How do growth factors affect the body?

Supply bottleneck for tumors

Tumors can only grow into a deadly threat if they manage to connect to the blood vessel system. To do this, the tumor cells release proteins that they use to attract blood vessels, which then supply them with oxygen and nutrients. For a long time, researchers have been looking for active substances that prevent this fatal new formation of blood vessels. Walter Nickel from the Biochemistry Center at Heidelberg University explains a new strategy with which tumor cells can be cut off from the supply and which could prove to be the basis for effective anti-cancer drugs.

Whether tumor cells grow into a life-threatening tumor depends largely on the access of the degenerated cells to the blood vessel system. The changed cells themselves release various growth factors that stimulate the blood vessels to contact the tumor cell accumulation and supply them with nutrients and oxygen. This process is technically referred to as angiogenesis (formation of new blood vessels).

For years there has been a search in laboratories around the world for active ingredients that suppress this process and that are suitable as drugs for cancer therapy. Today, many details of the complex process of angiogenesis are known: The growth factors (proangiogenic factors) secreted by tumor cells act on cells (endothelial cells) that line blood vessels from the inside. The endothelial cells then divide and the basement membrane on which the cells sit is broken down. This "clears the way" for a new vessel to grow out, which moves towards the tumor with astonishingly great precision. Smooth muscle cells stabilize the resulting vessel; Once in the tumor tissue, a loop is formed with which the new vessel connects to the blood vessel system. In this way, a network of blood vessels is formed which spans the tumor tissue. Well supplied with nutrients and oxygen, the degenerated cells can now continue to divide. Ultimately, tumor cells invade the surrounding vessels, allow the blood to carry them through the body, settle far away from their place of origin and grow into a metastasis, a secondary tumor, which displaces healthy tissue.

A large number of proangiogenic factors have now been identified. Classic examples are "Vascular Endothelial Growth Factor", VEGF for short, and "Fibroblast Growth Factor 2", FGF-2 for short. An antibody that is directed against VEGF is already being clinically tested and is considered a promising candidate for a new drug against cancer.

FGF-2 is also an excellent target for new active ingredients to target. FGF-2 is the prototype of a class of proteins also known as "direct acting molecules". The growth factor stimulates almost all sub-steps in angiogenesis; The main effect has been identified as the stimulation of endothelial cell division (proliferation), their organization into new blood vessels (assembly) and the targeted outgrowth of the vessel (migration) to the tumor tissue. In addition, FGF-2 enhances the effect of VEGF by ensuring that more receptors for VEGF growth factor are formed on the surface of the endothelial cells.

All studies to date show that FGF-2 plays a key role in angiogenesis, and it is therefore very promising to look for strategies that can help eliminate the factor. Our working group at the Biochemistry Center of the University of Heidelberg focuses on the question of the molecular way in which the protein FGF-2 is formed and released by tumor cells.

In order to understand this path, one must first consider how cells basically form and secrete proteins: The cells of all mammals have a membrane system (endomembrane system) inside that mediates the release of proteins that are essential for those outside the cell (extracellular ones) ) Space are determined. These proteins include growth factors as well as messenger substances of the immune system, antibodies or hormones. The separation between proteins that are released from the cell to the outside (secretory proteins) and those that are intended for the inside of the cell (cytoplasmic proteins) takes place in a membrane system, the so-called endoplasmic reticulum. The secretory proteins are provided with a signal peptide - a kind of address label - that identifies them for extracellular transport. This transport route, discovered by the cell biologist Günter Blobel, was awarded the Nobel Prize for Medicine in 1999.

Inside the endoplasmic reticulum, secretory proteins must pass a quality control, during which their correct structure and functionality is checked. Only those secretory proteins that pass the quality check are then packaged in small membrane-covered transport vesicles. The vesicles leave the endoplasmic reticulum in order to migrate into another membrane system of the cell, the "Golgi apparatus". There the proteins undergo further changes that are important for their correct function. Again as cargo in transport vesicles, the secretory proteins get from the Golgi apparatus to the plasma membrane, the outer covering of the cell. The membrane of the transport vesicle now fuses with the plasma membrane. The cargo of the transport vesicle - the secretory protein - is then released and released into the extracellular space. In this way, the blood sugar-lowering protein hormone insulin, for example, gets into the bloodstream from the place of its origin, the ribosomes inside the "beta cells" of the pancreas.

This "classic" mechanism has alternatives. After an unexpected discovery that initially puzzled them, the cell biologists suspected that there had to be alternative ways of releasing proteins. The protein interleukin 1b, a messenger substance of the immune system, is released by certain immune cells when needed - but this protein lacks the signal peptide that shows it the way. It soon became clear that this was by no means an exotic specialty of the interleukin protein. On the contrary, it was possible to identify other proteins which, even without a signal peptide, leave the cell via a path that is independent of the function of the endoplasmic reticulum. FGF-2 is one of these proteins.

It is still unclear why there are such alternative ways of releasing proteins. It may be a particularly original form of secretion that developed during evolution at a time when the endomembrane system of mammalian cells did not yet exist in its present form.

The fact that FGF-2 is able to leave the cell via a path independent of the endoplasmic reticulum has not only fascinated us from the perspective of basic research, but also because it results in interesting medical applications. It is conceivable, for example, to develop active substances that suppress the secretion of a certain factor - in this case FGF-2 - without "affecting" the endoplasmic reticulum, which is essential for many cell functions. Such active ingredients (inhibitors) would also block one of the first steps that create new blood vessels to supply tumors. Such early inhibitors promise a considerably more efficient effect than inhibitors that only intervene later in the process of angiogenesis.

But what do the molecular components of the alternative pathway to the secretion of FGF-2 look like? This question is the focus of our work. In order to be able to answer them, we first developed a model system that allows a quantitative and qualitative analysis of FGF-2 secretion both in living cells (in vivo) and in the test tube (in vitro). In order to make the formation (expression) of FGF-2 immediately visible, we fused an FGF-2 molecule with the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. The release of the "FGF-2-GFP molecule" can be determined with this experimental approach because the molecules secreted by the cell remain associated with the cell surface. The green fluorescence can be used to infer the expression rate of FGF-2. At the same time, the number of FGF-2 molecules on the surface of the cell can be determined with the aid of labeled antibodies that are directed against FGF-2. For quantitative analysis, we use flow cytometry, a method with which up to six different types of fluorescence can be precisely recorded simultaneously on the basis of individual cells. The experiment can be qualitatively analyzed with what is known as confocal laser scanning microscopy.

With this experimental system, we have made three major discoveries. Firstly, we were able to demonstrate that FGF-2 reaches the extracellular space directly from the cytoplasm via the plasma membrane (membrane translocation). In contrast to the classic secretion route, transport takes place without transport vesicles. Second, in contrast to other cellular membrane translocation processes, the release of FGF-2 occurs in the native state of the molecule. Thirdly, we were able to show that the release of FGF-2 depends on certain structures on the cell surface, the so-called heparan sulfate proteoglycans (HSPGs). They act as "exporters". Whether FGF-2 can interact with its exporters, in turn, depends on its state of folding. This could indicate a mechanism for quality control: only correctly folded and therefore exclusively functional FGF-2 molecules are transported from the cell into the extracellular space.

It is currently our aim to identify those molecules that are directly involved in the membrane translocation of FGF-2. Together with scientists from the "European Molecular Biology Laboratory" (EMBL) in Heidelberg, we are currently carrying out a functional genome analysis of FGF-2 secretion. We also use our model system to search drug collections for substances that are capable of preventing the release of FGF-2. Once the molecular mechanism and the molecular composition of the FGF-2 secretion machinery have been fully elucidated, the prerequisites are created to find active substances that prevent the first steps of angiogenesis and make it impossible for tumor cells to acquire new blood vessels for their survival.

Prof. Dr. Walter Nickel
Biochemistry Center of Heidelberg University (BZH)
Telephone (0 62 21) 54 54 25, email: [email protected]