A single protein can have many variants. This variability is achieved by a process known as splicing, which can introduce small modifications into the mRNA transcript of a gene. Prof. Dr. Elmar Stickeler from the University Women’s Hospital in Freiburg found that some of these splice variants can also induce cancer. Stickeler and his team are investigating how splicing factors change their target molecules and how this leads to breast cancer. This research is helping to increase our understanding of the molecular basis of cancer as well as potentially also leading to new strategies for the diagnosis and therapy of cancer.
One gene, one protein? This is not really true and it also greatly underestimates the real situation. The gene CD44, for example, can potentially be translated into up to ten thousand different proteins. Not all of the proteins are found in all cells, but it can safely be assumed that more than one is present. Extensive regions of the amino acid sequence can be exchanged. Does this variability also affect protein function? In many cases, it does. "There are CD44 protein variants that promote the progression and metastasising of breast tumours," said Prof. Dr. Elmar Stickeler, senior consultant at the University Women's Hospital in Freiburg and Director of the Comprehensive Cancer Centre Freiburg (CCCF). Stickeler's group of researchers are investigating the causes of the diversity of protein variants related to breast cancer, ovarian cancer or cancer of the uterus.
The basic splicing mechanism has been known for more than 15 years. In 1993, Richard John Roberts and Phillip A. Sharp were awarded the Nobel Prize in Physiology and Medicine for the discovery of splicing. It all started with the observation of biologist Susan Berget who found that the mRNA which occurred in the cell plasma of eukaryotes (organisms with cell nuclei) did not correspond 100% with the gene from which it originated. The mRNA lacked long stretches that seemed to have been excised in an intermediary step. This step, which still happens in the cell nucleus, is referred to as splicing. A protein complex, the so-called splicosome - excises introns and exons from the gene sequence, a process that is mediated by so-called splicing factors, proteins that recognise specific sequences on the pre-mRNA and guide them to the splicosome. The DNA consists of regions that are later translated into a protein, but there are other regions that are excised during the splicing process. The regions that are translated into a protein are the exons, the regions that are excised are the introns. The large number of protein variants is achieved because many proteins can be alternatively spliced. The splicosome cuts the gene at different sites and joins different exons to an mRNA that will eventually be used as the basis for the protein.
Stickeler learned about splicing in 1996 while he was working as a postdoc at the Baylor College of Medicine in Houston, Texas. His supervisor was Susan Berget who had previously discovered the intron/exon structure of DNA. In her laboratory, Stickeler investigated the mechanisms that regulated the alternative splicing of the CD44 gene. Nowadays, Stickeler and his team know that numerous groups of molecular factors contribute to the translation of cancer genes into different protein variants, with different effects on tumour growth. One of these groups consists of splicing factors that are rich in the amino acids serine and arginine (S/R group). Some of these molecules not only affect CD44, but also other tumour genes such as those encoding the oestrogen receptor or the receptor of the vascular endothelial growth factor (VEGF).
The Freiburg researchers have been able to show that the Tra2-ß1 splicing factor is specifically produced in breast cancer cells. Its presence leads to the alternative splicing of the gene CD44. The factor binds to certain regions in the sequence of the pre-mRNA of CD44, which causes the splicosome to cleave the pre-mRNA differently than it normally does. It combines other exons with each other, with the consequence that the resulting protein contains a completely new region, which seems to affect the aggressiveness of the disease.
“Alternative splicing is a very common phenomenon,” said Stickeler. “Previously it was assumed that only about 10 per cent of all genes are spliced alternatively. Nowadays, we know that about 70 to 80 per cent of all genes undergo alternative splicing.” The physician and his team use cell cultures and tissue samples of cancer patients to investigate the effects that the activity of different splicing factors have on the disease. The researchers transfer splicing factor genes into cancer cells and measure how the activity of the genes that affect tumour progression and metastasis alters. Such experiments might in future lead to the development of inhibitors that have the potential to prevent the generation of such harmful protein variants. However, too much should not be expected of such drugs. “Cancer is a very complex process, which involves a large number of different genes and proteins,” said Stickeler. “Splicing factor inhibitors might at best be one of many components that could be used in multimodal chemotherapy.”Alternative splicing, however, not only influences disease progression, but also how a particular patient responds to a certain breast cancer therapy. Besides doing basic research, Stickeler’s group is also working on the development of databases that link genome sequences of patients at risk of a certain disease course with the most suitable therapy. In future, a simple genetic test might be able to show whether a specific patient is particularly prone to developing metastases and which therapy might be best suited to this patient. The Freiburg physicians are working closely with researchers at the Institute of Microsystems Technology (IMTEK) and the Institute of Pathology at the Freiburg University Medical Centre to develop a chip based on laser technology that will enable them to produce a gene profile. The work is still in its infancy, but the researchers hope that cancer diagnostics will be able to benefit from their work within the next few years.
Prof. Dr. Elmar StickelerSenior ConsultantUniversity Women's HospitalComprehensive Cancer Centre Freiburg (CCCF)University Medical Centre FreiburgHugstetter-Str. 55 79106 FreiburgTel.: +49 (0)761/270-3148