C arcinogenesis results from the subversion o f the processes that control the norm al growth,
location and m ortality o f cells. This loss o f norm al control m echanism s arises from the
acquisition o f m utations in three broad categories o f genes:
1. Proto-oncogenes, the no nn al products o f w hich are com ponents o f signalling pathways
that regulate proliferation and w hich, in their m utated form, becom e dom inant oncogenes.
2. Tum our suppressor genes, w hich generally exhibit recessive behaviour, the loss o f
function o f w hich in cancer leads to deregulated control o f cell cycle progression, cellular
adhesion, etc.
3. DNA repair enzym es, m utations in w hich prom ote genetic instability.
Tum ours are thought to develop as a result o f the sequential (m ulti-step) accum ulation o f
alterations in the genes involved in the control o f cell proliferation, differentiation or death.
Therefore, a sequence o f som atic m utational events leads to the clonal expansion o f genetically
m odified cells that progressively show a selective grow th advantage over norm al non
transform ed cells and acquire an invasive and m etastatic potential, thus resulting in m alignant
transfonnation o f cells and tum our progression. The incidence o f hum an cancers suggests that
typically six to seven events are required over a span o f tw enty to forty years to induce tum our
growth. (Petro, 1997)
1.8.1 RET
The RET oncogene w as identified by Takahashi and colleagues in 1985 w ho reported a novel gene rearrangem ent w ith transfom iing activity in NIH 3T3 cells transfected w ith human lym phom a DNA. (Takahashi et al., 1985) The name RET stem s from “rearranged during /ransfection” . The hum an RET gene lies on the chrom osom e band l O q l l . 2 and com prises 21 exons. RET encodes a transm em brane receptor tyrosine kinase (TK) w ith a cadherin-related m o tif and a cysteine-rich dom ain that binds TG pp-related neurotrophic factors, including the glial cell line derived neurotrophic factor (G N D F) family. Binding o f the ligand causes receptor dim erization, autophosphor>iation o f tyrosine residues w ithin the intracellular domain, and activation o f the signalling cascade.
RET protein is com posed o f three dom ains; an extracellular ligand-binding dom ain w ith four cadherin-like repeats and a cysteine-rich region, a hydrophobic transm em brane dom ain, and a cytoplasm ic portion w ith the TK dom ain split by an insertion o f 27 am ino acids (Figure 1.16). (Arighi et al., 2005) Three isoform s o f RET are generated by alternative 3' splicing. The long, interm ediate and short RET isoform s, w hich differ by 51, 43 and 9 am ino acids in the C term inus, are referred to as RET51, RET43 and RET9 respectively. RET is norm ally expressed in cells o f neural crest origin such as neural and ganglion cells. Tum ours o f neural crest origin such as neuroblastom a, phaeochrom ocytom a and m edullary thyroid carcinom a also express RET. It is clear that RET plays a significant role during em bryogenesis, particularly w ith developm ent o f the neural and excretory systems. In 1996, glial cell line-derived neurotrophic factor (G DN F) was identified as a long sought after RET ligand by several groups. (D urbec et al., 1996; Jing et al., 1996; T reanor et al., 1996; Trupp et al., 1996)
C hapter 1 G eneral Introduction
M utations (both germ line and somatic) in the RET proto-oncogene are associated w ith several diseases including m ultiple endocrine neoplasia, types IIA and IIB (M EN 2A and M EN2B), H irschsprung disease, and m edullary thyroid carcinoma. Like m ost receptor tyrosine kinases, RET has the ability to activate a variety o f signalling pathways, including RA S/R A F/M EK /ERK , phosphatidylinositol 3-kinase (PI3K)/AK T, p38 M A PK and c-Jun N -tenninal kinase (JNK) pathways.
EX T I^\C E L L ^L A K
C adherin-Iike dum ain C ysteine-rich region T ra n sn ie m b ran e domainlBn
Tyrosine kinuse dumiiin
I
MEN2A C ys 609 Cys 611 Cys 618 Cys 620 Cys 6J4 IN T R A C E IJ.IT .A Rret protein ret exons
FM T C MEN2B
Va S04 S cr 891
532 : d u p lic a tio n de nucli'utides Cys 609 C ys 611 C ys 618 Cys 620 Cys 6J4 r;iu 768 790 791 8S.1 .Met 9 )8
1.8.2 ret/PTC oncogenes
In 1987 the dem onstration o f an activating oncogene specific for papillary thyroid carcinom a was shown using N IH 3T3 transfection assays. (Fusco et al., 1987) This oncogene w as show n to be a rearranged form o f RET called ret/PTC. To date, at least 16 chim eric m RN A s involving eleven distinct donor genes have been described. ret/PTC 1-9, PCM 1-ret, ELKS-ret, and R FP-ret have been isolated from sporadic and radiation associated PTCs (Table 1.1). In each case, the intracellular dom ain o f RET is fused to different activating genes, nam ely H4 (for ret/PTC-1), R Ia (ret/PTC2), R FG /ELE1/A R A 70 (ret/PTC3 and ret/PTC4), RFG5 (ret/PTC5), hTIF (ret/PTC- 6), R F G 7 /T F ly (ret/PTC7), kinectin (ret/PTC8), RFG9 (ret/PTC9), P C M l (PCM 1-ret), ELKS (ELK S-ret), and RFP (RFP-ret). This results in ligand-independent dim erization and constitutive activation o f these chim eric proteins. (Arighi et al., 2005)
ret/PTC rearrangem ents activate the transform ing potential o f RET by m ultiple m echanism s. First, by substituting its transcriptional prom oter with those o f the fusion partners, they allow the expression o f R ET in the epithelial follicular thyroid cells, w here it is norm ally transcriptionally silent. Secondly, the rearrangem ents generate constitutively active chim eric oncoproteins w hich are distributed in the cytosolic com partm ent o f the cell. Finally, activation o f the RET kinase is m ediated by fusion to dom ains that are capable o f dimerization.