Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues

Abstract

Overexpression of the oncogene HER2/neu (c-erbB-2) occurs in up to 30% of breast cancers and is correlated with reduced survival, especially in node-positive disease. The aim of this study was to identify genes associated with the aggressive phenotype of HER2/neu-positive breast cancer cells using cDNA microarrays. RNA was extracted from three HER2/neu-positive and three HER2/neu-negative breast cancer cell lines. Pooled RNA was hybridized in duplicate to the breast specific microarray filters from Research Genetics containing 5184 unique cDNAs. Subsequently, a similar comparison was performed for pooled RNAs from 10 node-positive, ER-positive invasive ductal carcinomas, half of which were HER2/neu overexpressers. In HER2/neu overexpressing breast cancer cell lines, 90 (1.7%) genes were up-regulated and 46 (0.9%) were down-regulated, compared to cell lines with low HER2/neu protein levels. In contrast, in HER2/neu overexpressing primary breast cancers, more genes were down-regulated (N = 132, 2.5%) than up-regulated (N = 19, 0.4%). Many of the differentially expressed genes have previously not been known to play a role in human neoplasia, and some of them may represent novel tumor suppressor or oncogenes. No genes were up-regulated, and only a small number of genes were down-regulated both in cell lines and in carcinomas with high HER2/neu protein levels. These included transforming acidic coiled-coil containing protein 1, glycogen phosphorylase BB, complement 1q and one EST. The differential expression of select genes was confirmed by Northern blotting (trefoil factor 3) or by immunocytochemistry (glycogen phosphorylase BB, vimentin, KAI1). In an extended validation study, 18 of 41 ER-negative, but none of 46 ER-positive, breast carcinomas were found to express vimentin, and all but one of the vimentin-positive tumors were confined to the HER2/neu-negative subgroup (P = 0.0019). Our findings support an important role of the mammary stroma in determining the clinical breast cancer phenotype.

Figure 1.

Representative comparison of gene expression profiles in pooled HER2/neu-negative and -positive breast cancer cell lines. A: Pathways generated green/red overlay. The green spots represent genes more highly expressed in pooled HER2/neu-negative breast cancer cell lines, the red spots represent genes more highly expressed in pooled HER2/neu-positive breast cancer cell lines, and the yellow spots indicate genes expressed at similar levels. B: Histogram derived from A. Cutoffs were determined by statistical analysis and are indicated by red lines. In this analysis, 202 genes (3.9%) were up-, and 94 genes (1.8%) were down-regulated in HER2/neu-overexpressing breast cancer cells in vitro.

Figure 2.

Representative comparison of gene expression profiles in pooled HER2/neu-negative and -positive breast cancers. A: Pathways generated green/red overlay. The green spots represent genes more highly expressed in pooled HER2/neu-negative breast cancers, the red spots represent genes more highly expressed in pooled HER2/neu-positive breast cancers, and the yellow spots indicate genes expressed at similar levels. B: Histogram derived from A. Cutoffs were determined by statistical analysis and are indicated by red lines. In this analysis, 40 genes (0.8%) were up-, and 219 genes (4.2%) were down-regulated in HER2/neu-overexpressing breast carcinomas.

Figure 3.

Northern blot analysis of trefoil factor 3 (TFF3) expression in breast cancer cell lines and carcinomas. Northern blots of HER2/neu-negative (-) and -positive (+) breast cancer cell lines and carcinomas (20 μg total RNA per lane) were hybridized with TFF3 and GAPDH probes. A: Pooled RNA from three HER2/neu-negative and three -positive breast cancer cell lines. TFF3 is only expressed in the latter. B: TFF3 expression in five of the six breast cancer cell lines. TFF3 is expressed in two of the three HER2/neu-positive, but in neither of the two HER2/neu-negative lines. C: Representative TFF3 expression patterns in randomly selected HER2/neu-negative and HER2/neu-positive breast carcinomas.

Figure 4.

Glycogen phosphorylase BB (GPBB) expression in HER2/neu-negative and -positive breast cancer cell lines and carcinomas. Immunohistochemical stains of cell lines MDA-MB-435 (A) and MDA-MB-361 (B) as well as a HER2/neu-negative (C) and -positive (D) primary breast carcinoma. There is strong cytoplasmic reactivity in the HER2/neu-negative breast cancer cells (A, C) but almost no staining in the HER2/neu-positive specimens (B, D). Original magnifications, ×400.

Figure 5.

Vimentin expression in HER2/neu-negative and -positive breast cancer cell lines and carcinomas. Immunohistochemical stains of cell lines MDA-MB-468 (A) and SKBR3 (B) as well as a HER2/neu-negative (C) and -positive (D) primary breast carcinoma. There is strong cytoplasmic reactivity in the HER2/neu-negative breast cancer cells (A, C) but absence of reactivity in the HER2/neu-positive tumor cells (B, D). Original magnifications, ×400.

Figure 6.

KAI1 down-regulation in a breast carcinoma. Representative immunohistochemical stain of a frozen section of an invasive ductal carcinoma. There is complete absence of membrane staining in the neoplastic cells. Admixed endothelial and inflammatory cells act as positive internal controls. Original magnification, ×400.