RAS in cellular transformation and senescence

Abstract

In 1964, a transforming retrovirus was described that produced tumours in mice [1]. The Harvey rat sarcoma virus, aptly named H-RAS after the discovering scientist, encoded an oncogene that had been hijacked from its host. A similar virus was isolated in 1970 and was named the Kirsten rat sarcoma virus or K-RAS [2]. In 1982, the human genes homologous to the viral genes were elucidated and were designated c-H-RAS and c-K-RAS [3], and subsequently the third and final RAS family member was isolated from human neuroblastoma samples and termed c-N-RAS. An examination of other human cancers also revealed RAS genes capable of transforming mammalian cells, and interestingly, these alleles were transforming due to point mutations that inactivated the proteins’ intrinsic guanine triphosphatase (GTPase) activity [4,5]. RAS functions downstream of mitogenic growth factor receptors. Following the ligation with growth factors, these receptor tyrosine kinases undergo autophosphorylation and thereby recruit adaptor proteins that bind guanine nucleotide exchange factors (GEFs) (for review, see [6]). GEFs catalyse the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) on small GTP-binding proteins, including RAS. GTP-bound RAS subsequently interacts with a number of effectors to regulate cellular proliferation and survival, notably including the RAF family of proteins and type I phosphoinositol-3-kinase (PI3K). RAS proteins mutated at codons 12, 13, or 61 are rendered constitutively GTP bound and consequently have increased affinity to RAF proteins and PI3K, leading to activation of downstream pathways. The RAF family of serine/threonine kinases consists of three members: ARAF, BRAF, and CRAF [6]. Following activation by RAS, RAF kinases phosphorylate Mitogen-Activated Protein Kinase (MEK) kinases (MEK1 and MEK2), resulting in their activation. MEK proteins subsequently phosphorylate Extracellular Regulated MAP Kinase (ERK) (ERK1 and ERK2) in the cytoplasm, resulting in their activation and nuclear translocation. ERK kinases have a variety of cytoplasmic and nuclear targets, importantly including ETS transcription factors to regulate the expression of many pro-proliferative genes. RAS-GTP also interacts with and stimulates the activity of PI3K [6]. Upon activation, PI3K catalyses the conversion of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to produce phosphatidylinositol-3,4,5trisphosphate (PtdIns(3,4,5)P3). Increased local concentrations of PtdIns(3,4,5)P3 result in recruitment of the kinase PDK1 via its pleckstrin homology domain, which phosphorylates and activates the kinase AKT. AKT in turn promotes cellular survival and activates mammalian target of rapamycin (mTOR) to increase ribosomal biogenesis and messenger RNA (mRNA) translation. Additionally, active PI3K also stimulates the GTP-binding protein RAC, a regulator of the actin cytoskeleton. Therefore, rather than relying upon a single pathway for cellular transformation, it is hypothesised that numerous alterations in multiple biochemical pathways collectively promote cellular transformation by oncogenic RAS. Oncogenic RAS mutations are the most common oncogenic events identified in human tumours, being found in 30% of human cancers. KRAS is the most commonly mutated member of the RAS family, present in over 90% of ductal pancreatic cancers, 40−50% colorectal cancers, and 30% of non-small cell lung cancers (6). A detailed characterisation of the cellular and molecular pathways altered by oncogenic RAS has been pursued in order to identify potential therapeutic targets for such cancers, but despite its expected role in promoting tumourigenesis, ectopic expression of oncogenic RAS in primary cell cultures induces a paradoxical irreversible growth arrest known as oncogene-induced senescence (OIS) [7]. OIS is characterised by a flattened cellular morphology, a large nucleus with a prominent nucleolus, chromatin reorganisation, and activation of the p53 and p16INK4a pathways [7]. Although ectopic expression of oncogenic RAS in primary cells induces OIS, the exact molecular mechanisms remain unclear. Studies have