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FOXO1 pathway in renal cell carcinoma cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells Zhenzhen Kong a, 1, Xinming Guo b, 1, Zhijian Zhao a, 1, Weizhou Wu a, Lianmin Luo a, Zhiguo Zhu a, Shanfeng Yin a, Chao Cai a, Wenqi Wu a, Ding Wang c, Yongda Liu a, **, Xiaolu Duan a, * a
Department of Urology, Minimally Invasive Surgery Center, The First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Guangzhou Institute of Urology, Guangzhou, China Department of Pharmacy, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China c The Third Affiliated Hospital of Guangzhou Medical University, Key Laboratory for Major Obstetric Diseases of Guangdong Province, Guangzhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 September 2019 Received in revised form 18 September 2019 Accepted 18 September 2019 Available online xxx
Serine palmitoyltransferase long chain-1 (SPTLC1), which is the rate-limiting enzyme for sphingolipid biosynthesis, has been indicated to be essential for carcinoma cell survival and proliferation in recent, but its role in the regulation of renal cell carcinoma (RCC) remains unknown. In the present study, we found that SPTLC1 expression was significantly decreased in RCC tissues compared to non-tumor tissues, and low SPTLC1 expression was associated with poor overall survival of RCC patients. In addition, our results revealed that forced expression of SPTLC1 could significantly inhibit cell growth in vitro and in vivo via, at least in part, modulating Akt/FOXO1 signaling pathway, thus representing a novel role of SPTLC1 in the regulation of tumor growth in RCC for the first time. © 2019 Elsevier Inc. All rights reserved.
Keywords: SPTLC1 Akt/FOXO1 pathway Cell growth Renal cell carcinoma
1. Introduction Renal cell carcinoma (RCC) is one of the most conmen malignancies in the world and the most frequent type of RCC is clear cell renal cell carcinoma (ccRCC), which accounts for about 75% of all primary kidney cancers. Despite improved techniques greatly promote the advances in diagnosis and treatments, therapeutic options for RCC remains be limited due to its resistance to chemotherapy and radiotherapy, as well as the low efficiency and toxicity of immunotherapy, especially for the patients are diagnosed with metastatic disease [1e4]. Further revealing the exact underlying mechanism and developing efficient therapeutic methods for RCC still are big challenges to date. SPTLC1 is one of the two major subunits of Serine
* Corresponding author. Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, 510230, China. ** Corresponding author. Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, 510230, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Duan). 1 These authors contribute this work equally.
palmitoyltransferase (SPT), which is the rate-limiting enzyme for sphingolipid biosynthesis [5]. Previous studies have revealed that sphingolipids play an important role in cell membrane formation, signal transduction and plasma lipoprotein metabolism, its dysregulated metabolism usually promotes the cancer cell survival, invasion and metastases, as well as therapy resistance [6e11]. Remarkably, SPT represents distinct effects in the regulation of tumorigenesis and progression in different tumors Some studies have revealed that inhibition of SPT activity can significantly inhibit the proliferations of mouse B16F10 cells and human U87MG cells [12,13]. Lee YS et al. also confirmed that the myriocin, a SPT inhibitor, can inhibit the growth of mouse melanoma. In addition, SPTLC1 activity is significantly increased in endometrial cancer tissues and involved in the regulation of tumor development and chemotherapy tolerance, suggesting that SPT may servers as an oncogene [14]. In contrast, Taouji S et al. demonstrated that inactivation of SPTLC1 could promote the survival of human K562 and LAMA-84 cells, indicating that SPTLC1 also can act as a tumor suppressor [11]. More recently, low SPTLC1 expression was indicated to predict poor outcomes in ccRCC patients [15], but its exact role and underlying mechanism in the regulation of RCC development remain unclear.
https://doi.org/10.1016/j.bbrc.2019.09.073 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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In the present study, we investigated the expression of SPTLC1 in RCC tissues and cell lines, and explored its role and possible underlying mechanism during RCC progress, thus representing a novel potential therapeutic target for RCC treatment. 2. Methods 2.1. Cell culture and drugs All RCC cell lines, including A498, 769-P, 786-O, OSRC-2 and Caki-1, were purchased from ATCC (USA) and cultured as ATCC's suggestion in a humidified incubator containing 5% CO2 at 37 C. HK-2 cells were maintained in Keratinocyte Serum Free Medium (K-SFM) supplemented with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml human recombinant epidermal growth factor (EGF), 1% penicillin/streptomycin and 10 mM Hepes buffer. A498 and OSRC-2 monoclonal cell lines expressing LV5-SPTLC1 (LV-SPT1) or empty vector LV5 were generated and selected in the presence of puromycin (2 mg/mL). 2.2. Lentivirus packaging and cell transfection Short hairpin RNA (shRNA) was synthesized by GenePharma (Shanghai, China) as previously described [16]. The shRNA sequence targeting human FOXO1 was 50 -GCCAGAUGCCUAUACAACATT-3’ (shRNA-FOXO1) and the sequence of unrelated shRNA was 5-TTCTCCGAACGTGTCACGT-3’ (shRNA-NC). The lentivirus expression plasmids containing SPTLC1 and Akt were constructed by GenePharma (Shanghai, China) using LV5 vector, respectively. For transient transfections, cells were seeded in 60-mm dishes and transfected at 70% confluence. The transfections were conducted with shRNAs using Lipofectamine LTX (Invitrogen) or lentivirus soup in the presence of 1 mg/ml polybrene according to the manufacturer's instructions. 2.3. Cell growth assay The cell growth was detected using trypan blue staining, MTS assay and colony formation methods as we previously described [16e18]. Briefly, cells were trypzinized and resuspended in a 1:1 mixture of PBS and 0.5% trypan blue and counted at indicated time points via using a hemocytometer. MTS assay was carried out according to the manufacturer's instruction via using the Cell Titer 96®AQueous One Solution Cell Proliferation Assay kit (Promega). For the colony formation assay, cells were seeded in six-well plates at a density of 2 103 per well and grown in a complete medium for 14 days, then the clones were stained with crystal violet and counted. 2.4. Western blot analysis Western blot analysis was carried out as our previous studies described [16e18]. In brief, cells were lysed in RIPA buffer and equal amounts of protein were separated on a 10% SDS polyacrylamide gel, transferred to a nitrocellulose membrane and immunoblotted with antibodies. The primary antibodies used included antibodies against SPTLC1(Abcam), phospho-Akt (Ser473), Akt, phosphoFOXO1(Ser256), FOXO1 (Cell Signaling Technology) and GAPDH (Santa Cruz). The secondary antibodies were IRDye® 800CW Goat anti-Mouse (or Rabbit) IgG and IRDye® 680RD Goat anti-Mouse (or Rabbit) IgG (LI-COR). The bands were detected via using Odyssey® Clx Imaging Systems and quantified with respect to GAPDH using ImageJ software.
2.5. Correlation test Public data of SPTLC1, FOXO1 and Akt expressions in ccRCC patients were obtained from The Cancer Genome Atlas Project (TCGA) online. After excluding patients with incomplete information or normal types, 493 ccRCC patients’ paired expression data were analyzed in total. 2.6. Nude mice xenograft experiment Twenty 6-week-old male nude mice were purchased from the Experimental Animal Center of Guangdong province (Guangzhou, China) and divided into two groups of five animals each. A total of 100 mL cells (2 106 cells/mL) were subcutaneously injected into the right side of axillary region of each mouse. Tumor size was measured twice a week with a vernier caliper. Tumor volume was calculated by formula 0.524 (length) (width)2. After 4 weeks observed, the mice were sacrificed and the tumors were dissected and weighted. All procedures were performed in accordance with the Animal Management Rules of the Ministry of Health of the People's Republic of China, and were approved by the Animal Care Commission of the First Affiliated Hospital of Guangzhou Medical University. 2.7. Immunohistochemical (IHC) staining Immunohistochemistry was performed on paraffin-embedded tissue sections from xenograft tumor as we described previously [18]. The tissue samples were fixed, paraffin-embedded, sectioned at 4-mm thickness and then stained according to standard IHC protocol. Images were obtained with a PathScope™ 4S scanner (DigiPath, USA). 2.8. Statistical analysis Survival curve was established using the KaplaneMeier method and the significance of difference between the curves was analyzed with the log-rank test. The data are reported as the means ± SD of at least three independent experiments. The mean differences were compared using ANOVA and the Student t-test. A P value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. SPTLC1 expression is decreased in RCC tissues and cell lines At first, we investigated the expression of SPTLC1 in ccRCC tissues and different RCC cell lines. As shown in Fig. 1A and B, a total of 493 ccRCC patients’ expression data (including 72 adjacent nonneoplastic and 493 tumor tissue specimens) of SPTLC1, as well as 72 paired non-neoplastic and tumor tissue specimens, in TGCA database were obtained and analyzed, and the results showed that SPTLC1 expression was significantly decreased in ccRCC tumor tissues compared to that in non-neoplastic tissues. In addition, patients with low SPTLC1 expression had significantly poorer overall survival (OS, P ¼ 0.004) than those with high SPTLC1 expression (Fig. 1C). Furthermore, compared to the normal human renal epithelial cell (HK-2 cell), the expressions of SPTLC1 in RCC cell lines, including A498, 769-P, 786-O, ACHN and OSRC-2, were also obviously decreased (Fig. 1D and E). 3.2. SPTLC1 inhibits the cell growth and clonogenicity of RCC cells As the infinite proliferation is a key factor of cancer progress, then we investigated the effect of SPTLC1 on the cell growth and
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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Fig. 1. SPTLC1 is decreased in RCC and inhibits the cell growth of RCC cells. (A) SPTLC1 expression in ccRCC patients' adjacent non-neoplastic tissues (ANT) and tumor tissues (T). (B) SPTLC1 expression in 72 paired ANT and tumor tissues. (C) KaplaneMeier analysis of overall survival according to the expression of SPTLC1 in ccRCC patients. (D and E) The expressions of indicated proteins in distinct kidney cell lines were detected using Western blot and quantified with respect to GAPDH using ImageJ software. (F) The expressions of the indicated proteins in A498 and OSRC-2 monoclonal cells that stably expressed LV-5 or LV5-SPTLC1 were detected using Western blot. (G) The cell growth curves of A498 and OSRC-2 monoclonal cells that stably expressed LV-5 or LV5-SPTLC1. (H) and (I) A498 and OSRC-2 monoclonal cells were seeded in six-well plate and cultured for 14 days, then cells were fixed and stained. The clone number was counted and expressed as a fold change of the LV5 group. * denotes P 0.05, **denotes P 0.01 and *** denotes P 0.001 and **** denotes P 0.0001.
clonogenicity in A498 and OSRC-2 cells. The results revealed that compared with the control group, forced overexpression of SPTLC1 significantly inhibited the cell growth and colony formation of A498 and OSRC-2 cells, suggesting that SPTLC1 plays an important role in the regulation of cell growth in RCC cells (Fig. 1FeI). 3.3. FOXO1 is required for SPTLC1-induced inhibition of cell growth in RCC cells As FOXO1 is a well-known tumor suppressor and plays a critical role in the regulation of cell cycle arrest and apoptosis [19e21], we then investigated its role in the regulation of SPTLC1-mediated inhibition of cell growth. As shown in Fig. 2A and B, shRNAmediated knockdown of FOXO1 expression markedly attenuated SPTLC1-induced inhibition of cell proliferation in A498 and OSRC2 cells. In addition, colony formation assay also represented similar
results to the cell proliferation detection (Fig. 2C and D). These results indicated that FOXO1 is, at least in part, required for SPTLC1induced inhibition of cell growth in RCC cells. 3.4. Correlation between the expression of SPTLC1 and FOXO1 in human RCC cell lines and tissues To assess the role of FOXO1 in SPTLC1-mediated cell growth, we then investigated their expression levels in RCC tissues and cell lines. As shown in Fig. 3A, Pearson correlation analysis clearly indicated a positive relationship between SPTLC1 and FOXO1 expressions (Pearson's coefficient ¼ 0.49; P value < 0.0001). In addition, similar to the SPTLC1 expression in RCC cells, FOXO1 expression was obviously decreased in RCC cells (except 769-P) than that in HK-2 cells and seemed to be positively correlated with SPTLC1 expression (Fig. 3B and C). Then we detected the effects of
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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Fig. 2. FOXO1 is required for SPTLC1-induced inhibition of cell growth. (A) A498 and OSRC-2 monoclonal cells were transfected with indicated shRNA, 72 h after transfection, the expressions of the indicated proteins were detected using Western blot. (B) Forty-eight hours after transfection, equal amounts of cells were seeded in a 96-well plate and cultured for 3 days, then the cell proliferation was detected using MTS array and expressed as a percent of the control group which transfected with LV5þsh-NC. (C) Colony formation assay was carried out as described in Fig.1 after transfection. (SPT1: SPTLC1). * denotes P 0.05, **denotes P 0.01.
Fig. 3. Akt modulates SPTLC1-induced FOXO1 activation and expression. (A) Correlation between SPTLC1 and FOXO1 expressions in ccRCC tissues. (B and C) The expressions of SPTLC1 and FOXO1 in distinct kidney cell lines were detected and quantified. (D) The expressions of the indicated proteins in A498 and OSRC-2 monoclonal cells were detected using Western blot. (E) A498 and OSRC-2 cells were infected with indicated lentivirus plasmids, respectively. Seventy-two hours after infection, the expressions of the indicated proteins were detected using Western blot. (F) Correlation between SPTLC1 and Akt expressions in ccRCC tissues. (G) Correlation between Akt and FOXO1 expressions in ccRCC tissues. * denotes P 0.05, **denotes P 0.01 and *** denotes P 0.001 vs HK-2.
SPTLC1 on FOXO1 activity and expression in RCC cells. The results revealed that overexpression of SPTLC1 significantly decreased the phosphorylation of FOXO1 and increased its expression, indicating that SPTLC1 may inhibit the cell growth via regulating the activation and expression of FOXO1 in RCC (Fig. 3D).
3.5. SPTLC1 increases FOXO1 activity and expression in an Aktdepended manner As Akt is a critical upstream of FOXO1 that mediating the activity and expression of FOXO1 [22e24], we then detected the effects of
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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SPTLC1 on Akt activation and expression. The results showed that SPTLC1 overexpression could obviously decreased the phosphorylation and expression of Akt (Fig. 3D), suggesting that Akt may involve in SPTLC-mediated FOXO1 activation and expression. To assess the role of Akt in SPTLC1-mediated activation and expression of FOXO1, the lentiviral expression plasmids LV5-Akt and LV5SPTLC1 were transfected alone or co-transfected into RCC cells. As shown in Fig. 3E, compared to the control group that transfected with LV5 vector, LV5-Akt significantly increased the phosphorylation of FOXO1 whereas decreased its expression. In addition, LV5Akt obviously attenuated the effects of LV5-SPTLC1 on the phosphorylation and expression of FOXO1. Furthermore, the expression correlations between the SPTLC1 and Akt, as well as Akt and FOXO1, in RCC tissues were calculated using Pearson correlations coefficients, and the results showed that the correlations between their expressions were significantly negative (Fig. 3F and G). Together, these data suggested that SPTLC1-mediated activation and expression of FOXO1 is, at least in part, Akt dependent. 3.6. SPTLC1 inhibits tumor growth of RCC in vivo Finally, nude mice xenograft tumor model assays were conducted to further confirm the effects of SPTLC1 on RCC cell proliferation in vitro. As shown in Fig. 4A, the tumor volumes were significantly smaller in the mice injected with A498-SPILC1 cells than that injected with A498-LV5 cells at the indicated time points, and the representative image of the tumors excised from each group was shown in Fig. 4B. In addition, the average weight of tumors in A498-SPILC1 group was markedly lower than that in A498LV5 group (Fig. 4C). Furthermore, IHC staining also revealed that FOXO1 expression in the tumors of LV-SPTLC1 group was significantly increased than that in LV5 group, whereas Akt expression was obviously decreased (Fig. 4D). Together, these results suggested that SPTLC1 could inhibit RCC growth via, at least in part, modulating Akt/FOXO1 signaling pathway in vivo.
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4. Discussion In the present study, our investigated the role and its underlying mechanism of SPTLC1 in the regulation of cell growth in RCC. Our data revealed SPTLC1 expression was significantly deceased in RCC tissues and cell lines, and SPTLC1 could inhibit the cell proliferation of RCC cells in vitro and in vivo via, at least in part, regulating the Akt/FOXO1 signaling pathway, thus representing a novel potential mechanism of RCC progress mediated by SPTLC1. As a key enzyme for sphingolipid biosynthesis, SPTLC1 plays an important role in establishing cell polarity and tissue integrity, and contributes to the cell proliferation, survival, therapy resistance, invasion and metastases in some cancers, but its exact role in the RCC progress remains unclear [5,9,10,25e27]. In this study, we investigated the expression of SPTLC1 in RCC tissues and cell lines at first. The results revealed that SPTLC1 expression was obviously decreased in tumor tissues and cell lines, and represented negative correlations with ccRCC patients’ overall survival, suggesting that low SPILC1 expression may predict poor prognosis of ccRCC patients, and these results were good consistent with a recent study which indicated that low SPTLC1 expression is obviously associated with disease progression and poor survival in ccRCC patients [15]. In contrast, SPILC1 expression in Merkel cell carcinoma (MCC) samples was obviously increased than that in non- MCC samples [28], suggesting that the roles of SPILC1 in the regulation of RCC and MCC may be distinct. Then we detected the effect of SPTLC1 on cell proliferation in RCC cells and found that overexpression of SPTLC1 could markedly inhibit cell proliferation and colony formation of RCC cells. These results were similar to SPTLC1’ effects on human K562 and LAMA84 cells [11], indicating that SPTLC1 acts as a tumor suppressor in RCC. In contrast, some studies reported that inhibitor- or siRNAmediated inhibition of SPT could inhibit the proliferation of certain cancer cell lines, including melanoma B16F10 cells, U87MG glioma cells, PL-21 acute myeloid leukemia cells [12,13,29]. These
Fig. 4. SPTLC1 inhibits tumor growth in xenograft mouse model. (A and B) The mean volume of xenograft tumor in A498-SPTLC1 group was obviously smaller than that in A498LV5 group (206.63 ± 69.02 mm3 vs 532.96 ± 57.05 mm3, n ¼ 5). (C) The average weight of xenograft tumors in A498-SPILC1 group was markedly lower than that in A498-LV5 group (0.184 ± 0.052g vs 0.598 ± 0.054g, n ¼ 5). (D) FOXO1 expression was significantly increased whereas Akt expression was markedly decreased in tumor samples of A498-SPTLC1 group compared with the A498-LV5 group. Magnification: 200. **denotes P 0.01 and *** denotes P 0.001.
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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data revealed that the effect of SPTLC1 on cell proliferation is cell context dependent and further study is needed to explore its underlying mechanism. As Akt/FOXO1 signaling pathway plays a critical role in the regulation of cell proliferation and RCC development [30e32], we then investigated its role in the regulation of SPTLC1-mediated cell growth. Consist with our hypothesis, the results indicated that FOXO1 is required for SPTLC1-mediated inhibition of cell growth in RCC cells. In addition, there was a significantly positive correlation between SPTLC1 and FOXO1 expression in RCC tissues, as well as cell lines. These data strongly suggested that FOXO1 plays an important role in SPTLC1-mediated cell growth. Since FOXO1 is a key downstream target of Akt and functionally inhibited by phosphorylated Akt [22,24], we subsequently investigated the role of Akt in the regulation of SPTLC1-mediated FOXO1 phosphorylation and expression. The results revealed that overexpression of SPTLC1 obviously decreased the phosphorylation and expression of Akt, though this data was different from a recent study which indicated that myriocin-induced pharmacological inhibition of SPTLC1 could induce cell apoptosis and inhibit cell proliferation via decreasing Akt phosphorylation in Merkel cell carcinoma cell lines, it should be noticed that the inactivation of SPTLC1 also can inhibit imatinibinduced cell apoptosis in K562 and LAMA-84 cells, suggesting the effect of SPTLC1 on cell survival and proliferation is cancer type dependent [11,28]. In addition, forced expression of Akt could clearly attenuate the effects of SPTLC1 on the phosphorylation and expression of FOXO1. Furthermore, the correlations between SPTLC1 and Akt expressions, as well as Akt and FOXO1 in ccRCC tissues, were significantly negative. These results strongly indicated that SPTLC1-induced inactivation and expression of FOXO1 is, at least in part, mediated by Akt. Unfortunately, previous studies have indicated that sphingolipids ceramide plays a crucial role in regulating SPT-mediated cell proliferation and survival, as well as Akt activation [25,33], the role of ceramide in SPTLC1-mediated RCC cell growth and Akt/FOXO1 signal pathway activation remains unclear and needs to be identified in future studies, this also is the deficiency of this study. In conclusion, our results revealed that SPTLC1 could inhibit cell growth of RCC cells in vitro and in vivo via, at least in part, modulating Akt/FOXO1 signal pathway, thus identifying the role and underlying mechanism of SPTLC1 in the development of RCC for the first time. We hope our findings on SPTLC1-mediated RCC cell proliferation will provide useful information for developing effective therapy methods against RCC.
[4] [5] [6]
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[8] [9] [10]
[11]
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[15]
[16]
[17]
[18]
[19] [20]
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Declarations [22]
The authors declare that they have no conflict of interest. [23]
Acknowledgement This work was financed by grants from the National Natural Science Foundation of China (NO. 81872437, NO. 81602541 and No. 81402430), the Guangzhou Science, Technology and Innovation Commission (No. 201604020001 and No. 201704020193), and the Science and Technology Planning Project of Guangdong Province (No.2017B030314108).
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[25]
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[27]
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Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells Zhenzhen Kong a, 1, Xinming Guo b, 1, Zhijian Zhao a, 1, Weizhou Wu a, Lianmin Luo a, Zhiguo Zhu a, Shanfeng Yin a, Chao Cai a, Wenqi Wu a, Ding Wang c, Yongda Liu a, **, Xiaolu Duan a, * a
Department of Urology, Minimally Invasive Surgery Center, The First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology, Guangzhou Institute of Urology, Guangzhou, China Department of Pharmacy, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China c The Third Affiliated Hospital of Guangzhou Medical University, Key Laboratory for Major Obstetric Diseases of Guangdong Province, Guangzhou, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 September 2019 Received in revised form 18 September 2019 Accepted 18 September 2019 Available online xxx
Serine palmitoyltransferase long chain-1 (SPTLC1), which is the rate-limiting enzyme for sphingolipid biosynthesis, has been indicated to be essential for carcinoma cell survival and proliferation in recent, but its role in the regulation of renal cell carcinoma (RCC) remains unknown. In the present study, we found that SPTLC1 expression was significantly decreased in RCC tissues compared to non-tumor tissues, and low SPTLC1 expression was associated with poor overall survival of RCC patients. In addition, our results revealed that forced expression of SPTLC1 could significantly inhibit cell growth in vitro and in vivo via, at least in part, modulating Akt/FOXO1 signaling pathway, thus representing a novel role of SPTLC1 in the regulation of tumor growth in RCC for the first time. © 2019 Elsevier Inc. All rights reserved.
Keywords: SPTLC1 Akt/FOXO1 pathway Cell growth Renal cell carcinoma
1. Introduction Renal cell carcinoma (RCC) is one of the most conmen malignancies in the world and the most frequent type of RCC is clear cell renal cell carcinoma (ccRCC), which accounts for about 75% of all primary kidney cancers. Despite improved techniques greatly promote the advances in diagnosis and treatments, therapeutic options for RCC remains be limited due to its resistance to chemotherapy and radiotherapy, as well as the low efficiency and toxicity of immunotherapy, especially for the patients are diagnosed with metastatic disease [1e4]. Further revealing the exact underlying mechanism and developing efficient therapeutic methods for RCC still are big challenges to date. SPTLC1 is one of the two major subunits of Serine
* Corresponding author. Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, 510230, China. ** Corresponding author. Kangda Road 1#, Haizhu District, Guangzhou, Guangdong, 510230, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Duan). 1 These authors contribute this work equally.
palmitoyltransferase (SPT), which is the rate-limiting enzyme for sphingolipid biosynthesis [5]. Previous studies have revealed that sphingolipids play an important role in cell membrane formation, signal transduction and plasma lipoprotein metabolism, its dysregulated metabolism usually promotes the cancer cell survival, invasion and metastases, as well as therapy resistance [6e11]. Remarkably, SPT represents distinct effects in the regulation of tumorigenesis and progression in different tumors Some studies have revealed that inhibition of SPT activity can significantly inhibit the proliferations of mouse B16F10 cells and human U87MG cells [12,13]. Lee YS et al. also confirmed that the myriocin, a SPT inhibitor, can inhibit the growth of mouse melanoma. In addition, SPTLC1 activity is significantly increased in endometrial cancer tissues and involved in the regulation of tumor development and chemotherapy tolerance, suggesting that SPT may servers as an oncogene [14]. In contrast, Taouji S et al. demonstrated that inactivation of SPTLC1 could promote the survival of human K562 and LAMA-84 cells, indicating that SPTLC1 also can act as a tumor suppressor [11]. More recently, low SPTLC1 expression was indicated to predict poor outcomes in ccRCC patients [15], but its exact role and underlying mechanism in the regulation of RCC development remain unclear.
https://doi.org/10.1016/j.bbrc.2019.09.073 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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In the present study, we investigated the expression of SPTLC1 in RCC tissues and cell lines, and explored its role and possible underlying mechanism during RCC progress, thus representing a novel potential therapeutic target for RCC treatment. 2. Methods 2.1. Cell culture and drugs All RCC cell lines, including A498, 769-P, 786-O, OSRC-2 and Caki-1, were purchased from ATCC (USA) and cultured as ATCC's suggestion in a humidified incubator containing 5% CO2 at 37 C. HK-2 cells were maintained in Keratinocyte Serum Free Medium (K-SFM) supplemented with 0.05 mg/ml bovine pituitary extract (BPE), 5 ng/ml human recombinant epidermal growth factor (EGF), 1% penicillin/streptomycin and 10 mM Hepes buffer. A498 and OSRC-2 monoclonal cell lines expressing LV5-SPTLC1 (LV-SPT1) or empty vector LV5 were generated and selected in the presence of puromycin (2 mg/mL). 2.2. Lentivirus packaging and cell transfection Short hairpin RNA (shRNA) was synthesized by GenePharma (Shanghai, China) as previously described [16]. The shRNA sequence targeting human FOXO1 was 50 -GCCAGAUGCCUAUACAACATT-3’ (shRNA-FOXO1) and the sequence of unrelated shRNA was 5-TTCTCCGAACGTGTCACGT-3’ (shRNA-NC). The lentivirus expression plasmids containing SPTLC1 and Akt were constructed by GenePharma (Shanghai, China) using LV5 vector, respectively. For transient transfections, cells were seeded in 60-mm dishes and transfected at 70% confluence. The transfections were conducted with shRNAs using Lipofectamine LTX (Invitrogen) or lentivirus soup in the presence of 1 mg/ml polybrene according to the manufacturer's instructions. 2.3. Cell growth assay The cell growth was detected using trypan blue staining, MTS assay and colony formation methods as we previously described [16e18]. Briefly, cells were trypzinized and resuspended in a 1:1 mixture of PBS and 0.5% trypan blue and counted at indicated time points via using a hemocytometer. MTS assay was carried out according to the manufacturer's instruction via using the Cell Titer 96®AQueous One Solution Cell Proliferation Assay kit (Promega). For the colony formation assay, cells were seeded in six-well plates at a density of 2 103 per well and grown in a complete medium for 14 days, then the clones were stained with crystal violet and counted. 2.4. Western blot analysis Western blot analysis was carried out as our previous studies described [16e18]. In brief, cells were lysed in RIPA buffer and equal amounts of protein were separated on a 10% SDS polyacrylamide gel, transferred to a nitrocellulose membrane and immunoblotted with antibodies. The primary antibodies used included antibodies against SPTLC1(Abcam), phospho-Akt (Ser473), Akt, phosphoFOXO1(Ser256), FOXO1 (Cell Signaling Technology) and GAPDH (Santa Cruz). The secondary antibodies were IRDye® 800CW Goat anti-Mouse (or Rabbit) IgG and IRDye® 680RD Goat anti-Mouse (or Rabbit) IgG (LI-COR). The bands were detected via using Odyssey® Clx Imaging Systems and quantified with respect to GAPDH using ImageJ software.
2.5. Correlation test Public data of SPTLC1, FOXO1 and Akt expressions in ccRCC patients were obtained from The Cancer Genome Atlas Project (TCGA) online. After excluding patients with incomplete information or normal types, 493 ccRCC patients’ paired expression data were analyzed in total. 2.6. Nude mice xenograft experiment Twenty 6-week-old male nude mice were purchased from the Experimental Animal Center of Guangdong province (Guangzhou, China) and divided into two groups of five animals each. A total of 100 mL cells (2 106 cells/mL) were subcutaneously injected into the right side of axillary region of each mouse. Tumor size was measured twice a week with a vernier caliper. Tumor volume was calculated by formula 0.524 (length) (width)2. After 4 weeks observed, the mice were sacrificed and the tumors were dissected and weighted. All procedures were performed in accordance with the Animal Management Rules of the Ministry of Health of the People's Republic of China, and were approved by the Animal Care Commission of the First Affiliated Hospital of Guangzhou Medical University. 2.7. Immunohistochemical (IHC) staining Immunohistochemistry was performed on paraffin-embedded tissue sections from xenograft tumor as we described previously [18]. The tissue samples were fixed, paraffin-embedded, sectioned at 4-mm thickness and then stained according to standard IHC protocol. Images were obtained with a PathScope™ 4S scanner (DigiPath, USA). 2.8. Statistical analysis Survival curve was established using the KaplaneMeier method and the significance of difference between the curves was analyzed with the log-rank test. The data are reported as the means ± SD of at least three independent experiments. The mean differences were compared using ANOVA and the Student t-test. A P value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. SPTLC1 expression is decreased in RCC tissues and cell lines At first, we investigated the expression of SPTLC1 in ccRCC tissues and different RCC cell lines. As shown in Fig. 1A and B, a total of 493 ccRCC patients’ expression data (including 72 adjacent nonneoplastic and 493 tumor tissue specimens) of SPTLC1, as well as 72 paired non-neoplastic and tumor tissue specimens, in TGCA database were obtained and analyzed, and the results showed that SPTLC1 expression was significantly decreased in ccRCC tumor tissues compared to that in non-neoplastic tissues. In addition, patients with low SPTLC1 expression had significantly poorer overall survival (OS, P ¼ 0.004) than those with high SPTLC1 expression (Fig. 1C). Furthermore, compared to the normal human renal epithelial cell (HK-2 cell), the expressions of SPTLC1 in RCC cell lines, including A498, 769-P, 786-O, ACHN and OSRC-2, were also obviously decreased (Fig. 1D and E). 3.2. SPTLC1 inhibits the cell growth and clonogenicity of RCC cells As the infinite proliferation is a key factor of cancer progress, then we investigated the effect of SPTLC1 on the cell growth and
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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Fig. 1. SPTLC1 is decreased in RCC and inhibits the cell growth of RCC cells. (A) SPTLC1 expression in ccRCC patients' adjacent non-neoplastic tissues (ANT) and tumor tissues (T). (B) SPTLC1 expression in 72 paired ANT and tumor tissues. (C) KaplaneMeier analysis of overall survival according to the expression of SPTLC1 in ccRCC patients. (D and E) The expressions of indicated proteins in distinct kidney cell lines were detected using Western blot and quantified with respect to GAPDH using ImageJ software. (F) The expressions of the indicated proteins in A498 and OSRC-2 monoclonal cells that stably expressed LV-5 or LV5-SPTLC1 were detected using Western blot. (G) The cell growth curves of A498 and OSRC-2 monoclonal cells that stably expressed LV-5 or LV5-SPTLC1. (H) and (I) A498 and OSRC-2 monoclonal cells were seeded in six-well plate and cultured for 14 days, then cells were fixed and stained. The clone number was counted and expressed as a fold change of the LV5 group. * denotes P 0.05, **denotes P 0.01 and *** denotes P 0.001 and **** denotes P 0.0001.
clonogenicity in A498 and OSRC-2 cells. The results revealed that compared with the control group, forced overexpression of SPTLC1 significantly inhibited the cell growth and colony formation of A498 and OSRC-2 cells, suggesting that SPTLC1 plays an important role in the regulation of cell growth in RCC cells (Fig. 1FeI). 3.3. FOXO1 is required for SPTLC1-induced inhibition of cell growth in RCC cells As FOXO1 is a well-known tumor suppressor and plays a critical role in the regulation of cell cycle arrest and apoptosis [19e21], we then investigated its role in the regulation of SPTLC1-mediated inhibition of cell growth. As shown in Fig. 2A and B, shRNAmediated knockdown of FOXO1 expression markedly attenuated SPTLC1-induced inhibition of cell proliferation in A498 and OSRC2 cells. In addition, colony formation assay also represented similar
results to the cell proliferation detection (Fig. 2C and D). These results indicated that FOXO1 is, at least in part, required for SPTLC1induced inhibition of cell growth in RCC cells. 3.4. Correlation between the expression of SPTLC1 and FOXO1 in human RCC cell lines and tissues To assess the role of FOXO1 in SPTLC1-mediated cell growth, we then investigated their expression levels in RCC tissues and cell lines. As shown in Fig. 3A, Pearson correlation analysis clearly indicated a positive relationship between SPTLC1 and FOXO1 expressions (Pearson's coefficient ¼ 0.49; P value < 0.0001). In addition, similar to the SPTLC1 expression in RCC cells, FOXO1 expression was obviously decreased in RCC cells (except 769-P) than that in HK-2 cells and seemed to be positively correlated with SPTLC1 expression (Fig. 3B and C). Then we detected the effects of
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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Fig. 2. FOXO1 is required for SPTLC1-induced inhibition of cell growth. (A) A498 and OSRC-2 monoclonal cells were transfected with indicated shRNA, 72 h after transfection, the expressions of the indicated proteins were detected using Western blot. (B) Forty-eight hours after transfection, equal amounts of cells were seeded in a 96-well plate and cultured for 3 days, then the cell proliferation was detected using MTS array and expressed as a percent of the control group which transfected with LV5þsh-NC. (C) Colony formation assay was carried out as described in Fig.1 after transfection. (SPT1: SPTLC1). * denotes P 0.05, **denotes P 0.01.
Fig. 3. Akt modulates SPTLC1-induced FOXO1 activation and expression. (A) Correlation between SPTLC1 and FOXO1 expressions in ccRCC tissues. (B and C) The expressions of SPTLC1 and FOXO1 in distinct kidney cell lines were detected and quantified. (D) The expressions of the indicated proteins in A498 and OSRC-2 monoclonal cells were detected using Western blot. (E) A498 and OSRC-2 cells were infected with indicated lentivirus plasmids, respectively. Seventy-two hours after infection, the expressions of the indicated proteins were detected using Western blot. (F) Correlation between SPTLC1 and Akt expressions in ccRCC tissues. (G) Correlation between Akt and FOXO1 expressions in ccRCC tissues. * denotes P 0.05, **denotes P 0.01 and *** denotes P 0.001 vs HK-2.
SPTLC1 on FOXO1 activity and expression in RCC cells. The results revealed that overexpression of SPTLC1 significantly decreased the phosphorylation of FOXO1 and increased its expression, indicating that SPTLC1 may inhibit the cell growth via regulating the activation and expression of FOXO1 in RCC (Fig. 3D).
3.5. SPTLC1 increases FOXO1 activity and expression in an Aktdepended manner As Akt is a critical upstream of FOXO1 that mediating the activity and expression of FOXO1 [22e24], we then detected the effects of
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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SPTLC1 on Akt activation and expression. The results showed that SPTLC1 overexpression could obviously decreased the phosphorylation and expression of Akt (Fig. 3D), suggesting that Akt may involve in SPTLC-mediated FOXO1 activation and expression. To assess the role of Akt in SPTLC1-mediated activation and expression of FOXO1, the lentiviral expression plasmids LV5-Akt and LV5SPTLC1 were transfected alone or co-transfected into RCC cells. As shown in Fig. 3E, compared to the control group that transfected with LV5 vector, LV5-Akt significantly increased the phosphorylation of FOXO1 whereas decreased its expression. In addition, LV5Akt obviously attenuated the effects of LV5-SPTLC1 on the phosphorylation and expression of FOXO1. Furthermore, the expression correlations between the SPTLC1 and Akt, as well as Akt and FOXO1, in RCC tissues were calculated using Pearson correlations coefficients, and the results showed that the correlations between their expressions were significantly negative (Fig. 3F and G). Together, these data suggested that SPTLC1-mediated activation and expression of FOXO1 is, at least in part, Akt dependent. 3.6. SPTLC1 inhibits tumor growth of RCC in vivo Finally, nude mice xenograft tumor model assays were conducted to further confirm the effects of SPTLC1 on RCC cell proliferation in vitro. As shown in Fig. 4A, the tumor volumes were significantly smaller in the mice injected with A498-SPILC1 cells than that injected with A498-LV5 cells at the indicated time points, and the representative image of the tumors excised from each group was shown in Fig. 4B. In addition, the average weight of tumors in A498-SPILC1 group was markedly lower than that in A498LV5 group (Fig. 4C). Furthermore, IHC staining also revealed that FOXO1 expression in the tumors of LV-SPTLC1 group was significantly increased than that in LV5 group, whereas Akt expression was obviously decreased (Fig. 4D). Together, these results suggested that SPTLC1 could inhibit RCC growth via, at least in part, modulating Akt/FOXO1 signaling pathway in vivo.
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4. Discussion In the present study, our investigated the role and its underlying mechanism of SPTLC1 in the regulation of cell growth in RCC. Our data revealed SPTLC1 expression was significantly deceased in RCC tissues and cell lines, and SPTLC1 could inhibit the cell proliferation of RCC cells in vitro and in vivo via, at least in part, regulating the Akt/FOXO1 signaling pathway, thus representing a novel potential mechanism of RCC progress mediated by SPTLC1. As a key enzyme for sphingolipid biosynthesis, SPTLC1 plays an important role in establishing cell polarity and tissue integrity, and contributes to the cell proliferation, survival, therapy resistance, invasion and metastases in some cancers, but its exact role in the RCC progress remains unclear [5,9,10,25e27]. In this study, we investigated the expression of SPTLC1 in RCC tissues and cell lines at first. The results revealed that SPTLC1 expression was obviously decreased in tumor tissues and cell lines, and represented negative correlations with ccRCC patients’ overall survival, suggesting that low SPILC1 expression may predict poor prognosis of ccRCC patients, and these results were good consistent with a recent study which indicated that low SPTLC1 expression is obviously associated with disease progression and poor survival in ccRCC patients [15]. In contrast, SPILC1 expression in Merkel cell carcinoma (MCC) samples was obviously increased than that in non- MCC samples [28], suggesting that the roles of SPILC1 in the regulation of RCC and MCC may be distinct. Then we detected the effect of SPTLC1 on cell proliferation in RCC cells and found that overexpression of SPTLC1 could markedly inhibit cell proliferation and colony formation of RCC cells. These results were similar to SPTLC1’ effects on human K562 and LAMA84 cells [11], indicating that SPTLC1 acts as a tumor suppressor in RCC. In contrast, some studies reported that inhibitor- or siRNAmediated inhibition of SPT could inhibit the proliferation of certain cancer cell lines, including melanoma B16F10 cells, U87MG glioma cells, PL-21 acute myeloid leukemia cells [12,13,29]. These
Fig. 4. SPTLC1 inhibits tumor growth in xenograft mouse model. (A and B) The mean volume of xenograft tumor in A498-SPTLC1 group was obviously smaller than that in A498LV5 group (206.63 ± 69.02 mm3 vs 532.96 ± 57.05 mm3, n ¼ 5). (C) The average weight of xenograft tumors in A498-SPILC1 group was markedly lower than that in A498-LV5 group (0.184 ± 0.052g vs 0.598 ± 0.054g, n ¼ 5). (D) FOXO1 expression was significantly increased whereas Akt expression was markedly decreased in tumor samples of A498-SPTLC1 group compared with the A498-LV5 group. Magnification: 200. **denotes P 0.01 and *** denotes P 0.001.
Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073
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data revealed that the effect of SPTLC1 on cell proliferation is cell context dependent and further study is needed to explore its underlying mechanism. As Akt/FOXO1 signaling pathway plays a critical role in the regulation of cell proliferation and RCC development [30e32], we then investigated its role in the regulation of SPTLC1-mediated cell growth. Consist with our hypothesis, the results indicated that FOXO1 is required for SPTLC1-mediated inhibition of cell growth in RCC cells. In addition, there was a significantly positive correlation between SPTLC1 and FOXO1 expression in RCC tissues, as well as cell lines. These data strongly suggested that FOXO1 plays an important role in SPTLC1-mediated cell growth. Since FOXO1 is a key downstream target of Akt and functionally inhibited by phosphorylated Akt [22,24], we subsequently investigated the role of Akt in the regulation of SPTLC1-mediated FOXO1 phosphorylation and expression. The results revealed that overexpression of SPTLC1 obviously decreased the phosphorylation and expression of Akt, though this data was different from a recent study which indicated that myriocin-induced pharmacological inhibition of SPTLC1 could induce cell apoptosis and inhibit cell proliferation via decreasing Akt phosphorylation in Merkel cell carcinoma cell lines, it should be noticed that the inactivation of SPTLC1 also can inhibit imatinibinduced cell apoptosis in K562 and LAMA-84 cells, suggesting the effect of SPTLC1 on cell survival and proliferation is cancer type dependent [11,28]. In addition, forced expression of Akt could clearly attenuate the effects of SPTLC1 on the phosphorylation and expression of FOXO1. Furthermore, the correlations between SPTLC1 and Akt expressions, as well as Akt and FOXO1 in ccRCC tissues, were significantly negative. These results strongly indicated that SPTLC1-induced inactivation and expression of FOXO1 is, at least in part, mediated by Akt. Unfortunately, previous studies have indicated that sphingolipids ceramide plays a crucial role in regulating SPT-mediated cell proliferation and survival, as well as Akt activation [25,33], the role of ceramide in SPTLC1-mediated RCC cell growth and Akt/FOXO1 signal pathway activation remains unclear and needs to be identified in future studies, this also is the deficiency of this study. In conclusion, our results revealed that SPTLC1 could inhibit cell growth of RCC cells in vitro and in vivo via, at least in part, modulating Akt/FOXO1 signal pathway, thus identifying the role and underlying mechanism of SPTLC1 in the development of RCC for the first time. We hope our findings on SPTLC1-mediated RCC cell proliferation will provide useful information for developing effective therapy methods against RCC.
[4] [5] [6]
[7]
[8] [9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19] [20]
[21]
Declarations [22]
The authors declare that they have no conflict of interest. [23]
Acknowledgement This work was financed by grants from the National Natural Science Foundation of China (NO. 81872437, NO. 81602541 and No. 81402430), the Guangzhou Science, Technology and Innovation Commission (No. 201604020001 and No. 201704020193), and the Science and Technology Planning Project of Guangdong Province (No.2017B030314108).
[24]
[25]
[26]
[27]
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Please cite this article as: Z. Kong et al., SPTLC1 inhibits cell growth via modulating Akt/FOXO1 pathway in renal cell carcinoma cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.073