3. Discuss Ras effector pathways and the role they play in tumour development.
The cancer develops because of genetic mutations in oncogenes and tumour suppressor genes which results in imbalance between cell proliferation and cell death. In complex multicellular organisms, growth factors, extracellular hormones and cytokines are responsible for cell proliferation, differentiation and survival. In the normal cells, these molecules act as ligands for cellular receptors resulting in intracellular signaling pathways, in order to enable communication with the nucleus of the cell. In tumour cells, overexpression or mutation of proto-oncogene may lead to dysregulated or improper cell signaling and proliferation. Ras is one such proto-oncogene that functions as a molecular switch in a large network of signaling pathways, and plays important role in checkingthe differentiation or proliferation of cells. The viral ras gene (v-ras) was the first viral oncogene of some particular acute transforming retroviruses to be discovered. NIH 3T3 cell transformation assay was demonstrated in which it was identified that normal cellular genes (c-ras) had been activated by point mutation. It was this discovery that helped in concluding that the retroviral oncogenes including v-ras are obtained from normal cellular genes (Lowy 1993). The mutation in proto-oncogene ras, leads to development of oncogenic form of ras and this mutated ras genes encode constitutively activated proteins. This forms the basis of tumorigenesis. Approximately 30% of all human cancers that are identified are due to mutations in ras gene, which makes this G protein an essential target in the field of development of anticancer drugs. (Bos 1989). The Ras proteins belong to a large superfamily of low molecular weight GTP-binding proteins, which canbe further sub-divided into several families according to the degree of sequence conservation. Each family has their peculiar role in different cellular processes. The Ras family is responsible for controlling the cell growth and the Rho family controls the actin cytoskeleton. Mutation in human tumours leads to activation of three members of the Ras family namely HRas, KRas and NRas (Lowy 1993). These three members have almost 85% identical amino acid sequence and, although they function in similar ways, some precise differences between them are recently identified. All three members of Ras proteins are widely expressed, along with KRas being expressed in almost all cell types (Downward 2003). Ras functions are better understood when their role in cell signaling is studied. The upstream and downstream Ras signaling cascades can be summarized as follows.
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When the Ras proteins are bound to GTP they get activated and can engage downstream target enzymes. When they bind to GDP, Ras proteins get inactivated and hence cannot interact with these effectors. Thus the activation of Ras proteins depends upon whether they are bound to GDP or GTP and in normal cells; the ratio of bound GTP to GDP4 controls the Ras protein activity as seen in figure 1.b. In vitro, a low level of intrinsic activity is possessed by purified Ras proteins in which the GTP that is bound is gradually converted into GDP. Followed by this, bound GDP is then slowly replaced by GTP indicating that there is slow rate of nucleotide exchange with its surrounding area. Catalysis of these reactions take place within the cell; wherein the guanine nucleotide exchange factors (GEFs) catalyze nucleotide exchange and GTPase activating proteins (GAPs) catalyze nucleotide hydrolysis. The balance between the large and divergent families of proteins involved in these activities determines the activation condition of Ras proteins and the downstream target pathways. The figure 1.a shows that once the receptor of tyrosine kinases like the epidermal growth factor receptor (EGFR) gets activated, the autophosphorylated receptor binds to the SH2 domain of the growth factor receptor bound protein 2 (GRB2 - the adaptor protein). GRB2 is bound to one of the upstream signaling elements SOS through its SH3 domain. As a result the activated receptor tyrosine kinase recruits SOS to the plasma membrane, where a Ras protein is localized due to farnesylation (a post-translational modification of proteins). When the nearness between SOS and Ras increases, the nucleotide exchange on Ras increases along with GDP being replaced by GTP, which is primarily guanine nucleotide in the cytosol. Many receptors coupled with G proteins, can activate Ras proteins through stimulation of exchange factors (Downward 2003). Also it has been identified that activation of Ras proteins through stimulation of exchange factors involves transactivation of growth factor receptor tyrosine kinase (Daub, et al., 1996). Figures 2.a and 2.b depict the signaling cascades downstream of Ras. Ras activates various factors that regulate cell cycle progression, gene transcription, cell survival, apoptosis, cell cycle arrest, etc.
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The functions of Ras proteins are controlled by nucleotide and they bring signals from outside the cell to the cytoplasm of the cell. The Ras proteins when in their active condition binds to the GTP, the GTP locks them, and results in high affinity interactions the downstream reaction targets known as effectors. At a later stage, Ras functions get inactivated due to phosphate group getting removed from slow intrinsic GTPase activity. Ras inactivation terminates the signals. GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs) tightly controls this activation-inactivation cycle. When the Ras binds GTP, it can activate effector enzymes and thus many pathways and cascade gets activated. It is through these pathways that Ras proteins controls cell proliferation, cell survival as well as other important features of cell behaviour that is responsible for transformed phenotype. Thus, therapies which directly target these effector enzymes can be beneficial in treating tumours in which the active Ras protein is mutated. The protein serine/threonine kinase Raf is the first mammalian Ras controlled effector to be identified and cheracterised. It is evident that GTP bound Ras has ability to bind to and activate three Raf proteins namely: c-RAF1, BRAF and ARAF. Followed by the interaction between them, the Raf protein gets relocated in the plasma membrane and this relocation seems to be important for it to be activated. The downstream process of cascade involves the phosphorylation of the activated Raf. It in turn activates mitogen activated protein kinases 1 and 2 (MEK1 & MEK2). MEK1 and MEK2 are dual specificity kinases and possess the ability to phosphorylate and activate the mitogen activated protein kinases (MAPKs) extracellular Signal-regulated kinases 1&2 (ERK1 and ERK2). ERK1/2 can be transported to the nucleus after they are activated. This can be done because their substrates include cytosolic and nuclear proteins. ERK in turn phosphorylates ETS family transcription factors including ELK1. ELK1 forms a part of serum response regulating the Fos expression. ERK also phophorylates c-Jun. It ultimately results in activation of AP1 transcription factor that comprises of fos-jun heterodimers (Yordy and Muise-Helmericks 2000). These activated transcriptional regulators, results in expression of key cell-cycle regulatory proteins like cyclinD that is responsible for leading the cell through G1 phase of the cell cycle. It can be said that Raf stimulation can promote cell cycle progression, along with the help of other signals (Downward 2003).
The above figure 2.b also shows that apart from Ras/MAPK pathway, the Ras protein also activates several other effector pathways. It possesses the ability to directly interact with the catalytic subunit of type I phosphatidylinositol 3-kinases (PI3Ks). This interaction leads to stimulation of lipid kinase due to its translocation to membrane and its structural changes. Activated PI3K further phosphorylates phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and leads to formation of a product phosphatidylinositol- 3,4,5-trisphosphate (PtdIns(3,4,5)P3). It is known as a second messenger and can bind to a large number of proteins through the pleckstrin homology and other domains. Thus PI3K is responsible in controlling the functions of enzymes participating in downstream signaling (Downward 2003).
Another protein kinase PDK1 (3-phosphoinositide-dependent protein kinase-1) is identified to be crucial for activation of many protein kinases of AGC family that includes AKT/PKB, few PKCs, p70S6K and RSK. By phosphorylating various targets AKT shows anti-apoptotic functions and helps in cell survival. PI3K stimulation also results in activation of Rac (a protein belonging to Rho family), that is involved in the regulation of actin cytoskeleton and transcriptional factor pathways, for instance, by activation of nuclear factor-?B (NF-?B). Rac activation, occurring through PI3K dependent and independent pathway, is crucial in transformation induced by Ras (Downward 2003).
Another effector family of Ras proteins consists of three exchange factors for the Ras related RAL proteins. The three exchange factors are as follows:RAL guanine nucleotide dissociation stimulator (RALGDS), RALGDS-like gene (RGL/RSB2) and RGL2/RLF. With the help of these proteins, Ras stimulates RAL, which leads to stimulation of phospholipase D1 and activation of the cdc42/Rac-GAP-RAL binding protein 1 (RALBP1). The RALGDS pathway along with AKT/PKB plays important role in inhibition of the Forkhead transcription factor of the FoxO family. This results in cell cycle arrest via induction of cyclin-dependent kinase inhibitor KIP1 (p27) and apoptosis through the expression of Bim and Fas ligand.
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Another Ras effector family that is identified is phospholipase Ce. Along with phospholipase C domain, it comprises of two Ras association domains and Ras-GEF domain. It promotes hydrolysis of PtdIns(4,5)P2 to diacylglycerol and inositol-1,4,5- trisphosphate (Ins(1,4,5)P3). Phospholipase Ce effector enables Ras to stimulate PKC and calcium mobilization. The expression of activated Ras in the cells that are mutated, promotes certain characteristics of malignant transformation via combined action of all the above mentioned Ras dependent signaling pathways. It comprises of increase in proliferation resulting from induction of cell cycle regulators like cyclin D1. This results in activation of the retinoblastoma (RB) pathway, the suppression of cell cycle inhibitors such as K1P1, densensitization of cells to the apoptosis via AKT/PKB signaling and less well defined mechanisms, which are downstream of Raf. Besides its impact on proliferation and survival of cell, Ras effector pathways also play important role in inducing angiogensis, via ERK-mediated transcriptional upregulation of angiogenic factors. It also results in increased invasiveness through the expression that is mediated by ERK of matrix metalloproteinases and the effects mediated through RAC on the cytoskeleton.
Ras gene is prone to mutation and there is aberrant signaling when this occurs. The following table shows the frequency of Ras mutations that lead to various types of cancer.
In human cancers, the mutations in the ras genes family are present in over one third of the tumours. However, tumours from some organs have Ras much more recurrently mutated. The above table 1 represents that about 90% of the adenocarcinomas those are present in the pancreas have mutated ras genes. Also, nearly half of the colon and thyroid, and 35% of the tumours in lungs consist of a ras mutation. Abnormal signalling via Ras pathways occur due to many various classes of mutational damage in the tumour cells. The mutation may be in GTPase activity of Ras, where GAPs can be prevented from promoting the GTP hydrolysis on Ras. As a result of which Ras gets accumulated in the GTP-bound activated condition. Maximum tumours in which Ras gets activated are due to mutations in the codon 12, 13 and 61 of ras gene.
Loss or deletion of GAPs can also contribute to activation of Ras in tumours. This type of tumour is seen in case of loss of neurofibromin. It is encoded by the NF1 gene32 showing al the characteristics of a tumoursuppressor. Type I neurofibromatosis is a dominant syndrome that is seen by many benign and few malignant tumours in tissues of neural crest origin and in people with such dominant syndrome one allele is lost. In people having malignant tumour, both the allele are lost and hence Ras gets activated.
It was also shown that the overexpression of growth factor receptor tyrosine kinase leads to activation and stimulation of Ras pathways, for instance, EGFR and ERBB2. As seen in table 1, these receptors are stimulated in various cancers like breast, ovarian and stomach carcinoma. When an EGFR gene gets mutated, truncated receptor in which extracellular domain is missing gets expressed and the receptor that is mutated is identified to be extra stimulated in crucial parts of glioblastomas as well as in some other tumour types. In tumours, EGFR-family tyrosine kinases in most cases are also stimulated by the autocrine production of EGF-like factors for example, transforming growth factor-a i.e. TGF-a. How frequently they get activated is yet not known but still they are identified in high frequency in tumours of epithelial origin.
As shown in table 1, BRAF a Ras effector is identified to be stimulated by mutation in human tumours. It is seen approximately 70% in case of melanomas and about 15% in colon carcinoma. When there are limited number of residues in kinase domain structure, mutations in BRAF gene takes place. From the table 1, it is also seen that the amplification of the p110a gene in a comparatively small amount of ovarian tumours and the amplification of its downstream target AKT2 gene in ovarian and breast tumour can lead to activation and stimulation of PI3K pathway. Also PI3K pathway in the case of tumours directly gets activated when the tumour suppressor gene phosphate and tensin homologue i.e. PTEN gets deleted or mutated. The lipid phosphatase is encoded by PTEN gene and this gene eradicates the phosphate group from the 3' end of PtdIns(3,4,5)P3 and PtdIns(3,4)P2. This causes reversing the accumulation of the second messengers that resulted from PI3K pathway. From the table 1 it is evident that the gene PTEN is deleted in approximately 30-40% of tumours caused in humans. Thus it is second most important tumour suppressor gene after TP53 gene (Downward 2003).
Rational therapies which target various Ras signalling pathways can be used that possesses ability to inhibit tumour growth, survival and proliferation. Some new therapeutic agents are promising in the clinic; whereas besides them many more are being processed (Downward 2003). As mentioned earlier, mutations in various MAPK pathways are responsible for the tumour development. Precisely, many studies and research have proved that stimulation of ERK is extremely crucial for the entry into the cell cycle and through cell cycle into the mitogenesis. Hence, Ras, Raf, and MEK forms the basis of present opportunities for the development of new anticancer drugs that can be made target specific. As a result of which, they can be expected to be less toxic as compared to available traditional chemotherapeutic agents. Some drugs and compounds that can be used as anti-cancer therapeutic agents are as follows: MEK inhibitors, PD98059 and U0126. When the combination of PD98059 and sodium butyrate (an inhibitor of histone deacetylase) is used, it results in increase in apoptosis more when compared to the effect resulted from the use of sodium butyrate alone. In addition, when there are changes caused in the MAPK enzymes like inhibiting ERK and activating JNK, it results in contribution to cell differentiation and apoptotic pathways in intestinal cells, indicating new strategies and therapies for preventing colorectal-cancer progression. (Fang and Richardson 2005). Other than these therapies, the major approaches that are taken are as follows. The inhibition of expression of Ras protein itself via ribozymes and antisense oligonucleotides can be done, in which specific RNA sequence is targeted in order to block the translation of RNA message into the proteins. Oligonucleotides being complementary to messenger RNA transcripts of activated v-ras are used to reduce the expression of the Ras protein. Secondly, the membrane localization of Ras can be prevented by using farnesyl protein transferase inhibitors. Farnesyl protein transferase inhibitors inhibit the farnesyl transferase and results in prevention of the effects of mutated ras genes. Also, downstream effectors of Ras function can be inhibited by the use of Raf kinase inhibitors (Adjei 2001).
It can be thus concluded that, right from the year of discovery of Ras (1964) to till date, huge leaps have been made in finding the biochemistry and molecular mechanisms underlying it. Many genes that are a part of signal transduction cascade are identified, but even after this pioneer discovery more studies into the intricate details of Ras regulation and its relationship to other pathways in the cell are required. Many factors that are responsible for altering the activity of Ras are discovered. But still the components through which Ras carry out its downstream events are poorly understood. It is crucial to some extent to know how Ras can promote growth signals in some case and differentiation specific signals in some other case (Lowy 1993). Till date it is known that in normal cells, the Ras activation is responsible for carrying out many crucial processes like cell growth, survival, and angiogenesis. Considering the fact that it plays important role in cell growth and proliferation, the regulation of its pathway has to be really strict and under control. However, any dysregulation of the upstream regulators, downstream regulators or mutation in the Ras protein itself can result in acute dis-balance of tissue homeostasis and could and can affect the hallmark of cancer that includes self-sufficiency in growth signalling, insensitivity to antigrowth signals, invasion & metastases, evasion of apoptosis and angiogenesis (Hanahan and Weinberg 2000). There is a challenge to discover and identify the factors that possesses ability to interfere with the growth and proliferation of tumor cells but care must be taken with sufficient specificity that the normal cells are comparatively not damaged. Present and future research on the ras signaling pathway may find out those steps which when inhibited, our goal might be achieved. (Lowy 1993).