An Escort Protein Discovered for Ubiquitous Ras, Whose Gene Mutates in 30 to 60% of All Cancers

In order to function properly, living organisms need to eliminate defective cells. This rule is however not always abided by, as evidenced by cancer cells which no longer carry out the tasks originally set for them and yet continue to proliferate, as though they were ” ignoring ” commands from their environment. Cancer can thus be defined, inter alia, as an ailment affecting signal transduction.

Cell signaling, as a focus for research, has already led to the discovery of a number of new anti-cancer drugs (Herceptin®, Iressa® and Glivec®) but much remains to be investigated. Developing basic knowledge on how cell signaling works is a prerequiste for the identification of therapeutic and diagnostic targets – one of the most promising approaches to cancer treatment.

(¹ Terms marked * are defined in the Cell Glossary box at the end of the text.)

Picture yourself at the helm of a vessel made up of several thousand billion individuals. An impossible scenario ? And yet this is a conundrum long since solved by all human beings. Every day inside our bodies, 6,000 billion cells carry out the tasks that have been assigned to them, all the while keeping an eye on their neighbours. A feat which can only be contemplated because of the quasi constant nature of intercellular communication. Cells are the recipients of large numbers of signals from their environment (i.e. from other cells, tissues and organs within the body). Once interpreted, these signals will allow cells to determine their position and role within the body. As such, these signals are indispensable to cell proliferation, differentiation, morphology, and mobility. At the organ level, such signals ensure the harmonious preservation of tissue size and function.

This is very finely tuned system where failure can lead to disaster : if a single cell evades these monitoring mechanisms, it can then become immortal and proliferate chaotically, and this can lead to the formation of a tumor.

A protein cascade to convey information

Cells can intercommunicate either through a number of factors (such as hormones, cytokines, growth factors) or directly, through contact with their neighbors. All cells have on their surface a number of membrane receptors* onto which external informative factors may lock.

The information must then be conveyed to its final destination within the cell, a target protein, most often located within the nucleus, where it modifies the expression of specific genes and thus, cell behaviour.

To this end, a whole set of proteins are going to act as messengers. Concretely, a message locking onto the cell surface will activate a protein within the cell which will in turn activate another protein, and so on and so forth, with proteins playing the part of team members in a relay race, passing the baton along to the next in line.
In order to activate other proteins, proteins can use a number of different mechanisms : most often they induce chemical changes in other proteins, thus changing their conformation* and thereby their function.

Cell reaction will depend firstly on membrane receptor features, and secondly on which proteins are present, and used to convey information. This means that a given informative molecule may have different consequences in different cells. (see graph 1)

Cancer, a signal transmission disease

Contrary to a number of other diseases which are linked to the development of an exogenous agent (a virus, or bacteria) within our bodies, cancer is a disease stemming from cellular dysfunction. When a number of genes involved in signal transmission and the monitoring of cell mechanisms prove defective, this triggers oncogenesis.
Cancer cells not only become immortal but further lose their ability to control proliferation and differentiation, as well as their specific function and that which is assigned to them within a given tissue; in short, they ” forget ” all the processes which rely on cell signaling. Hence the significance of better understanding signaling pathways in order to develop new diagnostic and therapeutic strategies for cancer.
Several drugs derived from this type of basic research have already been developed: Herceptin®, Iressa® and Glivec®² are all ” anti-signaling ” drugs to the extent that they inhibit membrane receptors or proteins involved in signaling. The mechanisms targeted may vary, but they all block the signaling cascade and entail cell death.
Basic knowledge on such mechanisms must be pursued in order to develop new ” anti-signaling ” molecules.

Ras, the cell signaling pivot

All signals involved in stimulating cell proliferation sooner or later ” transit ” through one of three proteins known as Ras. These proteins have therefore elicited considerable interest on the part of scientists working in oncology. A case in point is that of Inserm Unit 528 “Signal transduction and Oncogenesis” headed by Jean de Gunzburg, a CNRS Research Director working at the Institut Curie on cell response to signals and signaling dysfunction in oncogenesis.

A very extended family

Ras is both the generic name given to a family of proteins, and the individual name of a specific protein. This calls for an explanation.
The Ras superfamily is a very large group of proteins (close to a hundred in human beings) generally involved in controlling basic cell functions. All such proteins have a homologous structure and are classified, on the basis of their homologies, into one of five families: Ras, Ran, Rad/Gem, Rab and Rho³/Rac/Cdc42.
The Ras superfamily therefore includes the Ras family, the individual members of which are specifically involved in proliferation and differentiation.
Within the Ras family, there are three proteins actually called Ras (HRas, KRas et NRas) as well as a number of other proteins, including Rap proteins.
The rest of this press release will focus on these Ras proteins (without going into the differences between H, K and NRas), as well as on Rap proteins.

Ras proteins are biological switches

Anchored to the inner side of the cell’s plasma membrane, Ras proteins are activated by signals through membrane receptors. They can be activated or inactivated, and as such can be likened to switches. (see graph 2) When activated, Ras proteins trigger a number of protein cascades:

ß the MAP kinase pathway which is mainly involved in regulating proliferation,
ß the PI3kinase pathway, which inter alia inhibits apoptosis*,
ß the Ral pathway, which is mainly involved in exocytosis* and endocytosis*.
These pathways cooperate in order to coordinate those cell functions required for the smooth operation of the extremely complex processes of cell proliferation and differentiation.

When transmission goes awry…

A number of genetic mutations can lead to the production of abnormal proteins, which can be either overexpressed, truncated or mutated to forms that are constitutively active or inactive. When constantly active proteins are produced at any point in cell signaling pathways, they can play a role in cancer development. Genes coding for such proteins are known as oncogenes.
When one of the ras genes mutates, abnormal Ras proteins are produced which are constantly active. Such ras gene mutations, giving rise to constitutively active protein forms, have been found in 30 to 60% of all human malignancies. More specifically, the ras gene is mutated in 90% of all pancreatic cancers, and in about 60% of all colorectal cancers.
Per se, the mutation of a single oncogene will not lead to the development of a malignancy. Other genetic alterations must occur in order to keep cells from offsetting cell signaling dysfunctions : tumor suppressor genes (p53, Rb, p21, BRCA1, BRCA2Š) in particular, which supervise proliferation control and genome integrity must also be inactivated. According to current knowledge, oncogenesis requires four to five ” independent ” defects.

Rap, a close relative of Ras

Jean de Gunzburg’s team at the Institut Curie has been focusing more specifically on two branches of the Ras family, the Ras and Rap proteins. Identified in 1988, there are two Rap proteins: Rap 1 and Rap 2. Rap proteins are similar to Ras proteins in that they too are binary switches, active or inactive. They are however involved in distinct protein cascades.
The Institut Curie biologists are attempting to understand how these two proteins work, in order to elucidate a number of cell signaling mechanisms.

PDEd, an escort for proteins Ras and Rap

Jean de Gunzburg’s team has in particular recently evidenced that a protein previously identified by a US team, PDEd, interacts with Ras and Rap proteins, as well as with a number of other proteins from this same family. The team noted that PDEd escorts proteins Ras and Rap as they mature⁴, from the cytosol* to membranes (Ras heads for the plasma membrane while Rap locks onto intracellular membranes). Insofar as PDEd binds to immature Ras proteins, it must not be involved in the relay race mentioned earlier, but in processes upstream from protein cascades.

This is the first time a protein regulating the binding of proteins Ras and Rap to cell membranes has been evidenced.
This is a very significant discovery which opens the door to new strategies. PDEd may indeed be used to ” inactivate ” Ras proteins which, as we saw earlier, are stuck in an active position in 30 to 60% of all human malignancies.

The promise of new therapies

Scientists at the Institut Curie are also working on other proteins in the Ras superfamily in order better to understand the roles they play in structures as wonderful and complex as cells. Improved understanding of how normal cells function is a prerequisite for improved understanding of oncogenesis, as the processes involved in oncogenesis are closely related to the dysfunction of such normal mechanisms : and this is a highly promising approach to the identification of new therapeutic and diagnostic targets.

Anticancer therapies stemming from research into cell signaling have already started to appear, but many more are coming. Some are in development, others are in clinical trials and should therefore become available in the years to come.

Référence
The d subunit of retinal Rod cGMP Phosphodiesterase regulates the membrane association of Ras and Rap GTPases
Vanessa Nancy(1), Isabelle Callebaut(2), Ahmed El Marjou(3) et Jean de Gunzburg(1)
The Journal of Biological Chemistry, vol. 277, issue 17, pp. 15076-15084, april 26, 2002

(1) Laboratoire Transduction du signal et oncogenèse, Unité 528 Inserm
(2) Laboratoire de Minéralogie-Cristallographie, Système moléculaire et biologie structurale, UMR 7590 CNRS/Universités Paris 6 et 7
(3) Laboratoire Compartimentation et dynamique cellulaire, UMR 144 CNRS/Institut Curie

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Cell Glossary

Apoptosis : a form of cell suicide, apoptosis occurs in overly damaged cells. Also known as programmed cell death.
Conformation : the state of a protein, as defined by its shape in space and its functional status (active or inactive, for instance).
Cytosol : cytoplasm, excluding organelles such as endoplasmic reticulum and mitochondria.
Endocytosis : the process by which cells ingest materials by folding inward a portion of their plasma membrane with the resulting pouch turning into a membrane-enclosed vesicle.
Exocytose : the process by which most cell components are secreted. These components (or molecules) are wrapped in membrane-enclosed vesicles which fuse with the plasma membrane, releasing substances from the cell.
Membrane receptor : a protein binding to a specific extracellular informative molecule (ligand) and triggering a response within the cell.
Cell signaling : the process by which cells translate extracellular signals into intracellular responses.

(2) Glivec® (imatinibum) and Iressa® (ZD 1839) block the activity of tyrosine kinase-type membrane receptors: Glivec® impacts inter alia the bcr-Abl protein, featured in chronic myeloid leukemia (CML) while Iressa® is used against the EGFR protein (the EGF receptor) in a number of solid tumors. Herceptin® (trastuzumab) is a monoclonal antibody which specifically targets the HER2 protein expressed on the surface of malignant cells in 25 to 30 % of all breast cancers.

(3) Scientists at the University of Michigan have just shown that the RhoC protein (a member of the Rho family) can be detected in invasive breast cancer and that its presence is correlated to that of metastases. The RhoC protein could thus become a prognostic marker for potential tumor development. These preliminary results were presented at the 93rd Congress of the American Society of Cancer Research, held April 6-11, 2002 in San Francisco (Source : Quotidien du Médecin, April 10, 2002).

(4) Once synthethized, Ras and Rap proteins have to undergo a number of chemical changes in order to become “usable”, or mature; these changes will allow them to bind to the membranes of some intracellular compartments, a stage necessary to their becoming operational.

Background :

Ras proteins as biological switches

Ras proteins can take on one of two possible states :

ß An active state during which they trigger a number of protein cascades which convey signals from membrane receptors to the nucleus;
ß An inactive state

Ras proteins are switched on by proteins that can release guanine nucleotides (GEFs), and which in this specific case release the guanosine diphosphate (GDP) molecule which is bound to Ras, and replace it with a guanosine triphosphate molecule (GTP).
This process induces a change in the conformation of Ras proteins, making them active.
The switching off is handled by proteins known as GAPs (for GTPase activating proteins) which force Ras to transform GTP into GDP, by making it do away with GTP’s third phosphate. Ras proteins then revert to their initial conformation, and are inactivated.