Aromatase inhibitors (AIs) inhibit the conversion of testosterone and androstenedione to estradiol and estrone respectively in peripheral and breast tumor tissues. They exert this effect by inhibiting the cytochrome P450 aromatase enzyme (Nicholson, R.I. et al 2004). There are two types of Al: irreversible AIs such as exemestane, which bond and degrade the aromatase enzyme rendering it non-usable. There are also reversible AIs such as anastrozole, which inhibit the aromatase enzyme in a competitive manner allowing re-use of the enzyme.
1 3 3 Fulvestrant
Fulvestrant or faslodex is a non-steroidal, pure anti-estrogen developed to overcome the estrogenic actions seen in tamoxifen treatment. It has a binding efficiency of ~100 times that of tamoxifen (Robertson, J.F. et al 2001). It is thought to exert anti-estrogenic actions by downregulating ER and by inhibiting the nuclear/cytoplasm shuttling of the ER and by inhibiting receptor dimerization and DNA binding (McClelland, R.A. et al 2001). It is also suggested the faslodex may modulate the recruitment and interaction of co regulators.
Chapter 1 General Introduction
1.4 A n ti- h o r m o n e r e s is ta n c e a n d E G F R s ig n a llin g
The m echanism s b y w h ic h both p rim a ry and acquired resistance o ccur are o n ly vaguely understood and, so m ore w o rk needs to be done in th is area i f new therapies to overcom e th is obstacle are to be developed. It is n o w accepted, how ever, that g ro w th fa c to r s ig n a llin g is a key, b u t not sole, fa c to r in v o lv e d in anti-estrogen resistance. T h is has led to d ow n stream m ediators o f these pathw ays becom ing n ew targets fo r breast cancer therapy. Increased epiderm al g ro w th fa c to r receptor (E G F R ) s ig n a llin g is co m m o n ly seen both c lin ic a lly and in breast cancer c e ll lines that have developed endocrine resistance (N ich o lso n , R .I et al 2004).
Gives docking Sites for adaptor
Proteins and Signalling enzymes Link upstream MRK ’s to Downstream IP K ’s Dimerization TK sites phosphorylated Cell proliferation Apoptosis Angiogenesis
Figure 1.3 A flow chart to show EGFR signalling. TK - tyrosine kinase, MRK - membrane receptor kinase, IPK - intracellular protein kinase.
E piderm al g ro w th fa c to r receptors are a fa m ily o f 4 p ro te in tyrosine kinase receptors, named E G F R o r H E R -1 , H E R -2 , H E R -3 and H E R -4 (A ta la y , G. et al 2003). T h ey possess both a lig a nd b in d in g dom ain (except H E R -3 ) and a
Chapter 1 General Introduction
tyrosine kinase domain (except HER-2). Figure 1.3 shows a much simplified flow chart-type representation of EGFR signalling.
Taking into consideration that there are 10 known ligands, several dimer variations and many downstream effectors, the EGFR signalling network is complex and diverse and not fully understood (Atalay, G. et al 2003).
Activation of downstream EGFR mediators such as phosphatidylinositol-3- kinase (PI3K) and mitogen activated protein kinase (MAPK) can lead to ligand-independent activation of the estrogen receptor. PI3K and MAPK can phosphorylate key serine residues, serine 118 and 167 respectively on the estrogen receptor within the AF-1 domain. This phosphorylation activates the estrogen receptor in the absence of estrogen. In this instance, if tamoxifen is present the tamoxifen/ER complex is enhanced and results in tamoxifen displaying an estrogenic effect on breast cancer cells, (Nicholson, R.I. et al 2004). This highlights the attractiveness and benefit of targeting such kinase pathways in the fight against tamoxifen resistant breast cancer.
1.4.1 Phosphatidylinositol 3 kinase
Phosphatidylinositol 3-kinases are a sub-class o f the lipid kinase superfamily that catalyse the addition of phosphates to position 3 of the inositol ring of phosphoinositides. However, 3’ phosphorylated phosphoinositides only account for 0.025% o f all inositol-containing lipids, suggesting that they are not ubiquitous or homeostatic lipids, but play an important role in cell
Chapter 1 General Introduction
PI3K is heterodimer of two subunits; p85, which is a regulatory subunit and pi 10, a catalytic subunit. An inter SH (iSH) sequence separates the two SH2 domains of the p85 subunit and links this subunit to the catalytic subunit. The two SH2 domains provide binding sites for tyrosine kinases (TKs). The p i 10 subunit is also a dual specificity kinase that can phosphorylate serine and threonine residues in addition to phosphoinositides lipids. The regulatory subunit can interact with both the p i 10 subunit and TKs. This is proposed as the reason behind the membrane targeting of pi 10 and its subsequent complexation with lipid substrates (Krasilnikov, M.A. 2000). The PI3K enzymes can be activated in a number o f ways, by G-protein coupled receptors, proteins with tyrosine kinase activity or by tyrosine-phosphorylated proteins. PI3 kinase signalling revolves around the presence of lipid recognition modules found in target proteins. Two key recognition domains have been described to date; these are the pleckstrin homology (PH) domain and the FYVE domain (Vanhaesebroeck, B.et al 1999). 3-phosphorylated inositol lipids are produced in the target membrane upon activation of an appropriate PI3 kinase. This results in the recruitment of proteins containing high affinity binding sites for these lipids, such as Akt (Fry, M.J. 2001). Upon activation, these kinases phosphorylate inositol lipids at the ‘3 position. The PI3 kinase generated phosphoinositides recruit Akt to the plasma membrane through their affinity for the PH domain o f Akt (Scheid, M. et al 2002) where it undergoes independent phosphorylation at two residues, a threonine present in the activation loop and a serine at the carboxyl terminus. Once activated, Akt is
Chapter 1 General Introduction
capable o f p h o sp h o ry la tin g a num ber o f proteins in v o lv e d in m etabolism , translational c o n tro l, c o n tro l o f ce ll cycle b y a ffe c tin g c y c lin D i and cell s u rviva l by a ffe c tin g forkhead fa m ily tra n s c rip tio n factors, B A D and caspase- 9. (C a m p b e ll, R .A et al 2 001) The P I3 K pathw ay is one o f the pathw ays over activated in ta m o x ife n resistance. F igure 1.4 is a cartoon representation o f the PI3 kinase pathw ay.