Mol. Cells 2023; 46(6): 360-373
Published online January 24, 2023
https://doi.org/10.14348/molcells.2022.2242
© The Korean Society for Molecular and Cellular Biology
Correspondence to : henao22@xjtu.edu.cn
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/.
Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid carcinoma. Despite a good prognosis, approximately a quarter of PTC patients are likely to relapse. Previous reports suggest an association between S-phase kinase-associated protein 2 (SKP2) and the prognosis of thyroid cancer. SKP1 is related to apoptosis of PTC cells; however, its role in PTC remains largely elusive. This study aimed to understand the expression and molecular mechanism of SKP2 in PTC. SKP2 expression was upregulated in PTC tissues and closely associated with clinical diagnosis. In vitro and in vivo knockdown of SKP2 expression in PTC cells suppressed cell growth and proliferation and induced apoptosis. SKP2 depletion promoted cell autophagy under glucose deprivation. SKP2 interacted with PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1), triggering its degradation by ubiquitination. Furthermore, SKP2 activates the AKT-related pathways via PHLPP1, which leads to the cytoplasmic translocation of SKP2, indicating a reciprocal regulation between SKP2 and AKT. In conclusion, the upregulation of SKP2 leads to PTC proliferation and survival, and the regulatory network among SKP2, PHLPP1, and AKT provides novel insight into the molecular basis of SKP2 in tumor progression.
Keywords AKT, molecular mechanism, papillary thyroid carcinoma, PH domain leucine-rich repeat protein phosphatase-1, S-phase kinase-associated protein 2
Thyroid cancer is the most common cancer of the endocrine system. There are four subtypes: papillary thyroid carcinoma (PTC), follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer (Wang et al., 2015). PTC is the most common type, accounting for approximately 88% of all thyroid cancers (Aschebrook-Kilfoy et al., 2011). Reports suggest that the rise in PTC cases accounts for the increase in thyroid cancer cases in recent years (Cho et al., 2013; Elisei et al., 2010; McNally et al., 2012). PTC has a good prognosis after surgical treatment, with a 99% survival rate for a 20-year (Kakudo et al., 2004). Despite the high survival rate of PTC, the long-term follow-up shows a 25% high recurrence rate (Grogan et al., 2013). Surgical treatment and fear of recurrence are traumatizing and cause psychological distress and burden to PTC patients. Hence, understanding the underlying molecular mechanism of PTC development, progression, and recurrence will aid in enhancing treatment and preventing recurrences in PTC patients.
S-phase kinase-associated protein 2 (SKP2) is an F-Box protein (FBP) that contains at least one F-box domain. The F-box domain is a 50 amino acid protein motif that mediates the interaction between proteins. FBP is one of the components of the SCF (SKP1-cullin-F-box) ubiquitin-protein ligase complex (Bai et al., 1996), which plays an important role in signal transduction and cell cycle regulation (Craig and Tyers, 1999). SKP2 is a substrate recognition factor in the SCF complex (Nakayama and Nakayama, 2005). Recent studies have shown the role of SKP2 in various cancers, which makes SKP2 a potential therapeutic and drug target (Chan et al., 2010). Overexpression of SKP2 was reported in the initiation, growth, and metastasis of many cancers. Further
PTC tissues and their paired normal thyroid tissue (n = 98) were collected from PTC patients during surgical resection at The First Affiliated Hospital of Xi’An Jiaotong University. Written informed consent was obtained from all the participating patients. The patients who underwent chemotherapy or radiotherapy before the surgery were excluded from this study. All tissue samples were frozen in liquid nitrogen or paraffin-embedded immediately after surgical resection, and two independent pathologists evaluated the tissue biopsies. This study was approved by the institutional ethics committee of The First Affiliated Hospital of Xi’An Jiaotong University (No. 2021-1407). All the experiments were performed in accordance with the principles of the Declaration of Helsinki (2013).
Human embryonic kidney (HEK293T) cells were purchased from the National Infrastructure of Cell Line Resource (NICR, China), and Normal Human thyroid follicular epithelial cells (Nthy-ori 3-1) were purchased from American Type Culture Collection (ATCC, USA). Three human PTC cell lines, TPC-1, BCPAP, and IHH-4, were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Nthy-ori 3-1 cells and all three PTC cell lines were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). HEK293T cells were cultured in DMEM medium supplemented with 10% FBS.
Myc-tagged SKP2 and Flag-tagged PHLPP1 were cloned in pcDNA vectors using the Gateway recombination system (Invitrogen, USA).
Cell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Japan) method per the manufacturer’s protocol. Briefly, cells with the indicated treatment were seeded in 96-well plates at a density of 2 × 103 cells/well and cultured for 24, 48, and 72 h at 37°C. Further, 10 μl CCK-8 solution (Dojindo Molecular Technologies) was added to each well and incubated at 37°C for 2.5 h. The absorbance was measured at 450 nm wavelength using a microplate reader (Bio-Tek, USA).
For colony forming assay, cells in the logarithmic phase were digested using 0.25% trypsin. TPC-1 (1×103 cells) and IHH-4 (500 cells) cells were seeded in a 6-well plate and cultured for 10-14 days at 37°C. The colonies were fixed with methanol, stained with crystal violet, and manually counted.
5-ethynyl-2’-deoxyuridine (EdU) incorporation assay was used to study DNA synthesis. Cells were seeded in a 24-well plate, incubated with EdU solution at 37°C for 2 h, followed by incubation with Apollo 488 dye for another 30 min. The fluorescence was observed and captured using a fluorescence microscope (Nikon, Japan).
Cell apoptosis was measured using flow cytometry. Approximately 1 × 106 cells with the indicated treatment were collected and digested with trypsin and were stained using FITC Annexin V Apoptosis Detection Kit (BD Biosciences, USA) per the manufacturer’s instructions. After staining, the cells were sorted using a FACSCalibur flow cytometer (BD Biosciences). Apoptosis was analyzed using FlowJo software (TreeStar, USA).
BALB/c female nude pathogen-free mice (6-8 weeks old, 20-25 g) were purchased from Shanghai experimental animal center China. Mice were housed at 22°C ± 2°C in a light-dark cycle of 12 h. PTC cells (TPC-1 and IHH-4) transfected with
The animal study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Xi’An Jiaotong University prior to the experiments being conducted (No. 2020AM88). All the animal experiments were performed per the Guide for the Care and Use of Laboratory Animals.
Total RNA from the thyroid tissue or PTC cells was extracted using Trizol reagent (TaKaRa Bio, Japan) per the manufacturer’s protocol. One microgram RNA was used to carry out cDNA synthesis using ReverTra Ace qPCR-RT Master mix kit (Toyobo, Japan) according to the manufacturer’s protocol. PCR was performed using SYBR Green II kit (TaKaRa Bio). The PCR program was set as: denaturation at 94°C for 3 min, followed by 30 cycles including denaturation at 94°C for 30 s, annealing at 59.5°C for 30 s, and extension at 72°C for 60 s.The housekeeping gene
For protein extraction, ground tissue or cells were lysed using RIPA lysis buffer, and the debris was removed by centrifuge. The protein in the supernatant was collected and quantified using a bicinchoninic acid assay (BCA) kit (Beyotime, China). The extracted proteins were separated on 12% SDS-PAGE gels, the voltage was first 80 V for ~30 min and subsequent 120 V, for ~60 min, and then and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) (400 mA, 90 min). The membranes were blocked using skimmed milk in Tris-buffered saline Tween 20 (TBST) and probed using primary antibodies for 12 h at 4°C. After washing; the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for another 1 h at room temperature. Immunoreactive bands were detected using chemiluminescence detection kits (Millipore).
The primary antibodies used were as follows: SKP2 (ab183039, 1:500; Abcam, UK), β-actin (ab6276, 1:3,000; Abcam), LC3B (ab192890, 1:2,000; Abcam), p62 (#5114, 1:1,000; CST), PHLPP1 (ab135957, 1:1,000; Abcam), β-tubulin (#2146, 1:1,000; CST), AKT (#4691, 1:1,000; CST), phosphorylated-AKT (#4060, 1:2,000; CST), mTOR (#2972, 1:1,000; CST), phosphorylated-mTOR (#5536, 1:1,000; CST). Antibodies against HA tag (ab18181, 1:2,000; Abcam), Myc tag (ab9106, 1:1,000; Abcam), Flag tag (ab125243, 1:2,000; Abcam).
Four micrometers thick paraffin-embedded sections of human tissues or xenografts were prepared. The sections were dewaxed, incubated with citric buffer at 95°C for 10 min, and blocked using H2O2 at room temperature for 20 min. The sections were incubated with normal goat serum for another 30 min at room temperature, followed by overnight incubation at 37°C with primary antibodies (SKP2, 1:100, ab183039 [Abcam]; Ki-67, 1:150, ab16667 [Abcam]). The sections were washed using PBS and incubated with HRP-conjugated-secondary antibody. For peroxidase detection, the sections were stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) for approximately 15 s and counterstained with hematoxylin.
PTC cells after indicated treatment were washed using PBS and fixed with 4% polyformaldehyde (PFA) for 20 min. The excess PFA was removed by washing; the cells were permeabilized using 0.1% Triton X-100 for 10 min and incubated with 5% normal goat serum for 60 min to prevent nonspecific binding. Cells were probed with primary antibodies at 4°C overnight, followed by incubation for 1 h with fluorescent secondary antibodies (1:200, Alexa-488-conjugated goat anti-rabbit IgG, ab150077; Abcam). The cells were counterstained using 4’,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for 10 min at room temperature and visualized. The images were captured using a confocal microscope (Olympus, Japan). The primary antibodies used were anti-SKP2 (ab183039, 1:200; Abcam) and LC3B (ab192890, 1:100; Abcam).
Scrambled shRNA,
Data were represented as the mean ± SD. Statistical analysis was performed using IBM SPSS Statistics software (ver. 19.0; IBM, USA). Student’s
Expression of
To assess the effects of loss of
Additionally, a tumorigenicity assay performed in nude mice proved that knockdown of
These results suggested the oncogenic role of SKP2 in PTC cells as
Several recent studies show the role of SKP2 in autophagy. For instance, Gassen et al. (2019) demonstrated loss of
SKP2, an FBP, forms the SCF (Skp1-Cullin 1-F-box) complex to participate in the ubiquitination-mediated degradation of proteins (Wang et al., 2012b). Various proteins have been identified as the SKP2 substrate, which was degraded by ubiquitination, including p27 (Bochis et al., 2015), FOXO1 (Huang et al., 2005), PDCD4 (Li et al., 2019), etc. Previous studies have shown that β-TRCP (F-box family protein) degrades PHLPP1 via ubiquitination (Li et al., 2009). Therefore, the correlation between SKP2 and PHLPP1 was examined using HEK-293T cell overexpressing Myc-tagged SKP2 and Flag-tagged PHLPP1. Co-immunoprecipitation assay was performed to evaluate the interaction between SKP2 and PHLPP1. As shown in Fig. 4A, SKP2 interacted with PHLPP1. Immunoprecipitation on TPC-1 cell lysates and using SKP2 antibody revealed an endogenous interaction between SKP2 and PHLPP1 (Fig. 4B). Next, the effect of
The stability of PHLPP1 protein was investigated in the absence or presence of SKP2. PTC cells were incubated with a proteasome inhibitor (MG132), and the PHLPP1 expression was evaluated by western blot. As indicated in Fig. 5A, the PHLPP1 expression increased moderately over time and was independent of SKP2 expression, thus suggesting a proteasome-dependent degradation of PHLPP1. Next, the PTC cells were treated with a protein synthesis inhibitor (cycloheximide, CHX). A decrease in PHLPP1 expression was observed in cells treated with CHX. Similarly, in
PHLPP1 belongs to PH domain leucine‐rich repeat protein phosphatase family. Increasing evidence has suggested the role of PHLPP1 in various cancers (Wang et al., 2013). Multiple studies suggest that PHLPP1 regulates the AKT pathway by dephosphorylating AKT (Chen et al., 2011; Nitsche et al., 2012). Therefore, it is likely that SKP2 may regulate the AKT pathway via PHLPP1. Hence, AKT and mTOR phosphorylation was assessed in
The results indicate that the loss of SKP2 promotes autophagy without glucose. Hence, we next studied the mechanism associated with this phenomenon.
Similarly, an increase in expression of PHLPP1 was observed in
In this study, we show the overexpression of
SKP2 is widely known as the ubiquitin ligase subunit, involved in the substrate recognition by SCF complex and ubiquitin-dependent degradation of the proteins (Deshaies, 1999). Various proteins have been identified as the targets of SKP2, one of which is CDK inhibitor p27, which inhibits G1/S transition and promotes apoptosis (Yalcin et al., 2014). SCF-SKP2 complex recognizes and phosphorylated p27 at Thr-187 site (Bretones et al., 2011). Thus SKP2 triggered ubiquitin-dependent p27 degradation accelerates the cell cycle, leading to dysregulation in cell proliferation. SKP2 is also involved in post-transcriptional regulation of forkhead transcription factors; for instance, Akt-dependent phosphorylation of FOXO1at Ser-256 is mediated by SKP2 and degrades FOXO1 via ubiquitination (Huang et al., 2005). Further, FOXO3 undergoes deacetylation-dependent recognition by SKP2 and succedent ubiquitination (Wang et al., 2012a). Reports suggest SKP2 mediates ubiquitination and degradation of c-Myc. Meanwhile, SKP2 also acts as a transcriptional coactivator for c-Myc, which activates downstream genes (Kim et al., 2003; von der Lehr et al., 2003). This shows the dual role of SKP2 in c-Myc regulation, which suggests a peculiar feature of SKP2 in carcinogenesis. Interestingly, the role of SKP2-mediated ubiquitination extends beyond protein degradation. It also mediates YAP nuclear localization and Hippo-YAP pathway activation (Yao et al., 2018). Similarly, reports suggest SKP2 mediated polyubiquitination of LKB1 at K63 induced its binding to the STRAD-MO25 complex, which helps cell survival under stress (Lee et al., 2015). Interestingly, reports suggest membrane translocation of SKP2 activates AKT directly and leads to subsequent protumor signaling (Chan et al., 2012). Recent reports suggest PI3K independent activation of AKT, triggered by SKP2-mediated ubiquitination (Clement et al., 2018). Additionally, SKP2-mediated ubiquitination activates K63 which further interacts with HK2. Notably, the ubiquitination of AKT leads to mitochondrial translocation induced by EGF (Yu et al., 2019b). In this study, we demonstrated that SKP2 acts as a proto-oncogene in PTC and subsequently identified PHLPP1as a substrate for SKP2-mediated ubiquitination.
PHLPP1 is a serine/threonine phosphatase and plays a crucial role in tumor suppression by inactivating AKT. There are three AKT isoforms, AKT1, AKT2, and AKT3, encoded by different genes (Moc et al., 2015). Among these three isoforms, AKT2 and AKT3 are believed to be targets of PHLPP1, as PHLPP1 regulates dephosphorylation of AKT2 and AKT3, but not Akt1 (Dong et al., 2014). For instance, in pancreatic cancers, PHLPP1 selectively dephosphorylated and negatively correlated with AKT1 (Nitsche et al., 2012). Similarly, in metastatic melanoma, the selective regulation of AKT2 by PHLPP1 was shown (Yu et al., 2018). Despite multiple reports suggesting an indirect relationship between PHLPP1 and AKT2, few reports show a direct regulation between PHLPP1 and AKT1. A decrease in PHLPP1 expression increased the phosphorylation of AKT1 at P473 in lung cancer cells (Zhiqiang et al., 2012). Another study demonstrated that AKT1 binds to PHLPP1, which was verified by an increase in PHLPP1 and Scrib complex expression. This binding was not observed with other AKT isoforms (Li et al., 2011). Additionally, the blocking of the PHLPP1 catalytic site by a specific inhibitor promoted phosphorylation of AKT1 at Ser473 under serum starvation, suggesting the role of PHLPP1 in chaperone-mediated autophagy (Arias et al., 2015). These contradictory results indicate an alternative mechanism regulating the AKT pathway via PHLPP1. This study revealed that SKP2 indirectly regulated the AKT pathway triggered by ubiquitin-induced degradation of PHLPP1. However, our results indicate that
It is intriguing to note that the relationship between SKP2 and AKT is multi-directional. Previous studies propose a reciprocal regulation between SKP2 and AKT (Chan et al., 2012; Lin et al., 2009). Earlier studies show that AKT’s direct phosphorylation of SKP2 forms SCF complex and E3 ligase activity (Lin et al., 2009). Gao et al. (2009) have also described that AKT1, but not AKT2, phosphorylate SKP2 at Ser72. The Ser72 phosphorylation of SKP2 impedes its nuclear translocation and leads to its accumulation in the cytoplasm. Moreover, AKT1-mediated phosphorylation also endowed SKP2 resistance to degradation triggered by the APC-Cdh1 complex, enhanced SKP2 stability and cytoplasmic distribution. This mechanism may be responsible for the tumor progression (Gao et al., 2009). However, AKT1-mediated translocation of SKP2 to the cytoplasm raises questions about its role in cell cycle progression. The mechanism underlying the degradation of p27 via cytoplasmic SKP2 and subsequent dysregulates of the cell cycle remains elusive mainly (Zhang, 2010). Taking account of the proto-oncogenic role of SKP2, there is still much to be addressed.
Further, the association of AKT and SKP2 is established, where AKT regulates SKP2 activity indirectly. For instance, deactivating AKT suppressed SKP2 expression via FOXO3A, the latter consequently contributed to the expression of p27 by transcriptional regulation as well as repressing SKP2 (Li et al., 2018). Moreover, AKT regulated SKP2 expression at the translational level, along with mTORC1 and eIF4E (Nogueira et al., 2012). Based on these results, we conclude that survival stress induced a subcellular translocation of SKP2, but not its expression, which could be reversed by AKT inhibition, implying a possible role of AKT in SKP2 regulation.
However, the study has some limitations. The role of SKP2 as an oncogene in PTC was verified by downregulating the expression of
In summary, we identified a novel role of the SKP2/PHLPP1/AKT axis in PTC progression, including cell proliferation and survival under starvation. Further, activation of AKT was investigated to support the cytoplasmic accumulation of SKP2, as inhibition of AKT led to SKP2 nuclear location under glucose deprivation. The results added to the established understanding of SKP2 and would help to develop targeting therapy for PTC.
This work was supported by grants from the institutional Foundation of the First Affiliated Hospital of Xi’An Jiaotong University (No. 2019ZYTS-04 and No. 2019ZYTS-13), Xi’An Science and Technology Project (No. 2019114613YX001SF04(5)), Clinical Research Center for Thyroid Diseases of Shaanxi Province (No. 2017LCZX-03), the Clinical Research Award of the First Affiliated Hospital of Xi’An Jiaotong University (XJTU1AF-CRF-2020-020), The Basic Natural Science Research Program of Shaanxi Province (2021JQ-386 and 2021JQ-405), the Thyroid Research Project of Young and Middle-aged Physicians of Beijing Bethune Charitable Foundation (BQE-JZX-202103), the Key Research and Development Program of Shaanxi Province (2022SF-159) and the National Natural Science Foundation of China (No. 82103568).
Y.S., W.R., H.D., and C.X. designed the study, supervised the data collection, analyzed the data. F.Y., X.L., and S.Z. interpreted the data, and prepared the manuscript for publication. J.L., X.Y., Q.Z., X.S., and Z.Z. supervised the data collection, analyzed the data and reviewed the draft of the manuscript. All authors have read and approved the manuscript.
The authors have no potential conflicts of interest to disclose.
Relationship between
Variable | |||
---|---|---|---|
Low expression | High expression | ||
Age (y) | |||
≤45 | 29 | 27 | 0.683 |
>45 | 20 | 22 | |
Sex | |||
Male | 24 | 23 | 0.840 |
Female | 25 | 26 | |
Tumor size (cm) | |||
≤1 | 30 | 15 | 0.002* |
>1 | 19 | 34 | |
TNM stage | |||
I/II | 45 | 35 | 0.009* |
III/IV | 4 | 14 | |
Extra thyroidal | |||
Negative | 33 | 29 | 0.402 |
Positive | 16 | 20 | |
Lymph node metastasis | |||
Negative | 36 | 26 | 0.036* |
Positive | 13 | 23 | |
Nodular goiter | |||
Negative | 42 | 37 | 0.201 |
Positive | 7 | 12 |
Values are presented as median.
*P < 0.05.
Mol. Cells 2023; 46(6): 360-373
Published online June 30, 2023 https://doi.org/10.14348/molcells.2022.2242
Copyright © The Korean Society for Molecular and Cellular Biology.
Yuan Shao1,4 , Wanli Ren1,4
, Hao Dai1
, Fangli Yang1
, Xiang Li1
, Shaoqiang Zhang1
, Junsong Liu1
, Xiaobao Yao1
, Qian Zhao1
, Xin Sun2
, Zhiwei Zheng3
, and Chongwen Xu1,*
1Department of Otorhinolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Xi’An Jiaotong University, Xi’an, China, 2Department of Thoracic Surgery, The First Affiliated Hospital of Xi’An Jiaotong University, Xi’an, China, 3The Third Ward of General Surgery Department, Rizhao People’s Hospital, Rizhao, China, 4These authors contributed equally to this work.
Correspondence to:henao22@xjtu.edu.cn
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/.
Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid carcinoma. Despite a good prognosis, approximately a quarter of PTC patients are likely to relapse. Previous reports suggest an association between S-phase kinase-associated protein 2 (SKP2) and the prognosis of thyroid cancer. SKP1 is related to apoptosis of PTC cells; however, its role in PTC remains largely elusive. This study aimed to understand the expression and molecular mechanism of SKP2 in PTC. SKP2 expression was upregulated in PTC tissues and closely associated with clinical diagnosis. In vitro and in vivo knockdown of SKP2 expression in PTC cells suppressed cell growth and proliferation and induced apoptosis. SKP2 depletion promoted cell autophagy under glucose deprivation. SKP2 interacted with PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1), triggering its degradation by ubiquitination. Furthermore, SKP2 activates the AKT-related pathways via PHLPP1, which leads to the cytoplasmic translocation of SKP2, indicating a reciprocal regulation between SKP2 and AKT. In conclusion, the upregulation of SKP2 leads to PTC proliferation and survival, and the regulatory network among SKP2, PHLPP1, and AKT provides novel insight into the molecular basis of SKP2 in tumor progression.
Keywords: AKT, molecular mechanism, papillary thyroid carcinoma, PH domain leucine-rich repeat protein phosphatase-1, S-phase kinase-associated protein 2
Thyroid cancer is the most common cancer of the endocrine system. There are four subtypes: papillary thyroid carcinoma (PTC), follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer (Wang et al., 2015). PTC is the most common type, accounting for approximately 88% of all thyroid cancers (Aschebrook-Kilfoy et al., 2011). Reports suggest that the rise in PTC cases accounts for the increase in thyroid cancer cases in recent years (Cho et al., 2013; Elisei et al., 2010; McNally et al., 2012). PTC has a good prognosis after surgical treatment, with a 99% survival rate for a 20-year (Kakudo et al., 2004). Despite the high survival rate of PTC, the long-term follow-up shows a 25% high recurrence rate (Grogan et al., 2013). Surgical treatment and fear of recurrence are traumatizing and cause psychological distress and burden to PTC patients. Hence, understanding the underlying molecular mechanism of PTC development, progression, and recurrence will aid in enhancing treatment and preventing recurrences in PTC patients.
S-phase kinase-associated protein 2 (SKP2) is an F-Box protein (FBP) that contains at least one F-box domain. The F-box domain is a 50 amino acid protein motif that mediates the interaction between proteins. FBP is one of the components of the SCF (SKP1-cullin-F-box) ubiquitin-protein ligase complex (Bai et al., 1996), which plays an important role in signal transduction and cell cycle regulation (Craig and Tyers, 1999). SKP2 is a substrate recognition factor in the SCF complex (Nakayama and Nakayama, 2005). Recent studies have shown the role of SKP2 in various cancers, which makes SKP2 a potential therapeutic and drug target (Chan et al., 2010). Overexpression of SKP2 was reported in the initiation, growth, and metastasis of many cancers. Further
PTC tissues and their paired normal thyroid tissue (n = 98) were collected from PTC patients during surgical resection at The First Affiliated Hospital of Xi’An Jiaotong University. Written informed consent was obtained from all the participating patients. The patients who underwent chemotherapy or radiotherapy before the surgery were excluded from this study. All tissue samples were frozen in liquid nitrogen or paraffin-embedded immediately after surgical resection, and two independent pathologists evaluated the tissue biopsies. This study was approved by the institutional ethics committee of The First Affiliated Hospital of Xi’An Jiaotong University (No. 2021-1407). All the experiments were performed in accordance with the principles of the Declaration of Helsinki (2013).
Human embryonic kidney (HEK293T) cells were purchased from the National Infrastructure of Cell Line Resource (NICR, China), and Normal Human thyroid follicular epithelial cells (Nthy-ori 3-1) were purchased from American Type Culture Collection (ATCC, USA). Three human PTC cell lines, TPC-1, BCPAP, and IHH-4, were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Nthy-ori 3-1 cells and all three PTC cell lines were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). HEK293T cells were cultured in DMEM medium supplemented with 10% FBS.
Myc-tagged SKP2 and Flag-tagged PHLPP1 were cloned in pcDNA vectors using the Gateway recombination system (Invitrogen, USA).
Cell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Japan) method per the manufacturer’s protocol. Briefly, cells with the indicated treatment were seeded in 96-well plates at a density of 2 × 103 cells/well and cultured for 24, 48, and 72 h at 37°C. Further, 10 μl CCK-8 solution (Dojindo Molecular Technologies) was added to each well and incubated at 37°C for 2.5 h. The absorbance was measured at 450 nm wavelength using a microplate reader (Bio-Tek, USA).
For colony forming assay, cells in the logarithmic phase were digested using 0.25% trypsin. TPC-1 (1×103 cells) and IHH-4 (500 cells) cells were seeded in a 6-well plate and cultured for 10-14 days at 37°C. The colonies were fixed with methanol, stained with crystal violet, and manually counted.
5-ethynyl-2’-deoxyuridine (EdU) incorporation assay was used to study DNA synthesis. Cells were seeded in a 24-well plate, incubated with EdU solution at 37°C for 2 h, followed by incubation with Apollo 488 dye for another 30 min. The fluorescence was observed and captured using a fluorescence microscope (Nikon, Japan).
Cell apoptosis was measured using flow cytometry. Approximately 1 × 106 cells with the indicated treatment were collected and digested with trypsin and were stained using FITC Annexin V Apoptosis Detection Kit (BD Biosciences, USA) per the manufacturer’s instructions. After staining, the cells were sorted using a FACSCalibur flow cytometer (BD Biosciences). Apoptosis was analyzed using FlowJo software (TreeStar, USA).
BALB/c female nude pathogen-free mice (6-8 weeks old, 20-25 g) were purchased from Shanghai experimental animal center China. Mice were housed at 22°C ± 2°C in a light-dark cycle of 12 h. PTC cells (TPC-1 and IHH-4) transfected with
The animal study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Xi’An Jiaotong University prior to the experiments being conducted (No. 2020AM88). All the animal experiments were performed per the Guide for the Care and Use of Laboratory Animals.
Total RNA from the thyroid tissue or PTC cells was extracted using Trizol reagent (TaKaRa Bio, Japan) per the manufacturer’s protocol. One microgram RNA was used to carry out cDNA synthesis using ReverTra Ace qPCR-RT Master mix kit (Toyobo, Japan) according to the manufacturer’s protocol. PCR was performed using SYBR Green II kit (TaKaRa Bio). The PCR program was set as: denaturation at 94°C for 3 min, followed by 30 cycles including denaturation at 94°C for 30 s, annealing at 59.5°C for 30 s, and extension at 72°C for 60 s.The housekeeping gene
For protein extraction, ground tissue or cells were lysed using RIPA lysis buffer, and the debris was removed by centrifuge. The protein in the supernatant was collected and quantified using a bicinchoninic acid assay (BCA) kit (Beyotime, China). The extracted proteins were separated on 12% SDS-PAGE gels, the voltage was first 80 V for ~30 min and subsequent 120 V, for ~60 min, and then and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) (400 mA, 90 min). The membranes were blocked using skimmed milk in Tris-buffered saline Tween 20 (TBST) and probed using primary antibodies for 12 h at 4°C. After washing; the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for another 1 h at room temperature. Immunoreactive bands were detected using chemiluminescence detection kits (Millipore).
The primary antibodies used were as follows: SKP2 (ab183039, 1:500; Abcam, UK), β-actin (ab6276, 1:3,000; Abcam), LC3B (ab192890, 1:2,000; Abcam), p62 (#5114, 1:1,000; CST), PHLPP1 (ab135957, 1:1,000; Abcam), β-tubulin (#2146, 1:1,000; CST), AKT (#4691, 1:1,000; CST), phosphorylated-AKT (#4060, 1:2,000; CST), mTOR (#2972, 1:1,000; CST), phosphorylated-mTOR (#5536, 1:1,000; CST). Antibodies against HA tag (ab18181, 1:2,000; Abcam), Myc tag (ab9106, 1:1,000; Abcam), Flag tag (ab125243, 1:2,000; Abcam).
Four micrometers thick paraffin-embedded sections of human tissues or xenografts were prepared. The sections were dewaxed, incubated with citric buffer at 95°C for 10 min, and blocked using H2O2 at room temperature for 20 min. The sections were incubated with normal goat serum for another 30 min at room temperature, followed by overnight incubation at 37°C with primary antibodies (SKP2, 1:100, ab183039 [Abcam]; Ki-67, 1:150, ab16667 [Abcam]). The sections were washed using PBS and incubated with HRP-conjugated-secondary antibody. For peroxidase detection, the sections were stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) for approximately 15 s and counterstained with hematoxylin.
PTC cells after indicated treatment were washed using PBS and fixed with 4% polyformaldehyde (PFA) for 20 min. The excess PFA was removed by washing; the cells were permeabilized using 0.1% Triton X-100 for 10 min and incubated with 5% normal goat serum for 60 min to prevent nonspecific binding. Cells were probed with primary antibodies at 4°C overnight, followed by incubation for 1 h with fluorescent secondary antibodies (1:200, Alexa-488-conjugated goat anti-rabbit IgG, ab150077; Abcam). The cells were counterstained using 4’,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for 10 min at room temperature and visualized. The images were captured using a confocal microscope (Olympus, Japan). The primary antibodies used were anti-SKP2 (ab183039, 1:200; Abcam) and LC3B (ab192890, 1:100; Abcam).
Scrambled shRNA,
Data were represented as the mean ± SD. Statistical analysis was performed using IBM SPSS Statistics software (ver. 19.0; IBM, USA). Student’s
Expression of
To assess the effects of loss of
Additionally, a tumorigenicity assay performed in nude mice proved that knockdown of
These results suggested the oncogenic role of SKP2 in PTC cells as
Several recent studies show the role of SKP2 in autophagy. For instance, Gassen et al. (2019) demonstrated loss of
SKP2, an FBP, forms the SCF (Skp1-Cullin 1-F-box) complex to participate in the ubiquitination-mediated degradation of proteins (Wang et al., 2012b). Various proteins have been identified as the SKP2 substrate, which was degraded by ubiquitination, including p27 (Bochis et al., 2015), FOXO1 (Huang et al., 2005), PDCD4 (Li et al., 2019), etc. Previous studies have shown that β-TRCP (F-box family protein) degrades PHLPP1 via ubiquitination (Li et al., 2009). Therefore, the correlation between SKP2 and PHLPP1 was examined using HEK-293T cell overexpressing Myc-tagged SKP2 and Flag-tagged PHLPP1. Co-immunoprecipitation assay was performed to evaluate the interaction between SKP2 and PHLPP1. As shown in Fig. 4A, SKP2 interacted with PHLPP1. Immunoprecipitation on TPC-1 cell lysates and using SKP2 antibody revealed an endogenous interaction between SKP2 and PHLPP1 (Fig. 4B). Next, the effect of
The stability of PHLPP1 protein was investigated in the absence or presence of SKP2. PTC cells were incubated with a proteasome inhibitor (MG132), and the PHLPP1 expression was evaluated by western blot. As indicated in Fig. 5A, the PHLPP1 expression increased moderately over time and was independent of SKP2 expression, thus suggesting a proteasome-dependent degradation of PHLPP1. Next, the PTC cells were treated with a protein synthesis inhibitor (cycloheximide, CHX). A decrease in PHLPP1 expression was observed in cells treated with CHX. Similarly, in
PHLPP1 belongs to PH domain leucine‐rich repeat protein phosphatase family. Increasing evidence has suggested the role of PHLPP1 in various cancers (Wang et al., 2013). Multiple studies suggest that PHLPP1 regulates the AKT pathway by dephosphorylating AKT (Chen et al., 2011; Nitsche et al., 2012). Therefore, it is likely that SKP2 may regulate the AKT pathway via PHLPP1. Hence, AKT and mTOR phosphorylation was assessed in
The results indicate that the loss of SKP2 promotes autophagy without glucose. Hence, we next studied the mechanism associated with this phenomenon.
Similarly, an increase in expression of PHLPP1 was observed in
In this study, we show the overexpression of
SKP2 is widely known as the ubiquitin ligase subunit, involved in the substrate recognition by SCF complex and ubiquitin-dependent degradation of the proteins (Deshaies, 1999). Various proteins have been identified as the targets of SKP2, one of which is CDK inhibitor p27, which inhibits G1/S transition and promotes apoptosis (Yalcin et al., 2014). SCF-SKP2 complex recognizes and phosphorylated p27 at Thr-187 site (Bretones et al., 2011). Thus SKP2 triggered ubiquitin-dependent p27 degradation accelerates the cell cycle, leading to dysregulation in cell proliferation. SKP2 is also involved in post-transcriptional regulation of forkhead transcription factors; for instance, Akt-dependent phosphorylation of FOXO1at Ser-256 is mediated by SKP2 and degrades FOXO1 via ubiquitination (Huang et al., 2005). Further, FOXO3 undergoes deacetylation-dependent recognition by SKP2 and succedent ubiquitination (Wang et al., 2012a). Reports suggest SKP2 mediates ubiquitination and degradation of c-Myc. Meanwhile, SKP2 also acts as a transcriptional coactivator for c-Myc, which activates downstream genes (Kim et al., 2003; von der Lehr et al., 2003). This shows the dual role of SKP2 in c-Myc regulation, which suggests a peculiar feature of SKP2 in carcinogenesis. Interestingly, the role of SKP2-mediated ubiquitination extends beyond protein degradation. It also mediates YAP nuclear localization and Hippo-YAP pathway activation (Yao et al., 2018). Similarly, reports suggest SKP2 mediated polyubiquitination of LKB1 at K63 induced its binding to the STRAD-MO25 complex, which helps cell survival under stress (Lee et al., 2015). Interestingly, reports suggest membrane translocation of SKP2 activates AKT directly and leads to subsequent protumor signaling (Chan et al., 2012). Recent reports suggest PI3K independent activation of AKT, triggered by SKP2-mediated ubiquitination (Clement et al., 2018). Additionally, SKP2-mediated ubiquitination activates K63 which further interacts with HK2. Notably, the ubiquitination of AKT leads to mitochondrial translocation induced by EGF (Yu et al., 2019b). In this study, we demonstrated that SKP2 acts as a proto-oncogene in PTC and subsequently identified PHLPP1as a substrate for SKP2-mediated ubiquitination.
PHLPP1 is a serine/threonine phosphatase and plays a crucial role in tumor suppression by inactivating AKT. There are three AKT isoforms, AKT1, AKT2, and AKT3, encoded by different genes (Moc et al., 2015). Among these three isoforms, AKT2 and AKT3 are believed to be targets of PHLPP1, as PHLPP1 regulates dephosphorylation of AKT2 and AKT3, but not Akt1 (Dong et al., 2014). For instance, in pancreatic cancers, PHLPP1 selectively dephosphorylated and negatively correlated with AKT1 (Nitsche et al., 2012). Similarly, in metastatic melanoma, the selective regulation of AKT2 by PHLPP1 was shown (Yu et al., 2018). Despite multiple reports suggesting an indirect relationship between PHLPP1 and AKT2, few reports show a direct regulation between PHLPP1 and AKT1. A decrease in PHLPP1 expression increased the phosphorylation of AKT1 at P473 in lung cancer cells (Zhiqiang et al., 2012). Another study demonstrated that AKT1 binds to PHLPP1, which was verified by an increase in PHLPP1 and Scrib complex expression. This binding was not observed with other AKT isoforms (Li et al., 2011). Additionally, the blocking of the PHLPP1 catalytic site by a specific inhibitor promoted phosphorylation of AKT1 at Ser473 under serum starvation, suggesting the role of PHLPP1 in chaperone-mediated autophagy (Arias et al., 2015). These contradictory results indicate an alternative mechanism regulating the AKT pathway via PHLPP1. This study revealed that SKP2 indirectly regulated the AKT pathway triggered by ubiquitin-induced degradation of PHLPP1. However, our results indicate that
It is intriguing to note that the relationship between SKP2 and AKT is multi-directional. Previous studies propose a reciprocal regulation between SKP2 and AKT (Chan et al., 2012; Lin et al., 2009). Earlier studies show that AKT’s direct phosphorylation of SKP2 forms SCF complex and E3 ligase activity (Lin et al., 2009). Gao et al. (2009) have also described that AKT1, but not AKT2, phosphorylate SKP2 at Ser72. The Ser72 phosphorylation of SKP2 impedes its nuclear translocation and leads to its accumulation in the cytoplasm. Moreover, AKT1-mediated phosphorylation also endowed SKP2 resistance to degradation triggered by the APC-Cdh1 complex, enhanced SKP2 stability and cytoplasmic distribution. This mechanism may be responsible for the tumor progression (Gao et al., 2009). However, AKT1-mediated translocation of SKP2 to the cytoplasm raises questions about its role in cell cycle progression. The mechanism underlying the degradation of p27 via cytoplasmic SKP2 and subsequent dysregulates of the cell cycle remains elusive mainly (Zhang, 2010). Taking account of the proto-oncogenic role of SKP2, there is still much to be addressed.
Further, the association of AKT and SKP2 is established, where AKT regulates SKP2 activity indirectly. For instance, deactivating AKT suppressed SKP2 expression via FOXO3A, the latter consequently contributed to the expression of p27 by transcriptional regulation as well as repressing SKP2 (Li et al., 2018). Moreover, AKT regulated SKP2 expression at the translational level, along with mTORC1 and eIF4E (Nogueira et al., 2012). Based on these results, we conclude that survival stress induced a subcellular translocation of SKP2, but not its expression, which could be reversed by AKT inhibition, implying a possible role of AKT in SKP2 regulation.
However, the study has some limitations. The role of SKP2 as an oncogene in PTC was verified by downregulating the expression of
In summary, we identified a novel role of the SKP2/PHLPP1/AKT axis in PTC progression, including cell proliferation and survival under starvation. Further, activation of AKT was investigated to support the cytoplasmic accumulation of SKP2, as inhibition of AKT led to SKP2 nuclear location under glucose deprivation. The results added to the established understanding of SKP2 and would help to develop targeting therapy for PTC.
This work was supported by grants from the institutional Foundation of the First Affiliated Hospital of Xi’An Jiaotong University (No. 2019ZYTS-04 and No. 2019ZYTS-13), Xi’An Science and Technology Project (No. 2019114613YX001SF04(5)), Clinical Research Center for Thyroid Diseases of Shaanxi Province (No. 2017LCZX-03), the Clinical Research Award of the First Affiliated Hospital of Xi’An Jiaotong University (XJTU1AF-CRF-2020-020), The Basic Natural Science Research Program of Shaanxi Province (2021JQ-386 and 2021JQ-405), the Thyroid Research Project of Young and Middle-aged Physicians of Beijing Bethune Charitable Foundation (BQE-JZX-202103), the Key Research and Development Program of Shaanxi Province (2022SF-159) and the National Natural Science Foundation of China (No. 82103568).
Y.S., W.R., H.D., and C.X. designed the study, supervised the data collection, analyzed the data. F.Y., X.L., and S.Z. interpreted the data, and prepared the manuscript for publication. J.L., X.Y., Q.Z., X.S., and Z.Z. supervised the data collection, analyzed the data and reviewed the draft of the manuscript. All authors have read and approved the manuscript.
The authors have no potential conflicts of interest to disclose.
. Relationship between
Variable | |||
---|---|---|---|
Low expression | High expression | ||
Age (y) | |||
≤45 | 29 | 27 | 0.683 |
>45 | 20 | 22 | |
Sex | |||
Male | 24 | 23 | 0.840 |
Female | 25 | 26 | |
Tumor size (cm) | |||
≤1 | 30 | 15 | 0.002* |
>1 | 19 | 34 | |
TNM stage | |||
I/II | 45 | 35 | 0.009* |
III/IV | 4 | 14 | |
Extra thyroidal | |||
Negative | 33 | 29 | 0.402 |
Positive | 16 | 20 | |
Lymph node metastasis | |||
Negative | 36 | 26 | 0.036* |
Positive | 13 | 23 | |
Nodular goiter | |||
Negative | 42 | 37 | 0.201 |
Positive | 7 | 12 |
Values are presented as median..
*P < 0.05..
Sungjin Moon, and Yun Doo Chung
Mol. Cells 2013; 35(3): 261-268 https://doi.org/10.1007/s10059-013-0009-x