KIF5B-RET fusion kinase promotes cell growth by multilevel activation of STAT3 in lung cancer
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Lung cancer in nonsmokers tends to be driven by a single somatic mutation or a gene fusion. KIF5B-RET fusion is an oncogene identified in non-small cell lung cancers. In this study, we verified the oncogenic activity of KIF5B-RET fusion and investigated how KIF5B-RET activates the specific signaling pathways for cellular transformation. We aimed to provide a basis for the further development of the therapy for KIF5B-RET positive lung cancer patients.
RT-PCR was used to screen for KIF5B-RET fusions in Chinese lung cancer patients. To verify the oncogenic activity of KIF5B-RET kinase in lung cancer cells, we manipulated its expression genetically followed by colony formation and tumor formation assays. The mechanism by which KIF5B-RET kinase induces proliferation was investigated by western blot, coimmunoprecipitation, and administration of RET, MAPK and STAT3 inhibitors.
Our study identified a KIF5B-RET fusion in Chinese NSCLC patients and demonstrated that KIF5B-RET transfected cells showed a significantly increased proliferation rate and colony-forming ability. Furthermore, we found that KIF5B-RET fusion kinase induced multilevel activation of STAT3 at both Tyr705 and Ser727, and KIF5B-RET-STAT3 signaling related inhibitors repressed the proliferation and tumorigenicity of lung cancer cells significantly.
Our data suggest that KIF5B-RET promotes the cell growth and tumorigenicity of non-small cell lung cancers through multilevel activation of STAT3 signaling, providing possible strategies for the treatment of KIF5B-RET positive lung cancers.
KeywordsKIF5B-RET Lung cancer Cell growth STAT3 pathway
Non-small cell lung cancer
Kinesin family member 5B gene
Rearranged during transfection gene
Glial derived neurotrophic factor
Signal transducer and activator of transcription-3
Epidermal growth factor receptor
Kirsten rat sarcoma
Echinoderm microtubule associated protein like 4 –anaplastic lymphoma receptor tyrosine kinase
Tyrosine kinase inhibitors.
Lung cancer is a common malignancy and is the leading cause of cancer deaths in the world, and non-small cell lung cancer (NSCLC) is the most common tumor type. NSCLC in nonsmokers tends to be driven by a single somatic mutation or a gene fusion, such as mutated epidermal growth factor receptor (EGFR), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), the echinoderm microtubule associated protein like 4 (EML4) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes (EML4-ALK), etc. These have been proven to be “driver” genes in some subgroups of lung cancers. Recently, NSCLCs were reported to harbor novel gene fusions involving ROS1 and RET[7, 8]. RET is the receptor for members of the glial cell line-derived neurotrophic factor family (GDNF)[9, 10]. The RET gene is located on chromosome 10 and encodes a receptor tyrosine kinase, and the oncogenic potential of this gene product has been suggested in several tumors, especially in thyroid cancers.
KIF5B-RET is a novel fusion gene of the kinesin family member 5B gene (KIF5B) and the rearranged during transfection gene (RET) resulting from the chromosome inversion inv (10) (p11; q11). It was first identified in non-smoking Korean man as adenocarcinoma by whole-genome and transcriptome sequencing. KIF5B-RET is reported in a low percentage of lung cancers and is more frequent in non-smokers, in patients with adenocarcinoma, and exists exclusively with other mutations, such as EGFR, Kras, Braf, ErbB2 or EML4/ALK fusions[8, 12, 13]. It has been demonstrated that KIF5B-RET fusions may be oncogenic drivers and potential targets for existing small-molecule tyrosine kinase inhibitors (TKIs). However, it is unclear how KIF5B-RET activates the specific signaling pathways for cellular transformation. The incomplete understanding of its carcinogenic mechanisms leads to difficulties in selecting targeted treatment.
Signal transducer and activator of transcription-3 (STAT3) is a member of a gene transcription protein family that mediates a variety of biological processes including cell proliferation and carcinogenesis. Constitutive STAT3 activation has been found in multiple types of tumors[15, 16, 17, 18]. Conventional STAT3 activation mainly consists of phosphorylation on a single tyrosine residue Tyr705 resulting in dimerization of the STAT1/3 transcription factors, translocation into the nucleus and transcriptional activation of target genes.
Previous studies have demonstrated that STAT3 is required for efficient cellular transformation by an array of well-characterized oncogenes including EGFR, RAS, ALK, and even RET related oncogenes such as RET-MEN2A/2B, RET-FMTC and RET/PTC[25, 26]. Phosphorylation on the Ser727 residue enhances the transcriptional activity of STAT3. Different cellular systems may affect STAT3 Tyr705 or Ser727 phosphorylation as different protein kinases may be implicated. It is reported that RET-MEN2A mutant RETC634R induces both Tyr705 and Ser727 phosphorylation of STAT3 for cellular transformation in a process independent of JAKs and Src. By contrast, the FMTC-associated mutants RETY791F and RETS891A implicate Src and JAKs in constitutive activation of STAT3 only in Tyr705. While thyroid-specific rearrangement of RET/PTC phosphorylating STAT3 Tyr705 requires the intrinsic kinase activity of RET/PTC and JAKs, and c-Src kinase are not involved in the RET/PTC-mediated activation of STAT3.
Here, we screened for known KIF5B-RET fusions in Chinese NSCLC patients using real-time polymerase chain reaction (RT-PCR) and corroborated the oncogenic activity of this fusion kinase by colony formation and tumor formation assays. And we explored the hypothesis that STAT3 may be involved in the signaling triggered by KIF5B-RET. We aimed to provide a basis for the further development of the therapy for KIF5B-RET positive lung cancer patients.
A KIF5B-RET fusion gene is screened in Chinese LAD patients
This positive case was a poorly differentiated nodule subtype LAD. The patient was a countrywoman aged 58 years, and had no smoking history. The mass measured 4 cm in greatest dimension, and the right lung pleura and the bronchus were involved, but no lymph node metastasis or distant metastasis (pathologic stage I b). RET expression was observed as moderately intense cytoplasmic staining but no obvious perinuclear or paranuclear accentuation. The tumor cells also had strong expression of TTF1 and Napsin A. Characteristics are listed in Additional file1: Table S1 and Additional file1: Table S2. The clinicopathological characteristics of this case were consistent with previous researches, namely, more poorly differentiated tumors, younger (<65 years), non-smokers, and solid subtype.
KIF5B-RET fusion kinase is constitutively activated and enhances cell proliferation
Signaling pathways involved in the proliferation of KIF5B-RET positive cells
KIF5B-RET activates STAT3 both Tyr705 and Ser727 at different levels in positive cells
Down regulation of KIF5B-RET-STAT3 signaling suppresses the proliferation of positive lung cancer cells
In the era of personalized medicine, tailored treatment based on gene alterations, including gene mutations and gene fusions, called as “driver genes”, has become a standard practice. Gene alterations encode signaling proteins which are crucial for cellular proliferation and survival, and drive tumor formation and sustain tumorigenesis. Partial chromosomal translocation and the corresponding gene fusion are not unusual among malignancies and may serve as a driving force for carcinogenesis. Thanks to refinements in cytogenetic techniques, an increasing number of gene fusions are being discovered in NSCLCs, such as ALK rearrangement and ROS1 rearrangement[5, 6]. Most recently, the RET gene rearrangement has been identified and has been demonstrated to be a new driver mutation in a subset of lung adenocarcinomas in some studies[8, 29]. In contrast to 13 fusion partner genes of RET in thyroid carcinoma, only KIF5B-RET, CCDC6-RET, TRIM33-RET and NCOA4-RET have been reported in lung cancer, and KIF5B-RET is the most commonly identified gene fusion in NSCLCs to date[29, 32].
The overall prevalence of RET fusions is 1% to 2% in an unselected population of NSCLCs, however, this incidence increases substantially to 6% in never-smokers with lung adenocarcinomas that are pan-negative for other known driver mutations. Based on the huge number of global lung cancer patients, 6% is not a small figure. The identification of RET fusion genes represent a new addition to the growing list of actionable drivers in lung cancers, add to the knowledge of the underlying factors behind this malignancy, and more importantly, it might change today’s therapeutic landscape in lung cancers.
It has been observed in our study that KIF5B-RET fusion protein has higher levels of phosphorylation and enhances cell proliferation significantly. On the other hand, in the report in Journal of Clinical Oncology, Wang et al. found that all RET-positive adenocarcinomas had a small primary lesion but tended to present with N2 disease significantly more often than the other lung adenocarcinomas with small lesions (P < 0.024), which showed KIF5B-RET positive tumor may have stronger local invasion ability. The potential role of KIF5B-RET fusion genes in lung carcinogenesis deserves further study.
The KIF5B-RET fusion protein comprises the motor domain and the coiled-coil domain of KIF5B, and the juxtamembrane intracellular region of RET, including the entire tyrosine kinase domain. Because KIF5B is expressed ubiquitously, its active promoter may drive the expression of KIF5B-RET. Together with dimerization through the coiled-coil domain, the RET tyrosine kinase activity of the fusion protein may be activated aberrantly, thus facilitating oncogenesis in the lung. This hypothesis corroborates the oncogenic mechanism proposed for the fusion gene KIF5B-ALK, in which the coiled-coil domain of KIF5B is always preserved, and its constitutive expression in lung is believed to activate ALK and downstream oncogenic effects[30, 33].
Although information on signal transduction downstream of the KIF5B-RET is still limited, it has been demonstrated that several tyrosine residues are phosphorylated in RET with GDNFs stimulation, which serve as docking sites recruits the Grb2-Gab1 and Grb2-Sos complexes that then activate the PI3K/AKT, RAS/MAPK, p38MAPK and c-Jun N-terminal kinase (JNK) pathways respectively, which promote cell proliferation and survival in some endocrine tumors. In our study, KIF5B-RET fusion kinase was shown to possess marked oncogenic activity both in vitro and in vivo, and STAT3 signaling pathway might be the principal downstream mediator of the oncogenesis. Strong phosphorylation of STAT3 was presented in KIF5B-RET positive lung cancer cells. Here we provide several lines of evidence that show KIF5B-RET mediates continuous activation of STAT3. The fusion kinase could bind to STAT3, and directly phosphorylate and activate STAT3 Tyr705. It also can mediate activation of STAT3 Tyr705 in the JAKs/STAT3 dependent ways, and trigger Ser727 phosphorylation through the Ras/Raf/MEK1/2/ERK1/2 pathway. All in all, KIF5B-RET fusion protein regulates STAT3 activation at different levels which may target cyclinD1 and play a key role in oncogenesis.
Accumulating data shows that most tumors will depend on more than one signaling pathway for their growth and survival, which necessitates either the development of multitargeted agents or the combination of single targeted drugs to inhibit multiple signaling pathways or multiple steps in the same pathway. In our study, different inhibitors were used to suppress multiple steps of the KIF5B-RET-STAT3 pathway, such as MEK inhibitor (U0126), JAKs or Src-family tyrosine kinases inhibitor (AG490 and PP1), STAT3 inhibitor (S3I-201) and multi-targeted agent (ZD6474). Significantly, all these inhibitors reduced the cell proliferation of KIF5B-RET positive lung cancer cells in vitro. However, the use of a combination of different agents will also be less convenient to the patient and can result in more dosing mistakes, so further basic and clinical studies are warranted to assess the optimize target inhibition.
Our results have consolidated the role of KIF5B-RET fusion gene in the pathogenesis of NSCLC and identified STAT3 as a key mediator of the transforming activity of KIF5B-RET positive lung cancer cells. KIF5B-RET fusion protein regulates STAT3 activation at multilevels which may target cyclinD1 and play a key role in oncogenesis. Our results thus provide possible strategies for the treatment of KIF5B-RET positive lung cancer patients.
Materials and methods
A549, H1299, Beas-2b, and 293 T cell lines were all from the cell bank of Chinese academy of sciences. A549 and H1299 cells were cultured at 37°C in RPMI-1640 supplemented with 10% heat-inactivated FCS. Beas-2b and 293 T cells were cultured in DMEM with 10% FCS.
Chemicals and antibodies
Different inhibitors of specific signal transduction pathways, including Vandetanib (ZD6474), U0126, PP1, AG490 and S3I-201, were purchased from Selleck. Phosphor-Ret(Tyr905), Ret, phospho-STAT3 (Tyr705), Phospho-STAT3(Ser727), STAT3, phospho-ERK1/2(Thr202/Tyr204), ERK1/2, glyceraledehyde-3-phosphatedehydrogenase (GAPDH), and anti-Flag antibodies were purchased from Cell Signaling Technology. STAT3 recombinant protein was purchased from Abnova.
Primary lung cancers tissues were from Chinese patients who did not receive neoadjuvant therapy and who underwent resection at Zhejiang Provincial Cancer Hospital, Hangzhou, between 2008 and 2010. The corresponding non-neoplastic lung tissues were immediately frozen and stored at −80°C until assayed. Informed consent and ethics approval was obtained for research purposes. Ethics committee of the hospital approved the study.
Total RNA was extracted from lung cancer tissues or cultured cells with TRIzol Reagent (Invotrogen). Revert Aid First Strand cDNA Synthesis Kit (Fermentas) was used to construct the template cDNA for realtime PCR. The primer sequences for screening the KIF5B/RET fusion gene were as follows: forward primer, 5′-AGGAAATGACCAACCACCAG-3′, and reverse primer, 5′-TCCAAATTCGCCTTCTCCTA-3′. PCR was performed with initial denaturation at 95°C for5 min, followed by 40 cycles of amplification (at 98°C for 10 sec, 60°C for 15 sec, and 72°C for 3 min), and final extension at 72°C for 5 minutes.
Total protein lysates were obtained from cultured cells or tumor tissues using radio-immunoprecipitation assay. Cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% polyacrylamide gels) and then transferred to nitrocellulose membranes. The membranes were blocked overnight with TBS containing 0.1% Tween 20 (TBST) and 5% nonfat milk, and probed with the primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibody for 1 h at room temperature, washed with TBST 3 times, and reacted with Super Signal West Pico chemiluminescent substrate (Pierce).
Hematoxylin-eosin and immunohistochemical stain
Tumor samples were fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin. For hematoxylin-eosin stain, sections were reacted with hemalum for nuclear staining, which is followed by counterstaining with eosin for staining of other eosinophilic structures. For immunohistochemical stain, sections were reacted with primary antibodies for 24 h at 4°C, reacted with HRP–conjugated secondary antibody for 1 h, and diaminobenzidine (DAB) was used as the substrate to produce an observable brown color.
Plasmids and lentiviral packaging
A full-length KIF5B-RET cDNA was cloned into lentiviral vector for constitutive gene expression (pLenti6.3-MCS-IRES2 vector, Invitrogen). Lentiviral vector was cotransfected with pLP1, pLP2, and pLP/VSVG packaging vectors (Invitrogen) into 293 T cells. A549 and Beas-2b cells were infected with empty, KIF5B-RET-FLAG -expressing lentiviruses, and were treated with Blastcidin (10 μg/ml) for 2 weeks.
Colony formation assay
300 cells were cultured in a 6-well culture plate for 14 days. At the end of incubation, cells were fixed with methanol for 10 min and stained with crystal violet for 10 ~ 15 min. Three wells of cell colonies were scored (≥40 cells).
Xenograft tumor model
Four-week-old athymic nude mice (BALB/c nude) were used in these studies with the approval of the institution’s ethics committee. 1 × 106 control and A549 cells, carrying KIF5B-RET, were separately subcutaneously injected in the left and right flanks of mice. The tumors were measured 1 month later. The weight of the xenograft tumors was recorded and analyzed.
Flag and STAT3 antibodies were used for Co-IP. Cell lysates were incubated with primary antibodies or control IgG overnight at 4°C and the immune complex was precipitated by the ProteinG Magnetic Beads (Millipore).The beads were then washed, boiled, and subjected to SDS-PAGE.
Values are shown as means ± SEM. Statistical analysis was performed by Student’s t-test, and P value less than 0.05 was considered statistically significant.
We thank Prof. Xu for his thorough review of the manuscript and J. Peng, Y. Fang for their technical assistance.
This work was supported by grants from the Major Science and Technology Special Project of Zhejiang Province (No.2012C13022-1), the Zhejiang Provincial Natural Science Foundation of Distinguished Young Scientists (No.LR12H01001), the Science and Technology Department of Zhejiang Province (No.2012C23081, No. 2013C33130), the Health Department of Zhejiang Province (No. 201232200).
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