hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan

Abstract

hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003). Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Figure 1

hAG-2 and hAG-3 show close homology at the protein level and are adjacent to each other at chromosome position 7p21. (A) Protein sequence alignment of hAG-2 and hAG-3 (DNAStar MegAlign program–Jotun Hein alignment). The boxed regions on the hAG-3 sequence indicate exact amino-acid matches of hAG-3 with hAG-2. (B) Diagram illustrating the genomic locations, transcriptional orientation, and exon structures of hAG-2 and hAG-3 on chromosome 7p21.

Figure 2

Immunohistochemical analysis of hAG-2, hAG-3, and ER protein in three separate breast ductal carcinoma donor tissue sections. The boxed region in panels A, D, and G is magnified in panels J, K, and L, demonstrating cytoplasmic staining of hAG-2 (J) and hAG-3 (K) and nuclear staining of ER (L).

Figure 3

hAG-2, but not hAG-3, protein is expressed in malignant prostate epithelial cells. Immunohistochemical analysis of hAG-2 and hAG-3 in three prostate adenocarcinoma donor tissue sections.

Figure 4

(A) Diagrammatic illustration of DAG-1 and C4.4a proteins showing the extracellular hAG-2 and hAG-3 minimum binding domains identified by yeast two-hybrid analyses. Although both structures are shown attached to the same cell, it is likely that the interactions of the hAG proteins are autocrine in nature for C4.4a and paracrine for DAG-1. (B) Real-time quantitative RT–PCR analysis of C4.4a mRNA expression in seven donor-matched adjacent normal (N) and tumour (T) breast samples (indicated 1–7) and four human breast cancer-derived cell lines. C4.4a is upregulated in all of the tumour samples, relative to their matched normal tissue, and expressed in all the breast cancer cell lines used to create the yeast two-hybrid prey library. Expression values are described as copies per nanogram of cDNA derived from a standard curve of known dilutions of C4.4a DNA.