes. SP-C C-Raf BxB mice develop multifocal adenomas early in adulthood.

Results: We show that BAG-1 heterozygosity in mice impairs C-Raf oncogene-induced lung adenoma growth. Lung tumor initiation was reduced by half in BAG-1 heterozygous SP-C C-Raf BxB mice compared to their littermates. Tumor area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies. Whereas BAG-1 heterozygosity did not affect the rate of cell proliferation or signaling through the mitogenic cascade in adenoma cells, it increased the rate of apoptosis.

Conclusion: Reduced BAG-1 expression specifically targets tumor cells to apoptosis and impairs tumorigenesis. Our data implicate BAG-1 as a key player in oncogenic transformation by Raf and identify it as a potential molecular target for cancer treatment.

Figure 1

BAG-1 haplo-insufficiency delays C-Raf driven adenoma growth. (a) Adenoma initiation in SP-C C-Raf BxB mice (BAG-1^+/+) and their Bag-1 haplo-insufficient littermates (BAG-1^+/-) at 1, 2 and 4 months of age. Adenoma foci values represent mean ± s.e. from at least 4 mice of each genotype analyzed in a blinded fashion by two independent readers. (b) Adenoma area in the lungs of 4 months old mice. Each value represents mean ± s.e. from at least 4 mice of each genotype analyzed in a blinded fashion. (c-d) Examples of hematoxylin-eosin stained sections of lungs from SP-C C-Raf BxB transgenic mice wildtype for BAG-1 in comparison to a BAG-1 heterozygous littermate. Scale bar, 200 μm.

Figure 2

BAG-1 expression in the lung of SP-C C-Raf BxB transgenic mice (a) Lanes 1–8 show immunoblotting data for expression of BAG-1 in the lungs of 8 month old SP-C C-Raf BxB transgenic mice heterozygous (+/-) or homozygous (+/+) for BAG-1 as indicated below the lanes. Lane 9 shows absence of BAG-1 expression in a BAG-1 null (-/-) embryonic day 12,5 liver extract; lane 10 control liver. The markers along the left indicate relative molecular mass. The same blots were subsequently reacted with an antibody against GAPDH to demonstrate protein equal loading and are shown below. (b) BAG-1 immunostaining in SP-C C-Raf BxB transgenic mouse lung cancer tissue.

Figure 3

Increased apoptosis but no change in PCNA and p-ERK in tumor cells of SP-C C-RafBxB transgenic BAG-1 heterozygous mice (a-c) Quantification of immunohistochemical staining for apoptosis using an antibody that detects activated caspase-3 (a), PCNA (b) and phosphorylated ERK (p-ERK, c) of adenoma cells from 1-month-old SP-C C-Raf BxB transgenic mice of the indicated BAG-1 genotype. Each value represents mean ± s.e. from at least 4 mice of each genotype analyzed in three different experiments.

Figure 4

Model for cooperative action of BAG-1 and C-Raf in tumorigenesis A possible model combining the findings of this report and other data is shown. It indicates that BAG-1 functions as an activator of C-Raf at the outer mitochondrial membrane where enzymatically activated C-Raf finds apoptosis-related targets (for details see text).