Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants

Signaling by EGFR is frequently activated in cancer through activating mutations in the coding sequence of the EGFR gene, resulting in expression of a constitutively active mutant protein. <br><br>Epidermal growth factor receptor kinase domain mutants are present in ~16% of non-small-cell lung cancers (NSCLCs), but are also found in other cancer types, such as breast cancer, colorectal cancer, ovarian cancer and thyroid cancer. EGFR kinase domain mutants harbor activating mutations in exons 18-21 which code for the kinase domain (amino acids 712-979) . Small deletions, insertions or substitutions of amino acids within the kinase domain lock EGFR in its active conformation in which the enzyme can dimerize and undergo autophosphorylation spontaneously, without ligand binding (although ligand binding ability is preserved), and activate downstream signaling pathways that promote cell survival (Greulich et al. 2005, Zhang et al. 2006, Yun et al. 2007, Red Brewer et al. 2009). <br><br>Point mutations in the extracellular domain of EGFR are frequently found in glioblastoma. Similar to kinase domain mutations, point mutations in the extracellular domain result in constitutively active EGFR proteins that signal in the absence of ligands, but ligand binding ability and responsiveness are preserved (Lee et al. 2006). <br><br>EGFR kinase domain mutants need to maintain association with the chaperone heat shock protein 90 (HSP90) for proper functioning (Shimamura et al. 2005, Lavictoire et al. 2003). CDC37 is a co-chaperone of HSP90 that acts as a scaffold and regulator of interaction between HSP90 and its protein kinase clients. CDC37 is frequently over-expressed in cancers involving mutant kinases and acts as an oncogene (Roe et al. 2004, reviewed by Gray Jr. et al. 2008). <br><br>Over-expression of the wild-type EGFR or EGFR cancer mutants results in aberrant activation of downstream signaling cascades, namely RAS/RAF/MAP kinase signaling and PI3K/AKT signaling, and possibly signaling by PLCG1, which leads to increased cell proliferation and survival, providing selective advantage to cancer cells that harbor activating mutations in the EGFR gene (Sordella et al. 2004, Huang et al. 2007). <br><br>While growth factor activated wild-type EGFR is promptly down-regulated by internalization and degradation, cancer mutants of EGFR demonstrate prolonged activation (Lynch et al. 2004). Association of HSP90 with EGFR kinase domain mutants negatively affects CBL-mediated ubiquitination, possibly through decreasing the affinity of EGFR kinase domain mutants for phosphorylated CBL, so that CBL dissociates from the complex upon phosphorylation and cannot perform ubiquitination (Yang et al. 2006, Padron et al. 2007). <br><br>Various molecular therapeutics are being developed to target aberrantly activated EGFR in cancer. Non-covalent (reversible) small tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, selectively bind kinase domain of EGFR, competitively inhibiting ATP binding and subsequent autophosphorylation of EGFR dimers. EGFR kinase domain mutants sensitive to non-covalent TKIs exhibit greater affinity for TKIs than ATP compared with the wild-type EGFR protein, and are therefore preferential targets of non-covalent TKI therapeutics (Yun et al. 2007). EGFR proteins that harbor point mutations in the extracellular domain also show sensitivity to non-covalent tyrosine kinase inhibitors (Lee et al. 2006). EGFR kinase domain mutants harboring small insertions in exon 20 or a secondary T790M mutation are resistant to reversible TKIs (Balak et al. 2006) due to increased affinity for ATP (Yun et al. 2008), and are targets of covalent (irreversible) TKIs that form a covalent bond with EGFR cysteine residue C397. However, effective concentrations of covalent TKIs also inhibit wild-type EGFR, causing severe side effects (Zhou et al. 2009). Hence, covalent TKIs have not shown much promise in clinical trials (Reviewed by Pao and Chmielecki in 2010).

EGFR（表皮生长因子受体）的信号传导在癌症中常因EGFR基因编码序列的激活突变而被激活，导致持续活性突变蛋白的表达。表皮生长因子受体激酶结构域突变存在于约16%的非小细胞肺癌（NSCLC）中，但也见于其他癌症类型，如乳腺癌、结直肠癌、卵巢癌和甲状腺癌。EGFR激酶结构域突变携带外显子18-21中的激活突变，这些外显子编码激酶结构域（氨基酸712-979）。激酶结构域内氨基酸的小缺失、插入或替换将EGFR锁定在其活性构象中，在这种构象下，酶可以自发地二聚化并经历自磷酸化，无需配体结合（尽管保留了配体结合能力），并激活下游信号通路，促进细胞存活（Greulich等，2005年，Zhang等，2006年，Yun等，2007年，Red Brewer等，2009年）。<br><br>EGFR细胞外域的点突变在胶质母细胞瘤中较为常见。与激酶结构域突变相似，细胞外域的点突变导致EGFR蛋白持续活性，并在无配体存在的情况下进行信号传导，但保留了配体结合能力和反应性（Lee等，2006年）。<br><br>EGFR激酶结构域突变需要与热休克蛋白90（HSP90）伴侣保持关联以发挥正常功能（Shimamura等，2005年，Lavictoire等，2003年）。CDC37是HSP90的共伴侣，充当HSP90与其蛋白激酶客户的相互作用支架和调节因子。CDC37在涉及突变激酶的癌症中常过表达，并作为致癌基因发挥作用（Roe等，2004年，Gray Jr.等，2008年综述）。<br><br>野生型EGFR或EGFR癌症突变体的过表达导致下游信号级联反应异常激活，即RAS/RAF/MAP激酶信号传导和PI3K/AKT信号传导，以及可能的PLCG1信号传导，导致细胞增殖和存活增加，为携带EGFR基因激活突变的癌细胞提供了选择性优势（Sordella等，2004年，Huang等，2007年）。<br><br>虽然生长因子激活的野生型EGFR通过内化和降解迅速下调，但EGFR癌症突变体表现出持续的激活（Lynch等，2004年）。HSP90与EGFR激酶结构域突变体的结合负向影响CBL介导的泛素化，可能通过降低EGFR激酶结构域突变体对磷酸化CBL的亲和力，使得CBL在磷酸化后从复合物中解离，无法执行泛素化（Yang等，2006年，Padron等，2007年）。<br><br>正在开发各种分子治疗药物以靶向癌症中异常激活的EGFR。非共价（可逆）的小型酪氨酸激酶抑制剂（TKIs），如吉非替尼和厄洛替尼，选择性地结合EGFR的激酶结构域，竞争性地抑制ATP结合和随后的EGFR二聚体的自磷酸化。对非共价TKI敏感的EGFR激酶结构域突变体比野生型EGFR蛋白对TKI的亲和力更高，因此是非共价TKI治疗的首选靶点（Yun等，2007年）。携带细胞外域点突变的EGFR蛋白也表现出对非共价酪氨酸激酶抑制剂的敏感性（Lee等，2006年）。携带外显子20小插入或T790M二级突变的EGFR激酶结构域突变体对可逆TKI具有抗性，这是由于对ATP的亲和力增加（Yun等，2008年），因此是形成与EGFR半胱氨酸残基C397共价键的共价（不可逆）TKI的靶点。然而，共价TKI的有效浓度也会抑制野生型EGFR，导致严重的副作用（Zhou等，2009年）。因此，共价TKI在临床试验中并未显示出很大的前景（Pao和Chmielecki综述，2010年）。