Mol. Cells 2022; 45(10): 718-728
Published online August 23, 2022
https://doi.org/10.14348/molcells.2022.0037
© The Korean Society for Molecular and Cellular Biology
Correspondence to : leejh@catholic.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The in vivo expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.
Keywords non-small cell lung cancer, p21, p27, p53, SF3B4, UBE4B
Splicing factor B subunit 4 (SF3B4) is a component of the U2-pre-mRNA spliceosomal complex (Champion-Arnaud and Reed, 1994). Through interaction with another SF3b subunit, spliceosome-associated protein 145 (SAP145), SF3B4 mediates the tethering of the U2 complex to the branch site of pre-mRNA splicing (Gozani et al., 1996). The haploinsufficiency or mutation of SF3B4 is a major genetic cause of the Nager syndrome, characterized by a defective craniofacial formation and preaxial upper limb defect (Bernier et al., 2012; Cassina et al., 2017; Castori et al., 2014; Drozniewska et al., 2020; Hayata et al., 2019; Zhao and Yang, 2020). Even though the molecular basis by which SF3B4 mutation leads to the phenotypes of Nager syndrome is yet to be identified, a recent study indicates that SF3B4 is involved in the translational control of secretory proteins, such as collagen 1 or fibronectin, as cofactors for p180 in the endoplasmic reticulum (Ueno et al., 2019). Thus, the impaired synthesis of collagen by alteration of SF3B4 activity or expression might be a possible mechanism for the development of Nager syndrome.
The dysregulation of alternative splicing might contribute to tumorigenesis through alteration in the expression of key molecules involved in the cell cycle, apoptosis, migration, and invasion (Anczukow and Krainer, 2016). In addition to a mutation in splicing regulatory cis-elements in specific genes involved in survival or proliferation, the mutation or aberrant expression of splicing regulatory factors also frequently contributes to carcinogenesis. Several studies have demonstrated the oncogenic role of SF3B4 in hepatocellular carcinoma (HCC). SF3B4 expression is increased in HCC tissues compared to that in normal tissues and is frequently accompanied by an increased abundance of SF3B4 genes, which are associated with intrahepatic metastasis and poor prognosis (Iguchi et al., 2016; Wang et al., 2020; Xu et al., 2015). Recently, Shen et al. (2018) found that SF3B4 is one of the diagnostic factors for early-stage HCC. They also showed that the depletion or deletion of SF3B4 suppressed the proliferation and metastasis of HCC cells both
Ubiquitination factor E4B (UBE4B) is a member of the E4 ubiquitination factor class, a novel class of ubiquitination enzymes that catalyzes ubiquitin chain assembly, resulting in the elongation of a polyubiquitin chain (Koegl et al., 1999). Subsequent studies indicated that UBE4B can also function as an E3 ubiquitin ligase and that UBE4B mediates degradation of several selected targets including ataxin-2, Tau, and Tax (Matsumoto et al., 2004; Mohanty et al., 2020; Subramanian et al., 2021). Notably, while murine double minute 2 (MDM2) alone only catalyzes mono-ubiquitination of p53 (Lai et al., 2001), UBE4B promotes the poly-ubiquitination of p53, rendering p53 perceptible by the proteasome (Wu et al., 2011). Thus, UBE4B and MDM2 interdependently promote p53 degradation and inhibit p53-dependent transactivation (Du et al., 2016; Zhang et al., 2014). The negative regulatory role for p53 implicates the oncogenic feature of UBE4B, which was supported by aberrant expression of UBE4B in various types of cancers (Antoniou et al., 2019). However, the molecular mechanisms leading to the alterations in the UBE4B gene or in protein levels have not been clarified yet.
Lung cancer is one of the leading causes of cancer-associated death worldwide (Meza et al., 2015). The predominant histological subtype of lung cancer is the non-small cell lung cancer (NSCLC), accounting for 85% of all lung cancers. With regard to the relevance of the aberrant expression of splicing factors in lung cancer, overexpression or mutation of several splicing factors has been reported, including SRSF1, SRSF5, heterogeneous nuclear ribonucleoparticule A1, SRC associated with mitosis of 68 kDa (Sam68), and RNA binding motif protein 10 (de Miguel et al., 2014; Guo et al., 2013; Sumithra et al., 2020; Yan et al., 2019; Zhang et al., 2020). However, whether SF3B4 has oncogenic potential in NSCLC has not been studied yet. Hence, in the present study, we aimed to investigate the effects of SF3B4 silencing on the proliferation of A549 NSCLC cells and its underlying mechanisms. Our results demonstrated that SF3B4 depletion repressed the proliferation of A549 NSCLC cells, accompanied by the accumulation of p53, p21, and p27. We also provided evidence of the involvement of UBE4B in the SF3B4-mediated regulation of p53 stability.
The human NSCLC cell line A549 was provided by the American Type Culture Collection (USA). Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (GeneDEPOT Barker, USA), 1% penicillin-streptomycin (Biowest, France), and 0.1% gentamicin sulfate (Biowest) at 37°C in a humidified incubator with 5% CO2. The indicated proteins were knocked down using a small interfering RNA (siRNA) strategy using Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA). Cells were harvested 48 h after transfection for RNA isolation. The sequences of siRNAs used were as follows: SF3B4, 5’- GCAGUACCUCUGUAACCGU-3’; p53, 5’-CACUACAACUACAUGUGUA-3’; p27, 5’-GGAGCAAUGCGCAGGAAUA-3’; UBE4B, 5’-CCCUGUGUGCAAUUUGGUU-3’; control, 5’-CCUACGCCACCAAUUUCGU-3’.
For gene expression analysis, qRT-PCR was performed as previously described (Yun et al., 2021). Briefly, total RNA was isolated using RNAiso Plus (TaKara Biotechnology, Japan) and cDNA was synthesized using PrimeScript™ RT Master Mix (TaKara Biotechnology) according to the manufacturer’s instructions. RNA expression levels were measured by standard qPCR procedures with SYBR Premix Ex Taq™ (Takara Biotechnology) on an Applied Biosystems 7300 instrument (Applied Biosystems, USA). The relative values for the target mRNAs were calculated using the 2−ΔΔCT method after normalization to the Ct value of β-actin. The change in target mRNA expression level is presented as a fold change compared to the control cells. The primer sequences used are listed in Supplementary Table S1.
After treatment with the indicated siRNAs, cells (1 × 104/well) were seeded into 96-well plates. After incubation for the indicated times, cell proliferation was assessed using 100 μl/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) as previously described (Koh et al., 2020). Then, 100 μl 0.4 M hydrochloric acidic isopropanol and deionized water were added to each well. Optical density values at 570 nm were evaluated using an EpochTM microplate spectrophotometer (BioTek, USA). For the colony formation assay, cells were seeded at a density of 2,000 cells per 60-mm dish and incubated for eight days. Plates were then stained with 1 ml of 0.1% crystal violet (Sigma-Aldrich), and colonies were counted using a dissecting microscope.
The proportion of A549 cells in each phase of the cell cycle was determined by fluorescence-activated cell sorting (FACS) by labeling with bromodeoxyuridine (BrdU) and 7-aminoactinomycin D (7-AAD) using an APC BrDU flow kit (BD Biosciences, USA). Data acquisition and analysis were performed using a flow cytometer (FACS Calibur; BD Biosciences) and CellQuest Pro software (BD Biosciences).
The expression levels of specific proteins were determined by western blotting (Yun et al., 2020). The primary antibodies used are as follows: anti-SF3B4 (Cat. No. ab157117; Abcam, UK), anti-UBE4B (Cat. No. sc-100610; Santa Cruz Biotechnology, USA), anti-MDM2 (Cat. No. sc-965; Santa Cruz Biotechnology), anti-p53 (Cat. No. sc-126; Santa Cruz Biotechnology), anti-p27 (Cat. No. 610242; BD Biosciences), anti-p21 (Cat. No. ab109199; Abcam), anti-hemagglutinin (HA)-probe (Cat. No. sc-9133; Santa Cruz Biotechnology), and anti-β-Actin (Cat. No. A5441; Sigma-Aldrich). After incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Cat. No. GTX213110-01; Genetex, USA) or anti-mouse IgG (Cat. No. GTX213111-01; Genetex), the immunoreactive bands were detected with luminol enhancer solution and peroxide solution (Promega, USA) or SuperSignal™ West Femto Maximum Sensitivity Substrate (Sigma-Aldrich). Densitometric analysis of the specific bands was performed using ImageJ version 1.51 (NIH, USA). For the cycloheximide (CHX; Sigma-Aldrich) chase assay, CHX was added for the indicated times before harvesting cell lysates.
Cells were lysed with lysis buffer (Tris-HCl [pH 7.4], 5 M NaCl, 0.2% NP-40, and 0.5 M EDTA) and protease inhibitor cocktail (Roche, Switzerland). One microgram of normal rabbit IgG (Merck KGaA, Germany), p53 antibody (Cat. No. 10442-1-AP; Proteintech Group, USA), SF3B4 antibody (Abcam), or UBE4B antibody (Santa Cruz Biotechnology) were then added to the supernatant and incubated overnight at 4°C. Protein Agarose A/G (Merck KGaA) was added and the samples were mixed by rolling at 4°C for at least 3 h. The beads were washed three times with lysis buffer, and the pellets were dissolved in 2X SDS loading buffer, and then analyzed using western blot. For the polyubiquitination assay, HA-ubiquitin immunoprecipitation was performed. pRK5-HA-Ubiquitin-WT was a gift from Ted Dawson (Addgene plasmid #17608) (Lim et al., 2005). After 24 h of knockdown of p53 or UBE4B, A549 cells were transfected with pRK5-HA-Ubiquitin-WT using Lipofectamine 2000 reagent (Thermo Fisher Scientific). After 24 h, cells were harvested and polyubiquitination was analyzed by immunoprecipitation with anti-p53 antibody (Santa Cruz Biotechnology) and subsequent western blotting with anti-HA antibody (Santa Cruz Biotechnology).
The coding region of UBE4B was cloned into the BamHI and EcoR1 sites of a retroviral vector (pBabePuro; Cell Biolabs, USA) and transfected into HEK293T cells by the calcium phosphate method as previously described (Yang et al., 2018). After 36 h of transfection, the virus supernatants were collected and infected into A549 cells, which were then exposed to puromycin (Sigma-Aldrich) for 48 h for selection (A549-UBE4B). As a control, A549 cells were infected with retrovirus containing pBabe vector (A549-CON).
Data are expressed as the mean ± SD from three independent experiments. Statistical significance between two groups or multiple groups was analyzed by Student’s
The SF3B4 expression profile in patients with lung cancer or NSCLC was analyzed using GENT2 (http://gent2.appex.kr/gent2/) and the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo). As shown in Fig. 1A,
To verify the oncogenic potential of SF3B4 in lung cancer cells, we investigated the effect of SF3B4 knockdown on the proliferation of A549 lung cancer cells
Next, we determined the levels of p27 and p21, two representative cyclin-dependent kinase inhibitors, after SF3B4 depletion. Western blotting assay showed that p27 and p21 expression was notably increased by 3.6- and 5.5-fold, respectively, upon SF3B4 knockdown (Fig. 3A). Subsequent qRT-PCR analysis revealed that
As shown above, p53 induction by SF3B4 depletion was observed at the protein level but not the mRNA level. Hence, we examined whether p53 protein stability was affected by SF3B4 depletion using CHX chase experiments. As expected, the decay of p53 was delayed by SF3B4 depletion; the remaining p53 protein level was 30.6% of its level in the control, while it was 50.6% under SF3B4 depletion 1 h after CHX treatment (Fig. 4A). These findings indicate that, in the absence of SF3B4, p53 degradation was significantly delayed. MDM2 is a critical regulator of p53 protein stability and functions as an E3 ligase (Haupt et al., 1997). However, MDM2 levels increased both at the protein and mRNA levels upon SF3B4 depletion (Figs. 3A and 3B), suggesting that p53 accumulation is not the result of the quantitative reduction in MDM2. We also examined whether SF3B4 interacts with p53 through co-immunoprecipitation. We found that SF3B4 was neither bound to p53 nor MDM2, indicating that p53 stability is not directly affected by protein-protein interaction with SF3B4 (Fig. 4B).
Through a literature search, we recognized that UBE4B, an ubiquitin chain assembly factor, is predicted to be an SF3B4-binding protein based on high throughput complex fractionation and nanoflow liquid chromatography-tandem mass spectroscopy (LC-MS/MS) (Havugimana et al., 2012; Koegl et al., 1999). Furthermore, UBE4B has been previously reported to directly interact with p53 and MDM2, promoting p53 ubiquitination and degradation (Du et al., 2016; Wu et al., 2011; Zhang et al., 2014). Thus, we hypothesized that SF3B4 regulates p53 stability via UBE4B. To test this hypothesis, we investigated the interaction of endogenous SF3B4 with UBE4B in A549 cells by co-immunoprecipitation with UBE4B-specific antibody, followed by western blotting with SF3B4 antibody. As shown in Fig. 5A, SF3B4 interacted physically with UBE4B. We then examined whether UBE4B expression was altered by SF3B4 depletion. UBE4B expression was decreased by SF3B4 knockdown to 58.5% at the protein level but not at the mRNA level relative to their values in control (Fig. 5B). Subsequent CHX chase experiments revealed that, under SF3B4 depletion, UBE4B protein degradation was notably accelerated (Fig. 5C). Thus, SF3B4 affects stability of UBE4B, probably via protein–protein interaction. UBE4B depletion, in turn, increased p53 expression, which concomitantly increased p27 and p21 levels (Fig. 6A). Notably,
SF3B4 is a splicing factor whose mutation is a major cause of Nager Syndrome (Liu et al., 2018). SF3B4 has been reported as an oncogenic driver in several tumor types, including HCC and esophageal cancer, while in pancreatic cancer, it acts as a tumor-suppressor protein (Ding et al., 2021; Iguchi et al., 2016; Kidogami et al., 2020; Lee et al., 2020; Liu et al., 2018; Lv et al., 2020; Shen et al., 2018; Wang et al., 2020; Xu et al., 2015; Zhou et al., 2017). In this study, using a siRNA-based knockdown strategy with cell growth and cell cycle analysis, we demonstrated for the first time that SF3B4 exhibits oncogenic properties in A549 NSCLC cells. Our findings were consistent with public data showing a negative association between SF3B4 expression and survival probability in patients with lung cancer.
The molecular mechanisms underlying the oncogenic potential of SF3B4 are yet to be elucidated. Shen and Nam (2018) proposed that SF3B4 regulates p27 transcription level via alternative splicing of tumor suppressor KLF4 in HCC. Overexpression of SF3B4 promotes the generation of nonfunctional KLF4, resulting in the loss of function as a transcriptional regulator for p27 transcription. Likewise, we observed that SF3B4 depletion increased p27 expression in A549 cells with an accumulation of functional
We also provided evidence for the involvement of the p53/p21 axis in SF3B4-mediated cell cycle regulation. SF3B4 depletion induced the expression of
The stability of p53 is primarily controlled by MDM2, which targets p53, leading to proteasomal degradation (Haupt et al., 1997). p53 expression was upregulated at the protein but not at the mRNA level upon SF3B4 depletion. The major ubiquitin ligase for p53, MDM2, did not decrease but rather increased upon SF3B4 knockdown, excluding the possibility that the reduction in MDM2 level is the cause of p53 induction under SF3B4 depletion. The increase in
As far as the mechanism by which SF3B4 controls UBE4B expression, the splicing regulatory role of SF3B4 does not seem to be involved in this since the molecular weight of UBE4B protein is not altered by SF3B4 depletion. Given that UBE4B expression only decreased at the protein level by SF3B4 depletion and the decay rate of UBE4B was accelerated by SF3B4 depletion, UBE4B seems to be stabilized by interaction with SF3B4. However, in the case of the BMP receptor 1A, SF3B4 decreases its cell surface levels probably by facilitating the internalization of the receptor (Watanabe et al., 2007). Thus, SF3B4, which is known as a splicing factor, could participate in diverse cellular functions through interaction with specific proteins, resulting in the accumulation or degradation of partner proteins depending on the cellular milieu. Recently, UBE4B has been identified as a miR-9 target gene that promotes autophagy-mediated Tau degradation in miRNA libraries in
The role of UBE4B as a negative regulator of p53 implicates the oncogenic feature of UBE4B. This is supported by the aberrant expression of UBE4B in diverse cancers frequently associated with poor prognosis (Heuze et al., 2008; Huang et al., 2020; Zage et al., 2013; Zhang et al., 2014; 2016). Furthermore, low UBE4B expression sensitizes neuroblastoma cells to cetuximab or induces apoptosis in nasopharyngeal cancer cells (Memarzadeh et al., 2019; Weng et al., 2019). Thus, targeting either UBE4B, its upstream regulator SF3B4, or both, may have potential therapeutic implications in several types of cancers, including lung cancer.
In summary, our study reinforced the oncogenic properties of SF3B4 in A549 NSCLC cells using a siRNA strategy. We demonstrated for the first time, to our knowledge, that UBE4B is involved in regulating p53/p21 and p27 in SF3B4-mediated regulation of A549 cell growth. Therefore, SF3B4 and UBE4B could serve as molecular targets for the treatment of NSCLC harboring wild-type p53. Further, we discovered a novel role of SF3B4 in stabilizing specific proteins such as UBE4B, beyond its role as a splicing factor, in driving oncogenic processes.
This research was funded by grants from the National Research Foundation of Korea (NRF-2019R1A2C1086949).
J.-H.L. conceived and designed the experiments. H.K., J.L., S.-Y.J., and H.H.Y. performed experiments. J.-H.K. analyzed data. All authors have read and agreed to the published version of the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2022; 45(10): 718-728
Published online October 31, 2022 https://doi.org/10.14348/molcells.2022.0037
Copyright © The Korean Society for Molecular and Cellular Biology.
Hyungmin Kim1,2,3,5 , Jeehan Lee1,2,5
, Soon-Young Jung1,2
, Hye Hyeon Yun1,2
, Jeong-Heon Ko4
, and Jeong-Hwa Lee1,2,3,*
1Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea, 2Institute for Aging and Metabolic Diseases, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea, 3Department of Biomedicine & Health Sciences, Graduate School, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea, 4Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea, 5These authors contributed equally to this work.
Correspondence to:leejh@catholic.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Splicing factor B subunit 4 (SF3B4), a component of the U2-pre-mRNA spliceosomal complex, contributes to tumorigenesis in several types of tumors. However, the oncogenic potential of SF3B4 in lung cancer has not yet been determined. The in vivo expression profiles of SF3B4 in non-small cell lung cancer (NSCLC) from publicly available data revealed a significant increase in SF3B4 expression in tumor tissues compared to that in normal tissues. The impact of SF3B4 deletion on the growth of NSCLC cells was determined using a siRNA strategy in A549 lung adenocarcinoma cells. SF3B4 silencing resulted in marked retardation of the A549 cell proliferation, accompanied by the accumulation of cells at the G0/G1 phase and increased expression of p27, p21, and p53. Double knockdown of SF3B4 and p53 resulted in the restoration of p21 expression and partial recovery of cell proliferation, indicating that the p53/p21 axis is involved, at least in part, in the SF3B4-mediated regulation of A549 cell proliferation. We also provided ubiquitination factor E4B (UBE4B) is essential for p53 accumulation after SF3B4 depletion based on followings. First, co-immunoprecipitation showed that SF3B4 interacts with UBE4B. Furthermore, UBE4B levels were decreased by SF3B4 depletion. UBE4B depletion, in turn, reproduced the outcome of SF3B4 depletion, including reduction of polyubiquitinated p53 levels, subsequent induction of p53/p21 and p27, and proliferation retardation. Collectively, our findings indicate the important role of SF3B4 in the regulation of A549 cell proliferation through the UBE4B/p53/p21 axis and p27, implicating the therapeutic strategies for NSCLC targeting SF3B4 and UBE4B.
Keywords: non-small cell lung cancer, p21, p27, p53, SF3B4, UBE4B
Splicing factor B subunit 4 (SF3B4) is a component of the U2-pre-mRNA spliceosomal complex (Champion-Arnaud and Reed, 1994). Through interaction with another SF3b subunit, spliceosome-associated protein 145 (SAP145), SF3B4 mediates the tethering of the U2 complex to the branch site of pre-mRNA splicing (Gozani et al., 1996). The haploinsufficiency or mutation of SF3B4 is a major genetic cause of the Nager syndrome, characterized by a defective craniofacial formation and preaxial upper limb defect (Bernier et al., 2012; Cassina et al., 2017; Castori et al., 2014; Drozniewska et al., 2020; Hayata et al., 2019; Zhao and Yang, 2020). Even though the molecular basis by which SF3B4 mutation leads to the phenotypes of Nager syndrome is yet to be identified, a recent study indicates that SF3B4 is involved in the translational control of secretory proteins, such as collagen 1 or fibronectin, as cofactors for p180 in the endoplasmic reticulum (Ueno et al., 2019). Thus, the impaired synthesis of collagen by alteration of SF3B4 activity or expression might be a possible mechanism for the development of Nager syndrome.
The dysregulation of alternative splicing might contribute to tumorigenesis through alteration in the expression of key molecules involved in the cell cycle, apoptosis, migration, and invasion (Anczukow and Krainer, 2016). In addition to a mutation in splicing regulatory cis-elements in specific genes involved in survival or proliferation, the mutation or aberrant expression of splicing regulatory factors also frequently contributes to carcinogenesis. Several studies have demonstrated the oncogenic role of SF3B4 in hepatocellular carcinoma (HCC). SF3B4 expression is increased in HCC tissues compared to that in normal tissues and is frequently accompanied by an increased abundance of SF3B4 genes, which are associated with intrahepatic metastasis and poor prognosis (Iguchi et al., 2016; Wang et al., 2020; Xu et al., 2015). Recently, Shen et al. (2018) found that SF3B4 is one of the diagnostic factors for early-stage HCC. They also showed that the depletion or deletion of SF3B4 suppressed the proliferation and metastasis of HCC cells both
Ubiquitination factor E4B (UBE4B) is a member of the E4 ubiquitination factor class, a novel class of ubiquitination enzymes that catalyzes ubiquitin chain assembly, resulting in the elongation of a polyubiquitin chain (Koegl et al., 1999). Subsequent studies indicated that UBE4B can also function as an E3 ubiquitin ligase and that UBE4B mediates degradation of several selected targets including ataxin-2, Tau, and Tax (Matsumoto et al., 2004; Mohanty et al., 2020; Subramanian et al., 2021). Notably, while murine double minute 2 (MDM2) alone only catalyzes mono-ubiquitination of p53 (Lai et al., 2001), UBE4B promotes the poly-ubiquitination of p53, rendering p53 perceptible by the proteasome (Wu et al., 2011). Thus, UBE4B and MDM2 interdependently promote p53 degradation and inhibit p53-dependent transactivation (Du et al., 2016; Zhang et al., 2014). The negative regulatory role for p53 implicates the oncogenic feature of UBE4B, which was supported by aberrant expression of UBE4B in various types of cancers (Antoniou et al., 2019). However, the molecular mechanisms leading to the alterations in the UBE4B gene or in protein levels have not been clarified yet.
Lung cancer is one of the leading causes of cancer-associated death worldwide (Meza et al., 2015). The predominant histological subtype of lung cancer is the non-small cell lung cancer (NSCLC), accounting for 85% of all lung cancers. With regard to the relevance of the aberrant expression of splicing factors in lung cancer, overexpression or mutation of several splicing factors has been reported, including SRSF1, SRSF5, heterogeneous nuclear ribonucleoparticule A1, SRC associated with mitosis of 68 kDa (Sam68), and RNA binding motif protein 10 (de Miguel et al., 2014; Guo et al., 2013; Sumithra et al., 2020; Yan et al., 2019; Zhang et al., 2020). However, whether SF3B4 has oncogenic potential in NSCLC has not been studied yet. Hence, in the present study, we aimed to investigate the effects of SF3B4 silencing on the proliferation of A549 NSCLC cells and its underlying mechanisms. Our results demonstrated that SF3B4 depletion repressed the proliferation of A549 NSCLC cells, accompanied by the accumulation of p53, p21, and p27. We also provided evidence of the involvement of UBE4B in the SF3B4-mediated regulation of p53 stability.
The human NSCLC cell line A549 was provided by the American Type Culture Collection (USA). Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (GeneDEPOT Barker, USA), 1% penicillin-streptomycin (Biowest, France), and 0.1% gentamicin sulfate (Biowest) at 37°C in a humidified incubator with 5% CO2. The indicated proteins were knocked down using a small interfering RNA (siRNA) strategy using Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA). Cells were harvested 48 h after transfection for RNA isolation. The sequences of siRNAs used were as follows: SF3B4, 5’- GCAGUACCUCUGUAACCGU-3’; p53, 5’-CACUACAACUACAUGUGUA-3’; p27, 5’-GGAGCAAUGCGCAGGAAUA-3’; UBE4B, 5’-CCCUGUGUGCAAUUUGGUU-3’; control, 5’-CCUACGCCACCAAUUUCGU-3’.
For gene expression analysis, qRT-PCR was performed as previously described (Yun et al., 2021). Briefly, total RNA was isolated using RNAiso Plus (TaKara Biotechnology, Japan) and cDNA was synthesized using PrimeScript™ RT Master Mix (TaKara Biotechnology) according to the manufacturer’s instructions. RNA expression levels were measured by standard qPCR procedures with SYBR Premix Ex Taq™ (Takara Biotechnology) on an Applied Biosystems 7300 instrument (Applied Biosystems, USA). The relative values for the target mRNAs were calculated using the 2−ΔΔCT method after normalization to the Ct value of β-actin. The change in target mRNA expression level is presented as a fold change compared to the control cells. The primer sequences used are listed in Supplementary Table S1.
After treatment with the indicated siRNAs, cells (1 × 104/well) were seeded into 96-well plates. After incubation for the indicated times, cell proliferation was assessed using 100 μl/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) as previously described (Koh et al., 2020). Then, 100 μl 0.4 M hydrochloric acidic isopropanol and deionized water were added to each well. Optical density values at 570 nm were evaluated using an EpochTM microplate spectrophotometer (BioTek, USA). For the colony formation assay, cells were seeded at a density of 2,000 cells per 60-mm dish and incubated for eight days. Plates were then stained with 1 ml of 0.1% crystal violet (Sigma-Aldrich), and colonies were counted using a dissecting microscope.
The proportion of A549 cells in each phase of the cell cycle was determined by fluorescence-activated cell sorting (FACS) by labeling with bromodeoxyuridine (BrdU) and 7-aminoactinomycin D (7-AAD) using an APC BrDU flow kit (BD Biosciences, USA). Data acquisition and analysis were performed using a flow cytometer (FACS Calibur; BD Biosciences) and CellQuest Pro software (BD Biosciences).
The expression levels of specific proteins were determined by western blotting (Yun et al., 2020). The primary antibodies used are as follows: anti-SF3B4 (Cat. No. ab157117; Abcam, UK), anti-UBE4B (Cat. No. sc-100610; Santa Cruz Biotechnology, USA), anti-MDM2 (Cat. No. sc-965; Santa Cruz Biotechnology), anti-p53 (Cat. No. sc-126; Santa Cruz Biotechnology), anti-p27 (Cat. No. 610242; BD Biosciences), anti-p21 (Cat. No. ab109199; Abcam), anti-hemagglutinin (HA)-probe (Cat. No. sc-9133; Santa Cruz Biotechnology), and anti-β-Actin (Cat. No. A5441; Sigma-Aldrich). After incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Cat. No. GTX213110-01; Genetex, USA) or anti-mouse IgG (Cat. No. GTX213111-01; Genetex), the immunoreactive bands were detected with luminol enhancer solution and peroxide solution (Promega, USA) or SuperSignal™ West Femto Maximum Sensitivity Substrate (Sigma-Aldrich). Densitometric analysis of the specific bands was performed using ImageJ version 1.51 (NIH, USA). For the cycloheximide (CHX; Sigma-Aldrich) chase assay, CHX was added for the indicated times before harvesting cell lysates.
Cells were lysed with lysis buffer (Tris-HCl [pH 7.4], 5 M NaCl, 0.2% NP-40, and 0.5 M EDTA) and protease inhibitor cocktail (Roche, Switzerland). One microgram of normal rabbit IgG (Merck KGaA, Germany), p53 antibody (Cat. No. 10442-1-AP; Proteintech Group, USA), SF3B4 antibody (Abcam), or UBE4B antibody (Santa Cruz Biotechnology) were then added to the supernatant and incubated overnight at 4°C. Protein Agarose A/G (Merck KGaA) was added and the samples were mixed by rolling at 4°C for at least 3 h. The beads were washed three times with lysis buffer, and the pellets were dissolved in 2X SDS loading buffer, and then analyzed using western blot. For the polyubiquitination assay, HA-ubiquitin immunoprecipitation was performed. pRK5-HA-Ubiquitin-WT was a gift from Ted Dawson (Addgene plasmid #17608) (Lim et al., 2005). After 24 h of knockdown of p53 or UBE4B, A549 cells were transfected with pRK5-HA-Ubiquitin-WT using Lipofectamine 2000 reagent (Thermo Fisher Scientific). After 24 h, cells were harvested and polyubiquitination was analyzed by immunoprecipitation with anti-p53 antibody (Santa Cruz Biotechnology) and subsequent western blotting with anti-HA antibody (Santa Cruz Biotechnology).
The coding region of UBE4B was cloned into the BamHI and EcoR1 sites of a retroviral vector (pBabePuro; Cell Biolabs, USA) and transfected into HEK293T cells by the calcium phosphate method as previously described (Yang et al., 2018). After 36 h of transfection, the virus supernatants were collected and infected into A549 cells, which were then exposed to puromycin (Sigma-Aldrich) for 48 h for selection (A549-UBE4B). As a control, A549 cells were infected with retrovirus containing pBabe vector (A549-CON).
Data are expressed as the mean ± SD from three independent experiments. Statistical significance between two groups or multiple groups was analyzed by Student’s
The SF3B4 expression profile in patients with lung cancer or NSCLC was analyzed using GENT2 (http://gent2.appex.kr/gent2/) and the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo). As shown in Fig. 1A,
To verify the oncogenic potential of SF3B4 in lung cancer cells, we investigated the effect of SF3B4 knockdown on the proliferation of A549 lung cancer cells
Next, we determined the levels of p27 and p21, two representative cyclin-dependent kinase inhibitors, after SF3B4 depletion. Western blotting assay showed that p27 and p21 expression was notably increased by 3.6- and 5.5-fold, respectively, upon SF3B4 knockdown (Fig. 3A). Subsequent qRT-PCR analysis revealed that
As shown above, p53 induction by SF3B4 depletion was observed at the protein level but not the mRNA level. Hence, we examined whether p53 protein stability was affected by SF3B4 depletion using CHX chase experiments. As expected, the decay of p53 was delayed by SF3B4 depletion; the remaining p53 protein level was 30.6% of its level in the control, while it was 50.6% under SF3B4 depletion 1 h after CHX treatment (Fig. 4A). These findings indicate that, in the absence of SF3B4, p53 degradation was significantly delayed. MDM2 is a critical regulator of p53 protein stability and functions as an E3 ligase (Haupt et al., 1997). However, MDM2 levels increased both at the protein and mRNA levels upon SF3B4 depletion (Figs. 3A and 3B), suggesting that p53 accumulation is not the result of the quantitative reduction in MDM2. We also examined whether SF3B4 interacts with p53 through co-immunoprecipitation. We found that SF3B4 was neither bound to p53 nor MDM2, indicating that p53 stability is not directly affected by protein-protein interaction with SF3B4 (Fig. 4B).
Through a literature search, we recognized that UBE4B, an ubiquitin chain assembly factor, is predicted to be an SF3B4-binding protein based on high throughput complex fractionation and nanoflow liquid chromatography-tandem mass spectroscopy (LC-MS/MS) (Havugimana et al., 2012; Koegl et al., 1999). Furthermore, UBE4B has been previously reported to directly interact with p53 and MDM2, promoting p53 ubiquitination and degradation (Du et al., 2016; Wu et al., 2011; Zhang et al., 2014). Thus, we hypothesized that SF3B4 regulates p53 stability via UBE4B. To test this hypothesis, we investigated the interaction of endogenous SF3B4 with UBE4B in A549 cells by co-immunoprecipitation with UBE4B-specific antibody, followed by western blotting with SF3B4 antibody. As shown in Fig. 5A, SF3B4 interacted physically with UBE4B. We then examined whether UBE4B expression was altered by SF3B4 depletion. UBE4B expression was decreased by SF3B4 knockdown to 58.5% at the protein level but not at the mRNA level relative to their values in control (Fig. 5B). Subsequent CHX chase experiments revealed that, under SF3B4 depletion, UBE4B protein degradation was notably accelerated (Fig. 5C). Thus, SF3B4 affects stability of UBE4B, probably via protein–protein interaction. UBE4B depletion, in turn, increased p53 expression, which concomitantly increased p27 and p21 levels (Fig. 6A). Notably,
SF3B4 is a splicing factor whose mutation is a major cause of Nager Syndrome (Liu et al., 2018). SF3B4 has been reported as an oncogenic driver in several tumor types, including HCC and esophageal cancer, while in pancreatic cancer, it acts as a tumor-suppressor protein (Ding et al., 2021; Iguchi et al., 2016; Kidogami et al., 2020; Lee et al., 2020; Liu et al., 2018; Lv et al., 2020; Shen et al., 2018; Wang et al., 2020; Xu et al., 2015; Zhou et al., 2017). In this study, using a siRNA-based knockdown strategy with cell growth and cell cycle analysis, we demonstrated for the first time that SF3B4 exhibits oncogenic properties in A549 NSCLC cells. Our findings were consistent with public data showing a negative association between SF3B4 expression and survival probability in patients with lung cancer.
The molecular mechanisms underlying the oncogenic potential of SF3B4 are yet to be elucidated. Shen and Nam (2018) proposed that SF3B4 regulates p27 transcription level via alternative splicing of tumor suppressor KLF4 in HCC. Overexpression of SF3B4 promotes the generation of nonfunctional KLF4, resulting in the loss of function as a transcriptional regulator for p27 transcription. Likewise, we observed that SF3B4 depletion increased p27 expression in A549 cells with an accumulation of functional
We also provided evidence for the involvement of the p53/p21 axis in SF3B4-mediated cell cycle regulation. SF3B4 depletion induced the expression of
The stability of p53 is primarily controlled by MDM2, which targets p53, leading to proteasomal degradation (Haupt et al., 1997). p53 expression was upregulated at the protein but not at the mRNA level upon SF3B4 depletion. The major ubiquitin ligase for p53, MDM2, did not decrease but rather increased upon SF3B4 knockdown, excluding the possibility that the reduction in MDM2 level is the cause of p53 induction under SF3B4 depletion. The increase in
As far as the mechanism by which SF3B4 controls UBE4B expression, the splicing regulatory role of SF3B4 does not seem to be involved in this since the molecular weight of UBE4B protein is not altered by SF3B4 depletion. Given that UBE4B expression only decreased at the protein level by SF3B4 depletion and the decay rate of UBE4B was accelerated by SF3B4 depletion, UBE4B seems to be stabilized by interaction with SF3B4. However, in the case of the BMP receptor 1A, SF3B4 decreases its cell surface levels probably by facilitating the internalization of the receptor (Watanabe et al., 2007). Thus, SF3B4, which is known as a splicing factor, could participate in diverse cellular functions through interaction with specific proteins, resulting in the accumulation or degradation of partner proteins depending on the cellular milieu. Recently, UBE4B has been identified as a miR-9 target gene that promotes autophagy-mediated Tau degradation in miRNA libraries in
The role of UBE4B as a negative regulator of p53 implicates the oncogenic feature of UBE4B. This is supported by the aberrant expression of UBE4B in diverse cancers frequently associated with poor prognosis (Heuze et al., 2008; Huang et al., 2020; Zage et al., 2013; Zhang et al., 2014; 2016). Furthermore, low UBE4B expression sensitizes neuroblastoma cells to cetuximab or induces apoptosis in nasopharyngeal cancer cells (Memarzadeh et al., 2019; Weng et al., 2019). Thus, targeting either UBE4B, its upstream regulator SF3B4, or both, may have potential therapeutic implications in several types of cancers, including lung cancer.
In summary, our study reinforced the oncogenic properties of SF3B4 in A549 NSCLC cells using a siRNA strategy. We demonstrated for the first time, to our knowledge, that UBE4B is involved in regulating p53/p21 and p27 in SF3B4-mediated regulation of A549 cell growth. Therefore, SF3B4 and UBE4B could serve as molecular targets for the treatment of NSCLC harboring wild-type p53. Further, we discovered a novel role of SF3B4 in stabilizing specific proteins such as UBE4B, beyond its role as a splicing factor, in driving oncogenic processes.
This research was funded by grants from the National Research Foundation of Korea (NRF-2019R1A2C1086949).
J.-H.L. conceived and designed the experiments. H.K., J.L., S.-Y.J., and H.H.Y. performed experiments. J.-H.K. analyzed data. All authors have read and agreed to the published version of the manuscript.
The authors have no potential conflicts of interest to disclose.
Yong Jun Lee, Jii Bum Lee, Sang-Jun Ha, and Hye Ryun Kim
Mol. Cells 2021; 44(5): 363-373 https://doi.org/10.14348/molcells.2021.0044Dong-Seol Lee, Song Yi Roh, Hojae Choi, and Joo-Cheol Park
Mol. Cells 2020; 43(8): 739-748 https://doi.org/10.14348/molcells.2020.2272Hyeon Ju Lee, Yu-Jin Jung, Seungkoo Lee, Jong-Il Kim, and Jeong A. Han
Mol. Cells 2020; 43(4): 397-407 https://doi.org/10.14348/molcells.2020.2231