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)

Recently it has been shown that epithelial cell expression of the estrogen receptor (ER) and that of the proliferation-associated marker Ki-67 are almost mutually exclusive in the normal premenopausal human breast but that coexpression frequently occurs in estrogen receptor-positive (ER؉) breast cancers. This coexpression may indicate disordered expression of ER in the cell cycle or failure to suppress division of ER؉ cells and could be important in neoplastic transformation. The purpose of this study was to determine whether in situ proliferations known to be associated with different levels of risk for developing breast cancer contain these coexpressing cells and, if so, the stage at which they occur. We found that ER؉ proliferating cells were rare in premenopausal lobules but increased with age in the normal breast. There was no difference in nonlesional tissue between cancerous and noncancerous breasts. The percentage of dual-expressing cells was significantly increased , however , in all of the in situ proliferations and correlated positively with the level of risk of developing breast cancer. We suggest that development of at least some human breast cancers is associated with increasing failure to down-regulate ER as Estrogen is thought to be important in the pathogenesis of breast cancer and is associated with most of the epidemiological risk factors associated with the disease. 1 Furthermore, the antiestrogen tamoxifen decreases proliferation in breast cancers, 2,3 although its role in preventing the disease is in dispute at present. 4 Estrogen is also associated with epithelial proliferation in the noncancerous breast during the menstrual cycle 5,6 and in pregnancy 7 and acts on cells via the estrogen receptor (ER). Recently Clarke 8 has shown that there is almost mutual exclusion of steroid receptor expression and cell proliferation in normal human breast tissue, judged by the lack of dual immunostaining for ER and the human Ki-67 proliferation-associated nuclear antigen, which is usually demonstrable in the late G1, S, G2, and M phases of the cell cycle. This relationship, however, is lost in some breast cancers, in which a variable percentage of proliferating cells are estrogen receptor positive (ERϩ). This coexpression may therefore indicate disordered control of cell division in ERϩ cells or disordered regulation of ER in dividing cells and could represent an important pathogenetic step in the development of breast cancer. We have previously shown that the percentage of ERϩ cells within precancerous lesions correlates with the risk attributed to them in prospective studies. 9 Furthermore, a fundamental change appears to occur between hyperplasia of usual type (without atypia, HUT) and atypical ductal hyperplasia (ADH). The percentage of ERϩ cells in the former type of lesion increases with age, as it does in the normal breast, whereas in ADH it is high at all ages, as it is in ERϩ ductal carcinoma in situ (DCIS). This suggests that regulation of ER expression or control of numbers of ERϩ cells esca...

As oestrogen is associated with most of the epidemiological risk factors for breast cancer, the number and distribution of oestrogen receptor positive (ER+) cells could have a bearing on the development of the disease. ER+ cells were thus studied in the normal breast and in the spectrum of in situ proliferations which range from non‐atypical hyperplasia to in situ carcinoma and are associated with different levels of risk for developing breast cancer. In the normal pre‐menopausal breast, ER+ cells comprised the minority and were distributed singly, being surrounded by oestrogen receptor negative (ER−) cells. ER+ cells showed a statistically significant increase with age, reaching a plateau after the menopause, and the increase was associated with a tendency for positive cells to become contiguous in patches of variable size. A small proportion of lobules showing involutional change comprised over 90 per cent ER+ cells. The significance of this feature is not clear but no evidence was found that it was pre‐cancerous. The percentage of ER+ cells was slightly increased in hyperplasia of usual type (non‐atypical hyperplasia, HUT) and the relationship to age was maintained. The staining pattern was variable; in some lesions ER+ cells were surrounded by ER− cells whereas in others there were contiguous groups of positive cells sometimes accounting for more than 90 per cent of cells in the lesion. In contrast, all cases of atypical ductal hyperplasia (ADH), lobular in situ neoplasia (LIN) and ductal carcinoma in situ (DCIS) exhibited positivity of contiguous cells accounting for the majority in the lesions. Furthermore, the relationship between ER+ cell numbers and age was lost in these lesions, indicating autonomy of ER expression or of proliferation of cells expressing the receptor. It is hypothesized that this dysregulation of receptor expression or of ER+ cell numbers at the ADH stage may be the precursor of abnormal expression of cyclins and other cell cycle control proteins which have been shown first to appear in DCIS. Copyright © 1999 John Wiley & Sons, Ltd.