A suppression subtractive cDNA library representing mRNAs expressed at a higher level in the malignant human breast cancer cell line, MCF-7, relative to a benign breast tumorderived cell line, Huma 123, contained a cDNA, M36, which was expressed in estrogen receptor A (ERA)-positive breast carcinoma cell lines but not in cell lines from normal/benign/ ERA-negative malignant breast lesions. M36 cDNA had an identical coding sequence to anterior gradient 2 (AGR2), the human homologue of the cement gland-specific gene (Xenopus laevis). Screening of breast tumor specimens using reverse transcription-PCR and immunocytochemistry with affinitypurified anti-AGR2 antibodies showed that the presence of AGR2 mRNA and protein were both statistically significantly associated with ERA-positive carcinomas (P = 0.007, Fisher's exact test) and with malignancy (P V 0.025). When an expression vector for AGR2 cDNA was introduced into benign nonmetastatic rat mammary tumor cells, and three separate clones and two pools of cells were transferred to the mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. However, metastases occurred in the lungs of animals receiving the AGR2 transfectants in 77% to 92% of animals with primary tumors (P = 0.0001) compared with no metastases in the control groups. The AGR2 transfectants exhibited enhanced rates of adhesion to a plastic substratum and extracellular AGR2 enhanced the rate of attachment of AGR2-negative but not AGR2-positive cells. These experiments are the first to link mechanistically the developmental gene product, AGR2, with metastasis in vivo. (Cancer Res 2005; 65(9): 3796-805)

Anterior gradient 2 (AGR2) is a normal endoplasmic reticulum protein that has two important abnormal functions, amphibian limb regeneration and human cancer metastasis promotion. These normal intracellular and abnormal extracellular roles can be attributed to the multidomain structure of AGR2. The NMR structure shows that AGR2 consists of an unstructured N-terminal region followed by a thioredoxin fold. The protein exists in monomer-dimer equilibrium with a K(d) of 8.83μM, and intermolecular salt bridges involving E60 and K64 within the folded domain serve to stabilize the dimer. The unstructured region is primarily responsible for the ability of AGR2 to promote cell adhesion, while dimerization is less important for this activity. The structural data of AGR2 show a separation between potential catalytic redox activity and adhesion function within the context of metastasis and development.

The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor ␣, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and >216 months, respectively (P < 0.0001). Cox's multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9. Anterior gradient 2 (AGR2) protein is a secreted protein first described in Xenopus laevis embryos, where it induces the formation of the forebrain and the mucussecreting cement gland.1 Human AGR2 is also found co-expressed with estrogen receptor ␣ (ER␣) in breast cancer cell lines 2 and its presence significantly correlates with ER␣ in breast carcinoma specimens.3 Subsequent studies have found elevated expression of AGR2 in adenocarcinomas of the esophagus, pancreas, prostate, and non-small cell lung cancer, showing that it is a widely overexpressed protein in human carcinomas. 4 -10 We have shown that human AGR2 is expressed at higher levels in malignant, rather than in benign breast tumors, 11 and that, when introduced in an expression vector into the benign, nonmetastatic rat mammary cell line, Rama 37, 12 it causes metastasis in syngeneic rats. 11 These results suggest that AGR2's metastasis-inducing properties may contribute toward the malignant progression of some breast cancers. Certain molecules shown to induce metastasis in experimental breast cancer in rodents, for example, S100A4, S100P, and osteopontin (OPN), [13][14][15][16] provide a potential source for markers that may be useful as prognostic factors in predicting patient outcome in human breast cancer.

Epidermal growth factor (EGF) receptor (EGFR) modulates mitosis and apoptosis through signaling by its high-affinity (HA) and low-affinity (LA) EGF-binding states. The prevailing model of EGFR activation-derived from x-ray crystallography-involves the transition from tethered ectodomain monomers to extended back-to-back dimers and cannot explain these EGFR affinities or their different functions. Here, we use single-molecule Förster resonant energy transfer analysis in combination with ensemble fluorescence lifetime imaging microscopy to investigate the three-dimensional architecture of HA and LA EGFR-EGF complexes in cells by measuring the inter-EGF distances within discrete EGF pairs and the vertical distance from EGF to the plasma membrane. Our results show that EGFR ectodomains form interfaces resulting in two inter-EGF distances ( approximately 8 nm and < 5.5 nm), different from the back-to-back EGFR ectodomain interface ( approximately 11 nm). Distance measurements from EGF to the plasma membrane show that HA EGFR ectodomains are oriented flat on the membrane, whereas LA ectodomains stand proud from it. Their flat orientation confers on HA EGFR ectodomains the exclusive ability to interact via asymmetric interfaces, head-to-head with respect to the EGF-binding site, whereas LA EGFRs must interact only side-by-side. Our results support a structural model in which asymmetric EGFR head-to-head interfaces may be relevant for HA EGFR oligomerization.