SCYL 1 BP 1 has Tumor-suppressive Functions in Human Lung Squamous Carcinoma Cells by Regulating Degradation of MDM 2

Murine double minute 2 (MDM2) is involved in cell growth and differentiation through its interaction with other cellular proteins (Uldrijan et al., 2007; Wang et al., 2009). The MDM2 protein level is increased in a significant number of human tumors such as soft tissue sarcomas, osteosarcomas and breast tumors, underscoring its pivotal involvement in the development of human cancer (Cordon-Cardo et al., 1994; Liu et al., 2011; Ma et al., 2012; Wade et al., 2013). MDM2 belongs to the family of RING Finger ubiquitin ligases (Jackson et al., 2000; Huang and Tindall, 2011), and the principal function of MDM2 is to mediate the ubiquitination and proteasome-dependent degradation of the p53 tumor suppressor protein and other growth regulatory proteins (Grossman et al., 1998; Manfredi, 2010; Chansaenroj et al., 2013). Accordingly, the ligase activity of MDM2 can be abolished by mutation of either one of eight cysteine and histidine residues involved in zinc coordination within the RING finger domain. Besides p53, MDM2 also ubiquitinates Numb (Yogosawa et al., 2003; Sczaniecka et al., 2012), β-arrestin binding protein (Shenoy et al., 2001; Kang et al., 2014), histone acetyltransferase Tip60 (Legube et al., 2002) as well as


Introduction
Murine double minute 2 (MDM2) is involved in cell growth and differentiation through its interaction with other cellular proteins (Uldrijan et al., 2007;Wang et al., 2009).The MDM2 protein level is increased in a significant number of human tumors such as soft tissue sarcomas, osteosarcomas and breast tumors, underscoring its pivotal involvement in the development of human cancer (Cordon-Cardo et al., 1994;Liu et al., 2011;Ma et al., 2012;Wade et al., 2013).
MDM2 belongs to the family of RING Finger ubiquitin ligases (Jackson et al., 2000;Huang and Tindall, 2011), and the principal function of MDM2 is to mediate the ubiquitination and proteasome-dependent degradation of the p53 tumor suppressor protein and other growth regulatory proteins (Grossman et al., 1998;Manfredi, 2010;Chansaenroj et al., 2013).Accordingly, the ligase activity of MDM2 can be abolished by mutation of either one of eight cysteine and histidine residues involved in zinc coordination within the RING finger domain.Besides p53, MDM2 also ubiquitinates Numb (Yogosawa et al., 2003;Sczaniecka et al., 2012), β-arrestin binding protein (Shenoy et al., 2001;Kang et al., 2014), histone acetyltransferase Tip60 (Legube et al., 2002) as well as
SCY1-like 1-binding protein 1 (SCYL1BP1) is a newly identified regulator of Mdm2, which directly binds to Mdm2 and promotes Mdm2 self-ubiquitination, subsequently stabilizing p53 (Yan et al., 2010a;Yan et al., 2010b).SCYL1BP1, initially identified as a SCYL1 binding protein (Di et al., 2003;Hu et al., 2012), is an evolutionally conserved protein, existing in different organisms ranging from plants to animals.SCYL1BP1 protein is observed in many normal tissues and mainly localized in the cytoplasm, with a low level in the nucleus.However, the interaction between SCYL1BP1 and MDM2 in human lung squamous carcinoma remained unknown, and the effects of SCYL1BP1 on lung squamous carcinoma cell biological behaviors still remains poorly understood.
In the pre sent study, we find that SCYL1BP1 could promote MDM2 degradation, which may through an ubiquitin-dependent pathway.Overexpression of SCYL1BP1 inhibited the G1/S transition in lung squamous carcinoma cell lines.Functional studies confirmed the tumor-inhibitory effect of SCYL1BP1 both in vitro and in vivo.These findings suggested that SCYL1BP1 may function as an important tumor suppressor gene in lung squamous carcinoma development.

Reagents and cell lines
The following reagents and antibodies were used in this study: rabbit polyclonal anti-SCYL1BP1 (Abcam), goat polyclonal anti-Mdm2 (Abcam), rabbit polyclonal anti-β-actin (Santa-Cruz, CA, USA), goat polyclonal anti-HA (Abcam).Z-Leu-Leu-Leu-al (MG132) and Cycloheximide (CHX) were purchased from Sigma Aldrich.The human lung squamous carcinoma cell lines (NCI-H226 and LTEP-S) were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, and were passaged for fewer than 6 months.NCI-H226 and LTEP-S cells were cultured in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD, USA) and 1×antibiotics/antimycotics at 37°C in the presence of 5% CO 2 .

Plasmids, lentiviral production, and transduction
The full-length cDNA of human SCYL1BP1 was synthesized by GeneWiz (Beijing, China).The fragment was digested by the XhoI and NotI (Fermentas, Hanover, MD, USA) incision enzymes and was cloned into the lentiviral expression vector pLVX-IRES-neo (Clonetech Laboratories Inc., San Francisco, CA) (Hwang-Verslues et al., 2011;Zhou and Wang, 2011;Li et al., 2014).The resulting construct (pLVX-IRES-neo-SCYL1BP) was verified by sequencing.Replication-defective VSV-G pseudotyped viral particles were packaged in the human embryonic kidney cells LentiX 293T (Clonetech Laboratories Inc.) by a three plasmid transient cotransfection method (Lenti-T HT packaging mix, Clontech) using plus Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol.After 48 h of cotransfection, the lentivirus-containing supernatant was collected and passed through a 0.45-mm filter.For transduction, NCI-H226 and LTEP-S cells were infected with control lentivirus (empty vector, only lentivirus without SCYL1BP was inserted) and SCYL1BP lentivirus, respectively.After 48 h of transduction, the cells were stably selected with G418 (600μg/ml), and the drugresistant cell populations were used for subsequent studies.

Western blotting assay
Extracts corresponding to cells were prepared by detergent lysis buffer.Sixty micrograms of total proteins was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, Billerica, MA, USA).Antibodies for SCYL1BP1 (Abcam), MDM2 (Abcam) , or β-actin (Santa-Cruz, CA, USA) were used.The membranes were incubated overnight at 4°C with the primary antibody diluted 1:1000 and the proteins were detected with a Phot otope-horseradish peroxidase Western blot detection kit (Cell Sigaling Technology, Danvers, MA, USA).

Cell cycle analysis
NCI-H226-control, NCI-H226-SCYL1BP1, LTEP-S-control, and LTEP-S-SCYL1BP1 cells were plated at a concentration of 1×10 6 per 10cm dish and fixed with chilled 70% ethanol overnight at 4°C.Subsequently, we labeled single-cell suspensions with 50μg/ml PI (Sigma, St Louis, MO) and analyzed approximately 1×10 6 cells in 1ml by flow cytometry.Using the FlowJo Software (Treestar, Inc, Ashland, OR), the percentage of cells in the G2 phase for10, 000 cells was denoted by the 2N population phase.

Cell viability assay
NCI-H226-control, NCI-H226-SCYL1BP1, LTEP-Scontrol, and LTEP-S-SCYL1BP1 cells were collected and then seeded in 96-well flat-bottomed plates, and 2000 cells per 100μl of cell suspension were used to seed in each well.After 1, 2, 4, and 6 days of cultivation, cell viability was measured by Cell Counting Kit-8 (CCK-8) system (Dojindo Laboratory, Kumamoto, Japan) according to the manufacturer's instructions.Briefly, CCK-8 solution (10μl per 100μl of medium in each well) was added, the plates were then incubated at 37°C for 1h, and the absorbance of each well was read at 450 nm using a microplate reader (MRX; Dynex Technologies, West Sussex, United Kingdom).Six replicates for each group and the experiment were repeated at least 3 times.

Xenografts in mice
Female BALB/c nude mice, aged 4-5 weeks, were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China).Mice were allowed to acclimate to local conditions for at least 1 week and maintained under a 12h dark, 12h light cycle with food and water ad libitum.NCI-H226control, NCI-H226-SCYL1BP1, LTEP-S-control, and LTEP-S-SCYL1BP1 cells were diluted to a concentration of 1×10 6 /mL in physiological saline, Mice were injected subcutaneously with 0.1ml of the suspension into the back flank, each group consisted of 5 nude mice.When a tumor was palpable, tumor volume was measured by using a caliper every other day and calculated according to the following formula: V=L×W 2 ×0.5 (L, length; W, width) (Zheng et al., 2013).

Matrigel invasion assay
Cell migration and invasion ability were determined by Corning transwell insert chambers with 8μm pores (Corning) and BD BioCoat Matrigel Invasion Chamber (Becton Dickinson Biosciences), respectively.About 2×10 4 (migration assay) or 2×10 5 (invasion assay) transfected cells in 200μl serum-free RPMI 1640 medium were seeded in the upper well and the chambers were incubated for 48h at 37ºC to allow the cells to migrate to the lower well with RPMI 1640 medium plus 20% fetal bovine serum.Cells that had migrated or invaded through the membrane were fixed with methanol and stained with crystal violet (Invitrogen), imaged and counted.

Statistical analysis.
All analyses were performed in a two-sided fashion using SAS (version 9.1; SAS Institute), and p<0.05 was defined as statistically significant.For comparisons, oneway analyses of variance, Fisher's exact tests, chi-squared tests, and two-tailed Student's t-tests were performed, as appropriate.All data are presented as mean±SD.All experimental assays were performed in triplicated.

SCYL1BP1 regulates MDM2 gene expression in human lung squamous carcinoma cells
To evaluate the effects of SCYL1BP1 on cell biological behaviors, we constructed lung squamous carcinoma cell lines with SCYL1BP1 stable overexpression and control group using lentiviruses (Figure 1A).Western blotting analysis showed that the expression of MDM2 gene was diminished when SCYL1BP1 was overexpressed (Figure 1B), but we did not observe a reduction in MDM2 mRNA levels (Figure 1C).
To determine whether SCYL1BP1 reduced MDM2 protein stability, we treated SCYL1BP1-overexpressing and control NCI-H226 cells with the protein synthesis inhibitor, cycloheximide (CHX), or the proteasome inhibitor MG-132.As shown in Figure 2A, only MG-132 abolished the degradation of MDM2 protein levels in NCI-H226 cells overexpressing SCYL1BP1, and overexpressing SCYL1BP1 promoted the degradation of MDM2 protein levels Figure 2B.The same experiments were repeated using LTEP-S-control, and LTEP-S-SCYL1BP1 cells with similar results (Figure 2Aand B).

SCYL1BP1 regulates the cell cycle in human lung squamous carcinoma cells.
We further investigated the effects of SCYL1BP1 on cell cycle using fluorescence-activated cell sorting (FACS) analysis of propidium-iodide-stained lung squamous carcinoma cells.The representative results of the cellcycle distribution in empty vector, SCYL1BP1-transfected NCI-H226 and LTEP-S cells are shown in Figure 3A.Flow cytometry analysis revealed a statistically significant increase in the number of SCYL1BP1 overexpressed cells in the G1 phase (NCI-H226 cells: 11.08% increase, p=0.0043;LTEP-S cells: 8.93% increase, p=0.0023) accompanied by the decrease of S phase cells (NCI-H226 cells: 8.6% decrease, p=0.0005;LTEP-S cells: 11.48% decrease, p=0.0007), compared to control-transfected cells (Figure 3A).

Effects of SCYL1BP1 on human lung squamous carcinoma cells invasion
Using transwell chambers precoated with or without Matrigel, we examined the effect of SCYL1BP1 on cell invasion.Cells with higher expression of SCYL1BP1 (both NCI-H226 and LTEP-S cell lines) showed significantly decreased migration and invasion when compared to the control cells (Figure 4C).

Discussion
SCYL1BP1 was initially identified as an SCYL1binding protein (Di et al., 2003;Liu et al., 2012), but little is known about its biological function in human lung squamous carcinoma.Previous study showed that loss-of-function mutations of SCYL1BP1 caused gerodermia osteodysplastica (Hennies et al., 2008;Bexiga and Simpson, 2013), which suggested that SCYL1BP1 is an important pathogenic candidate gene.More recently, SCYL1BP1 was identified as a new regulator of the p53 pathway (Yan et al., 2010a).So it is perhaps not surprising that SCYL1BP1 has broader roles in physiology and pathology, such as the regulation of stress, metabolism, autophagy, tumors, and development.Here our specific aim is to investigate the potential roles of SCYL1BP1 in the development of lung squamous carcinoma.
In the present study, we found SCYL1BP1 can promote the degradation of MDM2 in lung squamous carcinoma cell lines.Overexpression of SCYL1BP1 decreased the expression of MDM2 at protein level but not at mRNA level.Protease inhibitor MG132 inhibited the degradation of MDM2 in SCYL1BP1 overexpressing cells.Recently, SCYL1BP1 was found to accelerate the self-ubiquitination of MDM2 and suppress the MDM2mediated p53 degradation (Momand et al., 1992;Yan et al., 2010b).Our result further confirmed this finding in lung squamous carcinoma.MDM2 is an important E3 ligase that may regulate the proteasomal degradation of p53 at different circumstances (Brooks and Gu, 2006;Sparks et al., 2013).It is possible that SCYL1BP1 may regulate the stability of p53 through this pathway.SCYL1BP1 may be a potential critical protein involved in the p53 pathway, for it can interact with and regulate the stability of MDM2.Since p53 is an important regulator of G1/S cell cycle check point (Martinez et al., 1991;Polyak et al., 1994;van der Laan et al., 2013), we next examined the effect of SCYL1BP1 on cell cycle progression.As expected, cell cycle analysis found that SCYL1BP1 could arrest cell cycle at G1/S checkpoint in NCI-H226 and LTEP-S cells.These data suggested that SCYL1BP1 may regulate cell cycle arrest mainly through the p53 pathway.To further explore the tumor-suppressive function of SCYL1BP1 in lung squamous carcinoma development, SCYL1BP1 was transfected into two lung squamous carcinoma cell lines.Functional studies found that the ectopic expression of SCYL1BP1 in NCI-H226 and LTEP-S cells dramatically inhibited cell growth, migration, invasion and tumor formation in nude mice, indicating that SCYL1BP1 had strong tumor-suppressive function during lung squamous carcinoma progression.
Based on these findings, we propose that SCYL1BP1 is a novel regulator of MDM2 stability in lung squamous carcinoma.Ectopic expression enhanced the degradation of MDM2and inhibited the G1/S transition of lung squamous carcinoma cell lines and exhibited strong tumorsuppressive function both in vitro and in vivo.The frequent downregulation may account for the overexpression of MDM2 in the development of lung squamous carcinoma.Further characterization of the tumor-suppressive function of SCYL1BP1 may greatly facilitate our understanding of the molecular mechanism of lung squamous carcinoma development and find new thera peutic targets for lung squamous carcinoma treatment.

Figure 1 .
Figure 1.A) The SCYL1BP1 expression levels in lung squamous carcinoma cells after SCYL1BP1 or control lentiviruses infection.B) Western blot analysis for MDM2 protein expression in SCYL1BP1 stable overexpressing and control cells.C) The relative mRNA levels of the MDM2 in SCYL1BP1 stable overexpressing, and control cell lines, data are mean±SEM, normalized to GAPDH

Figure 3 .
Figure 3. A) Cell cycle analysis of lung squamous carcinoma cells cells after transfected with SCYL1BP1 or Control lentiviruses.Results are represented as mean±SEM based on three independent experiments (*represents p<0.05 when compared to control).B) SCYL1BP1 stable overexpressing and control cells were seeded in 96-well plates, and cell proliferation was assessed daily for 4 days using the Cell Counting Kit-8 (CCK-8) assay (*represents p<0.05 when compared to control)

Figure 4 .
Figure 4. A) Subcutaneously implanted SCYL1BP1 stable overexpressing and control cells xenografted tumors were established.Bars, SEM.Mean tumor volume from 6 nude mice of each group are shown (*represents p<0.05 when compared to control).B) Matrigel invasion assays were performed in 24-well transwell chambers containing polycarbonate filters with 8μm pores coated with matrigel by using the SCYL1BP1 stable overexpressing and control cells.Results are represented as mean±SEM based on three independent experiments DOI:http://dx.doi.org/10.7314/APJCP.2014.15.17.7467SCYL1BP1 has Tumor-suppressive Functions in Human Lung SCC Cells by Regulating the Degradation of MDM2