Oncogenic activation of c-Abl in non-small cell lung cancer cells lacking FUS1 expression: inhibition of c-Abl by the tumor suppressor gene product Fus1 - PubMed (original) (raw)

Oncogenic activation of c-Abl in non-small cell lung cancer cells lacking FUS1 expression: inhibition of c-Abl by the tumor suppressor gene product Fus1

J Lin et al. Oncogene. 2007.

Abstract

In lung cancer, frequent loss of one allele of chromosome 3p is seen in both small cell lung cancer and non-small cell lung cancer (NSCLC), providing evidence of tumor suppressor genes (TSGs) in this chromosomal region. The mechanism of Fus1 tumor suppressor activity is unknown. We have found that a Fus1 peptide inhibits the Abl tyrosine kinase in vitro (IC(50) 35 microM). The inhibitory Fus1 sequence was derived from a region that was deleted in a mutant FUS1 gene (FUS1 (1-80)) detected in some lung cancer cell lines. Importantly, a stearic acid-modified form of this peptide was required for the inhibition, but stearic acid alone was not inhibitory. Two NSCLC cell lines, which lack expression of wild-type Fus1, contain activated c-Abl. Forced expression of an inducible FUS1 cDNA in H1299 NSCLC cells decreased levels of activated c-Abl and inhibited its tyrosine kinase activity. Similarly, treatment of c-Abl immune complexes with the inhibitory Fus1 peptide also reduced the level of c-Abl in these immune complexes. The size and number of colonies of the NSCLC cell line, H1,299, in soft agar was strongly inhibited by the Abl kinase inhibitor imatinib mesylate. Co-expression of FUS1 and c-ABL in COS1 cells blocked activation of c-Abl tyrosine kinase. In contrast, co-expression of mutant FUS1 (1-80) with c-ABL had little inhibitory activity against c-Abl. These findings provide strong evidence that c-Abl is a possible target in NSCLC patients that have reduced expression of Fus1 in their tumor cells.

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Figures

Figure 1

Figure 1. Stearic acid-modified Fus1 peptide inhibits constitutively activated commercial Abl tyrosine kinase

(a) Stearic acid-modified Fus1 peptide inhibits GST–Crk phosphorylation by a bacterially purified, constitutively active, Abl kinase (IC50 35uM). (b) Without the stearic acid modification, the Fus1 peptide does not inhibit GST–Crk phosphorylation by the bacterially purified, constitutively active, Abl kinase. (c) A stearic acid-modified Bcr peptide does not inhibit GST–Crk phosphorylation by the bacterially purified, constitutively active, Abl kinase. (d) Stearate Fus1 peptide inhibits phosphorylation of a known Abl kinase peptide target, EAIYAAPFAKKK, by full-length human Abl.

Figure 2

Figure 2. Detection of activated c-Abl tyrosine kinase in two NSCLC cell lines deficient of FUS1

(a) NSCLC cell lines A549 and H1299 contain an active c-Abl tyrosine kinase, as measured in an in vitro kinase assay. Cells were lysed and incubated with the monoclonal anti-Abl antibody P6D, raised against c-Abl 51–64 residues (Liu et al., 1993). The immune complex was pulled down using protein A beads and subjected to either kinase assays or western blotting. 32D cells expressing Bcr-Abl were used as a positive control. Normal human lung fibroblast, CCD16 and 32D cells expressing Bcr–Abl were used as negative and positive controls, respectively. (b) Anti-phosphotyrosine western blotting demonstrates that NSCLC cell lines A549 and H1299 have an active c-Abl tyrosine kinase while normal human lung fibroblast CCD16 lack an active c-Abl tyrosine kinase. Phosphotyrosine c-Abl was detected by anti-phosphotyrosine antibody 4G10 and c-Abl was detected by monoclonal anti-Abl antibody 8e9. Transformed Rat 1cells (Rat1 SH2) that express activated c-Abl was used a positive control (Ling et al., 2003). (c) NSCLC cell lines have increased levels of phosphotyrosine-containing proteins compared to normal lung fibroblast cells (CCD16) as measured by 4G10.

Figure 3

Figure 3. Wild-type full-length Fus1 down regulates c-Abl tyrosine kinase

(a) c-ABL was co-transfected with either wild-type full-length FUS1, truncated FUS1, or empty vector in COS1 cells. At 48 h post-transfection, COS1 cells were harvested and lysate was incubated with the monoclonal anti-Abl antibody P6D. The immune complex was pulled down using protein A beads and subjected to western blotting. Antibody specific for pTyr412 of Abl (Abcam) was used to show kinase active c-Abl. The same blot was stripped and re-probed with monoclonal anti-Abl antibody 8e9. N-terminal Fus1 antibody was used to detect Fus1. (b) The intensities of the c-Abl pTyr 412 bands were normalized against c-Abl expression. (c) C-ABL was co-transfected with either wild-type full-length FUS1, truncated FUS1 or a non-myristoylated mutant FUS1 (Gly2Ala) in COS1 cells. At 48 h post-transfection, COS1 cells were harvested and the lysate was incubated with the monoclonal anti-Abl antibody P6D. The immune complex was pulled down using protein A beads and subjected to western blotting. Phosphotyrosine c-Abl was detected by anti-phosphotyrosine antibody 4G10 and the same blot was stripped and re-probed with monoclonal anti-Abl antibody 8e9 . The band intensities were normalized for c-Abl expression. (d) C-ABL was co-transfected with either wild-type full-length FUS1 or truncated FUS1 in COS1 cells. At 48 h post-transfection, COS1 cells were harvested and lysate was incubated with a polyclonal N-terminal Fus1 antibody. The immune complex was pulled down using protein A beads and subjected to western blotting. Phosphotyrosine c-Abl was detected by anti-phosphotyrosine antibody 4G10 and the same blot was stripped and re-probed with monoclonal anti-Abl antibody 8e9.

Figure 4

Figure 4. Imatinib decreases colony formation of NSCLC cell line H1299 in soft agar

(a) A total of 1000 H1299 cells were plated in 0.35% soft agar containing either 0, 1, or 5 _μ_M imatinib. Agar cultures were grown for 18 days changing the media every 2 days containing the respective concentrations of imatinib. (b) Dose-dependent inhibition of H1299 colony formation in soft agar by imatinib. (c) A total of 1000 H1299 cells were plated in 0.35% soft agar either containing 10 _μ_M imatinib or untreated. Cells were grown for 18 days changing the media every 2 days containing the respective concentrations of imatinib.

Figure 5

Figure 5. Induction of Fus1 in NSCLC H1299 cells induces cell death and decreases c-Abl kinase activity

(a) Induction of FUS1 expression in H1299 NSCLC cells induces apoptosis. Stable clones of human NSCLC H1299 that express an inducible wild-type FUS1 (wt-FUS1) or myristoylation mutant FUS1 (mt-FUS1) were treated with of 20 _μ_M of PA for 48 h. Apoptosis was measured by flow cytometry using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-based fluorescence-activated cell sorter (FACS) analysis. DNA fragmentation was analysed by FACS. The relative apoptotic cells were calculated as the percentage of the TUNEL-positive cells in the total cell populations. (b) Induction of FUS1 expression inactivated c-Abl tyrosine kinase and reduced the level of the c-Abl protein. H1299 cells with inducible FUS1 were treated with PA for 24 and 36 h. Whole cell lysate was subjected to western blot and the membrane was probed with c-Abl pTyr 245 antibody (Abcam), c-Abl antibody 8e9 and N-terminal Fus1 antibody. COS1 cells transfected with c-Abl were used as a positive control. (c) FUS1 expression in H1299 cells reduces the level of phosphotyrosine-containing proteins. (d) Fus1 peptide decreases the level of the c-Abl protein in immune complexes of c-Abl from COS1. Immune complexes were treated with indicated amount of Fus1 peptide for 20 min at 30°C.

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