Oncogene (2008) 27, 4385–4391 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc REVIEW Activated c-Abl tyrosine kinase in malignant solid tumors J Lin and R Arlinghaus Department of Molecular Pathology, MD Anderson Cancer Center, University of Texas, Houston, TX, USA Mutant forms of the c-ABL gene are well known to be lesser degree in acute lymphocytic B-cell leukemia. Over involved in hematopoietic malignancies such as chronic 95% of CML patients have an abnormal chromosome myeloid leukemia (CML). CML patients possess a fused 22 known as the Philadelphia chromosome (Ph). The Ph BCR-ABL gene that activates the Abl tyrosine kinase fuses 50 portions of BCR to the second exon of the domain within Bcr-Abl. In general fusion proteins that c-ABL gene. The first exon of c-ABL encodes sequences cause oligomerization of Abl lead to activation of its involved in the autoinhibition of the c-Abl tyrosine tyrosine kinase activity. In this review, we highlight recent kinase, so deletion of these sequences is thought to discoveries indicating that the activated c-Abl tyrosine contribute to the activated state of the Abl tyrosine kinase, not as a fusion protein, plays an important role in kinase domain. malignant solid tumors of lung and breast. Our recent findings demonstrate that activated c-Abl Oncogene (2008) 27, 4385–4391; doi:10.1038/onc.2008.86; tyrosine kinase may also play a role in non-small cell published online 7 April 2008 lung cancer (NSCLC) (Lin et al., 2005, 2007). Activation of c-Abl tyrosine kinase in some solid tumors is further Keywords: c-Abl; FUS1; NSCLC; lung cancer; supported by studies from Plattner and co-workers imatinib; breast cancer (Srinivasan and Plattner, 2006; Srinivasan et al., 2008), whose findings indicate active c-Abl tyrosine kinase plays a role in aggressive human breast cancer cell lines. Introduction Activated c-Abl in non-small cell lung cancer The c-Abl protein is a tightly regulated nonreceptor tyrosine kinase that is involved in the regulation of cell Lung cancer, the leading cause of cancer-related proliferation, cell survival, cell adhesion, cell migration mortalities in the United States (US Cancer Statistics and apoptosis. The c-Abl protein is located both in the Working Group, 2004), is divided into two major nucleus and the cytoplasm. In the nucleus, c-Abl protein subgroups, small cell lung carcinomas (SCLC) and is known to be activated in response to DNA damage NSCLC. Depending on the type and stage of the and contributes to apoptosis (Wang, 2000). Cytoplasmic disease, treatment for lung cancer includes tumor c-Abl is associated with growth factor receptor signaling resection, chemotherapy and radiation therapy. that can affect cell mobility and cell adhesion (Taage- Recently, new approaches in the treatment of NSCLC pera et al., 1998). The oncogenic effects of Abl are have arisen from the discovery that the epithelial growth believed to reside in cytoplasmic forms of c-Abl. Several factor receptor (EGFR) is frequently overexpressed and studies have shown that c-Abl is downregulated in part activated in NSCLC (Bunn and Franklin, 2002; Dowell by intramolecular interactions that provide autoinhibi- and Minna, 2005). Importantly, loss of sequences within tory kinase effects (Pendergast, 2002; Pluk et al., 2002; chromosome 3p is frequently seen in both SCLC and Hantschel et al., 2003). Studies have also found that NSCLC, providing evidence of tumor suppressor genes c-Abl kinase activity is regulated by intermolecular (TSGs) in this chromosomal region (Girard et al., 2000; interactions with negative regulators, which include Zabarovsky et al., 2002). The FUS1 gene has been Bcr (Liu et al., 1996; Ling et al., 2003), PAG (Wen identified in the 3p21.3 critical chromosomal region and and Van Etten, 1997), F-actin (Woodring et al., 2003) is considered a novel TSG (Ji et al., 2002; Zabarovsky and phosphoinositides (Plattner and Pendergast, 2003). et al., 2002). The c-Abl protein is widely expressed in all tissues and Our studies showing that the c-Abl tyrosine kinase is until recently oncogenically activated forms of Abl were involved in NSCLC began during a screening of believed to be restricted to hematopoietic malignancies, synthetic peptides that had inhibitory activity towards principally chronic myeloid leukemia (CML) and to a the c-Abl tyrosine kinase in an assay involving the phosphorylation of a GST-Crk peptide. The c-Crk protein is a known target of the c-Abl kinase. Of Correspondence: Professor R Arlinghaus, Department of Molecular Pathology, The MD Anderson Cancer Center, University of Texas, interest, increased levels of c-Crk are associated with an 1515 Holcomb Boulevard, Houston, TX 77054, USA. aggressive phenotype in lung adenocarcinoma (Miller E-mail: [email protected] et al., 2003). This screening assay revealed that a small Activated c-Abl in lung and breast cancer cell lines J Lin and R Arlinghaus 4386 peptide derived from sequences deleted in a mutant 2008). The mechanism of Fus1 tumor suppressor FUS1, that lacks tumor suppressor activity, strongly activity is currently unknown. A deletion mutant of inhibited the recombinant 45 kDa Abl tyrosine kinase. FUS1 (FUS1 (1–80)) has been isolated and character- This Fus1 peptide was not functional unless a stearate ized from human NSCLC cell lines; it lacks the last 30 group was linked to the N terminus. (Lin et al., 2007). amino acids encoded by the FUS1 gene (Kondo et al., At the time Fus1 was not known to be involved with 2001; Ji et al., 2002). These deleted sequences encode the c-Abl, nor was it known that Fus1 may be an inhibitor Abl inhibitory peptide sequences described above. of the c-Abl tyrosine kinase. We verified Fus1’s ability Myristoylation of Fus1 at the N terminus is also to downregulate Abl kinase by showing that the above- required for the tumor suppressing function of FUS1. mentioned Fus1 peptide strongly inhibited a purified Myristoylation of Fus1 has been shown to increase the full-length human-activated c-Abl tyrosine kinase half-life of the Fus1 protein and appears to be involved (Lin et al., 2007). in the regulation of the subcellular localization of Fus1 It has been shown that normal lung tissue expresses to the cytoplasm (Uno et al., 2004) (Figure 1). The lack Fus1, whereas a majority of primary lung tumor of Fus1 myristoylation has been found in NSCLC samples do not express Fus1 (Uno et al., 2004). A new primary tumors (Uno et al., 2004). Of interest, the study has shown that 100% of human SCLC tissue myristic acid, which is bound to the N-terminal segment samples (22 samples) and 82% of human NSCLC tissue of c-Abl isoform 1b, is thought to be involved in samples (281) had a loss or reduction of Fus1, while in autoinhibition of c-Abl (Hantschel et al., 2003). contrast no normal or abnormal epithelial sites showed In our studies, two human NSCLC cell lines (A549 complete lack of Fus1 expression (Prudkin et al., 2008). and H1299) contained a tyrosine kinase active c-Abl In addition, for NSCLC they found that loss of Fus1 protein as determined by immune complex kinase expression is a significant independent adverse prog- assays. Importantly, both cell lines are defective for nostic factor for overall patient survival (Prudkin et al., the Fus1 protein expression (Kondo et al., 2001). In Tumor Suppressor Function + - - Fus1 sequence: 1 mgasgskarg lwpfasaagg ggseaagaeq 31 alvrprgrav ppfvftrrgs mfydedgdla 61 hefyeetivt kngqkraklr rvhknlipqg ivkldhprih 101 vdfpvilyev Figure 1 Structure–function relationships of the tumor suppressor gene FUS1. (a) Only myristoylated full-length wild-type Fus1 exhibits tumor suppressor activity. Myristoylation mutant and the C-terminal-truncated Fus1 proteins do not show tumor suppressor activity. Our findings show that only the myristoylated full-length Fus1 downregulates c-Abl tyrosine kinase (Lin et al., 2007). (b) The Fus1 peptide sequence. The underlined portion is the sequence of the full-length Fus1 peptide, which when co-valently linked to a stearate residue, has the ability to downregulate c-Abl kinase. Of note, amino acids 81–96, shown in the underlined portion, that are in the Fus1 peptide that inhibits c-Abl tyrosine kinase are part of the deleted sequences which are lacking in FUS1 (1–80) (Lin et al., 2007). Oncogene Activated c-Abl in lung and breast cancer cell lines J Lin and R Arlinghaus 4387 contrast, normal lung fibroblast cell line CCD16, which surrounding the myristolation site of Fus1 for effective expresses Fus1 tumor suppressor protein, did not c-Abl kinase inhibition (Figure 2). express an activated c-Abl tyrosine kinase. Lysates of the NSCLC cell lines also contained a tyrosine- phosphorylated c-Abl protein, as shown by immune complex western blotting (Lin et al., 2007). The presence Activated c-Abl in human breast cancer of a steady-state tyrosine-phosphorylated c-Abl protein is indicative of an activated c-Abl protein. Recent findings by Plattner and co-workers (Srinivasan Imatinib mesylate (TN Gleevec, Novartis Pharma- and Plattner, 2006; Srinivasan et al., 2008) indicate that ceuticals, East Hanover, NJ, USA) is widely known for c-Abl may play an important role in breast cancer. its ability to inhibit the activated tyrosine kinase activity Plattner’s previous studies have shown that endogenous of c-Abl and Bcr-Abl (Buchdunger et al., 1996). A c-Abl is activated downstream of growth factors, such previous study has shown that imatinib (Gleevec) as EFGR and platelet-derived growth factor receptor inhibited the growth of NSCLC cell line A549 in vitro (Plattner et al., 1999). Human breast cancers have (Zhang et al., 2003). We therefore tested the effects of deregulated constitutively activated EGFR family imatinib on colony formation of H1299 cells in soft members, including EGFR1 and ErbB2 (Ross and agar.