Colon Cancer : An alternative pathway of tumor development

Cancer develops from a single damaged cell subsequent to an accumulation of genetic errors in a number of its genes. The nature of these alterations and the order in which they occur differ from one cancer to another : hence, the “pathways” that may lead to cancer are numerous.

These findings were published in the August 2002 issue of Gastroenterology.

Cancer is a functional disorder of the body’s cells. The cells gradually lose control over their proliferation; they become immortal and develop erratically in the body.
This process is triggered by the alteration of the cell’s genetic material. All mutations do not necessarily lead to cancer. One of the genes that control the various vital cellular processes (cell division, differentiation, DNA repair or apoptosis) must be affected. Furthermore, a single mutation is not sufficient to transform a healthy cell into a cancer cell. Cancer is thus the result of a series of genetic accidents.

Mutations and cancer

The development of a tumor requires both the activation of an oncogene*(1), which triggers the uncontrolled proliferation of the cell, and the inactivation of a tumor-suppressor gene*, which prevents the destruction of damaged cells via apoptosis*.
To better understand this long and extremely complex process and to explore new therapeutic approaches, the various stages leading to the development of a tumor – from the initial mutation to the appearance of the lesion – must be elucidated.

An analysis of the molecular basis of the various types of colon cancer has revealed that a number of genes are implicated in the carcinogenetic process:
– The APC gene (Adenomatous Polyposis Coli), which is thought to play a crucial role in the control of cell proliferation in the colon, is mutated in 60 to 80% of all colon cancers;
– The oncogene K-ras, which controls cell proliferation and apoptosis, is found mutated in 50% of colon cancers;
– The DCC gene (Deleted in Colon Carcinoma), which encodes a transmembrane protein, has been altered in 70% of colon cancers yet is function is not fully understood;
– The ,tumor-suppressor gene p53 which plays a key role in apoptosis, is mutated in 70% of all colon cancers.
Although doubts persist as to the chronological order of these mutations, the alteration of the APC gene is often described as an initiator of carcinogenesis: once mutated, it triggers an excessive proliferation of cells, which marks the beginning of the tumor growth.

At the Institut Curie, the “Morphogenèse et signalisation cellulaires” team, under the direction of Professor Daniel Louvard (2), is working to better understand these phenomena in colon cancer and has focused its attention on the oncogene k-ras to clarify its role in the series of events that leads to this type of cancer.

A transgenic mouse as a model for tumor growth

To study the impact of the mutation of the k-ras gene in intestinal cells, Sylvie Robine (3) and Klaus-Peter Janssen, members of Daniel Louvard’s laboratory, decided to develop a transgenic animal model by introducing a mutated k-ras geneinto mice.

The mutated k-ras gene was associated with the regulatory region of the murine villin promoter (4) to achieve tissue-specific expression of the transgene in intestinal epithelial cells. With classical transgenic techniques, several mouse lines were generated that express oncogenic K-Ras protein throughout the intestinal epithelium, in differentiated enterocytes as well as in undifferentiated stem cells. The stem cells are required for the continuous renewal process that takes place in the intestinal mucosa throughout the life of an adult. The expression pattern of the oncogene in this new transgenic model constitutes a considerable advantage over previous models, since the stem cells remain anchored in the intestinal tissue and undergo several rounds of cell division. These are features that are crucial for the acquisition of additional epigenetic mutations which are necessary for tumorigenesis.

After several months, 80% of the transgenic mice developed malignant intestinal cancers despite the lack of any indication for APC mutations. This is, in fact, the first time that a transgenic “ras” mouse model had ever developed such cancers. This has cast doubt on the genetic model for colon cancer based on the assumption that k-ras mutations could only exert their oncogenic effects subsequent to mutations of the APC gene (5).

The results obtained by researchers at the Institut Curie have shattered this long-held assumption. The results of this rodent model have recently been confirmed by the genetic analysis of more than one hundred human cancerous growths. In a study published by a team of British researchers (6), it was demonstrated that in humans, k-ras gene mutations (as with the p53 gene) are also commonly present in colorectal cancers without any detectable mutation of the APC gene. In addition to confirming the utility of rodent models in studying carcinogenesis, these results further undermine the APC model.

An alternative “pathway” that leads to colon cancer

The researchers have shown that the expression of the mutated k-ras gene in the intestinal cells of the transgenic mice led to the continuous activation of the MAP kinases, a cascade of proteins triggering cellular proliferation. The hyperactivity of the MAP kinases has also been observed in human colon cancers.

Contrary to the conventional genetic model, the mutation of the k-ras gene may indeed be one of the initiating events in the carcinogenetic process of colon cancer – without any detectable alteration or mutation of the APC gene usually associated with colon cancer.

On the other hand, a mutation in the p53 gene was observed in 40% of these tumors. This would suggest that mutations in the k-ras oncogene and the tumor-suppressor gene p53 cooperate in the development of colon cancer.

Ras, the cell proliferation pivot

There are three isoforms of Ras proteins: K-ras, H-ras and N-ras. They play a key role in cell signaling by integrating extracellular signals and carrying it inside the cells. To insure this, they can take two possible states: activated or inactivated.
When activated, Ras proteins trigger cell proliferation – by the MAP kinase pathway – and apoptosis.

When one of the ras genes is mutated, abnormal Ras oncoproteins are produced which are constantly active. Then the cells divide continuously, increasing the likelihood of genetic errors.

Studying tumor growth and testing new medications in vivo

The research conducted by Daniel Louvard’s team has shed much light on the various stages of the carcinogenetic process and the pathways that lead to colon cancer. But there is still much to be learned from the k-ras transgenic mice. They provide an excellent model for long-term in vivo studies of tumor growth and are also well suited for analyzing the effects of certain environmental factors (like diet), and for testing new drug treatments. Furthermore, it is conceivable to analyze the cumulated effects of several genes by crossing the mice with other transgenic mice.

The mice are also an excellent pre-clinical model and as such would provide a basis for investigating new therapeutic targets.

This research is but one building block in a much wider project, in which researchers and physicians at the Institut Curie are cooperating to profile the genetic mutations specific to each cancerous tumor. A better understanding of the genetic alterations specific to each tumor will pave the road for tailored, more effective and better-targeted therapeutic treatments.

Notes
1 Terms marked * are defined in the Glossary below.
2 Pr Daniel Louvard is director of the research section of the Curie Institute. Reserch Director at the CNRS, his team is part of the UMR 144 CNRS/Curie Institute ” Subcellular structure and cellular dynamics” led by Jean Paul Thiery.
3 Reserch Director at the Inserm
4 « A genetic model for colorectal tumorigenesis » E. R. Fearon and B. Vogelstein. Cell, Vol 61, july 1990.
5 « Mutations in APC, Kirsten-ras and p53-alternative genetic pathways to colorectal cancer » Smith G. et coll. PNAS, Vol 99, 9 july 2002.
6 « Mutations in APC, Kirsten-ras and p53-alternative genetic pathways to colorectal cancer » Smith G. et coll. PNAS, Vol 99, 9 july 2002.

Reference

« Targeted expression of oncogenic K-ras in intestinal epithelium causes spontaneous tomorigenesis in mice »
Klaus-Peter Janssen1, Fatima El Marjou1, Daniel Pinto2, Xavier Sastre3, Dany Rouillard4, Coralie Fouquet5, Thierry Soussi5, Daniel Louvard1 and Sylvie Robine1
Gastroenterology, vol. 123, pp. 492-504, august 2002

1 Laboratoire Morphogenèse et signalisation cellulaires, UMR 144 CNRS/Institut Curie
2 Department of Immunology, UMC, Heidelberglaan, Utrecht, the Netherlands
3 Service d’Anatomo-pathologie, Institut Curie
4 Service de Cytopathologie et cytométrie clinique, Institut Curie
5 Laboratoire de Génotoxicologie des tumeurs, Institut Curie

Glossary

Oncogene : Mutated form of a gene (proto-oncogene) that controls cellular growth and division. It contributes to the transformation of a normal cell into a tumor cell. Only one mutation in one allele (gene copy) is needed to produce a mutated oncoprotein that stimulateshyperproliferation.

Tumor suppressor gene : Those genes supervise proliferation and genome integrity. They can induce the death of overly damaged cells. When inactived in tumoral cells, the cells can proliferate despite the presence of genetic errors. To be out of order, the two alleles (copies) of the gene have to be inactivated. p53, Rb, BRCA1, BRCA2, p21 belong to this category.

Apoptosis : A form of cell suicide that induces the death of a damaged cell that could prove harmful for the body. Also known as programmed cell death. When a cell is induced to commit suicide, a cascade of proteins is activated that provokes various biochemical and morphological changes. At the end, the cell is destroyed without consequence for its tissue environment.

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For your information

A tennis court as an intestine

The intestine is composed of the small intestine (7 meters in lenght) and of the large intestine (colon) about 1.4 meter in lenght. If the intestine is spread out, its surface is 300 m2, about a tennis court !
With an extremely simple organisation, this tissue is covered by millions of villi. Toward the base of the crypts are stem cells.
The more the cell divide, the more they differenciate and “migrate up” the villi. At the top of the villi, cells die.
This villi represent the different evolution steps of the cell; indeed it is a good model for biologist.
The intestine is being renewed in 3 to 5 days. This velocity involves a lot of cellular divisions and indeed a lot of genetic alterations. In addition, intestine is exposed to toxic substances in particular due to diet. This two facts could in part explain the high frequency of colo-rectal cancer.

Colorectal cancer

With approximately 35,000 new cases and 16,000 deaths every year, the incidence rate of colorectal cancer is second highest of all cancers diagnosed in France among both men and women. This rate increases with age; it is estimated that more than 50% of the population over the age of 70 has some form of colorectal cancer.
Colon cancer develops from a benign tumor, an adenoma more commonly referred to as a polyp. As colorectal cancer causes no pain, it is often diagnosed late, often requiring radical surgical procedures.

Towards widespread cancer screening

By 2003, colorectal cancer screening should become widespread among the general public between those 50 to 74 years of age. Current screening techniques rely on the Hemoccult® II test, which detects the presence of blood in the stools. If the test is positive, a colonoscopy is prescribed to establish a diagnosis. This procedure uses an instrument called a colonoscope, which is composed of an optic fiber, a camera, and a surgical tool for cutting and grasping that allows for the removal of suspicious intestinal tissue for subsequent analysis.

Trials are being conducted on two new tests

A British team has just reported encouraging results (1) in detecting the MCM-2 protein, specifically associated with divided cells, in stools. Preliminary studies indicate the MCM-2 protein may be a potential marker for the presence of colon cancer.
Meanwhile, an American team is refining a technique that enables the detection of mutations of the APC gene in the stools. This gene has shown to be a good colon cancer marker as it has mutated in 80% of such cases. Although this test appears to be reliable, according to data published earlier this year (2), it cannot detect all types of colon cancers.

Advances in treatment

Surgery is the primary line of treatment for colorectal cancer. Progress in surgical techniques now enables most patients to maintain basic intestinal functions.
If colorectal cancer is detected early enough – before it has had time to spread to neighboring tissues – the surgical removal of the tumor will suffice. Thanks to early detection, 55 to 60% of all patients are diagnosed before the cancer has metastasized or invaded the lymph nodes. When the tumor is too large, radiation therapy is often indicated prior to surgery in order to reduce the tumor’s size and render it more operable.
With more invasive forms of cancer, chemotherapy is used to improve the patient’s prognosis. In recent years, chemotherapy most often involves a regimen of 5-fluorouracil (5-FU) associated with other drugs to augment its efficacy and tolerance. All these advances have considerably improved the prognosis of colorectal cancer.

Improving prevention

Despite these advances, prevention is still the best cure. A low-fat diet rich in fruit and vegetables and a regimen of regular exercise appears to reduce the risk of colorectal cancer. Recent studies (3) have confirmed the impact of one’s diet on the development of colorectal cancer by demonstrating the protective role of vitamin D and its receptor.

Notes
1 « Analysis of minichromosome maintenance proteins as a novel method for detection of colorectal cancer in stool » R.J. Davies et coll. The Lancet, vol 359, 1er june 2002.
2 « Detection of APC Mutations in Fecal DNA from Patients with Colorectal Tumors » G. Traverso et coll. The New England Journal of Medicine, vol 346, 31 january 2002.
3 « Vitamin D receptors as an intestinal bile acid sensor ». Mangelsdorf DJ. et coll. Science, vol 17, 17 may 2002.