Differentially expressed genes regulating the progression of ductal carcinoma in situ to invasive breast cancer

Abstract

Molecular mechanisms mediating the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain largely unknown. We used gene expression profiling of human DCIS (n = 53) and invasive breast cancer (n = 51) to discover uniquely expressed genes that may also regulate progression. There were 470 total differentially expressed genes (≥2-fold; P < 0.05). Elevated expression of genes involved in synthesis and organization of extracellular matrix was particularly prominent in the epithelium of invasive breast cancer. The degree of overlap of the genes with nine similar studies in the literature was determined to help prioritize their potential importance, resulting in 74 showing overlap in ≥2 studies (average 3.6 studies/gene; range 2-8 studies). Using hierarchical clustering, the 74-gene profile correctly categorized 96% of samples in this study and 94% of samples from 3 similar independent studies. To study the progression of DCIS to invasive breast cancer in vivo, we introduced human DCIS cell lines engineered to express specific genes into a "mammary intraductal DCIS" xenograft model. Progression of xenografts to invasive breast cancer was dramatically increased by suppressing four genes that were usually elevated in clinical samples of DCIS, including a protease inhibitor (CSTA) and genes involved in cell adhesion and signaling (FAT1, DST, and TMEM45A), strongly suggesting that they normally function to suppress progression. In summary, we have identified unique gene expression profiles of human DCIS and invasive breast cancer, which include novel genes regulating tumor progression. Targeting some of these genes may improve the detection, diagnosis, and therapy of DCIS.

Figure 1. LCM of samples in Group 4

Tumor epithelial cells and immediately adjacent stroma were independently isolated by laser-capture micro dissection (LCM) to a purity of about 95%.

Figure 2. Hierarchical clustering

The top row shows dendrograms from unsupervised hierarchical clustering analyses based on genes expressed within each group of samples in this study (average=2866). The center row shows results from hierarchical clustering of the same samples using the refined profile of 74 differentially expressed genes determined in this study. The bottom row show results (dendrograms, heat maps, and genes) using the 74-gene profile in two independent cohorts from previous gene expression profiling studies comparing human DCIS and IBC. Note: analysis of the Scheutz study was restricted to 7 of the 9 total samples each of DCIS and IBC that were all evaluated using the same platform (U133A Plus 2.0 microarray chips).

Figure 3. Effect of Modulating Gene Expression in DCIS.COM Cells on MIND Xenografts

DCIS.COM cells were transduced with shRNAi corresponding to ten candidate invasion-suppressor genes (the cells originally expressed high levels of all genes). Four of the most efficiently suppressed genes (CSTA, DST, FAT1, TMEM45A) resulted in a highly significant increase in the progression of DCIS to IBC, consistent with the hypothesis that these genes function normally to suppress tumor invasion.

Figure 4. Effect of Modulating Gene Expression in h.DCIS.01 and SUM225 Cells on MIND Xenografts

h.DCIS.01 cells expressed high levels of CSTA, DST, and FAT1, and were transduced with the corresponding shRNAi which were most effective in suppressing expression in DCIS.COM cells. All three genes were highly suppressed, which resulted in significantly increased progression of DCIS to IBC xenografts, confirming results obtained with DCIS.COM. SUM225 cells expressed very low levels of all three genes. Further suppression using the same hairpins for CSTA and FAT1 were lethal, and further analysis was not possible. Further suppression of DST was not lethal and did not increase in the progression of DCIS to IBC xenografts.

Figure 5. Effects of Modulating Gene Expression in DCIS.COM Cells in 3-Dimensional (3D) Culture

To further characterize the functions of CSTA, FAT1, and DST observed in MIND xenografts, DCIS.COM.sh cells were evaluated in 3-dimensional cultures composed of Matrigel ± COL. All cells on Matrigel alone grew as small spheres. Control cells on Matrigel + COL1 formed small spheres which were occasionally connected by branching tubules. In contrast, shCSTA, shFAT1, and shDST cells spread and piled up on the gel surface, and invaded into the gels.