SRPK1/AKT axis promotes oxaliplatin-induced anti-apoptosis via NF-κB activation in colon cancer

Colorectal cancer is the third most common diagnosis. Oxaliplatin is used as first-line treatment of colon cancer. However, oxaliplatin resistance greatly reduces its therapeutic effect. SRPK1 involves in pre-mRNA splicing and tumorigenesis. How SRPK1 mediates drug resistance in colon cancer is unknown. The expression of SRPK1 was analyzed in the TCGA and the CPTAC pan-cancer samples and detected in colon cancer cell lines and tissues by IHC and western blot. The MTT and TUNEL assay were used to verify the anti-apoptosis ability of colon cancer cell. The activation of NF-κB was determined by luciferase assay and qRT-PCR. AKT, IKK, IκB and their phosphorylation level were verified by western blot. We found that SRPK1 expression was the second highest in TCGA and the CPTAC pan-cancer samples. The mRNA and protein levels of SRPK1 were increased in tissues from patients with colon cancer. SRPK1 was associated with clinical stage and TNM classifications in 148 cases of colon cancer patients. High SRPK1 levels correlated with poor prognosis (p < 0.001). SRPK1 overexpression enhanced the anti-apoptosis ability of colon cancer cells, whereas SRPK1 silencing had the opposite effect under oxaliplatin treatment. Mechanistically, SRPK1 enhances IKK kinase and IκB phosphorylation to promote NF-κB nuclear translocation to confer oxaliplatin resistance. Our findings suggest that SRPK1 participates in colon cancer progression and enhances the anti-apoptosis capacity to induce drug resistance in colon cancer cells via NF-κB pathway activation, and thus might be a potential pharmaceutically target for colon cancer treatment.

cancer types [29], which provides a potential strategy for reversing chemoresistance by targeting the NF-κB pathway.
In the present study, we found that SRPK1 is overexpressed in tissues from patients with colon cancer and in colon cancer cells. SRPK1 overexpression was significantly associated with clinical stage and the tumor-nodemetastasis (TNM) classification of paraffin embedded sections from patients with colon cancer' . Upregulation of SRPK1 expression increased the anti-apoptosis ability, whereas downregulation of SRPK1 expression increased the pro-apoptotic sensitivity of colon cancer cells. Moreover, we found that SRPK1 could enhance IKK and IκB phosphorylation to promote NF-κB translocation from the cytoplasm to the nucleus and activate the NF-κB pathway. Furthermore, AKT phosphorylation increased in SRPK1 overexpression cell lines. Conversely, silencing SRPK1 decreased AKT phosphorylation. In addition, the phosphorylation of IKK and IκB were inhibited by an AKT inhibitor in SRPK1 overexpression colon cancer cell lines. Taken together, these findings reveal that aberrant SRPK1 expression activates the NF-κB pathway via AKT, which participates in the anti-apoptosis process of colon cancer. Thus, our findings suggest that SRPK1 is significantly associated with colon cancer and might be a potential pharmaceutical target to treat colon cancer.

Cell culture
Colon cancer cell lines (SW620, LOVO, HCT-8, HT-29, SW480 and HCT-116) and 293 T cell were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the human normal intestinal epithelial cell line NCM-460 was obtained from Dr. Ying Ying (Shenzhen University, Shenzhen, China). All cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (15,140,122, Gibco) at 37 °C in a 5% CO 2 atmosphere. We used 0.25% trypsin (25,200,056,Gibco) to passage the cell lines when they grew to 90% confluence. All cell lines used in this study were authenticated using the short tandem repeat (STR) method (Additional file 1: Table S4) and verified as being free of mycoplasma contamination using polymerase chain reaction (PCR).

RNA extraction, reverse transcription, and qRT-PCR analysis
Total RNA from colon cancer cells was extracted using RNAiso Plus (9109, Takara, Shiga, Japan). Real-time PCR was performed using a Real-Time system (CFX96, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression data were normalized to the geometric mean of the expression of the housekeeping gene GAPDH and calculated using the 2 − ΔΔCt method [30]. The primer sequences are listed in Additional file 1: Table S6.

Patients, tissue specimens and immunohistochemistry (IHC) staining
This study was conducted on a total of 148 paraffinembedded colon cancer specimens, which were acquired from patients diagnosed from 2006 to 2010. Patient consent and approval from the Institutional Research Ethics Committee (No. 2018003) were obtained to use these clinical materials for research purposes. The tumors were staged according to the 7th edition of the Cancer Stage Manual written by the American Joint Committee on Cancer (AJCC) [31]. A section of the excised tumor was analyzed by IHC and H&E staining as described previously [32].

Dual-luciferase reporter assay
Stable cell lines infected with retroviruses were seeded in DMEM supplemented with 10% FBS in triplicate in 24-well plates (5 × 10 4 cells per well) and cultured for 12 h. The NF-kB luciferase reporter gene (pGL4.32 [luc2 NF-kB-RE Hyrgo], Promega, Madison, WI, USA) and pRL-TK Renilla plasmid were co-transfected into cells using Lipofectamine 3000 reagent (Invitrogen). At 48 h after transfection, dual luciferase reporter gene detection was performed using a Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer's protocol [33].

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay
Cells were seeded in DMEM supplemented with 10% FBS in triplicate in 96-well plates (5 × 10 3 cells per well) and cultured for 12 h. The medium was replaced with medium containing different concentrations of oxaliplatin and incubated for 24 h. According to CellTiter96 ® AQueous One Solution Cell Proliferation Assay Kit (Promega) instruction, the reagents were added to the culture medium, incubated for 1-4 h, and the absorbance value at 490 nm was read using a microplate reader. IC 50 values were calculated using GraphPad Prism software (version 7, GraphPad Software, Inc., San Diego, CA, USA) via nonlinear regression (curve fit) using the oxaliplatin concentration vs. the normalized response (variable slope) method.

Immunofluorescence assay
The immunofluorescence assay was performed as described previously [32]. Briefly, the cells were incubated with a primary monoclonal anti-p65 (1:100) antibody overnight at 4 °C and then incubated with a fluorescein-conjugated goat anti-rabbit secondary antibody (ab150077, Abcam) for 1 h at room temperature. The cells were visualized under a Dragonfly laser scanning confocal microscopy system (Andor, Belfast, UK).

Nuclear extract preparation
The indicated cells were washed with 5 mL PBS containing a protease inhibitor cocktail (B14011, Bimake, Houston, TX, USA) and a phosphatase inhibitor cocktail (B15001, Bimake) before adding 3 mL ice-cold PBS containing the same protease and phosphatase inhibitor cocktails. The cells were transferred to a pre-chilled 15-mL conical tube and centrifuged for 5 min at 200×g at 4 °C. A Nuclear Extract kit (40,010, Active Motif, Rixensart, Belgium) was then used to isolate the nuclear extracts from the cell pellets, according to the manufacturer's instructions.

Statistical analysis
All statistical analyses were carried out using SPSS version 22.0 statistical software (IBM Corp., Armonk, NY, USA). Comparisons between groups were performed using the two-tailed Mann-Whitney U-test. The relationship between SRPK1 expression and clinicopathological characteristics was assessed using Spearman's correlation test. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Survival data were evaluated using uni-and multivariate Cox regression analyses. Bivariate correlations between variables were calculated using Spearman's rank correlation coefficients. A p-value of less than 0.05 was considered statistically significant in all cases.

SRPK1 is overexpressed and associated with clinical stage, TNM classification, and prognosis of survival in patients with colon cancer
By analyzing the expression of SRPK1 in the Cancer Genome Atlas (TCGA) database, we found that the SRPK1 mRNA expression was the second highest in colon cancer, which was only lower than that in rectum adenocarcinoma (READ) among 33 types of cancer, and the SRPK1 mRNA expression was increased in the TCGA colon cancer tissues (n = 286) compared with that in normal tissues (n = 41) (p < 0.001) (Fig. 1a, b). In addition, the protein levels of SRPK1 in colon cancer were the second highest in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database, which was only lower than that in Ovarian serous cystadenocarcinoma (OV),and compared with that in normal tissues (n = 100), SRPK1 was also upregulated in CPTAC colon cancer tissues (n = 97) (p < 0.001) (Fig. 1c, d). Taken together, in colon cancer, SRPK1 was increased at both the mRNA and protein levels, and SRPK1 was highly expressed over other different cancer types. These findings suggested that SRPK1 might be a key oncoprotein in colon cancer. Next, we confirmed the findings from the public databases in six colon cancer cell lines (Fig. 1e, Additional file 2: Fig. S1a ), five colon cancer tissues (Fig. 1f, Additional file 2: Fig. S1b) and 148 paraffin-embedded, archived colon cancer tissues (Fig. 1g). Statistical analyses of IHC-stained sections showed that SRPK1 was overexpressed with increasing clinical stage of colon cancer (Fig. 1h) (Fig. 1i and Table 1). SRPK1 protein expression was strongly associated with the clinical stage (p < 0.001), as well as the T (p < 0.001), N (p = 0.005), and M (p = 0.006) classifications ( Fig. 1i and Additional file 1: Table S1). Spearman correlation analysis and univariate and multivariate analysis showed that SRPK1 protein expression correlated significantly with clinicopathological factors, including clinical stage and TNM classification (Additional file 1: Table S2 and S3).

Upregulated SRPK1 promotes the anti-apoptosis ability of colon cancer cell lines
To determine the role of SRPK1 in colon cancer, we stably overexpressed or inhibited SRPK1 in SW480 and HCT-116 cells (Fig. 3a, Additional file 3: Fig.  S2a). MTT assay was performed to evaluate the IC50 of oxaliplatin. SRPK1 overexpression increased the oxaliplatin IC 50 value (Fig. 3b), whereas SRPK1 downregulation decreased the IC 50 value (Fig. 3b). TUNEL assays also revealed that the percentage of TUNEL + cell among SW480 and HCT-116 SRPK1 overexpressing cells decreased compared with that in vector-transduced cells at each concentration, whereas the opposite effect was obtained in SRPK1 knock-down SW480 and HCT-116 cells (Fig. 3c). These data indicated that SRPK1 overexpression in these cells exhibited an obvious anti-apoptosis effect (Fig. 3c). Consistently, SRPK1knockdown led to apoptosis (Fig. 3c). Furthermore, we observed that and Bcl-xL levels increased, whereas Bcl-xS and cleaved PARP1 levels decreased, in SRPK1-overexpressing colon cancer cells (Fig. 3d, Additional file 3: Fig. S2b, c), and the opposite effect was observed in SRPK1 knockdown cell lines (Fig. 3d, Additional file 3: Fig. S2b, c). These results showed that elevated SRPK1 expression could enhance the anti-apoptosis capacity of colon cancer cells, whereas silencing SRPK1 expression enhanced apoptosis. Taken together, we demonstrated that SRPK1 increased anti-apoptosis capacity in colon cancer cell lines.

SRPK1 increased the phosphorylation of AKT to activate the NF-κB pathway
Both the in vitro and in vivo data presented above suggested that elevated levels of SRPK1 might enhance anti-apoptosis in colon cancer cells. Thus, we sought to determine the mechanisms underlying the antiapoptosis effect. Fu et al. reported that SRPK1 could interact with AKT to promote its phosphorylation [35]. Thus, we speculated SRPK1 could be involved in colon cancer pathway activation by enhancing AKT phosphorylation. Indeed, the level of phosphorylated AKT was upregulated in SRPK1 overexpressing SW480 and HCT-116 cells, while SRPK1 silencing decreased the level of phosphorylated AKT (Fig. 4a, Additional file 4: Fig. S3a). The NF-κB pathway, which is directly downstream of AKT, had been reported to regulate proteins that inhibit apoptosis, which is associated markedly with cancer [24]. Here, we performed a luciferase reporter assay and found that SRPK1 overexpression increased the transcriptional activity of the NF-κB pathway in SW480 and HCT-116 cells, whereas, the opposite results were obtained in SRPK1-silenced SW480 and HCT-116 cells (Fig. 4b). Analysis by qPCR also revealed that the levels of NF-κB pathway downstream target genes were markedly increased in SW480 and HCT-116 SRPK1-overexpressing cells and reduced in SW480 and HCT-116 SRPK1-silenced cells (Fig. 4c), suggesting that SRPK1 increases AKT phosphorylation to promote NF-κB pathway activation. Mechanistically, we further observed that SRPK1 overexpression promoted p65 nuclear translocation in SW480 and HCT-116 cells, whereas SRPK1 knockdown inhibited p65 nuclear translocation in SW480 and HCT-116 cells, compared with that in control cells (Fig. 4d, e, Additional file 4: Fig. S3b). In addition, we observed that oxaliplatin could decrease the level of phosphorylated AKT and the nuclear location of p65 (Additional file 5: Fig. S4). However, when the SRPK1 was overexpressed, oxaliplatin failed to regulate the level of phosphorylated AKT and p65 (Additional file 2: Fig. S1). These results indicated that SRPK1 could promote p65 translocation to activate the NF-κB pathway against oxaliplatin treatment.

SRPK1 induces IκB phosphorylation dependent on AKT activation
Among on NF-κB pathway, IKK is a downstream target of AKT, and when IKK and IκB are phosphorylated, NF-κB would be released, which activates the NF-κB pathway [26][27][28]. Thus, we detected the phosphorylation of IKKβ and IκBα. Immunoblotting analyses showed increased levels of phosphorylated IKKβ and IκBα in SRPK1 overexpressing SW480 and HCT-116 cells, whereas SRPK1 silencing decreased the levels of phosphorylated IKKβ and IκBα (Fig. 5a, Additional file 6: Fig. S5a, b). We then assessed whether SRPK1-mediated activation of the NF-κB pathway was AKT dependent. We demonstrated decreased levels of phosphorylated IκBα under AKT inhibitor treatment (Fig. 5b, Additional file 6: Fig. S5c, d) in cells overexpressing SRPK1 or control group, suggesting that SRPK1 activates the NF-κB pathway dependent on AKT. Collectively, these results indicated that elevated SRPK1 expression conferred an anti-apoptosis ability by activating the NF-κB pathway by increasing the phosphorylation of AKT in colon cancer (Fig. 5c).

Discussion
In the present study, the expression of SRPK1 in paraffinembedded tissues from 148 patients with colon cancer was greatly associated with clinical pathology stage and patient survival prognosis, which suggested that SRPK1 could be a potential biomarker and therapeutic target for colon cancer diagnosis, prognosis, and therapy. SRPK1 is maintained in the cytoplasm with chaperone HSP70/HSP90 and translocates to the nucleus to perform alternative splicing [36]. Bcl-X is a well-known apoptosis regulator modulated by alternative splicing. Anti-apoptosis protein Bcl-xL and pro-apoptosis protein Bcl-xS are the two inverse function isoforms formed from the BCL2L1 (Bcl-X) gene alternative splicing process [37]. However, whether SRPK1 could work as an oncoprotein to induce apoptosis via activation of key pathways is unclear. In the present study, Bcl-xL translation levels increased consequent to SRPK1 expression levels, which suggest that SRPK1 might be involve in the apoptosis process by participating in upstream pathway activation, besides its role as an SR splicing factor of Bcl-X splicing.
Oxaliplatin represent a major class of chemotherapeutic drugs in cancer cells [2]. It has been reported that NF-κB pathway may play an important role in tumorigenesis and the level of activation of NF-κB pathway is associated with chemotherapy effect [38], suggesting that combined NF-κB inhibitor with chemotherapy drugs might further suppress colon cancer proliferation. In addition, SRPK1 promotes cancer by regulating AKT phosphatase to induce AKT dephosphorylation by interacting with PHLPP1, which could dephosphorylate p-AKT, lead to AKT constitutive activation suggesting that SRPK1 plays a key role in signaling transduction [35]. Taken together, we assumed that SRPK1 overexpression might increase AKT phosphorylation to induce NF-κB pathway activation to enhance the ability of anti-apoptosis in colon cancer cells against oxaliplatin treatment.
Our data showed that after oxaliplatin treatment, the phosphorylation of AKT and the accumulation of NF-κB were both decreased in SW480 cell, this phenomenon was not observed in SRPK1 overexpression stable cell lines, suggesting that SRPK1 could maintain the AKT phosphorylation and the accumulation of NF-κB under oxaliplatin treatment (Additional file 2: Fig. S1). Furthermore, the data presented here show that SRPK1 participates in the activation of the NF-κB pathway by increasing IKK phosphorylation. IKK is a downstream target of AKT, which consists of the kinase subunits IKKα and IKKβ. Only when IKK phosphorylates IκB, could NF-κB be release from IκB's binding to accumulate in the nucleus and execute its biological function of increasing the transcription of its downstream target genes, including anti-apoptosis genes [26][27][28]. We found that the levels of phosphorylated AKT, IKK, and IκB increased in SRPK1 overexpressing colon cancer cell lines, which contribute to the activation of the downstream NF-κB pathway. These findings showed that SRPK1 could transduce anti-apoptosis signals directly as a downstream target of AKT to activate the NF-κB pathway, which suggested that SRPK1 participates in the anti-apoptosis process via the NF-κB pathway by activating AKT. Therefore, identifying new molecules that target in NF-κB pathway might contribute to overcoming chemoresistance, which is an ongoing project in our laboratory. Furthermore, targeting SRPK1's kinase activity would not suppress tumorigenesis completely; therefore, SRPK1's non-splicing-kinase activity should be considered in clinical treatment, which will help to better understanding chemoresistance.

Conclusions
Our finding suggested that SRPK1 enhances the antiapoptosis ability of colon cancer via the NF-κB pathway by activating AKT. In addition, specific inhibition of SRPK1 might represent a potential anti-drug resistance therapy, and SRPK1 might also be a prognostic biomarker for oxaliplatin resistance in colon cancer. Targeting SRPK1 might enhance the sensitivity of patients to oxaliplatin. . c A model of how SRPK1 enhances the anti-apoptosis ability of colon cancer via the NF-κB pathway by activating AKT. *p < 0.05, **p < 0.01, ***p < 0.001. SRPK1 Serine-arginine protein kinase 1, AKT protein kinase B, IκB inhibitor of nuclear factor kappa B, IKKβ IκB kinase beta subunit, IκBα IκB alpha subunit, NF-κB nuclear factor kappa B