By David N. Leff

Amid the welter of compounds in clinical trials to choke off a burgeoning tumor¿s blood supply, all of these anti-angiogenic drugs ¿ notably endostatin and angiostatin ¿ derive exogenously from the tumors themselves. So far, the mammalian body¿s own endogenous antitumor machinery has been spared this heavy lifting.

Now cell molecular biologist Luisa Iruela-Arispe asks this question: ¿What would happen to a mammary tumor if we artificially altered the levels of an endogenous angiogenesis inhibitor ¿ not by providing the protein ourselves from the lab, not by injecting it, but actually by making the body produce it at the relevant tumor site?¿

She answers her own question in a paper released electronically Oct. 16, 2001, by the Proceedings of the National Academy of Sciences (PNAS). Its title: ¿Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor.¿ Iruela-Arispe, a faculty member at the University of California at Los Angeles, is the article¿s senior author.

¿Thrombospondins,¿ she told BioWorld Today, ¿are among the first molecules to be acknowledged as an endogenous inhibitor of angiogenesis. Ten years ago it was recognized as such. Endostatin and angiostatin are molecules within our body with the ability to modulate the angiogenic response. They were identified from tumors. In our research, thrombospondin [TSP] is a physiological, endogenous inhibitor, which is present without having a tumor. We hypothesized that TSP was responsible for blocking tumor growth through an anti-angiogenic mechanism. The difference here is the genetic approach. We did not interfere; we didn¿t touch the animals. They made the protein themselves. We manipulated them at the genetic level, so that they would do that.

¿This is the first time the mechanism of TSP regulation has been demonstrated genetically in lab animals with cancer,¿ Iruela-Arispe said. ¿What we have reported in this PNAS paper is taking advantage of transgenic animals that will develop tumors 100 percent of the time. Then we exposed them to engineered mice that secrete either extremely high levels of TSP or no TSP at all.

¿What our finding revealed,¿ Iruela-Arispe observed, ¿was that even though the mammary cells have the oncogenic mutation, with the potential to become malignant, our manipulating the levels of the endogenous inhibitor, TSP, prevented their flourishing into a fully grown tumor.¿

Why 20 Percent Of Mice Escaped Cancer

¿In our in vivo experiments, by standard mouse embryonic stem cell homologous technology,¿ Iruela-Arispe recounted, ¿we generated mice that made higher levels of TSP than normal, and other mice that totally lacked TSP. Then we crossed these two populations with another strain of mouse that invariably develops mammary tumors. Thus, we had three animal study groups ¿ high TSP, zero TSP and wild-type normals, all with propensity to breast cancer. One hundred percent of them were supposed to develop tumors within their first five months. So we started following their clinical development for two years.

¿The result was that in the absence of TSP,¿ Iruela-Arispe went on, ¿tumors developed faster, bigger, more of them. And the opposite occurred in the presence of TSP. These outcomes were somehow expected. What wasn¿t expected was that 20 percent of the animals producing TSP did not develop tumors for up to 18 months, which was the end of the experiment.

¿Twenty percent of these mice being tumor-free,¿ Iruela-Arispe observed, ¿raises the question. Why no more? Our answer: We were forcing all these animals to go through a cancer pathway. We provided them with a very potent tumor promoter, the amplified neu oncogene.

¿All the mice had mammary lesions that we considered premalignant,¿ she continued. ¿So there was transformation, but in 20 percent of the animals which had overexpressed thrombospondin, not a single tumor formed. Which told us that if we can manipulate the endogenous levels of this angiogenesis inhibitor we might be able to regulate tumor development, even though the oncogenic transformations are there.

¿That would perhaps explain why,¿ Iruela-Arispe suggested, ¿for example, my father has smoked for over half a century, and he doesn¿t develop lung cancer while other people might. So there are clearly some genetic differences between us that make us more susceptible to cancer ¿ or less.

¿With this study we have brought to life what we now know: that outside of the endothelial blood vessel cells there are extracellular matrix tissue barriers, and in one of those is this angiogenesis inhibitor, TSP.

¿Anti-angiogenesis is not the only function of the TSP gene,¿ Iruela-Arispe pointed out, ¿but it seems to be an important one, particularly in settings such as the breast. For example, TSP is increased after lactation, when the mammary gland is going to reduce its size, and its tissue will regress. So any tissue expansion, for example, pregnancy, breast feeding, or when we go exercising ¿ increasing muscle mass ¿ is associated with increase in vascular supply, namely, angiogenesis. Conversely, any tissue shrinkage, as in the mammary gland after lactation ends, or when we lose weight, or so forth, is associated with concomitant loss of capillaries. And in the mammary gland, at time of regression is when the highest levels of TSP1 are made.¿

Secrets Of The Matrix Barrier

Two other critical proteins enter the PNAS paper¿s thrombospondin equation: matrix metalloproteinase (MMP) and vascular endothelial growth factor (VEGF).

¿What we really didn¿t suspect, and was revealed by this study,¿ Iruela-Arispe explained, ¿is that TSP regulates matrix metalloproteases, which in turn regulate VEGF ¿ in a cascade or domino effect. MMPs,¿ she added, ¿are enzymes, created by cells to degrade the extracellular matrix, so the endothelial cells can migrate. Cancer cells make a lot of MMPs, so they can metastasize. But in the course of chewing up this extracellular barrier, they also release critical growth factors ¿ VEGF being one of the most critical ones ¿ that would otherwise be sequestered by those enzymes. So it¿s like scissors, cut-cut, and then by cutting, MMP also releases molecules that are an angiogenic feedback loop for the growth of capillaries.

¿Our UCLA discovery,¿ Iruela-Arispe observed, ¿could lead to new treatments targeted at fighting malignancies. It could develop drugs that mimic TSP,¿ she concluded, ¿perhaps preventing tumors from developing independent blood supplies, starving themselves of oxygen and nutrients ¿ hopefully killing them.¿

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