Tumour cells organize into solid tumours and metastasize due to a change from the normal phenotype in the local environment toward an angiogenic one. This is a consequence of the prevalence of proangiogenic factors compared to the antiangiogenic ones. We demonstrated by means of RT–PCR and Northern blotting that pancreatic and colon adenocarcinoma cells express various angiogenic factors like the endothelial cell mitogens- VEGF and bFGF, ETS-1 (documented as an angiogenic factor by Oda et al, 1999a
), as well as haemoxygenase 1 (unpublished data), which has been recently found to induce angiogenesis (Deramaudt et al, 1998
). The presence of these factors is likely to mediate the growth and invasion of these adenocarcinomas by induction of angiogenesis. On the other hand, the expression of angiogenic factors in tumours is paralleled by the decrease of the antiangiogenic factors. One of them, the recently discovered BAI1 has now been detected in normal pancreatic, colon and lung tissue, both at transcriptional and protein level, as reported previously and in this study. However, there was no detectable BAI1 expression in any of the p53
defective pancreatic and colon cancer cell lines, nor in the pancreatic tumour tissue samples that were examined. Nevertheless, the wild-type 53 adenoviral-mediated transfer was able to restore the BAI1 expression in all tumour cell lines checked. These features displayed in pancreatic and colon cancer cells are in accordance with previous reports that the lack of BAI1 expression correlated with the p53
mutation-related tumour angiogenesis. However, additional mechanisms seem to play a role in the regulation of BAI1, as a wild-type p53
glioblastoma cell line – U87MG does not express BAI1 (Nishimori et al, 1997
). Nevertheless, expression of BAI1 induced by adenoviral transfer in these cells did strongly inhibit their growth in the transparent skin chamber compared to parental cells, a feature that was not found after LacZ transfer (unpublished observations).
Several lines of evidence presented in this study emphasize the importance of the recently described BAI1
gene. The presence of five thrombospondin type I repeats has been proposed as an explanation for its antiangiogenic effects, taking into consideration the effects on angiogenesis reported for other proteins containing these repeats such as TSP-1, TSP-2, METH-1, and METH-2 (Vazquez et al, 1999
). Of particular interest is the fact that the BAI1
gene transfer in tumour cells, similar to that of p53
, seemed to change the RNA level of the endothelial cell mitogen VEGF, but did it alter other factors (bFGF, uPA, and ETS-1) implicated in the local angiogenic and proteolitic balance. Moreover, as reported for wild-type p53, BAI1 over-expression inhibited the expression of collagenase-1 (MMP-1), which is known to be a positive regulator of tumour angiogenesis (Sang, 1998
), and was the only metalloproteinase we found to be expressed in Panc-1 cells. As yet, it is still unclear at what regulatory level this inhibition occurs, since BAI1 did not change the levels of ETS-1, which was implicated in the regulation of collagenase-1 and of other matrix metalloproteinases (Iwasaka et al, 1996
experiments revealed that BAI1 transfection resulted in no detectable cytotoxic effect on tumour cells, but confirmed a level of cytotoxicity for endothelial cells, consistent with previous reports (Nishizaki et al, 1999
). In vivo
, subcutaneous and skin chamber implantation of Panc-1/BAI1 in NK-depleted SCID mice strongly suppressed tumour growth, while wild-type and LacZ
-transfected tumours failed to do so. The immunostaining of tumour-related microvessels also showed a significant reduction of vessel density in Panc-1/BAI1, as compared to Panc-1 or Panc-1/Lac-Z tumour tissue. By capturing and off-line analysis of images from the transparent skin chamber, a decrease of vascularity in Panc-1/BAI1 tumour was directly observed and clearly indicated the inhibitory effect of BAI1 on angiogenesis.
Reported herein is also the observation that local overexpression of BAI1 disables any localized angiogenesis to such an extent that is able to induce an apparent state of dormancy in a transfected pancreatic adenocarcinoma cell line. Thus, even though the tumour cells were not killed, the tumour was unable to grow. One explanation for this phenomenon may involve an antiproliferative effect toward the endothelial cells, but the antitumour effects that occurs in vivo
may be also due to putative interactions of the extracellular region of BAI1, since it is known to contain five thrombospondin type I repeats and of a RGD motif that is recognized by integrins. In addition, BAI1 inhibition of interstitial collagenase expression may prevent the dissolution of the extracellular matrix. Matrix metalloproteinase-1 (MMP-1) was found in other cells to be a p53
-target gene, subject to p53 repression, mediated in part by associated protein 1 (Sun et al, 1999
). We are currently investigating the MMP-1 protein level and activity in relation to BAI1 and p53 expression.
Taken together, this data offers further evidence that BAI1 is the candidate for the glioma-derived angiogenesis inhibitory factor (GD-AIF). It appears that the presence of the integrin-recognition motif RGD plays an important role in antiangiogenesis mechanisms and therefore we are currently investigating the effects of the transfer of BAI1 with a point mutation within the RGD motif.
The loss of BAI1 expression plays a role in the tumour angiogenesis-related cascade of events not only in brain-specific tumours, but also in other tumours types. These events are generally associated with the loss of p53 integrity or function. On the other hand, the overexpression of BAI1 inhibited strongly the pancreatic tumour-related angiogenesis. Therefore, since pancreatic carcinoma has a very poor prognosis after surgery, chemotherapy, and radiation therapy, BAI1 should be considered as a relevant candidate for future trials using this type of molecules or peptides derived from them, that would employ targeting of tumour angiogenesis as adjunctive or adjuvant therapy.