Osteonectin Expression In Cancer Development And Progression

Osteonectin seems to be able to influence most of the biological properties of the cell that are highly relevant in the context of cancer development, growth, and dissemination. The most obvious reasons for this are that osteonectin is able to initiate changes in the ECM, cell adhesion and spreading, and also in the cytoskel-eton. It has been found to influence cell proliferation, and apparently, it can regulate vascularisation. The elucidation of these properties has inevitably led to the study of its expression in cancer development and progression.

Osteonectin reportedly occurs in a wide spectrum of cancerous tissues from human subjects. Porter et al. (1995) found high osteonectin immunoreactivity in invasive tumours of the GI tract, breast, lung, kidney, ovary, brain, and adrenal cortex. They state that normal tissues show low levels of reactivity. It is somewhat paradoxical that trophoblast cells, which are a highly invasive cell component of the placenta, show low levels of reactivity. Nevertheless, bone extracts and osteonectin itself have been found to enhance the motility in vitro of prostate epithelial cells as well as prostate cancer cells (Jacob et al. 1999). The presence of osteonectin has also been demonstrated in normal as well as adenoma of human thyroid (Burgi-Saville et al. 1997). However, there is a reasonable body of evidence that suggests a close association of osteonectin expression with the progression of human melanomas. Osteonectin is not expressed by normal melanocytes and it is weakly expressed in a small proportion (4/25) of nevocellular nevi. The level of osteonectin expression is moderate in a majority (13/14) of dysplastic nevi. However, the expression occurs invariably and at a very high level in both primary (7/7) and metastatic (29/29) melanomas (Ledda et al. 1997).

A few studies have also been carried out on the relevance of osteonectin as a marker for the progression of breast cancer. According to Bellahcene and Castronovo (1995), osteonectin is only weakly expressed in benign breast disease, but the expression is very strong in both in situ and invasive carcinomas. The presence of oestrogen receptors in breast cancer is regarded as an indicator of differentiation and good prognosis, and their absence is suggestive of clinically aggressive disease. Apparently, there is an inverse relationship between ER status and osteonectin expression. Tumour samples that were low in ER content tended to contain fourfold higher levels of osteonectin mRNA as compared with tumours with high ER levels (Graham et al. 1997). It would be of much interest, in this context, to examine whether osteonectin expression relates directly to the presence of EGFr, because ER-negative breast cancers often tend to be EGFr positive. There is thus an apparent relationship of osteonectin levels to disease progression. This has been confirmed in another study, in which Podhajcer et al. (1996) found that the osteonectin gene is expressed at a high level in invasive human breast carcinoma and also in metastatic lymph nodes. Osteonectin transcripts were found in the fibroblast stroma. Furthermore, high levels of expression of stromelysin-3 also accompanied high osteonectin expression. Osteonectin seems to be able to activate MMP2 in the invasive breast cancer cell lines MDA-MB231 and BT549, but not in the noninvasive MCF7 cells.

This ability was associated with the osteonectin peptide 1.1 (Gilles et al. 1998). Although Gilles et al. (1998) also found some attenuation of TIMP-2, they have suggested that the invasive propensity of the breast cancer cell lines is most likely attributable to osteonectin-mediated activation of MMP2.

There can be little doubt that metalloproteinase expression and the expression of their inhibitors corresponds closely with invasive potential of cancers (see Sherbet and Lakshmi, 1997b). The apparent relationship between osteonectin and MMP does not necessarily reflect a causal connection. There is the distinct possibility that a common effector, such as bFGF, may regulate both. Nonetheless, the outcome could be an alteration in the invasive nature of the tumour.

Bellahcene and Castronovo (1995) have stated also that high expression of osteonectin was associated with calcification of the lesions. However, contrary to the findings of Bellahcene and Castronovo (1995), Hirota et al. (1995) reported no correlation between osteonectin expression and the development of foci of calcification. It ought to be stated, nonetheless, that in other cellular systems a relationship does seem to subsist between osteonectin levels and calcification. Human dental pulp cells maintained in tissue culture express osteonectin, and they also contain calcified nodules. The level of osteonectin closely correlates with the level of calcification. When these cells are treated with bFGF, osteonectin expression is reduced and calcification of the ECM is abolished (H. Shiba et al. 1995). M. Sato et al. (1997) used a human salivary cancer cell line and have reached similar conclusions. These cells produce tumours when implanted into nude mice. When treated with VD3, tumour growth rate was reduced and calcified foci appeared in the tumours. In parallel, M. Sato et al. (1997) also found the expression of osteonectin mRNA in these treated tumours.

Whether the expression of osteonectin, together with other bone matrix components such as osteopontin and bone sialoprotein, could have some bearing on the propensity of breast tumours to metastasise to the bone, is currently being debated. Osteopontin has been implicated in tumour cell motility (Xuan et al. 1995; Sung et al. 1998). Oates et al. (1996, 1997) transfected Rama-37, a rat mammary epithelial cell line, with genomic DNA fragments from a human mammary carcinoma cell line. The transfectants were found to produce tumours with metastasising ability. They then isolated a cell line from a metastatic tumour and compared its mRNA profile with a control cell line, by subtractive hybridisation. One of the mRNAs strongly expressed, (nine-fold greater in the metastatic cell line as compared with the nonmetastatic parent line), was that for osteopontin. An increase in the level of expression does not constitute irrefutable evidence of a relationship to metastatic ability. Oates et al. (1996) did show that similar transfection of Rama-37 cells with DNA from benign tumours did not result in elevated expression of osteopontin. Some of these early studies have been confirmed recently. The levels of osteopontin have been found to be low in nontumorigenic cells and tumour cells with low metastatic ability. The osteopontin-transfectant cells as well as cells exposed to exogenous osteopontin have been reported to make marked gains in invasive ability (Tuck et al. 1999). What is even more interesting is the observation by these authors that, under both experimental conditions, the gain in invasive ability was accompanied by increases in the expression of uPA mRNA as well as the uPA protein. This has marked similarities with the effects of osteonectin on cell invasion. One should recognise, nonetheless, that VD3 can act independently of osteopontin, because although it can enhance the expression of both osteonectin and osteopontin, VD3 indeed inhibits cell proliferation and invasion. Its ability to inhibit invasion can be directly linked with the down-regulation of PAs and MMPs together with an up-regulation of the endogenous MMP inhibitors. These arguments simply emphasise the need to include osteopontin in studies of osteonectin involvement in cancer cell invasion and metastasis.

It would be relevant to cite here the observations of Jacob et al. (1999), who found that bone extracts as well as osteonectin enhanced the motility of tumour cells that normally metastasised to the bone, e.g., breast and prostate cancer. However, cell lines derived from tumours that do not normally metastasise to the bone did not respond in this way. Jacob et al. (1999) postulate, in consonance with the discussion above, that this apparent differential effect of osteonectin on cell motility could be due to the ability of osteonectin to induce tumour-associated metalloproteinase activity. An activation of uPA together with enhanced proliferative potential has been reported in MCF7 cells exposed to soluble factors secreted by the osteogenic cell line SaOS-2. MCF7 cells are normally only weakly invasive, but they appeared to become more invasive when cultured on an ECM produced by SaOS-2 cells. Furthermore, this acquisition of invasive potential seemed to be related to the ability of the ECM to induce uPA expression in MCF7 cells (Martinez et al. 1999). Although these findings are of considerable significance in the context of cancer invasion, it is necessary to take into account a number of related facts and factors, lest one be led into an alley of overinterpretation of the data. It should be recognised that both MCF7 and MDA cells do synthesise uPA, although MCF7 cells do so at a far lower level than do MDA-MB231 cells. It should be recognised also that ability to syn-thesise uPA is not itself directly related to the invasive ability of cancer cells. Urokinase receptors as well as PAIs enter into the equation. Undoubtedly there is a large body of correlative evidence derived from the study of plasminogen activator expression in a host of human tumours. Nonetheless, all potential interacting factors need to be checked before one can be certain that one is, indeed, dealing with uPA as the major instigator of invasive potential.

Martinez et al. (1999) state that the enhancement of invasive behaviour was found only in the ER-positive MCF7 cells, but not in the ER-negative MDA-MB231 cells. This is consistent with the experiments described by Hachiya et al. (1995), who found that oestrogen enhanced uPA as well as tPA expression and also enhanced the invasive ability of breast cancer cells. They also demonstrated that PA expression is regulated by oestrogen, because tamoxifen blocked the production of PA and also inhibited the invasive ability. Among other factors that come into the reckoning is the hepatocyte growth factor (HGF). HGF bears sequence homology to PA. PA is known to activate HGF (Mars et al. 1993). Furthermore, HGF is a potent mitogen and can induce angiogenesis as well as vascular invasion (Hildebrand et al. 1995). It would be worth recalling here that the ER status correlates inversely with EGFr in these cell lines. EGF is another modulator of the invasive behaviour of cancer cells. Whether the changes in the invasive behaviour might have been mediated by EGF receptors is a moot point.

As stated previously in this section, osteonectin expression is said to be inversely related to ER status, with high expression of osteonectin being found in ER-negative cells. Furthermore, it also has been claimed that osteonectin stimulates MMP expression in MDA cells, but not in MCF7 cells. It may be that uPA is more relevant in the context of breast cancer cell invasion than are MMPs. Overall, there is a reasonable body of evidence to suggest the ectopic expression of these bone matrix proteins might have serious implications for the osteotropic metastasis of breast cancer. However, the mechanisms involved remain to be elucidated.

Although the above discussion suggests a direct relationship between malignancy and levels of osteonectin expression, in ovarian epithelial cells an inverse relationship has been reported. Mok et al. (1996) found high osteonectin expression in normal ovarian epithelial cells, but this was markedly reduced in ovarian carcinoma cells. They also transfected osteonectin cDNA into SKOV3 cell, which is an ovarian carcinoma cell line. This resulted in reduced growth rate in vitro, and furthermore, these cells were less tumorigenic when implanted into nude mice. These results suggest and impute a tumour suppressor property to osteonectin. This is supported by recent work by Vial and Castellazzi (2000). The expression of osteonectin is down-regulated when cells are transformed by oncogenes. Such a down-regulation is noticed in chick embryo fibroblasts transformed by v-src or v-jun oncoproteins. When the protein is reexpressed, these cells retain the transformed phenotype but lose their ability to form fibrosarcomas in vivo (Vial and Castellazzi, 2000). In other words, in the experimental model osteonectin does seem to behave like a tumour suppressor. Nonetheless, it would be reasonable to expect further confirmation of the possible tumour suppressor function. Some of these uncertainties are compounded by the view expressed in some quarters that osteonectin might not be a reliable marker for osteosarcoma (Grundmann et al. 1995; Park et al. 1996).

An overall view of the present status of osteonectin in relation to cancer progression ought to be ambivalent. There is a need for far more extensive investigation of human tumour types. Above all, much more experimental work is needed to establish the various postulates that bring together the putative functions of osteonec-tin with the altered biological properties of the cancer cell. In a sense, therefore, it would be premature to dive into investigations of clinical material without appropriate groundwork. This is especially important with respect to osteonectin function, because the protein contains domains that reputedly possess antagonistic functions. The scientific community has not even begun to unravel the mechanisms by which these antagonistic functions become expressed in the physiological setting. Equally, it would be unreasonable to delay the investigation of the relevance of osteonectin in tumour classification, or its potential in clinical management of patients, if indeed it has even the semblance of predictive value.

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