FBXL18 promotes cell proliferation by inducing K63-linked ubiquitination of PTEN in NSCLC cells

doi:10.21203/rs.3.rs-4980695/v1

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FBXL18 promotes cell proliferation by inducing K63-linked ubiquitination of PTEN in NSCLC cells

https://doi.org/10.21203/rs.3.rs-4980695/v1

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Purpose: We aim to investigate the role of E3 ubiquitin ligase FBXL18 in promoting non-small cell lung cancer (NSCLC) progression through the K63-linked ubiquitination of PTEN, thereby activating the PI3K/AKT signaling pathway.

Methods: Forty-seven pairs of fresh NSCLC samples and adjacent para-cancerous tissues were collected from the patients at the time of surgical resection. The human NSCLC cell lines A549, H1299, H460, SPCA-1 were purchased from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China).

Results: The study's results demonstrate that FBXL18 is upregulated in NSCLC and correlates with poor patient prognosis. Mechanistically, FBXL18 interacts with PTEN, inhibits its activity by inducing its K63-linked ubiquitination and promotes AKT phosphorylation and activation. In human NSCLC specimens, the expression of FBXL18 was upregulated and correlated with poor prognosis. Taken together, we uncover a new potential therapeutic targeting molecular mechanism by which FBXL18 activates PTEN/PI3K/AKT signaling pathway in NSCLC.

Conclusions: FBXL18, an E3 ubiquitin ligase, upregulates in NSCLC and promotes tumor growth by activating the PTEN/PI3K/AKT pathway through K63-linked ubiquitination of PTEN. Clinical relevance is further supported by the positive correlation between FBXL18 expression and activation of the PI3K-AKT pathway in NSCLC patients.

non-small cell lung cancer (NSCLC) cells

PI3K/AKT pathway

FBXL18

K63-linked ubiquitination

Lung cancer is the leading cause of cancer-related mortality with a poor 5-year survival of about 21%[1]. Genomic mutations and methylation have been extensively investigated in NSCLC [2–6]. In parallel, the post-translational modification (PTM) such as ubiquitination are also involved in NSCLC progression [7–10]. Protein ubiquitination is one of the most prevalent PTMs in eukaryotic cells. Ubiquitination is catalyzed by a three-enzyme cascade consisting of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin protein ligase (E3). Ubiquitination is initially described as a process that induces protein degradation via 26s proteasome. Subsequently, emerging evidences have revealed that ubiquitination participates in nearly all kinds of biological processes, playing a vital role in protein stability, activity and subcellular localization [11, 12]. Dysregulated ubiquitination can induce various different types of diseases, such as cancer.

Considering the specificity and diversity, E3 ubiquitin ligases are regarded as potential therapeutic targets in cancer. There are hundreds of E3 ubiquitin ligases in human. Among them, the SKP1-Cullin1-F-box (SCF) E3 ligase family is the best characterized. There are 69 putative F-box protein genes identified in human genome [13]. These F-box proteins contain an F-box motif that is responsible for recruiting to SCF complex. F-box proteins can be broadly classified into three subgroups according to the substrate-recognition motif: F-box and leucine rich repeats (FBXL) family, F-box and WD40 repeats (FBXW) family and F-box only (FBXO) family. Some of the F-box proteins have been well characterized such as SKP2/FBXL1 and FBXW7 [14–16]. These F-box proteins act as either tumor suppressors or oncogenic factors regulating cancer progression. However, the biological functions of the other F-box proteins need further investigations.

The PI3K/AKT signaling pathway is involved in the regulation of various cellular functions such as proliferation, anti-apoptosis, differentiation, adhesion and invasion [17, 18]. Aberrant activation of this pathway is involved in lung tumorigenesis and associated with high grade tumors. The tumor suppressor phosphatase and tensin homolog (PTEN) is a PtdIns(3,4,5)P₃ phosphatase, which acts as a main negative regulator of PI3K/AKT pathway. Inactivation of PTEN occurs frequently in a variety of cancers including NSCLC and loss or mutations of PTEN leads to constitutive activation of PI3K/AKT pathway, and are associated with poor clinical outcomes [19].

In this study, we demonstrate that FBXL18 is upregulated and plays a pro-oncogenic role in NSCLC through activating PI3K/AKT pathway. FBXL18 interacts with PTEN and mediates its K63-linked ubiquitination. Moreover, FBXL18-induced K63-linked ubiquitination of PTEN promotes AKT phosphorylation and activation.

2.1. Cell culture

The human NSCLC cell lines A549, H1299, H460, SPCA-1 were purchased from the cell bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China). A549, H1299, H460, SPCA-1 cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% Penicillin-Streptomycin Liquid. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% FBS and antibiotics.

2.2 Patients’ specimens

Forty-seven pairs of fresh NSCLC samples and adjacent para-cancerous tissues were collected from the patients at the time of surgical resection. All of these patients have signed informed consents and agreed to donate their tissues for the research. Ethical approval for the project was provided by Research Ethics Committee of Liaoning cancer hospital and institute.

2.3 Plasmids and cloning

The plasmid constructs expressing FBXL18, PTEN and truncated PTEN were generated by polymerase chain reaction (PCR) and then sub-cloned into pDONR221 vector as the entry clones through Gateway recombination cloning technology according to the manufacturer’s instructions. Subsequently, the entry clones were recombined into gateway destination vector for the expression of fusion proteins with SFB tag (containing S tag, Flag tag and streptavidin binding peptide), Myc tag or V5 tag. FBXL18-shRNA1 (5’-AAACTGAAGTAGAACGGGTTG-3’) and FBXL18-shRNA2 (5’-ACGTTCAGAATCAGATCTGTG-3’) were designed and inserted into pLKO.1 lentiviral vector. The shRNA-FBXL18 sequences were shown in supplement Table 1. Ubiquitin expression vectors including HA-UB, HA-UB (K48), HA-UB (K63), HA-UB (K48R) and HA-UB (K63R) were purchased from Addgene.

2.4 SFB pull down assay and immunoblotting

SFB pull-down experiment was performed as described previously [20]. Briefly, cells were lysed in NETN lysis buffer (Tris-HCl, pH 8.0, 20 mM; NaCl 100 mM; EDTA 1 mM; NP-40, 0.5%), supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO, USA) for 30 min in ice. Lysates were subjected to centrifugation at 13500 rpm for 15 min at 4℃. Prepared S-protein beads (washed 3 times in NETN lysis buffer) were added into supernatants and rotated overnight at 4℃. Protein complexes were washed 4 times with NETN buffer before resolved by SDS-PAGE and analyzed by immunoblot. For direct immunoblotting analysis, cells were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein samples were separated by SDS-PAGE and blot onto a polyvinylidene fluoride (PVDF) membrane (Millipore). After blocking with 5% (w/v) milk, the membranes were incubated with specific antibodies overnight at 4°C and then with peroxidase-conjugated secondary antibodies.

2.5 Ubiquitination assay

HEK293T cells were co-transfected with indicated plasmids for 40 hours (h), then treated with MG132 (10 µM) for 6 h before harvest. Cells were lysed in NETN lysis buffer in ice and centrifuged. The supernatants were incubated with S-protein beads overnight at 4°C, then washed 4 times with NETN buffer. Proteins were eluted by boiling in 3×LDS loading buffer and subjected to SDS-PAGE for immunoblotting.

2.6 Cycloheximide (CHX) chase experiments

CHX chase experiments were carried out as described previously [20]. Briefly, cells were transfected with indicated plasmids for 36 h before CHX (50 µg/ml, #2112, cell signaling technology) was added. At indicated time points, cells were harvested and lysed, followed by immunoblotting analysis.

2.7 Cell proliferation assay and Wound-healing assay

Cells were plated in 6-well plates in triplicate with 3000 cells per well. After cultured for 2 days, 4 days, 6 days and 8 days, cells were fixed with 10% methanol and subjected to crystal violet staining. After staining for 15 min, wells were washed three times with PBS and destained using 10% acetic acid, and the absorbance was measured at 590 nm.

MTT assay was performed in accordance with manufacturer's protocol. Briefly, cells were seeded into a 96-well plate at density of 1000 cells/well, on the day of treatment, medium was replaced with fresh medium containing 10% MTT and incubated for 4 h at 37°C. After removing incubation medium, 200 µl DMSO was added to dissolve the formazan crystals and the light absorbance at 570 nm was measured.

2.8 TCGA data analysis

We obtained normalized RNA-seq data of NSCLC patients from The Cancer Genome Atlas (TCGA) by an R package-TCGAbiolinks, where the RNA-Seq data contained mRNA expressions genes across tumor and normal tissues. The median FBXL18 expression value was used as the cutoff for low and high expression. Normalized mutual information (NMI) between FBXL18 and genes in the RNA-seq data were calculated, and genes with NMIs larger than 0.98 were regarded as FBXL18-correlated items. Signaling pathways information were downloaded from KEGG and Fisher exact test was applied to recognize which pathways were significantly enriched by the FBXL18-correlated factors.

2.9 Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8 software. Unpaired t-test was used for normal distribution. One-way ANOVA was used to compare the means of three or more groups under normal distribution. The log-rank test was used to compare Kaplan-Meier survival curves. Statistical methods used for TCGA data analysis are described above. Results with *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001 were considered significant. All experimental data are reported as the mean and the error bars (mean ± standard deviation, SD).

3.1 FBXL18 promotes cell proliferation and colony formation of NSCLC cells

In order to explore the prognostic impacts and potential therapeutic targets of FBXL18 in NSCLC patients, we analyzed FBXL18 expressions from TCGA (https://portal.gdc.cancer.gov/) and KM-plotter database (http://kmplot.com/analysis/). The expression of FBXL18 was significantly increased in lung cancer compared to normal lung tissues (Fig. 1A). Meanwhile, higher FBXL18 expression (the median FBXL18 expression value was used as the cutoff for low and high expression) was significantly correlated with poor overall survival in lung cancer patients (Fig. 1B).

Next, to investigate the potential function of FBXL18 in NSCLC cells, we first examined FBXL18 protein expression in a panel of lung cancer cell lines and HEK293T (Fig. 1C). Consequently, we performed both gain-of-function and loss-of-function analyses of FBXL18 in human NSCLC cell lines (Fig. 1 and Fig. S1). Two independent FBXL18 short hairpin RNAs (shRNAs) both inhibited cell proliferation of A549 and SPC-A1 cells (Fig. 1D, 1E and Fig. S1A-C). In contrast, overexpression or reconstitution of FBXL18 in A549 cells increased the cell proliferation (Fig. 1F, 1G and Fig. S1D-F). Taken together, FBXL18 may function as an oncogenic driver in NSCLC progression.

3.2 FBXL18 activates PI3K-AKT signaling pathway

To investigate the molecular mechanism of FBXL18-mediated NSCLC cell proliferation, we performed signaling pathway enrichment analysis and found that FBXL18 was significantly correlated with PI3K-AKT signaling pathway in both LUAD and LUSC (Fig. 2A, 2B). In light of this, we analyzed the possible FBXL18 substrates using ESINetwork, an E3-substrate interaction prediction tool [20], and PTEN lied on the top one among the PI3K-AKT signaling pathway associated proteins (Fig. 2C). However, knock-down of FBXL18 expression did not alter the endogenous PTEN protein levels in various NSCLC cells (Fig. S2A), indicating FBXL18 failed to degrade PTEN.

Given that FBXL18 is significantly correlated with PI3K-AKT signaling in lung cancer, we investigated whether FBXL18 regulates PI3K-AKT pathway in NSCLC cells. As anticipated, knockdown of FBXL18 downregulated the expression of pAKT (S473) in A549 cells (Fig. 2D). Conversely, reconstitution of FBXL18 in shRNA knock-downed FBXL18 cells rescued the expression of pAKT (S473) (Fig. 2E). Furthermore, IGF-1 stimulation significantly induced the pAKT (S473) expression in A549 cells with FBXL18-overexpression compared to control cells (Fig. 2F).

3.3 FBXL18 interacts with PTEN

To examine whether FBXL18 interacts with PTEN, we performed a series of pull-down assays. The HEK293T cells were co-transfected with SFB-FBXL18 and Myc-PTEN or SFB-PTEN and Myc-FBXL18, respectively. Both exogenous SFB-fused FBXL18 or PTEN proteins could pull-down Myc-tagged PTEN or FBXL18 in HEK293T cells (Fig. 3A). In addition, exogenous SFB-fused FBXL18 or PTEN proteins interacted with endogenous PTEN or FBXL18 in HEK293T cells (Fig. 3B). Furthermore, we co-transfected full length (FL) and truncated PTEN expression vectors (PTEN-N: 1-185 AA, PTPase domain; PTEN-C2: 186–351 AA, C2 domain; PTEN-ΔN: 186–403 AA) with SFB-tagged FBXL18 in HEK293T cells. S-protein pull down assays demonstrated that FBXL18 interacted with full length of PTEN and PTEN-ΔN (Fig. 3C), indicating that C-tail or PDZ binding domain is essential for their physical interaction.

3.4 FBXL18 promotes K63-linked ubiquitination of PTEN

As CRL E3 ubiquitin ligase protein FBXL18 distinctly promoted the ubiquitination of PTEN in HEK293T cells (Fig. 4A). Furthermore, we assessed the effects of FBXL18 on PTEN protein turnover by manipulation of FBXL18 in the presence of cycloheximide (CHX) to block protein synthesis. Despite this, FBXL18 overexpression indeed prolonged phosphorylation and activation of pAKT (Fig. 4B), but the expression of PTEN was not altered by FBXL18 overexpression. Moreover, we examined the ubiquitination type of PTEN by co-transfecting various different types of HA-tagged ubiquitin vectors (wild type, K48, K63, K48R and K63R) with SFB-PTEN and Myc-FBXL18 together. Interestingly, the expression of FBXL18 significantly increased the polyubiquitylation level of PTEN, and this effect was abrogated when wild-type ubiquitin was substituted with a K48 or K63R mutant, but not a K48R or K63 mutant (Fig. 4C and 4D). These results indicate that FBXL18 mediates K63-linked ubiquitination of PTEN.

3.5 FBXL18 positively correlates with PI3K-AKT signaling activation in NSCLC patients

To investigate the relevance of our findings to human NSCLC, we analyzed transcriptomic and proteomic expression data from TCGA dataset of LUAD tumors (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). We found that patients with simultaneously high expression level of FBXL18 and low expression level of PTEN had worse overall survival in the LUAD cohort (Fig. 5A). On the other hand, the simultaneously high expression level of FBXL18 and high expression of pATK were also correlated with worse overall survival in the LUAD cohort (Fig. 5B). Next, to determine whether FBXL18 is dysregulated in NSCLC tissues, we performed immunoblotting analyses in 47 paired human NSCLC tissues and matched para-cancerous tissues. We observed that FBXL18 was significantly upregulated in NSCLC tissues compared with adjacent non-cancer tissues (Fig. 5C and D). Taken together, these findings suggested that upregulation of FBXL18 may serve as a potential prognosis biomarker in NSCLC.

FBXL family members are the core component of SCF complexes responsible for specific substrate recognition. Previously, several FBXLs have been reported to function as tumor suppressors or oncoproteins in various cancers, basically depending on their targeting substrates involved signaling pathways. For example, SKP2 (S-phase kinase-associated protein 2), a key cell cycle regulator, is capable of inhibiting apoptosis and promoting tumor growth [21, 22]. In contrast, FBXL2 induced mitotic arrest and suppressed tumor formation through its ability to ubiquitinate cyclin D3 and promote its degradation [23]. Unlike many other FBXLs, FBXL18 is an orphan F-box protein and has been poorly characterized. It was also reported that FBXL18 reduced mitotic arrest by targeting FBXL7 for polyubiquitination and degradation [24]. Zhang et al. revealed FBXL18 was upregulated in glioma and promoted AKT1-K63 linked-ubiquitination [25]. In addition, FBXL18 attenuates cell toxicity by selectively targeting phosphorylated LRRK2 for ubiquitination and proteasomal degradation [26]. Although previous studies indicated FBXL18 participated in cell growth, its role in lung cancer still remains unclear. Here, we conclude that FBXL18 may act as an oncogenic protein in NSCLC. The expression of FBXL18 was significantly upregulated in human NSCLC samples in both mRNA and protein levels, and was positively correlated with PI3K/AKT signaling activation. Mechanistically, FBXL18 inhibits PTEN activity by K63-linked ubiquitination, and this inhibition in turn activates PI3K/AKT signaling pathway in NSCLC cells (Fig. 5E).

PTEN/PI3K/AKT signaling pathway is one of the best characterized cancer related key pathways in human cancer progression. Pathway enrichment analysis revealed that FBXL18 was significantly correlated with PI3K-AKT signaling pathway in NSCLC (Fig. 2). FBXL18 substrate prediction assay showed that PTEN is the most significant interactor among PI3K/AKT signaling pathway associated proteins (Fig. 2C), indicating FBXL18 may regulate PI3K-AKT pathway through PTEN. In this study, we found that FBXL18 activated AKT phosphorylation by interacting with and ubiquitinating PTEN. FBXL18 interacts with C-terminal domain of PTEN, which comprises C-tail domain and PDZ-binding domain (PDZ-BD) (Fig. 3). The C-tail domain is important for PTEN regulation and stability. In addition, PTEN binds to PDZ-domain containing proteins through PDZ-BD to regulate its subcellular localization and enhance the ability of PTEN to suppress AKT activation. Therefore, the interaction between FBXL18 and PTEN may disrupt the binding ability of PTEN to associate its partner proteins and result in an inactive conformation.

Recently, it has been reported that the expression of PTEN can be controlled by genomic variations and various post-translational modifications such as acetylation, ubiquitination and SUMOylation [27]. In our current study, we found FBXL18 could function as an E3 ligase that promotes PTEN ubiquitination (Fig. 4A). However, our results showed that PTEN ubiquitination mediated by FBXL18 was K63-linked, rather than K48 (Fig. 4C). Commonly, K48-linked ubiquitination are proteolytic through proteasome in nature, whereas K63-linked type is associated with localization, activity and protein-protein interaction. In line with these findings, ectopic expression of FBXL18 failed to decrease the expression of PTEN in the half-life experiment (Fig. 4B). These results may explain that even FBXL18 could not alter PTEN expression but still could activate PI3K-AKT signaling pathway. This is an exciting result as it uncovers a new mechanism that has never been reported before for activating PI3K-AKT signaling pathway by FBXL18 through PTEN K63-linked ubiquitination.

In summary, our findings demonstrate that FBXL18 is upregulated in NSCLC tissues compared with adjacent non-cancer tissues and high expression of FBXL18 is associated with poor LUAD prognosis. And also, our study reveals that FBXL18, a critical upstream factor of PI3K/AKT signaling pathway, positively regulates ATK activity by promoting PTEN K63-linked ubiquitination in NSCLC. Consequently, inhibition of PI3K/AKT pathway in FBXL18-upregulated NSCLC patients, might improve clinical outcomes in lung cancer treatment.

This study identifies FBXL18 as a critical regulator in NSCLC, highlighting its role in promoting tumor cell proliferation through the novel mechanism of K63-linked ubiquitination of PTEN. The findings suggest that FBXL18 may serve as a potential therapeutic target for NSCLC, particularly for patients with high FBXL18 and altered PTEN/PI3K/AKT signaling. Further research is warranted to explore the clinical application of FBXL18 inhibition in lung cancer treatment.

Author Contribution

**Author Contributions:**Yu Liu and Xiaolong Liu conceived and designed the experiments. Di Chen, Yi Zhang, and Chao Duan performed the experiments. Yawei Wang, Haichao Sun, and Jinghan Li analyzed the data. Tian Xia and Huan Qi contributed reagents/materials/analysis tools. Hailong Piao and HongXu Liu provided guidance on the study and edited the manuscript. Yu Liu wrote the paper. All authors contributed to the discussion of the results and read and approved the final manuscript. Correspondence and requests for materials should be addressed to Yu Liu ([email protected]).

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No competing interests reported.

SupplementaryFig.1.pptx
Supplementary Figure 1 FBXL18 promotes the proliferation of NSCLC cells. A. Immunoblotting analysis of FBXL18 knockdown with two independent shRNAs in SPC-A1 cells. Crystal violet images (B) and quantification (C) of growth curves of cells described in (A). D. Immunoblotting analysis of FBXL18 knockdown and reconstruction in A549 cells. Crystal violet images (E) and quantification (F) of growth curves of cells described in (D). n=3 wells per group. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. **p < 0.01, ***p < 0.001, ****p < 0.0001.
SupplementaryFig.2.pptx
Supplementary Figure 2 FBXL18 has no effect on PTEN expression. A. Immunoblotting of FBXL18 and PTEN in A549 cells, SPC-A1 cells, H1299 cells and H460 cells after silencing FBXL18. B. Immunoblotting of FBXL18 and PTEN in A549 cells after overexpressing FBXL18.

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You are reading this latest preprint version

FBXL18 promotes cell proliferation by inducing K63-linked ubiquitination of PTEN in NSCLC cells

Status:

Version 1

Abstract

Figures

Introduction

Methods

2.1. Cell culture

2.2 Patients’ specimens

2.3 Plasmids and cloning

2.4 SFB pull down assay and immunoblotting

2.5 Ubiquitination assay

2.6 Cycloheximide (CHX) chase experiments

2.7 Cell proliferation assay and Wound-healing assay

2.8 TCGA data analysis

2.9 Statistical analysis

Results

3.1 FBXL18 promotes cell proliferation and colony formation of NSCLC cells

3.3 FBXL18 interacts with PTEN

3.4 FBXL18 promotes K63-linked ubiquitination of PTEN

3.5 FBXL18 positively correlates with PI3K-AKT signaling activation in NSCLC patients

Discussion

Conclusion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

Status:

Version 1