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PbS nanotube arrays with multi-heterojunctions for efficient visible-light-driven photoelectrochemical hydrogen evolution
Serine/arginine protein-specific kinase 2 promotes the development and progression of pancreatic cancer by downregulating Numb and p53 Guosen Wang, Weiwei Sheng, Xiaoyang Shi, Xin Li, Jianping Zhou and Ming Dong Department of Gastrointestinal Surgery, The First Hospital, China Medical University, Shenyang, China
Keywords Numb; p53; pancreatic cancer; progression; SRPK2 Correspondence M. Dong, Department of Gastrointestinal Surgery, The First Hospital, China Medical University, Shenyang, China Tel: +86 24 83282881 E-mail: [email protected] (Received 9 October 2018, revised 16 December 2018, accepted 4 February 2019) doi:10.1111/febs.14778
Serine/arginine protein-specific kinase 2 (SRPK2) plays a vital role in the progression of a range of different malignancies, including pancreatic cancer. However, the mechanisms are poorly understood. Previous studies have shown that in hepatocellular carcinoma, SRPK2 knockdown leads to the upregulation of the cell fate determining protein Numb, and in pancreatic cancer cells, Numb knockdown prevents ubiquitin-mediated degradation of p53. In this study, we investigated the relationship between SRPK2, Numb and p53 in the development of pancreatic cancer with or without chemical agent treatment in vitro. SRPK2 expression was upregulated in pancreatic cancer tissues and associated with decreased overall survival in pancreatic cancer patients, indicating that expression of this protein can be used as a marker of unfavourable prognosis. Expression of SRPK2 was positively associated with tumour T stage and Union for International Cancer Control (UICC) stage, and negatively associated with Numb expression in serial tissue sections. In pancreatic cancer cells, SRPK2 downregulation or overexpression led to modulation of Numb and wildtype p53 protein expression in response to oxaliplatin treatment. Furthermore, these three endogenous proteins could be coimmunoprecipitated as a triple complex. Numb or p53 knockdown reversed the upregulation of p53 that was induced by silencing SRPK2. SRPK2 overexpression promoted cell invasion and migration, and decreased chemosensitivity of cancer cells to gemcitabine or oxaliplatin treatment. Conversely, SRPK2 silencing decreased cell invasion and migration and increased chemosensitivity; these effects were reversed by silencing p53 in oxaliplatin-treated pancreatic cancer cells. Our data suggest that SRPK2 negatively regulates p53 by downregulating Numb under chemical agent treatment. Thus, SRPK2 promotes the development and progression of pancreatic cancer in a p53-dependent manner.
Introduction Pancreatic cancer (PC) is one of the most lethal malignancies, with an estimated 5-year survival rate of less than 5% [1,2]. The lethality of PC is mainly attributed
to its strong local invasion, high distant metastasis rate and chemotherapy resistance. Therefore, it is urgent to identify prognostic molecular markers to predict the
Abbreviations DAB, 3,30 -diaminobenzidine; HCC, hepatocellular carcinoma; mtp53, mutant p53; PC, pancreatic cancer; PDAC, pancreatic ductal adenocarcinoma; PTB, phosphotyrosine binding; PVDF, polyvinylidene difluoride; SRPIN340, N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl) isonicotinamide; SRPK2, SR protein-specific kinase 2; SR, serine/arginine; UICC, Union for International Cancer Control; wtp53, wild-type p53.
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malignant biology and combat the drug resistance of PC. Serine/arginine (SR) proteins are a highly conserved group of RNA-binding proteins [3] that are involved in the formation of spliceosomes and regulate alternative pre-mRNA splicing. SR protein-specific kinase 2 (SRPK2) phosphorylates SR domain-containing proteins and mediates the splicing activity of SR proteins [4,5]. Several studies have reported that SRPK2 plays a significant role in regulating the replication of multiple viruses including hepatitis [6–8], human immunodeficiency virus [9] and Epstein–Barr virus [10]. In addition, SRPK2 is also associated with the pathogenesis of Alzheimer’s disease [11,12] and lipogenesis [13]. Moreover, SRPK2 expression is elevated in many cancers, such as leukaemia [14], nonsmall cell lung carcinoma [15], head and neck squamous cell carcinoma [16], colon cancer [17] and prostate cancer [18]. Numb, a cell fate determinant, controls the function of the tumour suppressor p53 [19,20]. Our previous study showed that Numb could regulate wild-type p53 (wtp53) expression rather than mutant p53 (mtp53), and Numb downregulation increased chemoresistance in a p53-dependent manner in wtp53 PC cells [21]. In hepatocellular carcinoma (HCC) cell lines, SRPK2 knockdown upregulated Numb protein expression, and suppressed proliferation, colony formation, migration and invasion [22]. However, the significant roles of SRPK2 and its interaction with Numb and p53 in PC have not been explored to our knowledge, and are investigated in the current study.
Results Differential expression of SRPK2 protein in PC tissues Immunohistochemistry showed that the SRPK2 protein was located in the cytoplasm and nucleus in PC and pancreas tissues. SRPK2 expression in PC tissues was much higher than that in the paired normal pancreas (79.5%, 58/73 vs. 46.6%, 34/73; t = 5.976, P < 0.001) (Fig. 1A–C). Similarly, western blot and qRT-PCR also showed that SRPK2 protein and mRNA levels in 24 PC tissues were significantly increased compared with the paired normal tissues (t = 2.893, P = 0.008; t = 3.664, P = 0.001) (Fig. 2A,B). Association of SRPK2 expression with clinical data and survival of PC patients The clinicopathological data are summarized in Table 1. Briefly, SRPK2 expression was positively
SRPK2 promotes progression in PC
associated with tumour T stage (P = 0.017) and UICC stage (P = 0.044). Meanwhile, postoperative patients with SRPK2 expression had a significantly reduced overall survival than those without SRPK2 expression as determined by Kaplan–Meier analysis (P = 0.011) (Fig. 2C). Additionally, T stage (P = 0.010), lymph node metastasis (P < 0.001), UICC stage (P < 0.001) and postoperative liver metastasis (P = 0.007) were also associated with prognosis. However, only SRPK2 expression was an independent unfavourable prognostic indicator in multivariate analysis (P = 0.045) (Table 2). Relationship among SRPK2, Numb and p53 expression in PC tissues The expression of Numb and p53 in PC tissues was well described in our previous study [21,23]. The p53 detected by IHC was thought to be mtp53 due to the short half-life of the wtp53 that accumulated in the nucleus [24]. We first focused on investigating the relationship between SRPK2, Numb and p53 in PC tissues. Spearman’s rank correlation analysis showed that SRPK2 was negatively associated with Numb expression (r = 0.385, P = 0.023), but had no association with mtp53 expression (r = 0.207, P = 0.265) in 40 cases of PC serial sections (Table 3). Additionally, PC patients with Numb expression had a significantly better overall survival than those without Numb expression (P = 0.004) (Fig. 2D), which was opposite to the overall survival associated with SRPK2 expression. Moreover, PC tissues with high SRPK2 expression were associated with low Numb expression (Fig. 1D, E) and vice versa (Fig. 1G,H). This trend was not found between SRPK2 and mtp53 expression (Fig. 1F, I), which guided us to further investigate the expression of SRPK2, Numb and p53 in wtp53 PC cell lines. The association of SRPK2, Numb and p53 in wtp53 PC cell lines with or without oxaliplatin treatment The Wtp53 cells Capan-2 and SW1990 were used to construct stable cell lines with SRPK2 silenced and overexpressed. The SRPK2 protein level in the sgSRPK2 groups was significantly lower than that in the corresponding scrambled RNA groups in the SRPK2silenced stable cell lines (Fig. 3A,B), while SRPK2 expression in the SRPK2-GFP groups was significantly higher than that in the corresponding empty plasmid groups in the SRPK2-overexpressing stable cell lines (Fig. 3C,D). Without oxaliplatin treatment, silencing or overexpressing SRPK2 did not changed wtp53
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A
B
C
D
E
F
G
H
I
Fig. 1. Differential expression of SRPK2, Numb and p53 in PC and corresponding normal tissues as detected by immunohistochemistry. (A–C) SRPK2 expression in normal pancreatic tissues (A), highly differentiated PC (B) and poorly differentiated PC (C). (D–F) High SRPK2 (D), low Numb (E) and positive p53 (F) expression in one PC sample. (G–I) Low SRPK2 (G), high Numb (H) and positive p53 (I) expression in another PC sample. The sections were mounted under a microscope at 920 magnification. SRPK2 protein was located in the cytoplasm and nucleus, and Numb protein in the membrane and cytoplasm, and p53 protein in the nuclei was considered for scoring. The sum of the intensity and extent scores was used as the final staining score (0–7), and tumours with a final staining score >2 were considered to have positive expression. The differences in expression were compared with paired-sample t-tests.
protein levels. However, with the IC50 treatment of oxaliplatin for 24 h (our pre-experiment showed oxaliplatin was much more reliable to activate p53 protein expression), the expression of the p53 and p21 proteins was activated and significantly increased in the sgSRPK2 group compared with the scrambled RNA group, and conversely decreased in the SRPK2-GFP group compared with the empty plasmid group. Additionally, the Numb protein level was increased in the sg-SRPK2 group vs. the corresponding scrambled RNA group, and decreased in the SRPK2-GFP group vs. the empty plasmid group, regardless of treatment with oxaliplatin. The above results showed that SRPK2 negatively regulated Numb and wtp53 protein expression in response to treatment with oxaliplatin. 1670
We further showed that SRPK2, Numb and p53 were coimmunoprecipitated in a triple complex in both Capan2 and SW1990 cells, without or with the IC50 treatment of oxaliplatin for 24 h (Fig. 4). In addition, our prior studies showed that Numb can coimmunoprecipitate with p53 in PC cells [21,23]. These results indicated a close interaction among the SRPK2, Numb and p53 proteins in PC cells. SRPK2 regulated cell migration, invasion and chemosensitivity in PC cells SRPK2 silencing decreased cell migration and invasion in the sg-SRPK2 group compared with the scrambled RNA group in Capan-2 cells (Fig. 5A,B), whereas SRPK2 overexpression enhanced cell migration and
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invasion in the SRPK2-GFP group compared with the empty plasmid group (Fig. 5C,D). Similar results were also replicated in SW1990 cells (Fig. 5E–H). Furthermore, the CCK8 assays showed that the silencing and overexpression of SRPK2 enhanced and decreased, respectively, the chemosensitivity of Capan2 cells under the gemcitabine and oxaliplatin treatments, (Fig. 6A,C,E,G). Similar results were also observed in SW1990 cells (Fig. 6B,D,F,H). Numb or p53 knockdown reversed the increase in wtp53 protein induced by silencing SRPK2 in response to oxaliplatin SRPK2 silencing significantly enhanced the level of Numb protein in PC cells (Fig. 3A,B), and our
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previous studies showed that Numb knockdown downregulated the wtp53 protein during chemical agent treatment [21,23]. Thus, we inferred that silencing SRPK2 upregulated the oxaliplatin-induced wtp53 activation via upregulating Numb protein. Consistent with our previous studies [21,23], Numb knockdown significantly reversed the increase in wtp53 protein levels induced by silencing SRPK2 during oxaliplatin treatment in Capan-2 and SW1990 cells (Fig. 7). This trend was much more significant when p53 siRNA was used instead of Numb siRNA. Nevertheless, Numb or p53 knockdown did not change the SRPK2 protein level. Altogether, Numb or p53 knockdown reversed the upregulation of wtp53 induced by silencing SRPK2 in response to oxaliplatin.
Fig. 2. Expression of SRPK2 and Kaplan–Meier analysis of cumulative (Cmu) overall survival in PC patients. (A, B) Western blot and qRT-PCR analysis of SRPK2 protein (A) and mRNA (B) levels in 24 cases of PC (T) and corresponding normal tissues (N) respectively. (C) The presence (+) and absence () of SRPK2 expression was plotted against overall survival time (n = 73 PC patients). (D) The presence (+) and absence () of Numb expression was plotted against overall survival time (n = 40 PC patients). The differences in survival were evaluated by the log-rank test.
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Table 1. Association of SRPK2 expression with clinicopathological data in PC patients.
Parameters
No. of patients
Cases 73 Age (years) ≤65 59 >65 14 Gender Male 40 Female 33 Tumour location Head 50 Body-tail 23 Tumour size (cm) <3 16 ≥3 57 Differentiation Well 22 Moderate and poor 51 T stage T1 + T2 28 T3 + T4 45 Lymph nodes metastasis N0 (negative) 52 N1 (positive) 21 UICC stage I+IIA 50 IIB+III 23 Pretherapeutic CA19-9 level (U/mL) <37 16 ≥37 57 Postoperative liver metastasis Negative 47 Positive 26
SRPK2 P
Negative
Positive
15
58
12 3
47 11
1.000
9 6
31 27
0.774
12 3
38 20
0.445
4 11
12 46
0.882
3 12
19 39
0.519
10 5
18 40
0.017
14 1
38 20
0.072
14 1
36 22
0.044
4 11
12 46
0.882
9 6
38 20
0.766
It is unquestionable that p53 also plays a crucial role in the drug resistance of various cancers [27–29]. Thus, we investigated whether SRPK2 regulated drug resistance in a p53-dependent manner. Additional CCK8 assays showed that p53 knockdown significantly reversed the enhanced chemosensitivity induced by gemcitabine and oxaliplatin when SRPK2 was silenced (Fig. 9).
Discussion
p53 knockdown reversed the decrease in cell migration and invasion induced by SRPK2 silencing after treatment with oxaliplatin It is well known that p53 acts as a critical regulator in the malignant biology of various cancers [25,26]. Thus, we investigated whether SRPK2 promoted cell migration and invasion in a p53-dependent manner in PC cells. To induce p53 activation, transfected cells by lentivirus and siRNA were first pretreated with oxaliplatin for 6 h. Cell migration and invasion in the sgSRPK2 group were significantly decreased compared with that in the scrambled RNA group. However, p53 knockdown significantly reversed the decrease induced by silencing SRPK2 in Capan-2 cells (Fig. 8A,B). Similar results were found in SW1990 cells (Fig. 8C,D). Therefore, SRPK2 promoted migration and invasion in a p53-dependent manner in PC cells.
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p53 knockdown reversed the increased chemosensitivity induced by SRPK2 silencing in PC cells
SRPK2 phosphorylates SR proteins and plays an essential role in pre-mRNA alternative splicing, which may be linked to various diseases, including cancer, if splicing dysfunctions occur. Previous studies have reported that SRPK2 contributes to the development of haematologic and solid cancers by various signalling pathways. SRPK2 promotes the proliferation of leukaemia cells by upregulating the expression of Cyclin A1 [14]. Additionally, N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340), a SRPK inhibitor, has antileukaemia effects [30,31]. The elevated expression of SRPK2 promotes the growth and migration of colon cancer by activating ERK signalling [17]. SRPK2 knockdown elevates Numb expression, which inhibits proliferation, migration and invasion in HCC [22]. However, the definite role of SRPK2 in the development of PC is rarely reported to our knowledge. However, in a prior study, we found that Numb can prevent the ubiquitination-dependent degradation of wtp5 and increase chemosensitivity in a p53-dependent manner in PC [21]. Thus, we infer a close relationship between the SRPK2, Numb and p53 proteins in the development and progression of PC, which has not been previously investigated to our knowledge. We first observed the overexpression of SRPK2 in PC tissues, which was associated with tumour T stage, UICC stage and poor PC patient prognosis. In prostate cancer [18], the high expression of SRPK2 was significantly correlated with advanced pathological stage and a higher Gleason Score. Additionally, SRPK2 promoted cell invasion, migration and drug resistance in PC cells, in accordance with the findings of Radhakrishnan et al. [16], Wang et al. [17] and Lu et al. [22] in head and neck squamous cell carcinoma, colon cancer and hepatocellular carcinoma, respectively. Taken together, SRPK2 promoted the development and progression of PC.
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Table 2. Univariate and multivariate analysis of clinicopathological factors for survival in 73 postoperative PC patients.
Parameters Age (≤65/>65 years) Gender (male/female) Tumour location (head/body-tail) Tumour size (<3/≥3 cm) Well/Moderate and poor differentiation T stage (T1 + T2/T3 + T4) Lymph nodes metastasis (N0/N1) UICC stage (I + IIA/IIB + III) Pretherapeutic CA19-9 level (<37 UmL1/≥37 UmL1) Postoperative liver metastasis (negative/positive) SRPK2 (negative/positive)
Median survival (days)
Univariate analysis P (log rank)
257/239 270/219 296/234 270/256 296/256 369/249 310/180 315/180 270/256
0.642 0.455 0.089 0.447 0.555 0.010 < 0.001 < 0.001 0.731
299/200 480/249
Table 3. Correlation analysis of the relationship between SRPK2 with Numb and p53. SRPK2
Parameter Numb Negative (n = 23) Positive (n = 17) p53 Negative (n = 19) Positive (n = 21)
Negative (n = 9)
Positive (n = 31)
2 7 6 3
r rank
P
21 10
0.385
0.023
13 18
0.207
0.265
Mutations in the p53 gene are frequently detected in human cancers [32,33]. The activation of mtp53 promotes prosurvival signals and tumourigenesis, but does not result in the loss of wtp53 [34,35]. We first demonstrated that SRPK2 had no association with mtp53 in PC tissues. In the wtp53 PC cell line without stimulus, silencing or overexpressing SRPK2 alone altered Numb but not wtp53 expression. Thus, the enhanced expression of Numb induced by SRPK2 silencing does not change the basal p53 levels in the absence of external stress. Gemcitabine and oxaliplatin are widely used as chemotherapeutic agents for PC cells [36,37]. In oxaliplatin-treated wtp53 PC cells, wtp53 protein was significantly upregulated, and silencing or overexpressing SRPK2 induced an increase or decrease, respectively, in Numb and p53 levels. Furthermore, these 3 endogenous proteins could be coimmunoprecipitated as a triple complex. These findings suggest a close relationship between SRPK2 and Numb as well as p53, which
Multivariate analysis hazard ratio (95% CI)
P
1.251 (0.553–2.829) 1.231 (0.155–9.785) 2.983 (0.359–24.769)
0.590 0.845 0.311
0.007
1.945 (0.946–4.000)
0.070
0.011
2.594 (1.021–6.587)
0.045
might play an important role in the progression of PC. Generally, Numb contains a phosphotyrosine binding (PTB) domain that interacts with p53 [38]. Numb controls the function of p53 by interfering with MDM2dependent ubiquitination and degradation [20]. Based on these findings, SRPK2 downregulates the chemical agent-induced level of activated wtp53 by reducing Numb protein expression. Additionally, Numb knockdown significantly reversed the elevation of wtp53 protein levels induced by silencing SRPK2 in response to chemical agents. Moreover, our previous study found that Numb knockdown decreased chemosensitivity in a p53-dependent manner [21]. Taken together, SRPK2 negatively regulates wtp53 protein levels by downregulating Numb in the presence of chemical agents. Wtp53 is the terminal target protein of SRPK2. Undoubtedly, p53 plays an important role in the malignant progression and drug resistance in various cancers. We found that silencing SRPK2 decreased cell invasion and migration in PC cells, and increased chemosensitivity to gemcitabine and oxaliplatin, all of which were significantly reversed by p53 silencing. To date, the molecular mechanism of SRPK2-mediated drug resistance remains unclear. SRPIN340, the SRPK inhibitor, exhibited an antileukaemia effect by altering the expression of the MAP2K1, MAP2K2, VEGF and FAS genes [36,37]. For the first time, we demonstrated that SRPK2 promoted malignant biology and drug resistance of PC in a p53-dependent manner. In summary, SRPK2 promotes the development and progression of PC, and negatively regulates wtp53 expression by downregulating Numb protein expression in response to chemical agents. SRPK2 promotes the malignant biology and drug resistance of PC in a
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Fig. 3. The association of SRPK2, Numb, p53 and p21 in wtp53 Capan-2 and SW1990 cell lines. (A, B) Numb, p53 and p21 protein levels in sg-SRPK2 and scrambled RNA-transfected Capan-2 (A) and SW1990 (B) cells, with or without oxaliplatin treatment. (C, D) Numb, p53 and p21 protein levels in SRPK2-GFP and empty plasmid-transfected Capan-2 (C) and SW1990 (D) cells, with or without oxaliplatin treatment. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 vs. control.
Fig. 4. The close interaction between SRPK2, Numb and p53 in wtp53 PC cell lines, without or with oxaliplatin treatment. (A, B) SRPK2 coimmunoprecipitation with Numb and p53 without (A) or with (B) oxaliplatin treatment in Capan-2 cells. (C, D) SRPK2 coimmunoprecipitation with Numb and p53 regardless of whether the Numb (C) or p53 (D) antibody was used in Capan-2 cells. (E, F) SRPK2 coimmunoprecipitation with Numb and p53 without (E) or with (F) oxaliplatin treatment in SW1990 cells. (G, H) SRPK2 coimmunoprecipitated with Numb and p53 regardless of whether the Numb (G) or p53 (H) antibody was used in SW1990 cells. The input and IgG lanes show the positive and negative controls respectively.
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Fig. 5. Cell migration and invasion assays in PC cells. (A, B) Cell migration (A) and invasion (B) assays in sg-SRPK2 and scrambled RNAtransfected Capan-2 cells respectively. (C, D) Cell migration (C) and invasion (D) assays in SRPK2-GFP- and empty plasmid-transfected Capan-2 cells respectively. (E, F) Cell migration (E) and invasion (F) assays in sg-SRPK2 and scrambled RNA-transfected SW1990 cells respectively. (G, H) Cell migration (G) and invasion (H) assays in SRPK2-GFP- and empty plasmid-transfected SW1990 cells respectively. Cells were plated in upper modified Boyden chamber for 24 h. The photos of cells that had migrated or invaded to the bottom chambers were obtained with a microscope. The cells were counted under a microscope at 920 magnification and in five random fields per insert assay. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 compared with the control.
p53-dependent manner. Future research should further investigate the molecular mechanism of the interactions among SRPK2, Numb and p53.
of Union for International Cancer Control (UICC) classification.
Cell lines and culture
Materials and methods Tissue samples Specimen collection was approved by the Institutional Review Board of the China Medical University. Each patient enrolled in this study signed a consent form. Seventy-three formalin-fixed and paraffin-embedded pancreatic ductal adenocarcinoma (PDAC) and paired noncancerous tissues were collected from patients between 2006 and 2017 at the First Hospital of China Medical University. Furthermore, 24 cases of paired tumour and adjacent nontumour pancreatic tissues were used for protein extraction and RNA isolation. Patients with invasive intraductal papillary mucinous carcinoma, acinar cell carcinoma and endocrine carcinoma were excluded from the study. All diagnoses were confirmed by histopathological evaluation, and the data were analysed on the basis of the 8th edition
The Wtp53 Capan-2 cell line [39] was obtained from the American Type Culture Collection (ATTC; Manassas, VA, USA), and the wtp53 SW1990 cell line [39] was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the recommended growth medium supplemented with 10% FBS ( HyClone, Logan, UT, USA), and maintained at 37 °C in a humidified incubator under 5% CO2 atmosphere.
Immunohistochemistry assays Immunohistochemistry was performed as described previously [21,23]. Briefly, tissues were embedded in paraffin and cut into 4-lm consecutive sections. After deparaffinization, antigen retrieval was performed using sodium citrate buffer at a high pressure for 2 min, and cooled for
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Fig. 6. The CCK8 assays in wtp53 PC cells with silenced or overexpressed SRPK2 under chemotherapeutical treatment. (A, B) Silencing SRPK2 significantly enhanced the chemosensitivity of Capan-2 (A) and SW1990 (B) cells under gemcitabine treatment, compared with the scrambled RNA group. (C, D) Silencing SRPK2 significantly enhanced the chemosensitivity of Capan-2 (C) and SW1990 (D) cells under oxaliplatin treatment, compared with the scrambled RNA group. (E, F) SRPK2 overexpression significantly decreased the chemosensitivity of Capan-2 (E) and SW1990 (F) cells under gemcitabine treatment, compared with the empty plasmid group. (G, H) SRPK2 overexpression significantly decreased the chemosensitivity of Capan-2 (G) and SW1990 (H) cells under oxaliplatin treatment, compared with the empty plasmid group. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 compared with the control.
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Fig. 7. Numb or p53 knockdown reversed the increase in wtp53 protein induced by SRPK2 silencing in response to oxaliplatin. (A) The SRPK2, Numb and p53 protein levels in Capan-2 cells with the treatment combinations shown during oxaliplatin treatment. (B) The SRPK2, Numb and p53 protein levels in SW1990 cells with the treatment combinations shown during oxaliplatin treatment. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 compared with the control.
1 h at room temperature. Sections were incubated with 0.3% H2O2 for 15 min to block endogenous peroxidase activity. After blocking with goat serum for 20 min, sections were covered with anti-SRPK2 (Abcam, Cambridge, UK; 1 : 400), anti-Numb (Abcam; 1 : 400) or anti-p53 (Proteintech, Chicago, IL, USA; 1 : 100) overnight at 4 °C. Next, the sections were incubated with a secondary antibody for 15 min. The sections were then incubated with streptavidin–peroxidase reagent for 15 min. The signal was visualized using 3,30 -diaminobenzidine (DAB), and the sections were counterstained with haematoxylin, and then mounted for microscopy. Stained tissue sections were reviewed and scored as described by Masunaga et al. [40]. Staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The extent of staining was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), according to the percentage of the carcinoma that was positively stained. The final Immunohistochemistry staining scores were determined by three pathologists. The sum of the intensity and extent scores was used as the final staining score (0–7), and tumours with a final staining score >2 were considered to have positive expression.
Western blot assays Total cell lysates were harvested from PC tissues and cells. After protein quantification, the samples were separated by
10% SDS/PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary SRPK2 (Abcam), Numb (Abcam), p53 (Proteintech) or GAPDH (Proteintech) antibodies. The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit and anti-mouse (Santa Cruz, CA, UK). Immunoreactive protein bands were visualized using the ECL detection kit (Thermo Scientific, Rockford, IL, USA). Each experiment was repeated three times.
Immunoprecipitation As described previously [21,23], PC cells were lysed in lysis buffer [20 mM Tris/HCl (pH 7.4), 1.0% NP-40, 150 mM NaCl, 1 mM EDTA, 10 lgmL1 leupeptin, 50 lgmL1 PMSF], and the soluble supernatants of protein lysates were isolated by centrifugation at 4 °C. Briefly, magnetic beads (Bio-Rad, Hercules, CA, USA) were preincubated with SRPK2 (Abcam), Numb (Abcam), p53 (Proteintech) or IgG (Santa Cruz) antibodies at 4 °C for 4 h with agitation. The antibody-bead complexes were washed three times with lysis buffer and incubated with the soluble supernatants at 4 °C overnight. Next, the immunocomplexes were washed three times with lysis buffer. After elution in the sample loading buffer by boiling, the samples were separated by SDS/PAGE and subjected to western blot analysis with SRPK2 (Abcam), Numb (Abcam) and p53 (Proteintech) antibodies.
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qRT-PCR analysis Total RNA was extracted from PC tissues using Trizol reagent (Takara Bio, Otsu, Japan), and subjected to qRTPCR using the standard programme as described previously [21]. The primers used were as follows: SRPK2, 50 -GGA GATAGAAGAATTGGAGCGAGAAGC-30 (sense) and 50 -CCTCAGCCGCCTCCTCTAATCC-30 (antisense); GAD PH, 50 -CATGAGAAGTATGACAACAGCCT-30 (sense) and 50 -AGTCCTTCCACGATACCAAAGT-30 (antisense). All reactions were analysed using a Light Cycler 2.0 with the Light Cycler kit (Takara Bio).
Construction of stable cell lines and RNA interference The GV358-SRPK2-GFP plasmid (SRPK2-GFP) and corresponding empty plasmid (GFP) were synthesized and packaged into a lentivector by Genechem (GenePharma Co, Ltd, Shanghai, China). PC cells were transfected with plasmid for 24 h, and screened by puromycin (Sigma, St Louis, MO, USA) twice for 48 h. Lenti-cas9 and lenti-sgRNA were synthesized by Genechem (GenePharma Co). PC cells were infected with lenticas9 and selected by puromycin (Sigma) twice for 48 h.
Fig. 8. p53 knockdown reversed the decrease in cell migration and invasion induced by silencing SRPK2 after treatment with oxaliplatin in wtp53 PC cells. (A, B) Cell migration (A) and invasion (B) assays in Capan-2 cells transfected with the combinations shown. (C, D) Cell migration (C) and invasion (D) assays in SW1990 cells transfected with the combinations shown. Cells were plated in upper modified Boyden chamber for 24 h. The photos of cells that had migrated or invaded to the bottom chambers were obtained with a microscope. The cells were counted under a microscope at 920 magnification and in five random fields per insert assay. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 vs. control.
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Fig. 9. p53 knockdown reversed the increased chemosensitivity induced by SRPK2 silencing in PC cells. (A, B) Chemosensitivity of Capan-2 (A) and SW1990 (B) cells treated with gemcitabine, in the combined protein transfection and expression groups shown. (C, D) Chemosensitivity of Capan-2 (C) and SW1990 (D) cells treated with oxaliplatin, in the combined protein transfection and expression groups shown. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 vs. control.
Then, the stable sublines were infected with SRPK2sgRNA (sg-SRPK2) to specifically silence the target gene or an sgRNA control (scramble). All of the siRNAs were synthesized by GenePharma (GenePharma Co, Ltd, Shanghai, China). The target sequences used were as follows: Numb, 50 -CUGGAAA GAAAGCAGUUAATT-30 (sense) and 50 -UUAACUGCU UUCUUUCCAGTT-30 (antisense); p53, 50 -CUACUUC CUGAAAACAACGTT-30 (sense) and 50 -CGUUGUUU UCAGGAAGUAGTT-30 (antisense). Each siRNA sequ ence was selected from three effective sequences as described in our previous studies [21,23]. Targeting sequences for the control siRNA were as follows: 50 -U UCUCCGAACGUGUCACGUTT-30 (sense) and 50 -A CGUGACACGUUCGGAGAATT-30 (antisense). Pooled siRNAs (20 lM) were cotransfected transiently with stably infected cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Effective transfections were successfully verified by western blot analysis, as shown in the ‘Results section’.
Migration and invasion assays The migration of PC cells was evaluated using modified Boyden chamber (BD Biosciences, Sparks, MD, USA)
assays. Briefly, transfected cells were plated in upper chambers with FBS-free growth medium. The lower chambers contained growth media with 10% FBS as a chemoattractant. After 24 h, cells that had migrated to the bottom chambers were stained with crystal violet hydrate (Sigma), and cells that did not migrate were removed from the upper chambers. The invasiveness of cells was determined with a similar method, except the membrane separating the upper and lower chambers was coated with Matrigel (BD Biosciences) prior to the assay. Cell photos were obtained with a microscope (Nikon Microphot-FX, Tokyo, Japan), and the number of cells was counted in five random fields per chamber at 920 magnification.
CCK8 assays The chemosensitivity of transfected cells was determined using CCK8 assays. Briefly, cells were seeded in 96-well plates at 8 9 103 cells per well. After 8 h, the cells were incubated with gemcitabine (Abcam) and oxaliplatin (Abcam) for 48 h at the concentrations shown in the ‘Results section’. Next, 10 lL CCK8 (Dojindo, Kumamoto, Japan) was added and the cells were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm in an ELISA 96well microtitre plate reader (Bio-Rad 680, California,
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USA). Each experimental procedure was performed in triplicate, and the results are shown as the percentage of treated cells compared with control cells.
Statistical analysis Statistical analyses were performed using SPSS 13.0 software (Chicago, IL, USA). Continuous variables were expressed as the mean SD. The expression of SRPK2 in pairs of PDAC and their corresponding noncancerous tissues was compared with paired-sample t-tests. The association between SRPK2 expression and clinicopathological parameters was examined using a Chi-squared test. The relationships between SRPK2, Numb and p53 were analysed using Chi-squared and Spearman correlation tests. The Kaplan– Meier method was used to assess survival, and differences were evaluated by the log-rank test. Cox’s proportional hazards regression model was used to assess independent prognostic factors in a stepwise manner. Student’s t-tests were used to compare the differences in parameters of cell chemosensitivity, cell migration and invasion assays. Differences were considered statistically significant when P values < 0.05.
Acknowledgements We thank the Central Laboratory in the First Hospital of China Medical University for technical support. This work was supported by the Chinese National Science Foundation (No. 81672835 to MD).
Conflict of interest The authors declare no conflict of interest.
Author contributions MD and GW started the project, performed the research and analysed the data. WS and JZ collected the data. GW, XS and XL performed the statistical analyses and wrote the manuscript. GW, JZ and MD contributed to data analysis and revised the manuscript.
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Keywords Numb; p53; pancreatic cancer; progression; SRPK2 Correspondence M. Dong, Department of Gastrointestinal Surgery, The First Hospital, China Medical University, Shenyang, China Tel: +86 24 83282881 E-mail: [email protected] (Received 9 October 2018, revised 16 December 2018, accepted 4 February 2019) doi:10.1111/febs.14778
Serine/arginine protein-specific kinase 2 (SRPK2) plays a vital role in the progression of a range of different malignancies, including pancreatic cancer. However, the mechanisms are poorly understood. Previous studies have shown that in hepatocellular carcinoma, SRPK2 knockdown leads to the upregulation of the cell fate determining protein Numb, and in pancreatic cancer cells, Numb knockdown prevents ubiquitin-mediated degradation of p53. In this study, we investigated the relationship between SRPK2, Numb and p53 in the development of pancreatic cancer with or without chemical agent treatment in vitro. SRPK2 expression was upregulated in pancreatic cancer tissues and associated with decreased overall survival in pancreatic cancer patients, indicating that expression of this protein can be used as a marker of unfavourable prognosis. Expression of SRPK2 was positively associated with tumour T stage and Union for International Cancer Control (UICC) stage, and negatively associated with Numb expression in serial tissue sections. In pancreatic cancer cells, SRPK2 downregulation or overexpression led to modulation of Numb and wildtype p53 protein expression in response to oxaliplatin treatment. Furthermore, these three endogenous proteins could be coimmunoprecipitated as a triple complex. Numb or p53 knockdown reversed the upregulation of p53 that was induced by silencing SRPK2. SRPK2 overexpression promoted cell invasion and migration, and decreased chemosensitivity of cancer cells to gemcitabine or oxaliplatin treatment. Conversely, SRPK2 silencing decreased cell invasion and migration and increased chemosensitivity; these effects were reversed by silencing p53 in oxaliplatin-treated pancreatic cancer cells. Our data suggest that SRPK2 negatively regulates p53 by downregulating Numb under chemical agent treatment. Thus, SRPK2 promotes the development and progression of pancreatic cancer in a p53-dependent manner.
Introduction Pancreatic cancer (PC) is one of the most lethal malignancies, with an estimated 5-year survival rate of less than 5% [1,2]. The lethality of PC is mainly attributed
to its strong local invasion, high distant metastasis rate and chemotherapy resistance. Therefore, it is urgent to identify prognostic molecular markers to predict the
Abbreviations DAB, 3,30 -diaminobenzidine; HCC, hepatocellular carcinoma; mtp53, mutant p53; PC, pancreatic cancer; PDAC, pancreatic ductal adenocarcinoma; PTB, phosphotyrosine binding; PVDF, polyvinylidene difluoride; SRPIN340, N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl) isonicotinamide; SRPK2, SR protein-specific kinase 2; SR, serine/arginine; UICC, Union for International Cancer Control; wtp53, wild-type p53.
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malignant biology and combat the drug resistance of PC. Serine/arginine (SR) proteins are a highly conserved group of RNA-binding proteins [3] that are involved in the formation of spliceosomes and regulate alternative pre-mRNA splicing. SR protein-specific kinase 2 (SRPK2) phosphorylates SR domain-containing proteins and mediates the splicing activity of SR proteins [4,5]. Several studies have reported that SRPK2 plays a significant role in regulating the replication of multiple viruses including hepatitis [6–8], human immunodeficiency virus [9] and Epstein–Barr virus [10]. In addition, SRPK2 is also associated with the pathogenesis of Alzheimer’s disease [11,12] and lipogenesis [13]. Moreover, SRPK2 expression is elevated in many cancers, such as leukaemia [14], nonsmall cell lung carcinoma [15], head and neck squamous cell carcinoma [16], colon cancer [17] and prostate cancer [18]. Numb, a cell fate determinant, controls the function of the tumour suppressor p53 [19,20]. Our previous study showed that Numb could regulate wild-type p53 (wtp53) expression rather than mutant p53 (mtp53), and Numb downregulation increased chemoresistance in a p53-dependent manner in wtp53 PC cells [21]. In hepatocellular carcinoma (HCC) cell lines, SRPK2 knockdown upregulated Numb protein expression, and suppressed proliferation, colony formation, migration and invasion [22]. However, the significant roles of SRPK2 and its interaction with Numb and p53 in PC have not been explored to our knowledge, and are investigated in the current study.
Results Differential expression of SRPK2 protein in PC tissues Immunohistochemistry showed that the SRPK2 protein was located in the cytoplasm and nucleus in PC and pancreas tissues. SRPK2 expression in PC tissues was much higher than that in the paired normal pancreas (79.5%, 58/73 vs. 46.6%, 34/73; t = 5.976, P < 0.001) (Fig. 1A–C). Similarly, western blot and qRT-PCR also showed that SRPK2 protein and mRNA levels in 24 PC tissues were significantly increased compared with the paired normal tissues (t = 2.893, P = 0.008; t = 3.664, P = 0.001) (Fig. 2A,B). Association of SRPK2 expression with clinical data and survival of PC patients The clinicopathological data are summarized in Table 1. Briefly, SRPK2 expression was positively
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associated with tumour T stage (P = 0.017) and UICC stage (P = 0.044). Meanwhile, postoperative patients with SRPK2 expression had a significantly reduced overall survival than those without SRPK2 expression as determined by Kaplan–Meier analysis (P = 0.011) (Fig. 2C). Additionally, T stage (P = 0.010), lymph node metastasis (P < 0.001), UICC stage (P < 0.001) and postoperative liver metastasis (P = 0.007) were also associated with prognosis. However, only SRPK2 expression was an independent unfavourable prognostic indicator in multivariate analysis (P = 0.045) (Table 2). Relationship among SRPK2, Numb and p53 expression in PC tissues The expression of Numb and p53 in PC tissues was well described in our previous study [21,23]. The p53 detected by IHC was thought to be mtp53 due to the short half-life of the wtp53 that accumulated in the nucleus [24]. We first focused on investigating the relationship between SRPK2, Numb and p53 in PC tissues. Spearman’s rank correlation analysis showed that SRPK2 was negatively associated with Numb expression (r = 0.385, P = 0.023), but had no association with mtp53 expression (r = 0.207, P = 0.265) in 40 cases of PC serial sections (Table 3). Additionally, PC patients with Numb expression had a significantly better overall survival than those without Numb expression (P = 0.004) (Fig. 2D), which was opposite to the overall survival associated with SRPK2 expression. Moreover, PC tissues with high SRPK2 expression were associated with low Numb expression (Fig. 1D, E) and vice versa (Fig. 1G,H). This trend was not found between SRPK2 and mtp53 expression (Fig. 1F, I), which guided us to further investigate the expression of SRPK2, Numb and p53 in wtp53 PC cell lines. The association of SRPK2, Numb and p53 in wtp53 PC cell lines with or without oxaliplatin treatment The Wtp53 cells Capan-2 and SW1990 were used to construct stable cell lines with SRPK2 silenced and overexpressed. The SRPK2 protein level in the sgSRPK2 groups was significantly lower than that in the corresponding scrambled RNA groups in the SRPK2silenced stable cell lines (Fig. 3A,B), while SRPK2 expression in the SRPK2-GFP groups was significantly higher than that in the corresponding empty plasmid groups in the SRPK2-overexpressing stable cell lines (Fig. 3C,D). Without oxaliplatin treatment, silencing or overexpressing SRPK2 did not changed wtp53
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A
B
C
D
E
F
G
H
I
Fig. 1. Differential expression of SRPK2, Numb and p53 in PC and corresponding normal tissues as detected by immunohistochemistry. (A–C) SRPK2 expression in normal pancreatic tissues (A), highly differentiated PC (B) and poorly differentiated PC (C). (D–F) High SRPK2 (D), low Numb (E) and positive p53 (F) expression in one PC sample. (G–I) Low SRPK2 (G), high Numb (H) and positive p53 (I) expression in another PC sample. The sections were mounted under a microscope at 920 magnification. SRPK2 protein was located in the cytoplasm and nucleus, and Numb protein in the membrane and cytoplasm, and p53 protein in the nuclei was considered for scoring. The sum of the intensity and extent scores was used as the final staining score (0–7), and tumours with a final staining score >2 were considered to have positive expression. The differences in expression were compared with paired-sample t-tests.
protein levels. However, with the IC50 treatment of oxaliplatin for 24 h (our pre-experiment showed oxaliplatin was much more reliable to activate p53 protein expression), the expression of the p53 and p21 proteins was activated and significantly increased in the sgSRPK2 group compared with the scrambled RNA group, and conversely decreased in the SRPK2-GFP group compared with the empty plasmid group. Additionally, the Numb protein level was increased in the sg-SRPK2 group vs. the corresponding scrambled RNA group, and decreased in the SRPK2-GFP group vs. the empty plasmid group, regardless of treatment with oxaliplatin. The above results showed that SRPK2 negatively regulated Numb and wtp53 protein expression in response to treatment with oxaliplatin. 1670
We further showed that SRPK2, Numb and p53 were coimmunoprecipitated in a triple complex in both Capan2 and SW1990 cells, without or with the IC50 treatment of oxaliplatin for 24 h (Fig. 4). In addition, our prior studies showed that Numb can coimmunoprecipitate with p53 in PC cells [21,23]. These results indicated a close interaction among the SRPK2, Numb and p53 proteins in PC cells. SRPK2 regulated cell migration, invasion and chemosensitivity in PC cells SRPK2 silencing decreased cell migration and invasion in the sg-SRPK2 group compared with the scrambled RNA group in Capan-2 cells (Fig. 5A,B), whereas SRPK2 overexpression enhanced cell migration and
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invasion in the SRPK2-GFP group compared with the empty plasmid group (Fig. 5C,D). Similar results were also replicated in SW1990 cells (Fig. 5E–H). Furthermore, the CCK8 assays showed that the silencing and overexpression of SRPK2 enhanced and decreased, respectively, the chemosensitivity of Capan2 cells under the gemcitabine and oxaliplatin treatments, (Fig. 6A,C,E,G). Similar results were also observed in SW1990 cells (Fig. 6B,D,F,H). Numb or p53 knockdown reversed the increase in wtp53 protein induced by silencing SRPK2 in response to oxaliplatin SRPK2 silencing significantly enhanced the level of Numb protein in PC cells (Fig. 3A,B), and our
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previous studies showed that Numb knockdown downregulated the wtp53 protein during chemical agent treatment [21,23]. Thus, we inferred that silencing SRPK2 upregulated the oxaliplatin-induced wtp53 activation via upregulating Numb protein. Consistent with our previous studies [21,23], Numb knockdown significantly reversed the increase in wtp53 protein levels induced by silencing SRPK2 during oxaliplatin treatment in Capan-2 and SW1990 cells (Fig. 7). This trend was much more significant when p53 siRNA was used instead of Numb siRNA. Nevertheless, Numb or p53 knockdown did not change the SRPK2 protein level. Altogether, Numb or p53 knockdown reversed the upregulation of wtp53 induced by silencing SRPK2 in response to oxaliplatin.
Fig. 2. Expression of SRPK2 and Kaplan–Meier analysis of cumulative (Cmu) overall survival in PC patients. (A, B) Western blot and qRT-PCR analysis of SRPK2 protein (A) and mRNA (B) levels in 24 cases of PC (T) and corresponding normal tissues (N) respectively. (C) The presence (+) and absence () of SRPK2 expression was plotted against overall survival time (n = 73 PC patients). (D) The presence (+) and absence () of Numb expression was plotted against overall survival time (n = 40 PC patients). The differences in survival were evaluated by the log-rank test.
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Table 1. Association of SRPK2 expression with clinicopathological data in PC patients.
Parameters
No. of patients
Cases 73 Age (years) ≤65 59 >65 14 Gender Male 40 Female 33 Tumour location Head 50 Body-tail 23 Tumour size (cm) <3 16 ≥3 57 Differentiation Well 22 Moderate and poor 51 T stage T1 + T2 28 T3 + T4 45 Lymph nodes metastasis N0 (negative) 52 N1 (positive) 21 UICC stage I+IIA 50 IIB+III 23 Pretherapeutic CA19-9 level (U/mL) <37 16 ≥37 57 Postoperative liver metastasis Negative 47 Positive 26
SRPK2 P
Negative
Positive
15
58
12 3
47 11
1.000
9 6
31 27
0.774
12 3
38 20
0.445
4 11
12 46
0.882
3 12
19 39
0.519
10 5
18 40
0.017
14 1
38 20
0.072
14 1
36 22
0.044
4 11
12 46
0.882
9 6
38 20
0.766
It is unquestionable that p53 also plays a crucial role in the drug resistance of various cancers [27–29]. Thus, we investigated whether SRPK2 regulated drug resistance in a p53-dependent manner. Additional CCK8 assays showed that p53 knockdown significantly reversed the enhanced chemosensitivity induced by gemcitabine and oxaliplatin when SRPK2 was silenced (Fig. 9).
Discussion
p53 knockdown reversed the decrease in cell migration and invasion induced by SRPK2 silencing after treatment with oxaliplatin It is well known that p53 acts as a critical regulator in the malignant biology of various cancers [25,26]. Thus, we investigated whether SRPK2 promoted cell migration and invasion in a p53-dependent manner in PC cells. To induce p53 activation, transfected cells by lentivirus and siRNA were first pretreated with oxaliplatin for 6 h. Cell migration and invasion in the sgSRPK2 group were significantly decreased compared with that in the scrambled RNA group. However, p53 knockdown significantly reversed the decrease induced by silencing SRPK2 in Capan-2 cells (Fig. 8A,B). Similar results were found in SW1990 cells (Fig. 8C,D). Therefore, SRPK2 promoted migration and invasion in a p53-dependent manner in PC cells.
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p53 knockdown reversed the increased chemosensitivity induced by SRPK2 silencing in PC cells
SRPK2 phosphorylates SR proteins and plays an essential role in pre-mRNA alternative splicing, which may be linked to various diseases, including cancer, if splicing dysfunctions occur. Previous studies have reported that SRPK2 contributes to the development of haematologic and solid cancers by various signalling pathways. SRPK2 promotes the proliferation of leukaemia cells by upregulating the expression of Cyclin A1 [14]. Additionally, N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340), a SRPK inhibitor, has antileukaemia effects [30,31]. The elevated expression of SRPK2 promotes the growth and migration of colon cancer by activating ERK signalling [17]. SRPK2 knockdown elevates Numb expression, which inhibits proliferation, migration and invasion in HCC [22]. However, the definite role of SRPK2 in the development of PC is rarely reported to our knowledge. However, in a prior study, we found that Numb can prevent the ubiquitination-dependent degradation of wtp5 and increase chemosensitivity in a p53-dependent manner in PC [21]. Thus, we infer a close relationship between the SRPK2, Numb and p53 proteins in the development and progression of PC, which has not been previously investigated to our knowledge. We first observed the overexpression of SRPK2 in PC tissues, which was associated with tumour T stage, UICC stage and poor PC patient prognosis. In prostate cancer [18], the high expression of SRPK2 was significantly correlated with advanced pathological stage and a higher Gleason Score. Additionally, SRPK2 promoted cell invasion, migration and drug resistance in PC cells, in accordance with the findings of Radhakrishnan et al. [16], Wang et al. [17] and Lu et al. [22] in head and neck squamous cell carcinoma, colon cancer and hepatocellular carcinoma, respectively. Taken together, SRPK2 promoted the development and progression of PC.
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Table 2. Univariate and multivariate analysis of clinicopathological factors for survival in 73 postoperative PC patients.
Parameters Age (≤65/>65 years) Gender (male/female) Tumour location (head/body-tail) Tumour size (<3/≥3 cm) Well/Moderate and poor differentiation T stage (T1 + T2/T3 + T4) Lymph nodes metastasis (N0/N1) UICC stage (I + IIA/IIB + III) Pretherapeutic CA19-9 level (<37 UmL1/≥37 UmL1) Postoperative liver metastasis (negative/positive) SRPK2 (negative/positive)
Median survival (days)
Univariate analysis P (log rank)
257/239 270/219 296/234 270/256 296/256 369/249 310/180 315/180 270/256
0.642 0.455 0.089 0.447 0.555 0.010 < 0.001 < 0.001 0.731
299/200 480/249
Table 3. Correlation analysis of the relationship between SRPK2 with Numb and p53. SRPK2
Parameter Numb Negative (n = 23) Positive (n = 17) p53 Negative (n = 19) Positive (n = 21)
Negative (n = 9)
Positive (n = 31)
2 7 6 3
r rank
P
21 10
0.385
0.023
13 18
0.207
0.265
Mutations in the p53 gene are frequently detected in human cancers [32,33]. The activation of mtp53 promotes prosurvival signals and tumourigenesis, but does not result in the loss of wtp53 [34,35]. We first demonstrated that SRPK2 had no association with mtp53 in PC tissues. In the wtp53 PC cell line without stimulus, silencing or overexpressing SRPK2 alone altered Numb but not wtp53 expression. Thus, the enhanced expression of Numb induced by SRPK2 silencing does not change the basal p53 levels in the absence of external stress. Gemcitabine and oxaliplatin are widely used as chemotherapeutic agents for PC cells [36,37]. In oxaliplatin-treated wtp53 PC cells, wtp53 protein was significantly upregulated, and silencing or overexpressing SRPK2 induced an increase or decrease, respectively, in Numb and p53 levels. Furthermore, these 3 endogenous proteins could be coimmunoprecipitated as a triple complex. These findings suggest a close relationship between SRPK2 and Numb as well as p53, which
Multivariate analysis hazard ratio (95% CI)
P
1.251 (0.553–2.829) 1.231 (0.155–9.785) 2.983 (0.359–24.769)
0.590 0.845 0.311
0.007
1.945 (0.946–4.000)
0.070
0.011
2.594 (1.021–6.587)
0.045
might play an important role in the progression of PC. Generally, Numb contains a phosphotyrosine binding (PTB) domain that interacts with p53 [38]. Numb controls the function of p53 by interfering with MDM2dependent ubiquitination and degradation [20]. Based on these findings, SRPK2 downregulates the chemical agent-induced level of activated wtp53 by reducing Numb protein expression. Additionally, Numb knockdown significantly reversed the elevation of wtp53 protein levels induced by silencing SRPK2 in response to chemical agents. Moreover, our previous study found that Numb knockdown decreased chemosensitivity in a p53-dependent manner [21]. Taken together, SRPK2 negatively regulates wtp53 protein levels by downregulating Numb in the presence of chemical agents. Wtp53 is the terminal target protein of SRPK2. Undoubtedly, p53 plays an important role in the malignant progression and drug resistance in various cancers. We found that silencing SRPK2 decreased cell invasion and migration in PC cells, and increased chemosensitivity to gemcitabine and oxaliplatin, all of which were significantly reversed by p53 silencing. To date, the molecular mechanism of SRPK2-mediated drug resistance remains unclear. SRPIN340, the SRPK inhibitor, exhibited an antileukaemia effect by altering the expression of the MAP2K1, MAP2K2, VEGF and FAS genes [36,37]. For the first time, we demonstrated that SRPK2 promoted malignant biology and drug resistance of PC in a p53-dependent manner. In summary, SRPK2 promotes the development and progression of PC, and negatively regulates wtp53 expression by downregulating Numb protein expression in response to chemical agents. SRPK2 promotes the malignant biology and drug resistance of PC in a
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Fig. 3. The association of SRPK2, Numb, p53 and p21 in wtp53 Capan-2 and SW1990 cell lines. (A, B) Numb, p53 and p21 protein levels in sg-SRPK2 and scrambled RNA-transfected Capan-2 (A) and SW1990 (B) cells, with or without oxaliplatin treatment. (C, D) Numb, p53 and p21 protein levels in SRPK2-GFP and empty plasmid-transfected Capan-2 (C) and SW1990 (D) cells, with or without oxaliplatin treatment. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 vs. control.
Fig. 4. The close interaction between SRPK2, Numb and p53 in wtp53 PC cell lines, without or with oxaliplatin treatment. (A, B) SRPK2 coimmunoprecipitation with Numb and p53 without (A) or with (B) oxaliplatin treatment in Capan-2 cells. (C, D) SRPK2 coimmunoprecipitation with Numb and p53 regardless of whether the Numb (C) or p53 (D) antibody was used in Capan-2 cells. (E, F) SRPK2 coimmunoprecipitation with Numb and p53 without (E) or with (F) oxaliplatin treatment in SW1990 cells. (G, H) SRPK2 coimmunoprecipitated with Numb and p53 regardless of whether the Numb (G) or p53 (H) antibody was used in SW1990 cells. The input and IgG lanes show the positive and negative controls respectively.
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Fig. 5. Cell migration and invasion assays in PC cells. (A, B) Cell migration (A) and invasion (B) assays in sg-SRPK2 and scrambled RNAtransfected Capan-2 cells respectively. (C, D) Cell migration (C) and invasion (D) assays in SRPK2-GFP- and empty plasmid-transfected Capan-2 cells respectively. (E, F) Cell migration (E) and invasion (F) assays in sg-SRPK2 and scrambled RNA-transfected SW1990 cells respectively. (G, H) Cell migration (G) and invasion (H) assays in SRPK2-GFP- and empty plasmid-transfected SW1990 cells respectively. Cells were plated in upper modified Boyden chamber for 24 h. The photos of cells that had migrated or invaded to the bottom chambers were obtained with a microscope. The cells were counted under a microscope at 920 magnification and in five random fields per insert assay. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 compared with the control.
p53-dependent manner. Future research should further investigate the molecular mechanism of the interactions among SRPK2, Numb and p53.
of Union for International Cancer Control (UICC) classification.
Cell lines and culture
Materials and methods Tissue samples Specimen collection was approved by the Institutional Review Board of the China Medical University. Each patient enrolled in this study signed a consent form. Seventy-three formalin-fixed and paraffin-embedded pancreatic ductal adenocarcinoma (PDAC) and paired noncancerous tissues were collected from patients between 2006 and 2017 at the First Hospital of China Medical University. Furthermore, 24 cases of paired tumour and adjacent nontumour pancreatic tissues were used for protein extraction and RNA isolation. Patients with invasive intraductal papillary mucinous carcinoma, acinar cell carcinoma and endocrine carcinoma were excluded from the study. All diagnoses were confirmed by histopathological evaluation, and the data were analysed on the basis of the 8th edition
The Wtp53 Capan-2 cell line [39] was obtained from the American Type Culture Collection (ATTC; Manassas, VA, USA), and the wtp53 SW1990 cell line [39] was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the recommended growth medium supplemented with 10% FBS ( HyClone, Logan, UT, USA), and maintained at 37 °C in a humidified incubator under 5% CO2 atmosphere.
Immunohistochemistry assays Immunohistochemistry was performed as described previously [21,23]. Briefly, tissues were embedded in paraffin and cut into 4-lm consecutive sections. After deparaffinization, antigen retrieval was performed using sodium citrate buffer at a high pressure for 2 min, and cooled for
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Fig. 6. The CCK8 assays in wtp53 PC cells with silenced or overexpressed SRPK2 under chemotherapeutical treatment. (A, B) Silencing SRPK2 significantly enhanced the chemosensitivity of Capan-2 (A) and SW1990 (B) cells under gemcitabine treatment, compared with the scrambled RNA group. (C, D) Silencing SRPK2 significantly enhanced the chemosensitivity of Capan-2 (C) and SW1990 (D) cells under oxaliplatin treatment, compared with the scrambled RNA group. (E, F) SRPK2 overexpression significantly decreased the chemosensitivity of Capan-2 (E) and SW1990 (F) cells under gemcitabine treatment, compared with the empty plasmid group. (G, H) SRPK2 overexpression significantly decreased the chemosensitivity of Capan-2 (G) and SW1990 (H) cells under oxaliplatin treatment, compared with the empty plasmid group. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 compared with the control.
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Fig. 7. Numb or p53 knockdown reversed the increase in wtp53 protein induced by SRPK2 silencing in response to oxaliplatin. (A) The SRPK2, Numb and p53 protein levels in Capan-2 cells with the treatment combinations shown during oxaliplatin treatment. (B) The SRPK2, Numb and p53 protein levels in SW1990 cells with the treatment combinations shown during oxaliplatin treatment. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 compared with the control.
1 h at room temperature. Sections were incubated with 0.3% H2O2 for 15 min to block endogenous peroxidase activity. After blocking with goat serum for 20 min, sections were covered with anti-SRPK2 (Abcam, Cambridge, UK; 1 : 400), anti-Numb (Abcam; 1 : 400) or anti-p53 (Proteintech, Chicago, IL, USA; 1 : 100) overnight at 4 °C. Next, the sections were incubated with a secondary antibody for 15 min. The sections were then incubated with streptavidin–peroxidase reagent for 15 min. The signal was visualized using 3,30 -diaminobenzidine (DAB), and the sections were counterstained with haematoxylin, and then mounted for microscopy. Stained tissue sections were reviewed and scored as described by Masunaga et al. [40]. Staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The extent of staining was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), according to the percentage of the carcinoma that was positively stained. The final Immunohistochemistry staining scores were determined by three pathologists. The sum of the intensity and extent scores was used as the final staining score (0–7), and tumours with a final staining score >2 were considered to have positive expression.
Western blot assays Total cell lysates were harvested from PC tissues and cells. After protein quantification, the samples were separated by
10% SDS/PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary SRPK2 (Abcam), Numb (Abcam), p53 (Proteintech) or GAPDH (Proteintech) antibodies. The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit and anti-mouse (Santa Cruz, CA, UK). Immunoreactive protein bands were visualized using the ECL detection kit (Thermo Scientific, Rockford, IL, USA). Each experiment was repeated three times.
Immunoprecipitation As described previously [21,23], PC cells were lysed in lysis buffer [20 mM Tris/HCl (pH 7.4), 1.0% NP-40, 150 mM NaCl, 1 mM EDTA, 10 lgmL1 leupeptin, 50 lgmL1 PMSF], and the soluble supernatants of protein lysates were isolated by centrifugation at 4 °C. Briefly, magnetic beads (Bio-Rad, Hercules, CA, USA) were preincubated with SRPK2 (Abcam), Numb (Abcam), p53 (Proteintech) or IgG (Santa Cruz) antibodies at 4 °C for 4 h with agitation. The antibody-bead complexes were washed three times with lysis buffer and incubated with the soluble supernatants at 4 °C overnight. Next, the immunocomplexes were washed three times with lysis buffer. After elution in the sample loading buffer by boiling, the samples were separated by SDS/PAGE and subjected to western blot analysis with SRPK2 (Abcam), Numb (Abcam) and p53 (Proteintech) antibodies.
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qRT-PCR analysis Total RNA was extracted from PC tissues using Trizol reagent (Takara Bio, Otsu, Japan), and subjected to qRTPCR using the standard programme as described previously [21]. The primers used were as follows: SRPK2, 50 -GGA GATAGAAGAATTGGAGCGAGAAGC-30 (sense) and 50 -CCTCAGCCGCCTCCTCTAATCC-30 (antisense); GAD PH, 50 -CATGAGAAGTATGACAACAGCCT-30 (sense) and 50 -AGTCCTTCCACGATACCAAAGT-30 (antisense). All reactions were analysed using a Light Cycler 2.0 with the Light Cycler kit (Takara Bio).
Construction of stable cell lines and RNA interference The GV358-SRPK2-GFP plasmid (SRPK2-GFP) and corresponding empty plasmid (GFP) were synthesized and packaged into a lentivector by Genechem (GenePharma Co, Ltd, Shanghai, China). PC cells were transfected with plasmid for 24 h, and screened by puromycin (Sigma, St Louis, MO, USA) twice for 48 h. Lenti-cas9 and lenti-sgRNA were synthesized by Genechem (GenePharma Co). PC cells were infected with lenticas9 and selected by puromycin (Sigma) twice for 48 h.
Fig. 8. p53 knockdown reversed the decrease in cell migration and invasion induced by silencing SRPK2 after treatment with oxaliplatin in wtp53 PC cells. (A, B) Cell migration (A) and invasion (B) assays in Capan-2 cells transfected with the combinations shown. (C, D) Cell migration (C) and invasion (D) assays in SW1990 cells transfected with the combinations shown. Cells were plated in upper modified Boyden chamber for 24 h. The photos of cells that had migrated or invaded to the bottom chambers were obtained with a microscope. The cells were counted under a microscope at 920 magnification and in five random fields per insert assay. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 and **P < 0.01 vs. control.
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Fig. 9. p53 knockdown reversed the increased chemosensitivity induced by SRPK2 silencing in PC cells. (A, B) Chemosensitivity of Capan-2 (A) and SW1990 (B) cells treated with gemcitabine, in the combined protein transfection and expression groups shown. (C, D) Chemosensitivity of Capan-2 (C) and SW1990 (D) cells treated with oxaliplatin, in the combined protein transfection and expression groups shown. Experiments were performed in triplicate. Error bars SD. Statistical significance was determined by Student’s t-test. *P < 0.05 vs. control.
Then, the stable sublines were infected with SRPK2sgRNA (sg-SRPK2) to specifically silence the target gene or an sgRNA control (scramble). All of the siRNAs were synthesized by GenePharma (GenePharma Co, Ltd, Shanghai, China). The target sequences used were as follows: Numb, 50 -CUGGAAA GAAAGCAGUUAATT-30 (sense) and 50 -UUAACUGCU UUCUUUCCAGTT-30 (antisense); p53, 50 -CUACUUC CUGAAAACAACGTT-30 (sense) and 50 -CGUUGUUU UCAGGAAGUAGTT-30 (antisense). Each siRNA sequ ence was selected from three effective sequences as described in our previous studies [21,23]. Targeting sequences for the control siRNA were as follows: 50 -U UCUCCGAACGUGUCACGUTT-30 (sense) and 50 -A CGUGACACGUUCGGAGAATT-30 (antisense). Pooled siRNAs (20 lM) were cotransfected transiently with stably infected cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Effective transfections were successfully verified by western blot analysis, as shown in the ‘Results section’.
Migration and invasion assays The migration of PC cells was evaluated using modified Boyden chamber (BD Biosciences, Sparks, MD, USA)
assays. Briefly, transfected cells were plated in upper chambers with FBS-free growth medium. The lower chambers contained growth media with 10% FBS as a chemoattractant. After 24 h, cells that had migrated to the bottom chambers were stained with crystal violet hydrate (Sigma), and cells that did not migrate were removed from the upper chambers. The invasiveness of cells was determined with a similar method, except the membrane separating the upper and lower chambers was coated with Matrigel (BD Biosciences) prior to the assay. Cell photos were obtained with a microscope (Nikon Microphot-FX, Tokyo, Japan), and the number of cells was counted in five random fields per chamber at 920 magnification.
CCK8 assays The chemosensitivity of transfected cells was determined using CCK8 assays. Briefly, cells were seeded in 96-well plates at 8 9 103 cells per well. After 8 h, the cells were incubated with gemcitabine (Abcam) and oxaliplatin (Abcam) for 48 h at the concentrations shown in the ‘Results section’. Next, 10 lL CCK8 (Dojindo, Kumamoto, Japan) was added and the cells were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm in an ELISA 96well microtitre plate reader (Bio-Rad 680, California,
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USA). Each experimental procedure was performed in triplicate, and the results are shown as the percentage of treated cells compared with control cells.
Statistical analysis Statistical analyses were performed using SPSS 13.0 software (Chicago, IL, USA). Continuous variables were expressed as the mean SD. The expression of SRPK2 in pairs of PDAC and their corresponding noncancerous tissues was compared with paired-sample t-tests. The association between SRPK2 expression and clinicopathological parameters was examined using a Chi-squared test. The relationships between SRPK2, Numb and p53 were analysed using Chi-squared and Spearman correlation tests. The Kaplan– Meier method was used to assess survival, and differences were evaluated by the log-rank test. Cox’s proportional hazards regression model was used to assess independent prognostic factors in a stepwise manner. Student’s t-tests were used to compare the differences in parameters of cell chemosensitivity, cell migration and invasion assays. Differences were considered statistically significant when P values < 0.05.
Acknowledgements We thank the Central Laboratory in the First Hospital of China Medical University for technical support. This work was supported by the Chinese National Science Foundation (No. 81672835 to MD).
Conflict of interest The authors declare no conflict of interest.
Author contributions MD and GW started the project, performed the research and analysed the data. WS and JZ collected the data. GW, XS and XL performed the statistical analyses and wrote the manuscript. GW, JZ and MD contributed to data analysis and revised the manuscript.
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