Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression study of MCF7/tet-off clones expressing different HER2 isoforms

ABSTRACT: HER2 is a tyrosine kinase receptor causally involved in cancer. A subgroup of breast cancer patients with particularly poor clinical outcome expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTFs). However, since the CTFs lack the extracellular domain that drives dimerization and subsequent activation of full-length HER2, they are in principle expected to be inactive. Here we present evidence that at low expression levels one of these fragments, 611-CTF, activated multiple signaling pathways because of its unanticipated ability to constitutively homodimerize. A transcriptomic analysis revealed that 611-CTF specifically controlled the expression of genes that we found correlated with poor prognosis in breast cancer. Among the 611-CTF-regulated genes were several that previously have been linked to metastasis, including MET, EPHA2, MMP1, IL11, ANGPTL4 and different Integrins. Transgenic mice overexpressing HER2 in the mammary gland develop tumors only after acquisition of activating mutations in the transgene. In contrast, we show that expression of 611-CTF led to development of aggressive and invasive mammary tumors without the need for mutations. These results demonstrate that 611-CTF is a potent oncogene capable of promoting mammary tumor progression and metastasis. Affymetrix Gene Array expression study, using MCF7/tet-off clones stably transfected with vectors encoding HER2 and different truncated forms of the receptor, were used to elucidate the activity of the various protein isoforms. MCF7 clones were selected, maintain and expanded in the presence of doxycycline to avoid expression of the HER2 receptor isoforms. The experiments were started by seeding equal numbers of cells in two dishes, one with and one without doxycycline. 15 or 60 hours later total RNA was isolated in parallel from the two dishes. In the dishes without doxycycline the cloned HER2 isoforms were expressed from a CMV promoter. In total 50 arrays were included in the analysis. For key clones and time points biological replicates were included.

ORGANISM(S): Homo sapiens

SUBMITTER: Kim Pedersen

PROVIDER: E-GEOD-68256 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The nuclear LIM-only protein LMO4 is upregulated in breast cancer, especially estrogen receptor negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, Co-factor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both datasets was BMP7, whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its co-factor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity. Experiment Overall Design: We used the Tet-off system to establish several distinct MCF-7 cell clones, referred to as MCF7-LMO4-TetOff and MCF7-DN-Clim-TetOff cells, in which the expression of Myc-tagged LMO4 or DN-Clim was repressed by the presence of doxycycline in the medium. Total RNA was isolated from three distinct MCF7-LMO4-TetOff cell lines and three distinct MCF7-DN-Clim-TetOff cell lines in presence and absence of doxycycline after 6 days. Treatment with doxycycline To decrease variability, we pooled RNAs from three experiments for each of the three cell lines.

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Gene expression study of MCF7/tet-off clones expressing different HER2 isoforms

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