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. 2022 Apr 16;79(5):242. doi: 10.1007/s00018-022-04275-5

Molecular dissection on inhibition of Ras-induced cellular senescence by small t antigen of SV40

Dongsheng Shang 1, Tianchu Zhou 1, Xinying Zhuang 2, Yanfang Wu 1, Hanqing Liu 3,✉, Zhigang Tu 1,✉
PMCID: PMC11072472  PMID: 35429286

Abstract

Simian virus 40 (SV40) is a potentially oncogenic virus of monkey origin. Transmission, prevalence, and pathogenicity rates of SV40 are unclear, but infection can occur in humans, for example individuals with high contact with rhesus macaques and individuals that received contaminated early batches of polio vaccines in 1950–1963. In addition, several human polyomaviruses, proven carcinogenic, are also highly common in global populations. Cellular senescence is a major mechanism of cancer prevention in vivo. Hyperactivation of Ras usually induces cellular senescence rather than cell transformation. Previous studies suggest small t antigen (ST) of SV40 may interfere with cellular senescence induced by Ras. In the current study, ST was demonstrated to inhibit Ras-induced cellular senescence (RIS) and accumulation of DNA damage in Ras-activated cells. In addition, ST suppressed the signal transmission from BRaf to MEK and thus blocked the downstream transmission of the activated Ras signal. B56γ knockdown mimicked the inhibitory effects of ST overexpression on RIS. Furthermore, KSR1 knockdown inhibited Ras activation and the subsequent cellular senescence. Further mechanism studies indicated that the phosphorylation level of KSR1 rather than the levels of the total protein regulates the activation of Ras signaling pathway. In sum, ST inhibits the continuous hyperactivation of Ras signals by interfering with the normal functions of PP2A-B56γ of dephosphorylating KSR1, thus inhibiting the occurrence of cellular senescence. Although the roles of SV40 in human carcinogenesis are controversial so far, our study has shown that ST of polyomaviruses has tumorigenic potential by inhibiting oncogene-induced senescence (OIS) as a proof of concept.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-022-04275-5.

Keywords: Cellular senescence, Ras, Braf, Small T, PP2A-B56γ, KSR1

Introduction

Cellular senescence, a state of irreversible growth arrest of cells, was first formally described 6 decades ago [1]. Oncogene-induced senescence (OIS), by definition, is a type of premature cellular senescence caused by hyperactivation of oncogenes, such as H-RasG12V and BRafV600E [2, 3]. Remarkable progress has been made over the past 2 decades in understanding the causes and characteristics of OIS. Mounting evidence strongly supports OIS as a mechanism of tumor suppression in vivo [4, 5].

Simian virus 40 (SV40) is a potentially oncogenic virus originated in monkeys. Transmission, prevalence, and pathogenicity rates of SV40 are unclear, but infection can occur in humans, for example individuals with high contact with rhesus macaques. In addition, infection also occurred in individuals that received contaminated early batches of polio vaccines in 1950–1963. Besides that, DNAs of SV40 were detected in a few human tumors [6–8]; the human polyomaviruses, such as polyomavirus BK (BKPyV), JC (JCPyV), and MC (MCPyV) are highly common in global populations. For example, MCPyV has a very high seroprevalence at around 60–90% in adults according to different studies [9, 10]. Most people are subclinically infected with BKPyV in childhood, and thus are often established as a lifelong latent infection in the renourinary tract [11]. More importantly, MCPyV has been proven carcinogenic and associated with about 80% of Merkel cell carcinomas [12], while BKPyV infection is potentially associated with the occurrence of renourinary tumors [13].

As a well-known proto-oncogene, Ras (H-, K-, and N-Ras) mutations are identified in about 30% of human tumors. The incidence of H-Ras mutations in bladder cancers ranges from 10 to 30% according to different studies [14–16]. In addition, the mutation frequency of fibroblast growth factor receptor 3 (FGFR3), upstream of Ras, was as high as 54–64% [15, 16]. If both the mutations of FGFR3 and Ras were taken into account, activation of Ras signaling occurs in about 75% of bladder cancers. Interestingly, the infection of human polyomaviruses is widespread in the human population, and such viruses may become active during renourinary tumorigenesis [17]. For example, patients who developed BKPyV-associated diseases after kidney transplantation had a fourfold to 11-fold increased risk of bladder cancer compared to transplant recipients without BKPyV diseases [18, 19]. These coincidences led us to wonder whether hyperactivation of Ras and reactivation of human polyomavirus synergistically induce bladder cancer.

Although the roles of SV40 in human carcinogenesis remain controversial, people have used SV40 as a research tool for more than 30 years. The mechanistic studies have revealed that a number of crucial intracellular signaling pathways involving SV40 are associated with tumorigenesis [7]. SV40 induces mammalian cell transformation via the expression of large t (LT) and small t (ST) antigens [20–22], especially with H-Ras activation [23]. Based on these findings, we hypothesized that overexpression of ST may increase cell transformation through the inhibition of OIS driven by H-Ras. In this current study, we seek to test this hypothesis and try to explore the underlying mechanisms.

Materials and methods

Antibodies and other reagents

The antibodies used in the current study are listed in Table S1. The reagents used are as follows: MTT (3-[4]-2,5- diphenyltetrazolium bromide thiazolyl blue), X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), crystal violet, BrdU (5-bromo-2-deoxyuridine), DAPI (4′, 6-diamidine-2′-phenylindole dihydrochloride), puromycin, and G418 (Sigma-Aldrich, St. Louis, MO, USA); Lipofectamine 2000, Opti-MEM medium, and Halt Phosphatase Inhibitor Cocktail EDTA-free 100X (Thermo Fisher Scientific, Waltham, MA, USA); Recombinant EGF (PeproTech, Cranbury, NJ, USA); BCA protein assay kit (Beyotime Biotech., Shanghai, China). The other reagents were purchased from Sinopharm (Shanghai, China) unless otherwise specified.

Cell culture

IMR90 and Phoenix cells were generous gifts from Prof. Rugang Zhang at the Wistar Institute. IMR90 and 293FT cells were cultured as described previously [24]. Phoenix cells were cultured in DMEM, supplemented with 10% FBS, penicillin, and streptomycin (Thermo Fisher).

Plasmid construction, retrovirus and lentivirus infections

pBabe-puro, pBabe-puro-H-RasG12V, pBabe-puro-BRafV600E, pBabe-puro-MEKDD, pQCXIN, pQCXIN-neo-H-RasG12V, pQCXIN-neo-SV40 LT, pQCXIN-neo-SV40 ST, pLKO.1 and pcDNA3.1 were generous gifts from Prof. Rugang Zhang. pcDNA3.1 neo-KSR1, pBABE puro-PP2A-B56γ, and pBABE puro-KSR1 were built using standard protocols. shRNAs of B56γ and KSR1 in vector pLKO.1 were purchased from BGI (Shenzheng, Guangdong, China) and the sequences are listed in Table S2.

Phoenix cells were used to package the infectious retroviruses as described previously [25, 26]. Lentiviruses were produced and transducted as described previously [24]. 1 μg/ml of puromycin and/or 200 μg/ml of G418 were used following the routine protocol for screening.

Colony formation assays and growth curves

Colony formation assays were performed as previously described [25]. Equal numbers of cells (3000 cells for each well) after screening were seeded into six-well plates and cultured for 2 weeks. Then, the plates were stained with 0.05% crystal violet in PBS. The experiment was independently repeated at least three times, and the representative results are presented. Equal numbers of cells (5 × 104) after screening were plated and cultured in six-well plates. Then, the numbers of cells were counted at the indicated time points for plotting growth curves.

KSR1 rescue experiments

IMR90 cells were co-infected with lentivirus with shKSR1 plasmids which target 3´-noncoding regions or vectors and retrovirus with plasmids of H-RasG12V or vectors as indicated. After screening, electroporation was performed as previously described [27]. Briefly, cells (1.0 × 105 cells/well) were transfected via electroporation using Etta X-Porator H1 (Etta Biotech, Jiangsu, China) at a final amount of 1 μg of pcDNA3.1 or pcDNA3.1 neo-KSR1. The manufacturer’s protocol was followed and the electroporation program was optimized in our laboratory as: voltage at 250 V, pulse duration at 100 μs, pulse number at 6, interval at 1000 ms.

Immunofluorescence, SAHF, SA-β-gal, and BrdU staining

Cells after screening (50,000 cells/well) were seeded into 24-well flat-bottomed plates with coverslips and cultured for 1 day. Then, immunofluorescence, SAHF, SA-β-gal, and BrdU staining were performed as previously described [24, 28]. Images were captured using Nikon Eclipse microscope (Tokyo, Japan).

qRT-PCR

The qRT-PCR experiments were performed as previously described [24, 28] and the primers in qRT-PCR experiments are listed in Table S3. β-Actin was used as the internal control. Data shown are the mean values (± SD) from at least three independent experiments.

Immunoblotting

To extract total proteins in immunoblotting experiments, cells were lysed with lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1% Halt Phosphatase Inhibitor Cocktail EDTA-free 100X) for 10 min, and then the supernatants were collected after centrifugation at 10,000×g for 1 min at 4 ℃. To extract chromatin proteins, cells were first lysed with buffer A (10 mM HEPES–KOH (pH = 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100) for 5 min, and then the supernatant was removed after centrifugation at 1300×g for 5 min at 4 ℃. The nuclei pellets were washed once with buffer A and then lysed with buffer B (3 mM EDTA (pH = 8.0), 0.2 mM EGTA) for 30 min, and the supernatant was removed after centrifugation at 1700×g for 4 min at 4 ℃. The chromatin pellets were washed once with buffer B and then lysed with lysis buffer as mentioned above. Then, immunoblotting was performed following the routine protocols in our laboratory [24, 28]. β-Actin or Histone H3 was used as the internal controls as indicated.

Statistical analysis

Data are presented as mean ± S.D. and were analyzed for significance between groups using Student's t tests (two-tailed), unless otherwise specified. P < 0.05 was considered statistically significant (#P ≥ 0.005; *P < 0.05; **P < 0.001; ***P < 0.0001).

Results

ST antigen inhibits cellular senescence induced by Ras

To test our hypothesis, oncogenic H-Ras was co-expressed with SV40-ST in cells (Fig. 1A, B), while LT was used as a positive control since it had been proved to inhibit cellular senescence [29, 30]. As shown in Fig. 1C, D, both LT and ST significantly prevented growth arrest induced by Ras activation, manifested by the results of colony formation assays and growth curves of cells. At day 7, while only about 3% of Ras/vector cells (only H-Ras overexpressed) still proliferated as shown by BrdU incorporation assays, significantly larger percentages of cells with both overexpressed H-Ras and LT or ST proliferated (Fig. 1E, F). SAHF (senescence-associated heterochromatic foci) is one of the most prominent markers of cellular senescence. Consistently, while more than 40% of Ras/vector cells were positive in SAHF staining, significantly smaller portions of cells with both overexpressed H-Ras and LT or ST were SAHF positive (Fig. 1G, H). In addition, SA-β-gal staining also showed that LT and ST dramatically inhibited OIS induced by Ras (Fig. 1I, J). Because cell culture conditions, such as persistent stress, can often affect β-galactosidase activities in cells [31], we used the molecular algorithm based on the expression of Ki67, RPS6, and β-galactosidase activity to identify the status of the cells more accurately [32]. Consistently, the results showed that both SV40 LT and ST can significantly inhibit cellular senescence induced by Ras overexpression manifested by increased cycling cells and decreased SA-β-gal-positive senescent cells (Table S4). Based on the above results, the function of ST in inhibiting cellular senescence seems similar to that of LT.

Fig. 1.

Fig. 1

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ST antigen inhibits cellular senescence induced by Ras. A The scheme of general experiment settings. Here, IMR90 cells were infected with retrovirus with H-RasG12V plasmids or vectors together with plasmids of SV40 LT, ST antigens or vectors as indicated. Cells in which Ras, LT, and ST were successfully expressed were selected using puromycin or G418. B Protein levels of Ras, LT, and ST in co-expressed IMR90 cells. C Representative images of colony formation assays at the indicated conditions. Equal numbers of IMR90 cells (3 × 103 cells/well) after antibiotics screening were plated in six-well plates and cultured for 2 weeks, and the cells were stained with 0.05% crystal violet in PBS. D The growth curves of IMR90 cells in the indicated groups. Equal numbers of IMR90 cells (5 × 104 cells/well) after antibiotics screening were plated in six-well plates, and the number of cells was counted at the indicated time. Data shown are the mean values (± SD) from three independent experiments. E Representative images of BrdU staining. F Statistical analysis results of (E). Data shown are the mean values (± SD) from three independent experiments. Positive cells of BrdU staining have green fluorescence in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. G Representative images of DAPI staining. H Statistical analysis results of (G). Data shown are the mean values (± SD) from three independent experiments. Positive cells of SAHF formation have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. I Representative images of SA-β-gal staining. J Statistical analysis results of (I). Data shown are the mean values (± SD) from three independent experiments. The percentages of positive cells in five different fields from each well were counted and calculated for each condition. Statistically significant differences with P < 0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001)

ST antigen inhibits accumulation of DNA damage in Ras-activated cells

Since elevated DNA damage in cells is one of the key characteristics of OIS, the effects of ST overexpression on accumulation of DNA damage in Ras-activated cells was also tested. As shown in Fig. 2A–C, ST overexpression dramatically inhibited the accumulation of DNA damage in cells with Ras overexpression as manifested by decreased positive cells of γH2AX or 53BP1 foci. Consistent with the IF results, the protein levels of chromatin-bound γH2AX and 53BP1 decreased in ST overexpressed cells compared to Ras/vector cells (Fig. 2D). Previously, we reported that in Ras-activated cells, BRCA1 dissociates from chromatin while its cellular expression is unaffected, and this process may contribute to the accumulation of DNA damage in Ras-activated cells [25]. In this current study, it was found that ST overexpression blocked BRCA1 dissociation from chromatin which was induced by Ras activation (Fig. 2D, E). Interestingly, we also found that ST overexpression reduced the phosphorylation levels of Erk1/2, the downstream executor of Ras signals (Fig. 2E).

Fig. 2.

Fig. 2

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ST antigen inhibits accumulation of DNA-damage in Ras-activated cells. IMR90 cells were infected with retrovirus with H-RasG12V plasmids or vectors together with plasmids of SV40 ST or vectors as indicated. A Representative images of immunofluorescence staining of γH2AX and 53BP1. B, C Statistical analysis results of (A). Data shown are the mean values (± SD) from three independent experiments. Positive cells of γH2AX (B) or 53BP1 (C) staining have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. D Statistical quantification and representative immunoblotting images of BRCA1, 53BP1, and γH2AX in chromatin extracts, respectively. E Statistical quantification and representative immunoblotting images of BRCA1, p-Erk1/2, total-Erk1/2, ST, and Ras in whole lysates, respectively. For quantitative analysis, the histograms on the left of the dotted line uses the left Y axis, and the histograms on the right of the dotted line uses the right Y axis. Statistically significant differences with P < 0.05 were considered significant (#P ≥ 0.05; *P < 0.05; **P < 0.01)

ST antigen suppresses the signal transmission from BRaf to MEK

Classically, the activated Ras signals are transmitted downstream through the sequential phosphorylation of BRaf, MEK, and ERK1/2. In addition, overexpression of constitutively activated mutants of BRaf and MEK1, e.g., BRaf (V600E, BRafV600E) and MEK1 (S218D, S222D, MEKDD), have been demonstrated to induce cellular senescence [33–36]. Since ST can inhibit cellular senescence induced by Ras, we wondered whether ST can also affect the senescence induced by overexpression of BRafV600E or MEKDD.

It was found that ST inhibited cellular senescence induced by BRafV600E overexpression when BRafV600E and ST were overexpressed simultaneously (Fig. 3A), as manifested by the decreased SAHF formation and SA-β-gal activity (Fig. 3B, C) as well as the decreased protein levels of P16 and P21, the direct and critical regulators of cellular senescence (Fig. 3A). However, when MEKDD and ST were overexpressed concomitantly, we were surprised to find that ST cannot inhibit cellular senescence induced by MEKDD overexpression, since there were no significant changes in SAHF or SA-β-gal assays (Fig. 3D–F). More importantly, the results showed that ST overexpression inhibited phosphorylation of ERK caused by BRafV600E overexpression, but left phosphorylation of ERK unaltered after MEK overexpression (Fig. 3A, D). The above results indicated that ST inhibits the transmission of activated Ras signals by blocking the signal transmission from BRaf to MEK, therefore inhibiting the cellular senescence induced by Ras activation.

Fig. 3.

Fig. 3

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ST antigen suppresses the signal transmission from BRaf to MEK. IMR90 cells were infected with retrovirus with BRafV600E plasmids, MEKDD plasmids, or vectors together with plasmids of SV40 ST or vectors as indicated. A Statistical quantification and representative immunoblotting images of p-Erk1/2, total-Erk1/2, p21, p16, and BRaf. For quantitative analysis, the histograms on the left of the dotted line uses the left Y axis, and the histograms on the right of the dotted line uses the right Y axis. B Statistical analysis results of positive cells from DAPI staining assays in the indicated groups. Positive cells of SAHF formation have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. C Statistical analysis results of positive cells from SA-β-gal staining experiments in the indicated groups. The percentages of SA-β-gal-positive cells in five different fields from each well were counted for each condition. D Statistical quantification and representative immunoblotting images of p-Erk1/2, total-Erk1/2, and MEK1/2. For quantitative analysis, the histograms on the left of the dotted line uses the left Y axis, and the histograms on the right of the dotted line uses the right Y axis. E Statistical analysis results of positive cells from DAPI staining assays in the indicated groups. Positive cells of SAHF formation have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. F Statistical analysis results of positive cells from SA-β-gal staining experiments in the indicated groups. The percentages of SA-β-gal-positive cells in five different fields from each well were counted for each condition. Data shown are the mean values (± SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001; #P ≥ 0.05)

PP2A-B56γ is necessary in Ras-induced senescence, but is not capable of inducing senescence

The function of ST was largely attributed to its ability to bind serine/threonine-protein phosphatase 2A (PP2A) in cells [37–39]. PP2A is one of the major Ser/Thr phosphatases composed of three subunits and engages in many different cellular functions via the complex structure and regulatory mechanisms. The results of structural biology studies showed that ST plays a role by hindering the assembly of the B56 subunit in PP2A holoenzyme [40, 41]. More importantly, Chen et al. reported that the suppression of PP2A-B56γ imitated the function of ST to increase cell transformation driven by Ras activation [42]. Therefore, it is interesting to find out the roles of PP2A-B56γ in Ras-induced cellular senescence (RIS).

As shown in Fig. 4A–C, B56γ knockdown recovered the growth of cells arrested by Ras activation. In addition, both shRNA knockdown constructs of B56γ significantly inhibited SAHF formation and SA-β-gal activity in Ras-activated cells (Fig. 4D, E). More importantly, B56γ knockdown also hindered accumulation of DNA damage in Ras-activated cells (Fig. 4F–H) as manifested by the decreased percentages of positive cells with γH2AX and 53BP1 foci and decreased protein levels of chromatin-binding γH2AX and 53BP1. Probing the molecular mechanism, we found that B56γ knockdown reduced the protein levels of p21 and p16 (Fig. 4I). More importantly, it was demonstrated that the phosphorylation of ERK1/2 was also inhibited by B56γ knockdown (Fig. 4I). Our results showed that B56γ knockdown mimicked the inhibitory effects of ST overexpression on RIS. These results suggest that ST may inhibit RIS by interfering with certain functions of B56γ.

Fig. 4.

Fig. 4

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PP2A-B56γ knockdown impedes cellular senescence induced by Ras, but its overexpression is incapable of inducing senescence. IMR90 cells were co-infected with lentivirus with shB56γ plasmids or vectors and retrovirus with plasmids of H-RasG12V or vectors as indicated. A Representative immunoblotting results of Ras and PP2A-B56γ. B Representative images of colony formation assays at the indicated conditions. Equal numbers of IMR90 cells (3 × 103 cells/well) after antibiotics screening were plated in six-well plates and cultured for 2 weeks, and the cells were stained with 0.05% crystal violet in PBS. C Statistical analysis results of positive cells of IMR90 cells after BrdU staining. Data shown are the mean values (± SD) from three independent experiments. The percentages of positive cells of BrdU staining in five different fields from each well were counted and calculated for each condition. D Statistical analysis results of positive cells from SAHF formation assays. Positive cells of DAPI staining have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. E Statistical analysis results of positive cells from SA-β-gal staining. The percentages of positive cells of SA-β-gal staining in five different fields from each well were counted and calculated for each condition. F, G Statistical analysis results of positive cells of γH2AX (F) or 53BP1 (G) in immunofluorescence experiments. γH2AX or 53BP1 positive cells have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. H Representative immunoblotting images of 53BP1 and γH2AX in chromatin proteins. I Representative immunoblotting images of p-Erk1/2, total-Erk1/2, p21, and p16 in whole cell lysates. J Statistical quantification and representative immunoblotting images of p-Erk1/2, total-Erk1/2, PP2A-B56γ, and Ras in whole cell lysates. For quantitative analysis, the histograms on the left of the dotted line uses the left Y axis, and the histograms on the right of the dotted line uses the right Y axis. IMR90 cells were infected with retrovirus with H-RasG12V, B56γ, or vectors. K Statistical analysis results of positive cells from SAHF formation assays. Positive cells of DAPI staining have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. L Statistical analysis results of positive cells from SA-β-gal staining. The percentages of positive cells of SA-β-gal staining in five different fields from each well were counted and calculated for each condition. Data shown are the mean values (± SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001; #P ≥ 0.05)

Since B56γ knockdown can attenuate RIS, whether B56γ overexpression is sufficient to induce cellular senescence appears intriguing. To ensure satisfactory experimental setup, Ras was used as a positive control (Fig. 4J). Unexpectedly, B56γ overexpression increased neither SAHF formation nor SA-β-gal activity of the cells (Fig. 4K, L). In addition, B56γ overexpression could not increase the phosphorylation level of ERK1/2 (Fig. 4J). Therefore, B56γ overexpression can neither activate the Ras signaling pathway nor induce cellular senescence.

KSR1 is necessary in Ras-induced senescence, but not sufficient to induce senescence

The above results indicated that ST blocked the signal transmission from BRaf to MEK, and meanwhile PP2A-B56γ participated in this process. After careful literature search and years of experimental trials, KSR1 was selected as a candidate for further studies. As a scaffold protein, KSR1 promotes Raf-mediated phosphorylation of MEK and is required in optimal Ras-mediated ERK activation [43–45]. While the KSR1/MEK interaction is constitutive [46], the KSR1/Raf interaction is dynamic [47]. Activated ERK disrupts KSR1 scaffold complexes via negative feedback phosphorylation on KSR1 and Raf [48]. Studies have shown that the phosphorylation of KSR1 hinders its binding to Raf [49, 50]; therefore, KSR1 is crucial for sustained signal activation of the Raf–MEK–ERK pathway [49, 51]. On the other hand, PP2A is responsible for dephosphorylation of KSR1 [50]. Based on these literatures, we speculated that ST regulates the phosphorylation of KSR1 by inhibiting PP2A, then interferes with the formation of the BRaf–KSR1–MEK complex, and finally hinders the signal transmission from BRaf to MEK.

As shown in Fig. 5A, we engineered KSR1 knockdown constructs while Ras was overexpressed. As expected, KSR1 knockdown decreased SAHF formation, SA-β-gal activity and the protein levels of P21 and P16 when Ras was over-expressed (Fig. 5B–D). At the same time, KSR1 knockdown reduced the levels of intracellular DNA damage caused by Ras activation, as shown by the decreased positive cells of γH2AX or 53BP1 foci (Fig. 5E, F). In addition, KSR1 knockdown significantly reduced ERK phosphorylation caused by Ras activation (Fig. 5D). These results suggest that KSR1 knockdown may achieve these effects by impeding the downstream transmission of Ras activation signals.

Fig. 5.

Fig. 5

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KSR1 is necessary in Ras-induced senescence, but overexpression of KSR1 is not sufficient to induce senescence. IMR90 cells were co-infected with lentivirus with shKSR1 plasmids or vectors and retrovirus with plasmids of H-RasG12V or vectors as indicated. A Representative immunoblotting results of Ras and KSR1. B Statistical analysis results of positive cells from SAHF formation assays. Positive cells of DAPI staining have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. C Statistical analysis results of positive cells from SA-β-gal staining. The percentages of positive cells of SA-β-gal staining in five different fields from each well were counted and calculated for each condition. D Representative immunoblotting images of p-Erk1/2, total-Erk1/2, p21 and p16. E, F Statistical analysis results of positive cells of γH2AX (E) or 53BP1 (F) in immunofluorescence experiments. γH2AX or 53BP1-positive cells have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. G Representative immunoblotting images of p-Erk1/2, total-Erk1/2, and KSR1 in whole lysates. IMR90 cells were infected with retrovirus with KSR1 or vectors. H Statistical analysis results of positive cells from SAHF formation assays. Positive cells of DAPI staining have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. I Statistical analysis results of positive cells from SA-β-gal staining. The percentages of positive cells of SA-β-gal staining in five different fields from each well were counted and calculated for each condition. J The scheme of the experiment setting. IMR90 cells were co-infected with lentivirus with shKSR1 plasmid (targeting 3´-noncoding region) or vectors and retrovirus with plasmids of H-RasG12V or vectors as indicated. After drug-screening, KSR1 rescue is achieved by electroporation using plasmid coding wt-KSR1. K Representative immunoblotting images of p-Erk1/2, total-Erk1/2, p21, p16, KSR1, and Ras. L–M Statistical analysis results of positive cells from SAHF formation assays and SA-β-gal staining. Positive cells of DAPI staining have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. Data shown are the mean values (± SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001; #P ≥ 0.05)

Then the effects of KSR1 overexpression on cells were also examined. Again, overexpression of KSR1 cannot induce cellular senescence (Fig. 5G–I). To further verify our hypothesis, we performed KSR1 rescue experiments (Fig. 5J). As expected, KSR1 knockdown inhibited cellular senescence induced by Ras overexpression, while expression of KSR1 restored the function of Ras (Fig. 5K–M).

Phosphorylation of KSR1 regulates Ras-induced cellular senescence

To further explore the molecular mechanism, we screened PP2A-B56 family genes by detecting their mRNA levels. The data showed that only the mRNA level of B56γ significantly increased after Ras activation (Fig. 6A). Consistently, the protein level of B56γ also increased mildly (Fig. 6B). As expected, while the total amounts of KSR1 did not change, the protein level of phosphorylated KSR1 decreased (Fig. 6B). These results indicated that the amount of dephosphorylated KSR1, which ensures activated Ras signal continuously transmitted downstream, increased after Ras activation. Consistently, when B56γ was overexpressed, KSR1 was also significantly dephosphorylated (Fig. 6C).

Fig. 6.

Fig. 6

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Phosphorylation of KSR1 controls Ras-induced cellular senescence. A mRNA levels of B56α, β, γ, δ, and ε by qRT-PCR with or without Ras overexpression. IMR90 cells were infected with retrovirus with plasmids of H-RasG12V or vectors as indicated. B Statistical quantification and representative immunoblotting images of p-KSR1, KSR1, B56γ, and Ras with or without Ras overexpression using the cells from (A). C Statistical quantification and representative immunoblotting images of p-KSR1, KSR1, and B56γ with or without B56γ overexpression. D Statistical analysis results of SAHF formation with B56γ overexpression and treatment with 100 nM EGF as indicated. Positive cells of SAHF formation have > 5 foci in the nuclei, and the percentages of positive cells in five different fields from each well were counted and calculated for each condition. E Statistical analysis results of SA-β-gal staining with B56γ overexpression and/or treatment with 100 nM EGF as indicated. The percentage of positive cells in five different fields from each well were counted and calculated for each condition. F Statistical quantification and representative immunoblotting images of p-KSR1 and KSR1 at the indicated conditions. IMR90 cells were infected with lentivirus with shB56γ plasmids or vectors and later infected with retrovirus with plasmids of H-RasG12V or the vectors as indicated. Data shown are the mean values (± SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001; #P ≥ 0.05)

In previous studies, we found that overexpression of both B56γ and KSR1 could not induce cellular senescence. However, we speculated that this is due to the lack of initial mitogen activation in the whole signal system, although the dephosphorylation of KSR1 will facilitate the transmission and amplification of the signal system. In a recent study, we showed that EGF stimulation can transiently but potently activate Ras and induce cellular senescence to a certain degree [24]. Here, we used EGF to treat cells with or without B56γ overexpression. Excitingly, we found that the cells with B56γ overexpression were more sensitive to EGF treatment (Fig. 6D, E). This result suggested that when Ras is activated, the dephosphorylation of KSR1 can indeed amplify its effects. At the same time, it was also found that B56γ knockdown can increase the phosphorylation of KSR1 when Ras was activated (Fig. 6F). That B56γ knockdown inhibits Ras activation and RIS indicates that phosphorylated KSR1 inhibits the sustained activation of Ras.

Discussion

In this study, we first found that ST overexpression can effectively inhibit cellular senescence and the accumulation of DNA damage induced by Ras activation (Figs. 1 and 2). Then, we found that ST overexpression affected cellular senescence caused by BRaf overexpression, while it did not significantly alter cellular senescence induced by MEK overexpression (Fig. 3). These results suggest that ST blocks the downstream transmission of activated Ras signals between BRaf and MEK. In addition, we found that knockdown of PP2A-B56γ well imitated the functions of ST overexpression. Since KSR1 is in charge of transmitting the activation signals of Ras downstream and also regulated by B56γ, we then further explored the molecular mechanism involving KSR1. Indeed, we found that KSR1 knockdown inhibited the downstream transmission of Ras activation signal and the cellular senescence.

But on the other hand, we found that neither overexpression of B56γ nor that of KSR1 induced cell senescence (Figs. 4 and 5). Studies have shown that the activation of the Raf–MEK–ERK signaling pathways receives negative feedback from the downstream molecule ERK. Specifically, activated ERK phosphorylates Raf and KSR1, thereby leading to the dissociation of the Raf–KSR1 complex. Since PP2A can dephosphorylate KSR1 and promote the formation of the Raf–KSR1 complex [50], ST can thus indirectly inhibit the sequential activation of the Ras–Raf–MEK–ERK pathway by inhibiting the functions of PP2A (Fig. 7). This model can rationally explain our experimental results, and also explains why overexpression of neither B56γ nor KSR1 can induce cellular senescence. Because KSR1 acts on the negative feedback mechanism mediated by ERK, the whole system is in a resting status in the absence of activation signals in the upstream, such as overexpression of RasG12V and BRafV600E. Indeed, the cells with more dephosphorylated KSR1 (caused by B56γ overexpression) were more sensitive to EGF treatment, which can transiently but potently activate Ras (Fig. 6). So far, there are only two publications from the same group studying the roles of KSR1 in cellular senescence [52, 53]. Similarly, these authors reported that KSR1 is necessary for RasG12V-induced senescence in primary mouse embryonic fibroblasts. But unfortunately, the authors did not study the effects of KSR1 overexpression on cellular senescence in either paper; therefore, it is impossible to compare the current results to those from these two papers.

Fig. 7.

Fig. 7

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Schematic illustration of SV40-ST inhibiting Ras-induced cellular senescence by interfering PP2A–B56γ–KSR1 signaling pathway

It is interesting that BRaf is phosphorylated by activated ERK and the phosphorylation state of BRaf, which is also regulated by PP2A [54], affects its binding to KSR1 [47]. Our current study does not address the effects of inhibitory phosphorylation of BRaf on RIS. This leaves us interesting questions, which are what mechanism mediated by PP2A affects the phosphorylation of BRaf and how this mechanism affects RIS, for the future.

The roles of PP2A as a tumor suppressor in cancers have attracted great attention. However, the correlation between PP2A and cellular senescence, a major mechanism of cancer prevention in vivo, has been rarely studied except that Mannava et al. studied the roles of PP2A–B56α in OIS in normal and tumor human melanocytic cells [55]. They actually found overexpression of B56α induced cellular senescence, while knockdown of B56α suppressed OIS to various degrees. The authors further demonstrated that these results are driven by inhibition of B56α on c-myc expression. Here, we studied B56γ rather than B56α. Although they belong to the same protein family, these two proteins have great differences in sequences, structures, and functions. In addition, we used IMR90 cells rather than melanocytic cells, and the histological differences between these two types of cells further prevented us from carefully comparing our results with those from the above paper.

The studies on the ST functions in cancers mainly focused on its roles in cell transformation [42, 56, 57]. Until recently, there has been only one paper discussing the roles of ST in cellular senescence. In this study, Oshikawa et al. reported that ST, cooperating with LT, caused cells to escape from OIS by inhibiting SASP [58]. However, the authors claimed that suppression of SASP by ST was independent of PP2A, but associated with HP1BP3. The viewpoint of this paper is obviously quite different from ours. Firstly, ST alone can inhibit OIS in our experiment; secondly, ST works via its inhibition on PP2A in our study.

Despite the controversy, the possibility of ST and its human homologous viruses (such as BKPyV and MCPyV) to be carcinogenic is still worrisome due to their prevalence [59, 60]. In 2014, the International Agency for Research on Cancer judged MCPyV to be a probable human carcinogen. BKPyV was also ruled a possible carcinogen. Specifically, patients with BKPyV viremia or polyomavirus associated nephropathy after transplantation have a higher risk (increased by 4–11 times) to develop bladder cancer in contrast to the transplant recipients without BKPyV diseases [17, 61]. On the other hand, the mutations or overexpression of H-Ras and its upstream regulators (such as FGFR3) are highly prevalent in bladder cancer [62, 63]. The co-activation of the Ras signaling pathway and BKPyV in bladder cancer makes us reason that BKPyV may inhibit cellular senescence induced by Ras activation, thereby promoting tumorigenesis. It is known that mild Ras activation will not cause cellular senescence while causing cell proliferation [24]. The mechanism proposed by us may provide a point of view for the study of the above questions.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 58 kb) (58.2KB, pdf)
Supplementary file2 (PDF 39 kb) (39.3KB, pdf)
Supplementary file3 (PDF 50 kb) (50.6KB, pdf)
Supplementary file4 (PDF 99 kb) (99.2KB, pdf)

Acknowledgements

We would like to thank Prof. Rugang Zhang at the Wistar Institute for his great help and suggestions.

Author contributions

Project administration, supervision, funding acquisition and provision, and writing the original draft were accomplished by ZT and HL. Conceptualization and methodology were accomplished by ZT, HL, and XZ. DS, TZ, and YW were responsible for investigation, data curation, and analysis.

Funding

This work was supported by the National Natural Science Foundation 31771521 (to Z. Tu) and 81672582 (to H. Liu); Top Talent of Innovative Research Team of Jiangsu Province (to H. Liu and Z. Tu); Senior Talent Start-up Funds of Jiangsu University 14JDG050 (to H. Liu) and 14JDG011 (to Z. Tu).

Availability of data and material

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Declarations

Conflict of interests

The authors have no conflict of interest to disclose about this study.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Footnotes

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Contributor Information

Hanqing Liu, Email: hanqing@ujs.edu.cn.

Zhigang Tu, Email: zhigangtu@ujs.edu.cn.

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Supplementary Materials

Supplementary file1 (PDF 58 kb) (58.2KB, pdf)
Supplementary file2 (PDF 39 kb) (39.3KB, pdf)
Supplementary file3 (PDF 50 kb) (50.6KB, pdf)
Supplementary file4 (PDF 99 kb) (99.2KB, pdf)

Data Availability Statement

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