Inhibition of EGFR signaling with Spautin-1 represents a novel therapeutics for prostate cancer
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Prostate cancer (PCa) remains a challenge worldwide. Due to the development of castration-resistance, traditional first-line androgen deprivation therapy (ADT) became powerlessness. Epidermal growth factor receptor (EGFR) is a well characterized therapeutic target to treat colorectal carcinoma and non-small cell lung cancer. Increasing studies have unraveled the significance of EGFR and its downstream signaling in the progression of castration-resistant PCa.
MTS, colony formation and Edu staining assays were used to analyze the cell proliferation of PCa cells. Flow cytometry was used to analyze PCa cell cycle distribution and cell apoptosis. Western blot was used to measure the expression of key proteins associated with cell cycle progression, apoptosis and EGFR signaling pathways. Transfection of exogenous small interfering RNA (siRNA) or plasmid was used to intervene specific gene expression. Nude mouse model was employed to test the in vivo effect of Spautin-1.
The current study reveals that Spautin-1, a known inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13, inhibits EGFR phosphorylation and the activation of its downstream signaling. Inhibition of EGFR signaling induced by Spautin-1 leads to cell cycle arrest and apoptosis of PCa in a USP10/USP13 independent manner. The application of Spautin-1 reduces the expression of glucose transporter 1 (Glut1) and dramatically induces cell death under glucose deprivation condition. In vivo experiments show a potent anti-tumor effect of Spautin-1 alone and in combination with Enzalutamide.
This study demonstrates the therapeutic potential of EGFR signaling inhibition by the use of Spautin-1 for PCa treatment.
KeywordsProstate cancer EGFR Spautin-1 Glut1 Apoptosis
Androgen deprivation therapy
Epidermal growth factor receptor
Glucose transporter 1
c-Jun N-terminal kinase
Mitogen-activated protein kinase
Ubiquitin-specific peptidase 10
Prostate cancer (PCa) is the second most frequently diagnosed carcinoma among males with a high fatality rate worldwide . Although the application of androgen deprivation therapy (ADT) makes a great achievement in PCa treatment, many patients are not sensitive to this treatment or inevitably progress to the castration-resistant state, which renders PCa incurable even to the present time [2, 3]. Therefore, identifying novel therapeutics for patients with ADT-insensitive PCa or castration-resistant PCa (CRPC) warranted.
Epidermal growth factor receptor (EGFR) is crucial for the development and proliferation of multiple cancers, including colorectal carcinoma, non-small cell lung cancer, and PCa [4, 5, 6]. Hence, EGFR stands out as a target to treat these cancers. Recent studies have also been revealed that aberrant activity of EGFR may drive the development of CRPC, possibly due to the deprivation of androgen signaling [7, 8]. EGFR is a well-documented member of the transmembrane receptor tyrosine kinase family . The binding of ligands, such as EGF, TNFα, etc. leads to the transphosphorylation of EGFRs, which provides a docking site for SRC homology 2 (SH2) domain-containing signaling proteins . Activation of these molecules subsequently regulate their downstream effectors, including mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K)/−AKT pathways [11, 12], and thereby mediating multiple physiological and pathological processes, including cell cycle progression and cell survival and invasion that promote the development of multiple cancers [13, 14, 15].
Spautin-1 acts as an inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13 via promoting the degradation of Vps34, and has been documented as an autophagy inhibitor . Subsequent studies showed that Spautin-1 enhances the anticancer activity of targeted-therapy or traditional chemotherapy via autophagy inhibition in several cancers [17, 18, 19]. Recent study also showed that Spautin-1 activates c-Jun N-terminal kinase (JNK) and thereby inducing immunogenic cell death . These findings provided alternative evidence that Spautin-1 may serve as a novel agent with potential therapeutic implication for certain cancers. However, whether Spautin-1 could be used as a therapeutic agent to treat PCa or not and its working mechanism remain unknown.
In the current study, we report that Spautin-1 significantly suppressed the growth of PCa by arresting cell cycle progression and triggering apoptosis. Mechanistically, Spautin-1 inhibits EGFR and its downstream signaling pathways, leading to activation of the MKK4/JNK/Bax axis and inactivation of the MEK1/2/ERK/Cyclin D1 axis. The downregulation of Glut1 induced by Spautin-1 notably increased cell death in glucose deprivation condition. Moreover, the application of Spautin-1 surprisingly enhanced the anti-cancer activity of Enzalutamide both in vitro and in vivo. Together, these findings reveal that Spautin-1 could be developed as a clinically available compound against CRPC via inhibition of EGFR signaling pathways.
Spautin-1(S7888), 3-methyladanine (3-MA, S2767), Z-VAD-FMK (S7023), SP600125 (S1460), SB230580 (S1076), LY3214996 (S8534), Gefitinib (S1025) and Enzalutamide (MDV3100, S1250) were purchased from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was obtained from Xcessbio Biosciences, Inc. (San Diego, CA). USP10 (sc-76,811), USP13 (sc-76,815) and Glut1 (sc-35,493) siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies: anti-Glut1 (ab652), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630), anti-Ki67 (BS1454) (Bioworld Technology, Inc., Louis Park, MN, USA); anti-USP10(#8501), anti-SKP2(#2652), anti-p27(#3686), anti-CDK4(#12790), anti-CDK2(#2546), anti-CyclinD1(#2978), anti-p15(#4822), anti-p21(#2947), anti-PARP(#9532), anti-Bim(#2933), anti-Bax(#14796), anti-Bcl-2(#15071), anti-activated Caspase-3(#9664), anti-phospho-JNK (#9255), anti-JNK(#9252), anti-phospho-ERK1/2(#4370), anti-ERK1/2(#4695), anti-phospho-p38(#4511), anti-p38(#8690), anti-phospho-MKK4(#4514), anti-MKK4(#9152), anti-phospho-MEK1/2 (#9154), anti-MEK1/2(#4694), anti-phospho-EGFR (Tyr1173)(#4407), anti-phospho-EGFR (Tyr1068)(#3777), anti-EGFR(#2085) (Cell Signaling Technology, Beverly, MA, USA).
The following cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA): WPMY-1, 22Rv1, LNCaP, PC3, C4–2 and DU145. WPMY-1 cells were grown in DMEM supplemented with 10% FBS. 22Rv1, LNCaP and C4–2 cells were grown in RPMI 1640 supplemented with 10% FBS. PC3 and DU145 cells were grown in Hyclone DMEM/F-12 supplemented with 10% FBS. Cultured cells were maintained at 37 °C and 5% CO2.
Cell viability assay
MTS (catalog no. G111) was purchased from Promega Corporation (Madison, WI, USA) and used to test cell viability as we reported . Briefly, exponentially growing PCa cells were seeded at 2000 cells/well in a 96-well plate. After incubation for 24 h, cells were treated with increasing doses of Spautin-1 for 24, 48 or 72 h. 20 μl MTS reagent was directly added to each well and cells were incubated for additional 3 h. The absorbance of optical density (OD) was measured with a microplate reader (Sunrise, Tecan, Mannedorf, Switzerland) at wavelength 490 nm.
This assay was performed as previously described [22, 23]. PCa cells were exposed to Spautin-1 at 10 μM or control solvent for 24 h. Cells post Spautin-1 or control solvent treatment were then digested, resuspended and seeded in 60 mm dishes supplemented with 10% FBS medium. Cells were then cultured in an atmosphere of 5% CO2 for 2 weeks. For crystal violet staining of colonies, cells were fixed with 4% paraformaldehyde for 15 min, then washed with PBS twice, followed by crystal violet solution incubation for 5 min. Colonies > 60 μm were counted from three independent experiments.
Cell cycle and proliferation assay
For cell cycle assay, 22Rv1, PC3 and LNCaP cells treated with Spautin-1 or SKP2-C25 treatment for 24 h were digested, harvested and washed with cold PBS twice. After discarding supernatant, cells were resuspended with 500 μl PBS and 2 ml 70% ethanol at 4 °C overnight. And then the cells were washed with 4 °C PBS twice again, followed by incubation with PI (50 μg/ml) (Keygen, Nanjing, China), RNase A (100 μg/ml) and 0.2% Triton X-100 mixtures for 30 min at 4 °C in dark. Cell cycle distributions of each group were ultimately analyzed with flow cytometry. For cell proliferation assay, PC3 cells post Spautin-1, SKP2-C25, 3-MA, USP10 siRNAs or USP13 siRNAs treatment were incubated with EdU (5-ethynyl-2′-deoxyuridine, Ribobio, Guangzhou, China) at 50 μM for 2 h. Apollo was used to probe EdU. DAPI was used to indicate the nucleus. Nuclear EdU level indicates the activity of DNA replication and cell proliferation.
Cell death assay
Analysis of cell death was performed as we described [24, 25]. Briefly, PCa cells post Spautin-1, USP10 KD or USP13KD treatment were digested, collected, and washed with cold PBS for three times. After discarding supernatant, cells were resuspended with 500 μl annexin V-FITC binding buffer, 5 μl annexin V-FITC, and 5 μl PI mixture (Keygen, Nanjing, China) in each group. Cells were then incubated with annexin V-FITC/PI mixture for 30 min, followed by the analysis of stained cells using flow cytometry.
SiRNA and plasmid transfection
Transfection of nucleic acids was performed as we reported [22, 24]. PCa cells were randomly seeded in 60 mm dishes or 96-well plates for 24 h. For siRNAs transfection, RPMI opti-MEM (Gibco), lipofectamine RNAiMax (Invitrogen) reagent and siRNAs (Santa Cruz, CA) targeting human USP10/USP13/Glut1 siRNAs (Santa Cruz, CA), or control siRNAs (non-specific sequences) mixtures were prepared, respectively. After incubation for 15 min, the mixtures was added in each group. Cells were cultured for 48 h and 72 h for further analysis. For plasmid transfection, RPMI opti-MEM (Gibco), lipofectamine 2000 (Invitrogen) reagent and eukaryotic expression vector pcDNA3.1(+)-SKP2-HA (NM005983), pENTER-CDK2-HA (NM_001798.4), pENTER-CyclinD1-FLAG (NM_053056.2), pEGFP-N1-Glut1-HA (NM_006516) plasmids or their control vector mixtures were prepared respectively. After incubation for 15 min, the mixtures was added in each group and cultured for 48 h and 72 h for further analysis. Fresh medium was replaced appropriately after transfection for 6 h.
Western blot analysis was performed as we described [26, 27]. In brief, equal amounts of total proteins extracted from PCa cells exposed to Spautin-1 or USP10/USP13/Glut1 siRNAs were fractionated using 12% SDS–PAGE. The separated proteins on SDS–PAGE were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% milk for 1 h. Primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were each incubated for 1 h. The bounded secondary antibodies were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan).
This assay was performed as we described [28, 29]. Briefly, PCa cells post Spautin-1 treatment were fixed with 4% paraformaldehyde for 15 min. 0.5% Triton-X was used to permeabilize cells for 5 min. 5% BSA (bovine serum albumin, Sigma) was used to block for 30 min. And then the cells were incubated with Phospho-JNK, Phospho-ERK or Glut1 primary antibodies overnight at 4 °C. Next the cells were incubated with anti-mouse IgG H&L (Alexa Fluor 488, Abcam) or anti-rabbit IgG-cy3 (Bioworld) secondary antibodies for 1 h. DAPI (4′,6-diamidino-2-phenylindole, Abcam) was used to indicate the nucleus. Images were acquired using an Olympus fluorescence microscope with 400× magnification.
Nude mouse xenograft model
Nude Balb/c mice were bred and housed at the animal center of Guangzhou Medical University in accordance with ethical treatment of animals. The xenograft models were prepared as previously reported [30, 31]. Briefly, 2 × 106 22Rv1 or PC3 cells were inoculated subcutaneously on the flanks of 5- to 6-week-old male nude mice. After inoculation for 3 days, the mice successfully bearing xenografts were bred and treated with either Spautin-1 (20 mg/kg/day, i.p.), Enzalutamide (25 mg/kg/day, p.o.) or vehicle for a total of 30 days. The size of xenografts was measured and calculated as previously reported .
Xenografts were fixed with formalin and sectioned according to standard techniques. MaxVision kit (Maixin Biol) was used to immunostain xenograft sections (4 μm) according to a previous report . Primary antibodies were against Ki67. MaxVisionTM reagents were added one by one on the slide in 50 μl according to the instruction. Color was developed with 0.05% DAB and 0.03% H2O2 in 50 mM Tris-HCl (pH 7.6), and the slides were counterstained with hematoxylin.
Data are presented as mean ± SD from three independent experiments where applicable. To determine statistical probabilities, unpaired Student’s t test or one way ANOVA is used where appropriate. Statistical analysis was performed by GraphPad Prism5.0 software (GraphPad Software) and SPSS 16.0. A P value of < 0.05 was considered statistically significant.
Spautin-1 suppressed the proliferation of PCa cells independent of autophagy inhibition and the USP10/USP13-SKP2-p27 axis
Spautin-1 arrested the G0/G1 to S phase transition via decreasing cyclin D1 expression
Spautin-1 in high dose induced caspases-dependent apoptosis of PCa cells
JNK and ERK mediated Spautin-1-induced growth inhibition
Spautin-1 inhibited EGFR activation of PCa cells
Spautin-1 inhibited cell survival in glucose deprivation condition via down-regulating Glut1
Spautin-1 suppressed the growth of PCa xenografts
Spautin-1 enhanced the sensitivity of PCa cells to enzalutamide in vitro and in vivo
Androgen receptor (AR) is the main driver of PCa development and progression. Androgen-AR binding triggers AR translocation from cytoplasm to the nucleus and activates the transcription of multiple androgen-responsive genes essential to supporting the proliferation and survival of PCa cells . Traditional ADT aims to block the androgen-AR binding by diminishing androgens and is initially effective in PCa treatment. However, castration-resistance developed over time in most cases impedes its further application [44, 45]. Several mechanisms, including EGFR, intratumoral androgens, AR overexpression or mutation and AR cofactors, were proposed to contribute to AR signaling maintenance in an androgen-poor condition [43, 45, 46]. Increasing evidence shows the significance of EGFR in the progression of CRPC [7, 8, 47, 48, 49]; thus, inhibition of EGFR may represent a novel therapy to overcome CRPC.
In the current study, we found that Spautin-1 remarkably suppressed the proliferation of PCa cells, regardless of AR expression. Spautin-1 was primarily reported as an selective inhibitor of USP10/USP13 as well as autophagy inhibitor . USP10 and USP13 are critical in the development of several cancers because they regulate some key tumor promoters or suppressors. Our previous study showed that Spautin-1 inhibits the growth of chronic myeloid leukemia via promoting the degradation of the oncoprotein, SKP2. Recent studies also have revealed that Spautin-1, alone or in combination with some classic anticancer agents, inhibits multiple cancers, such as ovarian cancer, osteosarcoma, chronic myeloid leukemia and colon cancer [17, 18, 19, 20]. However, there is no definite mechanism by which Spautin-1 inhibits these cancers, possibly due to complexity of this chemical. Our further experiments showed that the proliferation inhibition effect induced by Spautin-1 was independent of USP10/USP13 and autophagy blockage. This phenomenon prompted us to address the underlying mechanisms. By the use of flow cytometry, we showed that Spautin-1 significantly induced G0/G1 phase arrest and cell death. Further investigation unraveled that Cyclin D1 is the key protein mediated Spautin-1 induced cell cycle arrest and that higher doses of Spautin-1 induced a caspase-dependent apoptosis in PCa.
MAPKs are critical to cell survival and proliferation in cancers. Mammals have three major groups of MAPKs: JNK, ERK and p38. Our findings indicate that Spautin-1 induces apoptosis through JNK activation and ERK inactivation in PCa cells. This is consistent with the general agreement that JNK activation can cause apoptosis while ERK activation contributes to cell survival . In this study, we showed that the activation of JNK induced by Spautin-1 increased the expression of Bax and activated caspase-3, and eventually led to apoptosis. Additionally, activation of ERK was considered a driving force of G0/G1 cell cycle progression, possibly due to its induction of Cyclin D1 expression [51, 52]. Supporting the hypothesis that Spautin-1 induces cell cycle arrest through suppressing the ERK/Cyclin D1 axis, our further investigations showed that Spautin-1 activated MKK4 but not MKK7 (both the upstream kinase of JNK) and decreased the expression of phospho-MEK1/2 which directly controls the activation of ERK.
In general, MAPKs are highly regulated by EGFR. This study further explored the responsiveness of EGFR to Spautin-1 in PCa. We found that Spautin-1 treatment rapidly decreased the phosphorylation of EGFR, before the decrease of phospho-MEK1/2 and phospho-ERK1/2 and the increase of phospho-MKK4 and phospho-JNK became discernible. These findings indicate that the cell cycle arrest and apoptosis triggered by Spautin-1 through interfering MAPKs may root in the inhibition of EGFR. Inhibition of EGFR signaling decreases the expression of Glut1, possibly via decreasing the transcription and enhancing the degradation of Glut1, which was associated with ERK/PKM2/c-myc and AKT status [41, 42]. Therefore, Glut1 was regulated by EGFR in cancers. In the current study, we explored the effects of Spautin-1 on Glut1 expression in PCa on the basis of EGFR inhibition triggered by Spautin-1. We showed that Spautin-1 notably reduced the expression of the downstream effecter of EGFR, Glut1, that controls glucose uptake and cell survival in glucose deprivation condition. To combine previous studies and the current findings, we hypothesize that Spautin-1 induced Glut1 downregulation likely through EGFR inhibition. The downregulation of Glut1 induced by Spautin-1 notably enhanced cell death of PCa upon glucose removal, a state frequently occurs in the core of solid tumors .
Our in vivo experiments demonstrate that Spautin-1 has a potent inhibitory effect on the growth of PCa xenografts. The application of Spautin-1 did not apparently decrease the body weight of mice for one month, suggesting that Spautin-1 may have little undesired cytotoxicity toward normal tisues. Enzalutamide is the conventional anti-androgen agent for the treatment of advanced PCa. To explore whether Spautin-1 may enhance the efficacy of enzalutamide or even overcome enzalutamide resistance (or castration resistance) is significant for clinical translation of Spautin-1. The current study preliminarily try to address this question and hoping this combination therapy may benefit the patients with PCa in future. Therefore, further investigations were performed to determine the synergistic effects of Spautin-1 with Enzalutamide. Our results show that Spautin-1 notably sensitizes PCa cells to Enzalutamide both in vitro and in vivo. Hence, we suggest that Spautin-1 may serve as not only a standalone anticancer agent but a readily available adjuvant for Enzalutamide to treat PCa without obvious undesired cytotoxicity toward normal tissues.
In summary, this study provides preclinical evidence that Spautin-1 inhibits EGFR signaling and thereby suppresses the growth of PCa. Inhibition of EGFR with Spautin-1 inactivates the MEK/ERK/Cyclin D1 axis and decreases Glut1 expression, while activating the MKK4/JNK/Bax axis, which collectively induced cell cycle arrest and apoptosis of PCa cells (Fig. 8h).
We thank Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University for flow cytometry analysis.
The study was supported by the National Natural Science Foundation of China (81472390, 81872151, 81773213), The National Funds for Developing Local Colleges and Universities (B16056001), Natural Science Foundation research team of Guangdong Province (2018B030312001), the Science and Technology Program of Guangzhou (201604020001), Innovative Academic Team of Guangzhou Education System (1201610014), the Project of Department of Education of Guangdong Province (2016KTSCX119), the Research Team of Department of Education of Guangdong Province (2017KCXTD027), Guangzhou key medical discipline construction project fund.
Availability of data and materials
All the data and materials supporting the conclusions were included in the main paper.
HBH and JBL designed the experiments. YNL, ZQG, XHX, YL, CYH, and LLJ performed the experiments, HBH, JBL and XJW wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The use and care of experimental animals were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.
Consent for publication
The authors declare no conflicts of interest.
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