Tumor Suppressor FUS1 Signaling Pathway

Abstract

FUS1 is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region where allele losses and genetic alterations occur early and frequently for many human cancers. Expression of FUS1 protein is absent or reduced in the majority of lung cancers and premalignant lung lesions. Restoration of wt-FUS1 function in 3p21.3-deficient non-small cell lung carcinoma cells significantly inhibits tumor cell growth by induction of apoptosis and alteration of cell cycle kinetics. Here we present recent findings indicating that FUS1 induces apoptosis through the activation of the intrinsic mitochondrial-dependent and Apaf-1-associated pathways and inhibits the function of protein tyrosine kinases including EGFR, PDGFR, AKT, c-Abl, and c-Kit. Intravenous administration of a nanoparticle encapsulated FUS1 expression plasmid effectively delivers FUS1 to distant tumor sites and mediates an antitumor effect in orthotopic human lung cancer xenograft models. This approach is the rationale for an ongoing FUS1-nanoparticle-mediated gene delivery clinical trial for the treatment of lung cancer.

Cytogenetic and allelotyping studies of fresh tumors and tumor cell lines have shown that allele losses and genetic alterations on the short arm of chromosome 3p (3p25, 3p21–22, 3p14, and 3p12–13) are among the most frequent and earliest genomic abnormalities involved in a wide spectrum of human cancers, including lung^{1– 6} and breast.^7–9 Multiple overlapping homozygous deletions have also been found in the 3p21.3 region, spanning a 120 kb genomic locus in human lung and breast cancer cell lines.^10,11 Chromosomal abnormalities in the 3p21.3 region have been frequently detected in smoke-damaged respiratory epithelium and preneoplastic lesions.^10,12–13 These findings suggest that one or more putative 3p21.3 tumor suppressor genes function as “gatekeepers” in the molecular pathogenesis of lung and other human cancers.^10,12,14 The novel FUS1 gene is one of the nine candidate TSGs (CACNA2D2, PL6, 101F6, FUS1, BLU, RASSF1, NPRL2, HYAL2, and HYAL1) that were identified in this region.^{1,14 –17} In this review, we will describe a pathway involved in FUS1-mediated tumor suppression and discuss potential translational applications of the FUS1 TSG for human lung cancer therapy.

Inactivation of FUS1 In Lung Cancer Pathogenesis

The FUS1 gene may be inactivated in human cancer cell lines and primary tumors by haploinsufficiency.^16,17 Although single allele loss is common, only a few missense mutations and C-terminal deletion mutations have been identified in primary lung cancer samples, and there is no evidence for promoter hypermethylation.^6,16,17 FUS1 mRNA transcripts could be detected on Northern blots of RNAs prepared from some lung cancer cell lines, but no endogenous FUS1 protein could be detected in a majority of non-small cell lung carcinoma (NSCLC) cells and almost all of the small-cell lung cancer (SCLC) cell lines tested.^6,16,17 Myristoylation of the FUS1 N-terminus is required for tumor suppressor activity.¹⁷ A loss of expression coupled with a myristoylation defect of the FUS1 protein was detected in primary lung cancers. The myristoylation defective FUS1 protein has a greatly reduced half-life and is subject to rapid proteosomal degradation.¹⁷ Using a tissue microarray of 303 lung cancers, loss or reduction of FUS1 expression was detected in 100% of SCLCs and 82% of NSCLCs.¹⁸ In NSCLCs, loss or reduction of FUS1 expression was associated with significantly worse overall patient survival. Squamous metaplasia and dysplasia expressed significantly lower levels of FUS1 than did normal and hyper-plastic bronchial epithelia. Lee et al.¹⁹ showed the translation of FUS1 was significantly down-regulated by microRNA-378 targeting the 3′UTR of FUS1 mRNA and the ectopic expression of miR-378 enhanced cell survival, tumor growth, and angiogenesis. A genetically engineered mouse with a targeted disruption of the FUS1 gene developed signs of autoimmune disease, showed an increased frequency of spontaneous vascular tumor formation, and had defects in natural killer cell maturation coupled with IL-15 insufficiency.²⁰ These findings suggest that loss of FUS1 expression may play an important role in the early pathogenesis of lung cancer.

The Role of FUS1 in the Intrinsic Apoptotic Signaling Pathway

We previously used recombinant adenoviruses or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol nanoparticle-complexed plasmid vectors to introduce FUS1 and other genes into lung cancer cells.^{14,15,17,21,22} FUS1 showed the most potent proapoptotic activity in human lung cancer cells among these candidate 3p21.3 TSGs.^15–17,23 To identify the pathway involved in FUS1-mediated apoptosis, we used a ProteinChip array-based SELDI-MS spectrometry to analyze all of the protein species in complexes immunoprecipitated by anti-FUS1-antibodies. The apoptotic protease-activating factor 1 (Apaf-1) was identified as a potential cellular target of FUS1 protein by its direct protein–protein interaction (Figure 1). A computer-based analysis of the functional domains and signaling motifs within the amino acid sequence of FUS1 and Apaf-1 proteins reveals Class II and Class I PDZ^24,25 protein–protein interaction motifs at the C-termini of FUS1 and Apaf-1 proteins, respectively, providing a structural bases for FUS1-Apaf-1 protein–protein interaction. Apaf-1 plays an important role in the mitochondria-dependent apoptotic pathway.^{26 –28} A relatively high level of endogenous Apaf-1 protein was universally detected in lung cancer cells. These Apaf-1 proteins appeared to be functionally inactive, as indicated by their lack of intrinsic ATPase activity, which is essential for Apaf-1-mediated caspase activation and apoptosis induction in both cancer cells deficient in FUS1 expression and in normal cells with low level of endogenous FUS1 expression.^28,29 We showed that activation of endogenous FUS1 in normal cells in response to stress, such as UV irradiation, and the forced expression of FUS1 in FUS1-deficient tumor cells can trigger cytochrome C release from mitochondria into the cytosol and cause FUS1 binding to Apaf-1 and recruit it to critical cellular locations, thus, activating Apaf-1 in situ, initiating Apaf-1-mediated caspase activation, and inducing apoptosis.^17,30,31 Although our proposed mechanism remains to be validated by identifying all of the components in this complicated apoptotic apparatus and their dynamic interactions, our findings support a role for loss of FUS1 expression as a critical event in lung cancer pathogenesis.

FIGURE 1.

Schema of the FUS1 pathway. Activation of FUS1 in normal cells in response to apoptotic stimuli, stress, or restoration of wt-FUS1 function by ectopic gene transfer in FUS1-deficient tumor cells activates the intrinsic mitochondrial apopto-sis pathway. Activation of FUS1 triggers cytochrome c (Cyt C) release from the inner membrane of mitochondria to the cy-tosol, selectively and directly interacts with Apaf-1 and recruits it to a critical subcellular location, and activates Apaf-1 by induction of its ATPase activity in situ thus facilitating downstream Apaf-1-mediated apoptosome assembly, caspase activation, and apoptosis induction. Activation of FUS1 may also block MDM2-associated proteolytic degradation of p53 and enhance the p53-dependent apoptotic pathway. The potent tumor suppressor activity of FUS1 is also in part mediated by its inhibition of multiple oncogenic protein tyrosine kinases (PTKs) including EGFR, PDGFR, c-Abl, c-Kit, and AKT that are up-regulated in cancer cells. FUS1-mediated inactivation of these oncogenic PTKs leads to induction of apoptosis and inhibition of tumor cell proliferation and survival.

Inhibition of Tyrosine Kinase Signaling by FUS1

We found that reactivating FUS1 in 3p21.3-deficient lung cancer cells inhibited their growth and induced apoptosis, in part, by inhibiting protein tryrosine kinases (PTKs) such as EGFR, PDGFR, c-Abl, c-Kit, and AKT (Figure 1). A computer-based homology modeling of the FUS1 protein sequence and structure³² predicts a potential protein kinase A activation site, and an A kinase anchoring protein homology motif.¹⁷ It has been shown that a FUS1 peptide derived from FUS1 protein sequence in a region that was deleted in a mutant FUS1 gene detected in some lung cancer cell lines inhibits a constitutively active recombinant c-Abl tyrosine kinase and the full length c-Abl kinase in vitro.³³ Platelet-derived growth factors (PDGFs) play a crucial role in cell migration, proliferation, apoptosis, and cell survival. Forced expression of wt-FUS1 by nanoparticle-mediated gene transfer in the PDGFRβ-expressing SCLC H69 and H417 cell lines inactivated PDGFRβ and its downstream targets, PI3K and AKT kinases, as shown by marked reduction in PTK phosphorylation.

We explored the ability of FUS1 expression to overcome gefitinib resistance in NSCLC cells. We found that reexpression of wt-FUS1 by FUS1-nanoparticle-mediated gene transfer into FUS1-deficient and gefitinib-resistant NSCLC cell lines that have wt-EGFR sensitized them to gefitinib treatment and synergistically induced apoptosis. FUS1 nanoparticle treatment alone or with gefitinib in gefitinib-resistant NSCLC cells markedly inactivated EGFR and AKT, as shown by decreased phosphorylation levels of these proteins, and activated caspase-3, caspase-9, and PARP, as shown by the increased cleavage of their precursor proteins on Western blots. Together, these results suggest that combination treatment with FUS1 and PTK inhibitors may be a useful therapeutic strategy for human lung cancer.

Translational Applications of FUS1 for Lung Cancer Therapy

We initiated a dose escalation Phase I clinical trial of FUS1-nanoparticles in patients with chemotherapy refractory stage IV lung cancer. In this clinical trial, a FUS1 expression plasmid in a nanoparticle is injected intravenously in stage IV lung cancer patients who had progressed after cisplatin combination chemotherapy. The trial continues to accrue patients.

We have also explored the combined effects of the FUS1-nanoparticles with conventional chemotherapy and external beam radiotherapy.³¹ Forced expression by FUS1-nanoparticle-mediated gene transfer sensitized NSCLC cells to cisplatin or γ-radiation, resulting in a 3- to 8-fold increase in inhibition of tumor cell viability and induction of apoptosis in FUS1-transfected cells. Systemic treatment with a combination of FUS1 nanoparticles and cisplatin in a human lung cancer orthotopic mouse model synergistically enhanced the therapeutic efficacy of cisplatin.

We evaluated the combined effects of FUS1 and the TSG p53 on tumor cell growth and apoptosis induction in NSCLC cells cotransfected with FUS1- and p53.³⁰ We found that coexpression of wt-p53 with wt-FUS1, but not the myristoylation mutant (mt-FUS1), synergistically inhibited cell proliferation and induced apoptosis in human NSCLC cells. We also found that coexpression of FUS1 and p53 enhanced the sensitivity of NSCLC cells to treatments with the DNA-damaging agents γ-radiation and cisplatin. We found that the observed synergistic tumor suppression by FUS1 and p53 correlated with FUS1-mediated down-regulation of MDM2 expression resulting in the accumulation and stabilization of p53 protein and the up-regulation of Apaf-1 expression with activation of the caspase cascade (Figure 1). Our results demonstrate an important role for FUS1 in modulating chemo- and radiosensitivities of lung cancer cells and suggest that an optimal combination of molecular therapeutics, such as the proapoptotic tumor suppressor FUS1-nano-particle and conventional anticancer agents, such as cisplatin, may be an effective treatment strategy for human lung cancer.