Gene expression profiling of cancer progression reveals intrinsic regulation of transforming growth factor-beta signaling in ErbB2/Neu-induced tumors from transgenic mice

Abstract

Upregulation of HER2/ErbB2/Neu occurs in 15-30% of human breast cancers and correlates with poor prognosis. Identification of ErbB2/Neu transcriptional targets should facilitate development of novel therapeutic approaches. Development of breast cancer is a multistep process; thus, to identify the transcriptomes associated with different stages of progression of tumorigenesis, we compared expression profiles of mammary tumors and preneoplastic mammary tissue from MMTV-Neu transgenic mice to expression profiles of wild-type mammary glands using Affymetrix microarrays. We identified 324 candidate genes that were unique to ErbB2/Neu-induced tumors relative to normal mammary gland tissue from wild-type controls. Expression of a subset of these genes (82) was also changed in the preneoplastic mammary glands compared to wild-type controls, indicating that they may play a pivotal role during early events of ErbB2/Neu-initiated mammary tumorigenesis. Further analysis of the microarray data revealed that expression of several known transforming growth factor (TGF)-beta target genes was altered, suggesting that the TGF-beta signaling cascade is downregulated in ErbB2/Neu-induced tumors. Western blot analysis for TGF-beta-Receptor-I/ALK5 and immunohistochemistry for TGF-beta-Receptor-I/ALK5 and phosphorylated/activated Smad2 confirmed that the Smad-dependent TGF-beta signaling cascade was inactive in these tumors. Although absent in most of the tumor, phosphorylated Smad2 was present in the periphery of tumors. Interestingly, presence of phosphorylated/activated Smad2 correlated with expression of Activin-Receptor-IB/ALK4, suggesting that although Smad-dependent TGF-beta signaling is absent in ErbB2/Neu-induced tumors, Activin signaling may be active at the leading edge of these tumors. Cumulatively, these data indicate that the TGF-beta pathway is intrinsically suppressed in ErbB2/Neu tumors via a mechanism involving loss of TGF-beta-Receptor-I/ALK5.

Figure 1

Identification of an ErbB2/Neu mammary tumor molecular signature. (a) Experimental design. Three mammary gland tissue types were isolated from mice for gene expression microarray analyses including: age-matched wild-type (Wild-Type), ErbB2/Neu tumor, and tissue adjacent to, but not including, the tumor. For the ErbB2/Neu-expressing tissues, the entire mammary gland was removed from the MMTV-Neu mouse. The tumor plus the 2 mm border of tissue immediately surrounding the tumor were cut out. This 2 mm bordering peritumoral tissue was removed and discarded. The adjacent ErbB2/Neu gland and the tumor were retained for analysis. (b) Northern blot analysis confirms ErbB2/Neu transgene expression in adjacent ErbB2/Neu tissue. In total, 20 μg of total RNA from microarray samples were separated by gel electrophoresis, transferred to nylon membrane, and then hybridized with a PCR-generated probe for Rat neu, cytokeratin-8 (epithelial cell marker), and cyclophilin (loading control). (c) Identification of genes with altered expression using the Affymetrix Change Call Algorithm. Samples were analysed by Affymetrix MGU74Av2 microarrays. The gene expression profile of each tumor (n = 5) was compared to the expression profile of each pooled wild-type sample (n = 3 profiles; five pooled tissues per profile). Only genes that were called changed in every comparison using the Affymetrix change call algorithm were retained. The intersection of these 15 comparisons (five tumors × three wild-type samples) contained 821 genes and ESTs. Removal of genes that were called absent on all of the microarrays reduced the list to 818 genes and ESTs. These genes represent the global tumor expression profile for ErbB2/Neu-induced mammary tumors. (d) Comparison of the Affymetrix subset of genes to the subset of genes acquired using Welch's approximate t-test with the Benjamini and Hochberg multiple testing correction. Statistical analysis of the entire 12 448 genes identified 849 genes that were statistically different in expression levels when comparing the ErbB2/Neu tumor partial transcriptomes to the wild-type partial transcriptomes. A total of 324 genes met both the Affymetrix algorithm and the statistical criteria, as indicated by the Venn diagram. These genes represent a filtered ErbB2/Neu tumor expression profile

Figure 2

The adjacent ErbB2/Neu tissue has preneoplastic characteristics. (a) Mammary gland tissue adjacent to ErbB2/Neu-induced tumors displays focal hyperplasia. Representative sections of wild-type control mammary gland and tissue adjacent to an ErbB2/Neu-induced tumor are shown (H&E, × 40). (b) Adjacent ErbB2/Neu mammary epithelia demonstrate active ErbB2/Neu signaling. Representative sections ( × 400) of immunohistochemical staining for phosphorylated ErbB2/Neu (Tyr-877) in wild-type mammary gland tissue (top panel) and adjacent ErbB2/Neu tissue (bottom panel) are shown. Note the selective membrane staining in the adjacent ErbB2/Neu tissue. (c) Known targets of ErbB2/Neu and previously characterized ErbB2/Neu tumor markers are expressed in the adjacent ErbB2/Neu samples. The signal intensity from microarray data is depicted on the y-axis with the tissue type on the x-axis. The expression values for individual samples are represented with the mean value for each group being indicated by a horizontal line

Figure 3

The molecular profile of the adjacent ErbB2/Neu tissue is intermediate between the profiles of tumors and control mammary tissues. (a) A dendrogram derived by two-way hierarchical clustering analysis of 7976 genes that were called ‘present’ or ‘marginal’ on at least one microarray is shown. Tissue types are grouped vertically, and genes are grouped horizontally. The arms of the tree are color coded by tissue type (pink = wild-type control (n = 3); green = adjacent ErbB2/Neu (n = 4); blue = ErbB2/Neu tumor (n = 5)). Expression levels are displayed as red = high, yellow = intermediate, and blue = low expression. (b) Self-organizing Map (SOM) analysis reveals progressive alterations of gene expression that correlate with tumorigenic stage. The y-axis represents the signal intensity derived from microarray analyses. For individual genes, values were normalized to the median value across all the samples and expression of individual genes is represented by a thin vertical line for each gene. Each bar represents a compression of all genes in an individual sample. The sample type is color coded (pink = wild-type control (n = 3); green = adjacent ErbB2/Neu (n = 4); blue = tumor (n = 5)). The clusters have been numbered from 1 to 4 (i.e. Cluster 1 to Cluster 4), with the total number of genes represented in each cluster indicated in the top right corner. Genes within individual clusters are identified in Tables 1 and 2 and in Supplementary Tables 1 and 2

Figure 4

Western blot analysis confirms alterations in components of the TGF-β pathway in ErbB2/Neu-induced mammary tumors. Whole-cell lysate (150 μg) from mammary gland tissue was resolved by SDS–PAGE and transferred to PVDF membrane. Membranes were sequentially evaluated for the presence/absence of several TGF-β pathway components. E-cadherin was included as a marker for epithelial cell content and the IgG band represents a loading control

Figure 5

Smad2 is inactive throughout ErbB2/Neu-induced mammary tumors except at the tumor/stroma interface. (a) Immunohistochemical analysis of phosphorylated and total Smad2 in ErbB2/Neu-induced tumors. The top panels are representative immunohistochemical analyses for phosphorylated Smad2 and total Smad2/3 on ErbB2/Neu tumor sections ( × 40). The bottom panels are a higher magnification ( × 200) of the boxed area from the panels above. Note the selective nuclear staining (brown) in the enlarged sections, identifying cells with activated Smad2. Sections were counterstained with Gill's hematoxylin (blue). Seven independent tumors from ErbB2/Neu mice were examined. These tumors were comparable to the size of tumors that were evaluated by microarray analyses and utilized for subsequent immunohistochemical analysis. (b) Assessment of DNA synthesis and apoptosis reveals cycling cells throughout ErbB2/Neu-induced mammary tumors. The top panel ( × 100) is representative staining for BrdU (green). Nuclei were counterstained with DAPI (blue). The bottom panel ( × 200) is representative in situ analysis for apoptosis. The brown cells (indicated by arrows) are apoptotic cells. (c) Smad2 is activated throughout small ErbB2/Neu tumors. The left panel is a representative immunohistochemical analysis for phosphorylated Smad2 on sections of small ErbB2/Neu tumors ( × 40). The right panel is a higher magnification ( × 200) of the boxed area from the panel on the left. Note the selective nuclear staining (brown) in the enlarged section, identifying cells with activated Smad2

Figure 6

Immunohistochemical analysis demonstrates heterogeneous activation of the ErbB2/Neu receptor in ErbB2/Neu tumor sections. The top panels ( × 40) are representative ErbB2/Neu tumor sections that were incubated with antibodies to total ErbB2/Neu and phosphorylated ErbB2/Neu (P-877 and P-1248). The bottom panels ( × 400) are higher magnifications of the boxed areas in the top panels. Note the selective membrane staining (brown). Sections were counterstained with Gill's hematoxylin (blue)

Figure 7

Immunohistochemical assessment reveals loss of detectable TGF-β-Receptor-I in adjacent ErbB2/Neu mammary gland and ErbB2/Neu tumor epithelia. The panels contain representative immunohistochemical staining for TGF-β-Receptor-I/ALK5 in wild-type control tissue sections (a, × 200), adjacent ErbB2/Neu tissues (b, × 200), and the ErbB2/Neu tumor tissues (c, × 200). Note the brown epithelial cells (arrow) in wild-type glands and the loss of this staining in epithelial cells in adjacent glands and tumors (arrows)

Figure 8

Activin-Receptor-IB expression correlates with active Smad2 signaling. The panels contain representative immunohistochemical staining for Activin-Receptor-IB on wild-type mammary glands (a, × 200), adjacent ErbB2/Neu mammary glands (b, 200), and ErbB2/Neu tumor sections (c, × 40). The bottom panel is a higher magnification (d, × 200) of the boxed area from the ErbB2/Neu tumor panel above. Sections were counterstained with Gill's hematoxylin (blue)