The LIM domain gene LMO4 inhibits differentiation of mammary epithelial cells in vitro and is overexpressed in breast cancer

Abstract

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

Figure 1

Lmo4 is abundantly expressed in the lobuloalveolar units during pregnancy. (A) A single layer of ductal epithelium expressing Lmo4 transcripts is evident in the adult mammary gland. RNA expression was assessed by in situ hybridization by using sense (control) and antisense digoxigenin-labeled riboprobes corresponding to the full-length mouse Lmo4 sequence. Young adult virgin, 12-day pregnant, 2-day lactating, and 4-day involuting mammary glands were analyzed. The sense probe was hybridized to sections of 3-day involuting (shown), and to sections of lactating and pregnant mammary glands. (Original magnification ×50.) (B) Northern blot analysis of mammary glands from virgin, pregnant, lactating, and force-weaned (involuting) mice. Filters containing 20 μg of total RNA were hybridized with a mouse Lmo4 probe. The lower molecular weight bands may represent either an alternatively spliced variant of Lmo4 RNA or a cross-hybridizing species. The ethidium bromide-stained gel showing 28S and 18S ribosomal RNA provides a loading control.

Figure 2

Lmo4 and Ldb1 inhibit β-casein and whey acidic protein (Wap) RNA synthesis in SCp2 mammary cells induced to differentiate. (A) Reverse transcription-PCR analysis was performed by using total RNA derived from stably transfected SCp2 pools that were stimulated (+) with prolactin, insulin, and hydrocortisone or unstimulated (−) for 96 h. β-casein and Hprt were used as markers of differentiation and loading, respectively. At least five independent transfections were performed. PCR products were fractionated by gel electrophoresis, blotted, and hybridized with internal oligonucleotide probes. The ethidium bromide- stained gel for Flag-Ldb1 is shown Right. (B) Reverse transcription-PCR analysis was performed on the same pools of transfectants as shown in A, using primers specific for Wap and Hprt. (C) Immunoprecipitation (IP) and Western blot analysis (W) confirmed expression of Flag-Lmo4 and Flag-Ldb1 in SCp2 transfectants. Lysates from cells expressing either gene or containing empty vector were subjected to immunoprecipitation by using mouse anti-Flag antibody and then blotted with rabbit anti-Lmo4 or rabbit anti-Ldb1 antibody. Arrows indicate relevant proteins; HPRT, hypoxanthine phosphoribosyltransferase.

Figure 3

LMO4 is overexpressed in several human breast cancer cell lines. Northern blot analysis of poly(A)⁺ RNA (3 μg) from human and mouse (SCp2) breast epithelial cell lines. Filters were hybridized sequentially with mouse Lmo4, Ldb1, and Gapdh cDNA probes. The lower molecular weight LDB1 transcript in human breast cancer cell lines may represent a cross-hybridizing species. Sizes of the Lmo4 and Ldb1 transcripts were lower in the mouse SCp2 cell line relative to those in human cell lines. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Overexpression of LMO4 RNA in primary breast cancer. In situ hybridization by using full-length human LMO4 sense and antisense riboprobes labeled with digoxigenin. Low magnification of tumor specimens displaying low (A and E), moderate (B and F), and high (C and G) levels of LMO4 mRNA. The LMO4 sense riboprobe gave negligible staining (D and H). Sense controls (D and H) correspond to the tumors shown in C and G, respectively.

Figure 5

Overexpression of LMO4 RNA and LMO4 protein in primary breast cancer. In situ hybridization (with digoxigenin-labeled human LMO4 riboprobes) and immunohistochemistry (with a rat anti-LMO4 monoclonal antibody) were performed on tissue arrays containing archival breast specimens. High LMO4 RNA expression in an infiltrating lobular carcinoma (A), and two infiltrating ductal carcinomas (B and C) (antisense LMO4 probe); (D) benign fibroadenoma, displaying low expression of LMO4 RNA. Abundant LMO4 protein expression was detected in the corresponding infiltrating lobular and ductal cancer samples (E–G). Low levels of LMO4 protein were detected in the benign sample (H). Corresponding negative controls (Ig) for immunostaining are shown (I–K); (L) ductal carcinoma in situ, showing high expression of LMO4 RNA. Sense LMO4 probe gave no signal for any of the tumor samples. (Original magnification: A–D, ×100; E–K, ×200; L, ×50.)