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Novel effect of B-Raf inhibitors against bowel cancer discovered

Colorectal carcinoma is the second most common tumour disease in men and women in Germany. It accounts for 95 per cent of all malignant bowel cancers and its incidence has increased significantly in industrialised countries over the past decades. A particularly aggressive form occurs when a mutation is present in the proto-oncogene BRAF. As part of the Collaborative Research Centre 850 at the Institute of Molecular Medicine and Cell Research at the University of Freiburg, Dr. Ricarda Herr and Dr. Tilman Brummer are trying to find out how a mutated BRAF gene contributes to the development and growth of colorectal cancer. Their research has revealed that standard clinically applied B-Raf inhibitors have a new, previously unknown effect on cancer cells. As more information becomes available on the pathogenesis of cancer, the likelihood of developing more appropriately targeted pharmacological therapies will increase.

In most cases, malignant colon tumours develop from benign colon polyps. However, it is difficult to diagnose in the early stages due to an absence of obvious disease-related symptoms. At a more advanced stage, i.e. when the tumour starts to bleed heavily and restrict the intestinal lumen, blood appears in the stool and there is an onset of cramps and nausea. Risk factors for colorectal cancer include age and a diet characterised by the frequent consumption of red meat, elevated calorie intake and limited fibre content. Other risk factors include smoking, alcohol, lack of sunlight and lack of physical activity. Moreover, epigenetic factors and gut flora are increasingly being suggested as important colorectal cancer risk factors.

Superficially, a tumour is a heterogeneous mass of morphologically and phenotypically different cells that change over time. "For several years, attempts have been made to classify cancers according to molecular factors in order to improve patient selection for therapy," says Dr. Tilman Brummer from the Institute of Molecular Medicine and Cell Research at the University of Freiburg. Brummer has been studying the biology of colorectal carcinoma for many years. Molecular classification of cancer can improve cancer treatment for individual patients. It goes without saying that many different factors contribute to the development of cancer. Many different mutations need to accumulate before an invasive tumour develops from epithelial cells.

The protein B-Raf is involved in cellular regulation

What kind of signals are needed for precancerous lesions to become a colorectal cancer? Dr. Ricarda Herr and Dr. Tilman Brummer are seeking answers to this question. © Dr. Ricarda Herr, Dr. Tilman Brummer, University of Freiburg

A cell that has accumulated mutations becomes hyperproliferative, meaning that it is switched into cell division mode. This initiates a sequence of events that eventually turns precancerous lesions into malignant tumours. "A mutated BRAF gene alone does not lead to cancer," says Tilman Brummer. In addition to a mutated BRAF gene, tumours usually also contain mutated forms of the KRAS gene or the tumour suppressor gene p53. It is therefore worth taking a close look at the mutations, as they contribute to classifying a tumour and stratify therapy. For example, a patient with a tumour characterised by KRAS gene mutations is very unlikely to benefit from anti-EGFR (epidermal growth factor receptor) therapy.

Tilman Brummer and Ricarda Herr are studying the molecular basis of the BRAF oncogene and its involvement in the growth and invasiveness of colorectal cancer cells. For their research, they are using three-dimensional cell cultures in which they expose the B-Raf protein to clinical B-Raf inhibitors. The signal transducer B-Raf is the most potent activator of the MAP kinase pathway and is also a sensor for determining whether sufficient growth factors are present for the cell to divide. Yet, B-Raf seems to play a more important role during embryogenesis than at the adult stage of development. The MAP kinase signalling pathway consists of a number of multistage transduction pathways that are involved in cell differentiation and cell growth. This pathway is known to be overactive in 30 per cent of all cancers. Under normal circumstances, a growth factor binds to the EGF receptor, to name but one example, which triggers a signalling chain that consecutively activates B-Raf, MEK and ERK. ERK initiates cell division and cell survival programmes. Negative feedback ensures that the system does not overreact: ERK also phosphorylates the EGF receptor, which is subsequently switched off, resulting in the production of smaller quantities of active ERK. The lack of ERK in turn re-sensitises the receptor.

Mutation in the proto-oncogene BRAF leads to a permanently active B-Raf protein

A point mutation in the proto-oncogene BRAF means that the amino acid valine is exchanged with glutamate at a crucial site in the molecule's catalytic centre. This amino acid substitution turns the B-Raf protein into an onco-protein. "Glutamate has a negative charge; it thus imitates phosphate groups that normally activate the enzyme B-Raf," says Ricarda Herr. At this point, the enzyme becomes permanently active, and phosphorylation is no longer necessary. The cells therefore receive excessive growth signals and constantly multiply, ultimately leading to cancer. The constant signal that B-Raf sends at this point is independent of external events. The EGF receptor is also no longer relevant.

The involvement of BRAF in various cancers has already been shown. Amongst other things, the mutated BRAF variant has been shown to be involved in the pathogenesis of lung and thyroid cancer types as well as in malignant melanoma. The comparison of oncogenic BRAF signalling in different tumours could potentially provide important information about the molecular mechanisms of tumorigenesis.

Langerhans cell histiocytosis used to be treated as an autoimmune disease, and this only changed when a BRAF gene mutation was discovered in the diseased cells, which suggested that the disease was a cancer. Outstanding success can be achieved in patients who are given an inhibitor against the mutated B-Raf protein. "Moreover, patients with rare diseases may benefit from research on signalling pathways in tumours," says Tilman Brummer.

Differentiation upon the loss of the BRAF oncogene

In a three-dimensional cell culture of colorectal cancer cells, inhibitors of mutated B-Raf lead to a huge increase in the expression of cell adhesion molecules. © Dr. Ricarda Herr, Dr. Tilman Brummer, University of Freiburg

In their experiments with down-regulated B-Raf in three-dimensional cell cultures with colorectal cancer cells, Ricarda Herr and Tilman Brummer identified a novel effect of B-Raf inhibitors. They showed that colorectal cancer cells that lacked the BRAF mutation and the pharmacological inhibition of the B-Raf oncoportein did more than just reduce the division rate of cancer cells and slow down tumour growth. The researchers were surprised to find that the loss of the oncoprotein also led the cancer cells to differentiate into cells with more mature features. Tumour cell characteristics are similar to those of undifferentiated embryonic cells or stem cells with less matured features. However, they have the ability to divide rapidly and to invade other tissues.

Cancer cells often lack the transcription factor CDX2, which is responsible for the differentiation of intestinal cells. Inhibition of B-Raf leads to the upregulation of CDX2, resulting in the spontaneous differentiation of the cells: Tilman Brummer and his team unexpectedly discovered glandular tissue in their sample. "The B-Raf inhibitors have two effects. On the one hand, the inhibitors induce differentiation into cells with more mature features and that are specialised to fulfil a specific function, and on the other hand, more differentiated cells often have less aggressive behaviour as they tend not to spread into other organs as much as undifferentiated cancer cells," says Brummer. The latter is due to the fact that differentiated cells adhere more strongly to each other than undifferentiated cells.

There have already been initial successes with the use of B-Raf inhibitors for the treatment of melanomas. Unfortunately, the trials also showed that colorectal cancer cells did not respond effectively to B-Raf inhibitors on their own, due to the complexity of colorectal cancer. Other international studies involving cell culture experiments, preclinical animal experiments and initial clinical studies have, however, shown that a combination of B-Raf and EGRF inhibitors led to a better treatment outcome and was able to inhibit metastasis.

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