Osteopontin
(OPN)

2ar
Secreted phosphoprotein-1, Spp-1

Gene: maps to 4q21-q25 and comprises seven exons, six of which contain coding sequence (Crosby A.H. et al., 1995, 1996).
mRNA: size: 1.5 kb.
Protein: a secreted, adhesive, glycophosphoprotein. Contains a RGD (Arg-Gly-Asp) sequence and binds to integrins, including alpha_vbeta₃. In normal breast tissue, OPN is expressed by secretory phase ductal epithelium, occasionally by non-lactating breast epithelial cells, and is seen to be localized on the apical (luminal) aspect of the cells (Brown L.F. et al., 1992).

Although the functional consequences of OPN in breast cancer cells have not yet been completely elucidated, there is evidence from cell culture that at least some breast cancer cells (BCC) can adhere to and show increased migration in response to OPN (Bautista D.S. et al., 1994; Senger D.R. and Peruzzi C.A., 1996), suggesting one potential mechanism for increased aggressive behavior of OPN positive tumors.

Cell lines:
- OPN is produced by MDA-MB-435 breast cancer cells. These cells show RGD-dependent adhesion to and migration toward OPN in culture (Bautista D.S. et al., 1994).

- To study the effect of OPN on cellular invasiveness, basal OPN expression was first assessed in members of a progression series of human mammary epithelial cell lines (21PT: immortalized, non-tumorigenic; 21NT: weakly tumorigenic; 21MT-1: tumorigenic, weakly metastatic; MDA-MB-435 cells: tumorigenic, highly metastatic). The two lines which expressed lowest basal levels of OPN (21PT, 21NT) showed increased invasiveness through Matrigel when human recombinant (hr)OPN was added to the lower chamber of transwells. Both also showed a cell migration response to hrOPN. Populations of 21PT and 21NT cells stably transfected with an OPN-expression vector showed higher levels of cell invasiness than control vector transfectants. Examination of transfectants for mRNA of a number of secreted proteases showed that only urokinase-type plasminogen activator (uPA) expression was closely associated with OPN expression and cellular invasiveness. Treatment of the parental 21PT and 21NT cells with exogenous hrOPN resulted in increased uPA mRNA expression and increased urokinase activity of the conditioned media. Both increased cell migration and induction of uPA expression are thus potential mechanisms of increased invasiness of breast epithelial cells in response to OPN.

- Bone sialoprotein (BSP) expression was not detected in a panel of 11 human BCC lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB-231, MDA-MB-436, BT549, MCF-7ADR, Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts (Sharp J.M. et al., 1999).

Breast tumors:
- In a series of 14 tumors, expression of OPN protein (immunohistochemistry), but not mRNA (in situ hybridization), by breast cancer cells has been reported, suggesting that OPN secreted by tumour infiltrating macrophages may bind to and be taken up by the tumor cells (Brown L.F. et al., 1994).

- A majority of both in situ and invasive breast carcinoma lesions were found to express OPN. A high OPN expression was frequently associated with microcalcification deposition in the lesions (Bellahcène A. and Castronovo V., 1995).

- Plasma OPN level was found significantly elevated in metastatic breast cancer; it was higher for 3 or more sites of involvement versus 1 or 2 metastatic sites (Singhal H. et al., 1997).

- 18 primary breast cancers were studied by RT-PCR, for expression of calcitonin receptors (CTR) and of the bone proteins osteopontin (OPN) and bone sialoprotein (BSP). OPN and CTR were expressed by each of the tumours, and 7 (39%) additionally expressed an alternate form of CTR, whilst BSP was expressed by 13 tumours (72%). In situ hybridisation confirmed that expression of OPN and CTR was confined to the tumour cells (Gillespie M.T. et al., 1997).

- OPN protein and mRNA levels were examined in tumors from 154 patients with lymph node negative breast cancer. OPN protein was found (by immunohistochemistry) in tumor infiltrating macrophages and lymphocytes in 70% of tumors, and in carcinoma cells themselves in 26%. OPN mRNA was found (by in situ hybridization) in groups of tumor cells, individual tumor cells and tumor infiltrating macrophages and lymphocytes. Thus, breast cancer cells have the ability to either synthesize OPN or to bind and sequester OPN from the microenvironment (Tuck A.B. et al., 1998).

- Bone sialoprotein (BSP) transcripts were detected in 65% and OPN transcripts in 77% of a cohort of archival, primary invasive breast carcinoma examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages (Sharp J.A. et al., 1999).

Brown L.F. et al. (1992) Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces. Mol. Biol. Cell. 3, 1169-1180.
Bautista D.S. et al. (1994) Inhibition of RGD-mediated cell adhesion to osteopontin by a monoclonal antibody against osteopontin. J. Biol. Chem. 269, 23280-23285.
Brown L.F. et al. (1994) Osteopontin expression and distribution in human carcinomas. Am. J. Pathol. 145, 610-623.
Bellahcène A. and Castronovo V. (1995) Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer. Am. J. Pathol. 146, 95-100.
Crosby A.H. et al. (1995) Genomic organization of the human osteopontin gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II. Genomics 27, 155-160.
Crosby A.H. et al. (1996) Mapping of the human and mouse bone sialoprotein and osteopontin loci. Mammalian Genome 7, 149-151.
Gillespie M.T. et al. (1997) Calcitonin receptors, bone sialoprotein and osteopontin are expressed in primary breast cancers. Int. J. Cancer 73, 812-815.
Senger D.R. and Peruzzi C.A. (1996) Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin. Biochim. Biophys. Acta 1314, 13-24.
Sharp J.A. et al. (1999) Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer. Lab. Invest. 79, 869-877.
Singhal H. et al. (1997) Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival. Clin. Cancer Res. 3, 605-611.
Tuck A.B. et al. (1998) Osteopontin expression in a group of lymph node negative breast cancer patients. Int. J. Cancer 79, 502-508.
Tuck A.B. et al. (1999) Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene 18, 4237-4246.
Young M.F. et al. (1990) cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN). Genomics 7, 491-502.