The Ras-association domain family (RASSF) members and their role in human tumourigenesis

Abstract

Ras proteins play a direct causal role in human cancer with activating mutations in Ras occurring in approximately 30% of tumours. Ras effectors also contribute to cancer, as mutations occur in Ras effectors, notably B-Raf and PI3-K, and drugs blocking elements of these pathways are in clinical development. In 2000, a new Ras effector was identified, RAS-association domain family 1 (RASSF1), and expression of the RASSF1A isoform of this gene is silenced in tumours by methylation of its promoter. Since methylation is reversible and demethylating agents are currently being used in clinical trials, detection of RASSF1A silencing by promoter hypermethylation has potential clinical uses in cancer diagnosis, prognosis and treatment. RASSF1A belongs to a new family of RAS effectors, of which there are currently 8 members (RASSF1-8). RASSF1-6 each contain a variable N-terminal segment followed by a Ras-association (RA) domain of the Ral-GDS/AF6 type, and a specialised coiled-coil structure known as a SARAH domain extending to the C-terminus. RASSF7-8 contain an N-terminal RA domain and a variable C-terminus. Members of the RASSF family are thought to function as tumour suppressors by regulating the cell cycle and apoptosis. This review will summarise our current knowledge of each member of the RASSF family and in particular what role they play in tumourigenesis, with a special focus on RASSF1A, whose promoter methylation is one of the most frequent alterations found in human tumours.

Fig. 1

Ras signalling pathways. Ras transmits signals from receptor tyrosine kinases (RTK) to the nucleus and regulate a diverse array of biological functions. Ras functions as a molecular switch, being inactive when bound to GDP and active when bound to GTP. Activated Ras acts by regulating the cellular response through distinct Ras effectors proteins and their complex signal transduction cascades, such as mediated by the Raf serine/threonine kinases, the lipid kinase, phosphatidylinositol 3-kinase (PI3-K) and the Ras association domain family 1, RASSF1A. The best-characterised signal transduction pathway of Ras is by the Raf kinases. Activated Raf phosphorylates MAPK/ERK kinase (MEK) and the activated MEK phosphorylates the mitogen-activated protein kinase (MAPK), which becomes activated and translocates to the nucleus where it phosphorylates a set of transcription factors. For example, the activation of Elk-1 leads to the transcription of Fos, which together with the MAPK-activated Jun, forms the activation protein 1 (AP-1), which has been shown to induce cyclin D1 and therefore stimulate proliferation. Another cascade of Ras-activated signalling is by anti-apoptotic PI3-K, which can stimulate the activity of the protein kinase B, Akt. Akt subsequently phosphorylates BAD, a pro-apoptotic member of the Bcl-family, and thus inhibits apoptosis (inactivating BAD enables BCL to promote cell survival by blocking the release of mitochondrial cytochrome c and therefore inhibiting caspase activation). Additionally, Ras regulates a pro-apoptotic pathway by binding to the Ras effectors NORE1 and RASSF1A and RASSF1A can also block cell cycle progression.

Fig. 2

Schematic representation of the protein domains of members of the RASSF family described in the literature. Putative DAG-binding (C1, green), Ras association (RA, red) and Sav/RASSF/Hpo interaction (SARAH, blue) domains are shown, predicted using Prosite (release 20.9) . The Genbank ID ‘accession’ number is listed for each protein.

Fig. 3

Map of the RASSF1 gene. Through alternative promoter usage and splicing of the exons, 7 transcripts have been reported to be produced from the RASSF1 locus. RASSF1A (Vega: BCM:RASSF1-001, OTTHUMT00000264806), RASSF1B (Vega: BCM:RASSF1-003, OTTHUMT00000264808; Ensembl: Q9NS23-3, ENST00000362008), RASSF1C (Vega: BCM:RASSF1-005 and -006, OTTHUMT00000264808 and OTTHUMT00000264807; Ensembl: Q9NS23-4, ENST00000327761), RASSF1D (Vega: BCM:RASSF1-002, OTTHUMT00000264810; Ensembl: RASF1_HUMAN, ENST00000357043), RASSF1E (Ensembl: Q9NS23-5, ENST00000359365), RASSF1F (Ensembl: Q9NS23-6, ENST00000273611), and RASSF1G (Vega: BCM:RASSF1-004, OTTHUMT00000264809; Ensembl: Q9NS23-7, ENST00000266020). The Vega program also predicts 3 additional transcripts, however, these have yet to be experimentally verified and have not been shown (BCM:RASSF1-007, OTTHUMT00000264812, a 601-bp transcript that produces a 133-amino acid protein with no recognisable domains; BCM:RASSF1-008, OTTHUMT00000264813, a 571-bp transcript that produces a 41-amino acid protein with no recognisable domains; and BCM:RASSF1-009, OTTHUMT00000264814, a 488-bp transcript that produces a 158-amino acid protein containing a C1 domain). UTR regions are depicted by open boxes, exons by black boxes, promoters by black arrows and CpG islands by grey bars (as predicted by Ensembl). The domain structure of the protein products are predicted using Prosite: putative ATM kinase phosphorylation consensus sequence motif (orange), DAG-binding (C1) domain (green), Ras association (RA) domain (red), and Sav/RASSF/Hpo (SARAH) interaction domain (blue) domains. Position of the extra 4 amino acids in RASSF1D and RASSF1E (black asterisk). Ensembl (release 45) , Vega (release 24) , Prosite (release 20.9) .

Fig. 4

A summary of the reported RASSF1A interactions and RASSF1A-mediated biological functions. RASSF1A can regulate the microtubule network, cell cycle progression and apoptosis by recruiting effectors and their signalling pathways. Proteins that directly interact (bind) with RASSF1A are shown in green, with downstream proteins affected by this interaction shown in blue. RASSF1A induces apoptosis through its interaction with Ras, the Ras effector NORE1, the connector enhancer of KSR (CNK1), the pro-apoptotic kinase MST1, and the modulator of apoptosis-1 (MAP-1; activated K-Ras, RASSF1A, and MAP-1 synergize to induce Bax activation and cell death). RASSF1A regulates proliferation through its interactions with the microtubules and Cdc20 (by inhibiting the APC–Cdc20 complex and its degradation of cyclins A and B), the microtubule-associated protein 1B (MAP1B), Aurora-A (which phosphorylates RASSF1A), C19ORF5 (the C19ORF5–RASSF1A interaction at the centrosome is thought to be required for the proper control of the APC–Cdc20 complex during mitosis), the transcription factor p120^E4F (RASSF1A-induced G1 cell cycle arrest and S-phase inhibition was enhanced by p120^E4F) and inhibition of cyclin D1 accumulation. RASSF1A also inhibits the epidermal growth factor-dependent activation of Erk through the plasma membrane calmodulin-dependent calcium ATPase 4b (PMCA4b). Taken together, these activities all support a tumour suppressor role for RASSF1A.

Fig. 5

Transcripts produced from the RASSF2 locus at chromosome 20p13. Three transcripts are produced from the RASSF2 locus, namely RASSF2A (Vega: RASSF2-001, OTTHUMT00000077828; Ensembl: RASF2_HUMAN, ENST00000379400), RASSF2B (Vega: RASSF2-002, OTTHUMT00000077829), and RASSF2C (Vega: RASSF2-003, OTTHUMT00000253005; Ensembl: novel, ENST00000379376). However, the Vega program only defines RASSF2A as a coding transcript (RASSF2B and RASSF2C are defined as unclassified non-coding transcripts). UTR regions are depicted by open boxes, exons by black boxes and CpG islands by grey bars (as predicted by Ensembl). Ensembl (release 45) , Vega (release 24) , Prosite (release 20.9) .

Fig. 6

Transcripts produced from the RASSF3 locus at chromosome 12q14. Three transcripts are predicted to be produced from the RASSF3 locus. RASSF3A is a 1377-bp transcript composed of 5 exons and translating a 238-residue protein (Vega: RASSF3-001, OTTHUMT00000261784; Ensembl: RASF3_HUMAN, ENST00000336061). RASSF3B is a 1099-bp transcript which splices from exon 2 to exon 4 causing a shift in the reading frame and resulting in a protein containing neither a RA nor SARAH domain (Ensembl: Q86WH2-2, ENST00000283172). Similarly, RASSF3C contains only 2 exons and produces a transcript of 444 bp that translates a 75-residue protein containing neither a RA nor SARAH domain (Vega: RASSF2-002, OTTHUMT00000077829). UTR regions are depicted by open boxes, exons by black boxes and CpG islands by grey bars (as predicted by Ensembl). Protein domains were predicted by Prosite. Ensembl (release 45) , Vega (release 24) , Prosite (release 20.9) .

Fig. 7

Transcripts produced from the RASSF4 locus at chromosome 10q11. Multiple transcripts are predicted from the RASSF4 locus. RASSF4A is a 2472-bp transcript that translates a 321-amino acid protein (Vega: RASSF4-001, OTTHUMT00000047745; Ensembl: RASF4_HUMAN, ENST00000374411 [note: the transcript predicted by Ensembl is only 2344 bp as it does not contain the 5′UTR]). RASSF4B and RASSF4C are very similar to RASSF4A, only differing in the N-terminal exons, with the C-terminal RA and SARAH domains of the translated transcript being identical (Ensembl: Q9H2L5-2 and Q9H2L5-3, ENST00000334940 and ENST00000374417, respectively). RASSF4D (Ensembl: Q5T737_HUMAN, ENST00000374414), RASSF4E (Vega: RASSF-006, OTTHUMT00000047750) and RASSF4F (Vega: RASSF4-008, OTTHUMT00000047752) transcripts are much shorter than the other isoforms due to premature truncation at exon 5, and as such the translated proteins contain no RA or SARAH domains. There are an additional 9 transcripts predicted by the Vega program, however they are alternatively spliced transcripts believed to contain intronic sequence relative to other coding variants or are unclassified non-coding transcripts, so for the sake of clarity these have not been shown. UTR regions are depicted by open boxes, exons by black boxes and CpG islands by grey bars (as predicted by Ensembl). Protein domains were predicted by Prosite. Ensembl (release 45) , Vega (release 24) , Prosite (release 20.9) .

Fig. 8

Transcripts produced from the RASSF5 locus at chromosome 1q32. Several transcripts are produced from the RASSF5 locus. NORE1A_α is a 2048-bp transcript that translates a 418-amino acid protein (Vega: RASSF5-001, OTTHUMT00000088469; Ensembl: RASF5_HUMAN, ENST00000367118) and NORE1B is a 3496-bp transcript that translates a 265-amino acid protein (Vega: RASSF5-003, OTTHUMT00000088471; Ensembl: Q8WWW0-2, ENST00000304534). additional 2 transcripts are predicted, namely NORE1A_β (Vega: RASSF5-002, OTTHUMT00000088470; Ensembl: Q8WWW0-3, ENST00000355294), which splices from exon 4 to exon 6 causing a shift in the reading frame that results in premature termination and NORE1A_γ (Vega: RASSF5-004, OTTHUMT00000088472), which has a C-terminal truncation of exon 6, and; translation of these transcripts produces proteins lacking the C-terminal SARAH domain. Additional transcripts have been predicted by the Vega program but as they are non-coding they have not been shown (Vega: RASSF5-005 and -006, OTTHUMT00000088473 and OTTHUMT00000088474, respectively). UTR regions are depicted by open boxes, exons by black boxes and CpG islands by grey bars (as predicted by Ensembl). Protein domains were predicted by Prosite. Ensembl (release 45) , Vega (release 24) , Prosite (release 20.9) .

Fig. 9

Transcripts produced from the RASSF6 locus at chromosome 4q13. Three transcripts are produced from the RASSF6 locus. RASSF6A is a 5872-bp transcript that translates a 337-amino acid protein (Vega: RASSF6-001, OTTHUMT00000252278; Ensembl: Q6ZTQ3-2, ENST00000307439; the Ensembl transcript is actually predicted to be slightly smaller at 5123 bp due to a shorter 3′UTR sequence). RASSF6B is a 4331-bp transcript that translates a 369-amino acid protein (Vega: RASSF6-002, OTTHUMT00000252279; Ensembl: RASF6_HUMAN, ENST00000342081; the Ensembl transcript is actually predicted to be slightly larger at 4902 bp due to a longer 3′UTR sequence). RASSF6C is 1360 bp and translates a 325-amino acid protein (Vega: RASSF6-003, OTTHUMT00000252280; Ensembl: Q6ZTQ3-3, ENST00000335049) that differs from RASSF6A and B only at the N-terminus due to use of an alternative exon 2 and splicing around exon 3. UTR regions are depicted by open boxes, and exons by black boxes (no CpG islands were identified; as predicted by Ensembl). Ensembl (release 45) , Vega (release 24) .

Fig. 10

Transcripts produced from the RASSF7 locus at chromosome 11p15. Three transcripts are produced from the RASSF7 locus due to use of different N-terminal and C-terminal exons. RASSF7A (Vega: RASSF7-003, OTTHUMT00000254972; Ensembl: RASF7_HUMAN, ENST00000344375) is a 1902-bp transcript that initiates transcription from the ATG present in exon 2, utilises different C-terminal exons from the other RASSF7 transcripts and corresponds to the HRC1 type I transcript identified by Weitzel and colleagues . RASSF7B (Vega: RASSF7-002, OTTHUMT00000254971) uses an alternative first exon to RASSF7A to generate a 1745-bp transcript that translates a 377-amino acid protein. RASSF7C (Vega: RASSF7-001, OTTHUMT00000254970) differs from RASSF7B only in the N-terminal exon and is a 1731-bp transcript that translates a 366-amino acid protein. UTR regions are depicted by open boxes, exons by black boxes and CpG islands by grey bars (as predicted by Ensembl). Each of these proteins contain an N-terminal RA domain (as predicted by Prosite). Ensembl (release 45) , Vega (release 24) , Prosite (release 20.9) .

Fig. 11

Transcripts produced from the RASSF8 locus at chromosome 12p12. Multiple transcripts are predicted to be produced from the RASSF8 locus. RASSF8A (Ensembl: RASF8_HUMAN, ENST00000381352) and RASSF8B (Vega: RASSF8-001, OTTHUMT00000260394; Ensembl: Q8NHQ8-2, ENST00000282884) are very similar, differing only in their C-terminal exon, and both encode proteins containing an N-terminal RA domain. RASSF8C-E transcripts (Vega: RASSF8-003, -004 and -005, OTTHUMT00000260396, OTTHUMT00000260397, and OTTHUMT00000260398, respectively) prematurely terminate at the 5′ end of exon 4 and translate a 91-, 107- and 134-amino acid protein, respectively, that almost consists entirely of the RA domain. RASSF8F (Vega: RASSF8-002, OTTHUMT00000260395) uses an alternative third exon however, as most of this is part of the 5′UTR, the transcript generates a 24-amino acid protein with no recognisable domains. Similarly, RASSF8G (Vega: RASSF8-006, OTTHUMT00000260399) only translates a 40-amino acid protein containing no recognisable domains. UTR regions are depicted by open boxes, exons by black boxes and CpG islands by grey bars (as predicted by Ensembl). Protein domains were predicted by Prosite. Ensembl (release 45) , Vega (Release 24) , Prosite (Release 20.9) .