Abstract
Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1.
Highlights
Breast cancer is one of the most common malignancies and is increasing fatal worldwide
To identify candidate genes involved during mammary tumorigenesis, we examined the following seven independent microarray datasets: “Ductal carcinoma in situ (DCIS) epithelium versus normal breast epithelium” and “invasive ductal breast carcinoma versus normal breast epithelium” (GSE14548) [8]; “DCIS versus normal tissue”, “invasive ductal breast carcinoma versus normal tissue” and “invasive lobular breast carcinoma versus normal tissue” (GSE1477) [9]; and “invasive ductal breast carcinoma versus normal tissue” and “invasive breast carcinoma versus normal tissue” (TCGA)
To validate the differential expression of the genes, we employed Quantitative RT-PCR (qRT-PCR) analysis of the following samples: as for human mammary glands, we used RNA samples extracted from breast cancer tissues of two independent cases and their corresponding normal mammary tissues as a control (Figure 1B); as for human cultured cells, we used RNA samples extracted from eleven human breast cancer cell lines of distinct intrinsic subtypes [10] and primary culture of normal mammary epithelium (HMEC), and immortalized normal mammary epithelium (HMEC4htertshp16) as a control (Figure 1C)
Summary
Breast cancer is one of the most common malignancies and is increasing fatal worldwide. Hedgehog (Hh) signaling is key for cell fate determination and tissue patterning during embryonic development and adult tissue restoration. Malfunction of this signaling pathway causes numerous developmental anomalies and in turn its dysregulated activation impacts on the development and progression of many human malignancies, including breast cancer [1, 2]. The expression of GLI1, a key transcription factor in the downstream of Hh signaling, clinically associates with unfavorable prognosis of breast cancer patients [3], and experimentally the conditional expression of GLI1 in a transgenic mouse model induced mammary tumors [4], suggesting a crucial role of GLI1 in breast cancer development
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