DOI: 10.1111/exd.12515
Original Article
www.wileyonlinelibrary.com/journal/EXD
Activation of autophagic pathways is related to growth inhibition and senescence in cutaneous squamous cell carcinoma So Ra Choi1*, Bo Young Chung1*, Seong Who Kim2, Chang Deok Kim3, Woo Jin Yun1, Mi Woo Lee1, Jee Ho Choi1 and Sung Eun Chang1 1 Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 2Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 3Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea Correspondence: Sung Eun Chang, Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, 86 Asanbyeongwon-gil, Songpa-gu, Seoul 138-736, Korea, Tel.: +82-2-3010-3467, Fax: +82-2-486-7831; e-mail: [email protected] *These authors contributed equally to this work.
Abstract: Cutaneous squamous cell carcinoma (SCC) is a very common resectable cancer; however, cutaneous SCC is highly resistant to chemotherapy if metastasis develops. Activating transcription factor 3 (ATF3) has been suggested as a marker of advanced or metastatic cutaneous SCC. Autophagy is one of the most important mechanisms in cancer biology and commonly induced by in vitro serum starvation. To investigate the role of autophagy activation in cutaneous SCC, we activated autophagic pathways by serum starvation in SCC13 and ATF3-overexpressing SCC13 (ATF3-SCC13) cell lines. ATF3-SCC13 cells demonstrated high proliferative capacity and low p53 and autophagy levels in
comparison with control SCC13 cells under basal conditions. Intriguingly, autophagic stimulation via serum starvation resulted in growth inhibition and senescence in both cells, while ATF3SCC13 cells further demonstrated growth inhibition and senescence. Apoptosis was not significantly induced by autophagy activation. Taken together, autophagy activation may be a promising antitumor approach for advanced cutaneous SCC.
Introduction
ATF3 expression is elevated in human breast cancer (11) and prostate cancer (13) and significantly elevated in cutaneous SCCs that are metastatic or arise in organ transplant recipients (14). ATF3 is a well-known suppressor of p53-dependent senescence and enhances tumorigenic potential in cutaneous SCC (15). In a previous study, ATF3-overexpressing SCC13 cells (ATF3-SCC13) were far more advanced and metastatic than control SCC13 cells (15). The role of autophagy remains unknown in skin cancers. Recent studies have reported that autophagic activation was correlated with the disease progression and prognosis of cutaneous SCC and malignant melanoma (16,17). Autophagy has been described as a process that induces non-apoptotic cell death and as an antitumor mechanism (18–21). Autophagy, originally known as a survival mechanism, is a lysosomal-associated catabolic process that degrades and recycles cellular constituents, such as longlife proteins and organelles (22). Experimental tools in vitro for autophagy activation include serum starvation and/or rapamycin treatment, a well-known mammalian target of rapamycin (mTOR) inhibitor. To investigate the role of autophagy activation in cutaneous SCC, we activated autophagic pathways in SCC13 and ATF3-overexpressing SCC13 (ATF3-SCC13) cell lines. Our current study was aimed to elucidate how autophagy activation in localized cutaneous and advanced cutaneous SCCs functions as an antitumor mechanism.
Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers worldwide, and SCC is the second most common skin cancer (1). Early-stage cutaneous SCC is highly curable and demonstrates a relatively low rate of metastasis (3–5%) (2). However, cutaneous SCC with lymph node or distant metastasis demonstrates poor prognosis regardless of treatment (3). Standard first-line treatments for advanced cutaneous SCC currently include surgical resection, radiation therapy or both. Chemotherapy has been integrated into standard treatment regimens for advanced cutaneous SCC in an attempt to improve survival outcomes. However, current therapies for advanced cutaneous SCC demonstrate poor response and high recurrence rates, demonstrating the need for new therapeutic targets (2,4). The incidence of cutaneous SCC is increasing, partly due to new therapeutic regimens involving for example BRAF inhibitors (5,6). Patients treated with BRAF inhibitors, targeted therapeutics for advanced melanomas harbouring certain BRAF mutations, frequently develop non-melanoma skin cancers including cutaneous SCC. The cause of the keratinocyte proliferation underlying these SCC lesions has been suggested to be paradoxical hyperactivation of the mitogen-activated protein kinase (MAPK) pathway (7). As another mechanism of cutaneous SCC development and progression, a recent study has reported the involvement of p53 family members and their signalling via Notch (8). Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding family of transcription factors (9). ATF3 is significantly increased by a variety of stress signals, including anoxia, DNA damage and carcinogenesis (10–12).
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Key words: activating transcription factor 3 – autophagy – cutaneous squamous cell carcinoma – growth inhibition – senescence
Accepted for publication 17 July 2014
Materials and methods Ethics statement All patient biopsies were obtained with written informed consent and in accordance with the Helsinki declaration. The study was
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Role of autophagy in cutaneous squamous cell carcinoma
approved by the institutional review board of the Asan Medical Center (file reference: 2014-0403).
nyl acetate and lead citrate and examined at 50 kV using a JEM 1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan).
Immunohistochemistry
Cell growth analysis and apoptosis assay
Five sets of biopsy tissues were obtained from each patient, fixed in 10% buffered neutral formalin and embedded in paraffin. These specimens were cut to 4-lm-thick sections, and serial sections were prepared for immunohistochemistry. For antigen retrieval, sections were autoclaved in antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) and immunostained using Vector Elite ABC Kit (Vector Laboratories) according to the manufacturer’s instructions. Sections were treated with diaminobenzidine (0.5 mg/ml) and hydrogen peroxide. The primary antibodies included an ATF3 antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p62 antibody (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA). Staining positivity was classified into five grades: 0, negative; +, 50%. All slides were evaluated independently by two dermatologists with no knowledge of the identity of the patient or clinical outcome.
Following treatment with rapamycin, BFA1 or serum starvation, cell growth was determined using the EZ-Cytox cell growth assay kit (ITSBIO, Seoul, Korea) according to the manufacturer’s guidelines. Absorbance was measured at 450 nm using an enzyme immunosorbent assay (ELISA) reader (Molecular Devices Co., Sunnyvale, CA, USA). Apoptosis was measured by flow cytometry using the annexin V-FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Annexin V-FITC and propidium iodide (PI) were added according to the manufacturer’s instructions. After 15 min of incubation at room temperature, cells were acquired by flow cytometry (BD Biosciences) and the presence of viable cells (annexin V- and PI-negative), early apoptotic cells (annexin V-positive and PI-negative) and late apoptotic cells (annexin V- and PI-positive) were assessed using Cell Quest software (BD Biosciences).
Reagents Rapamycin and bafilomycin A1 (BFA1) were purchased from Sigma-Aldrich (St Louis, MO, USA). The following primary antibodies against human proteins were used: anti-b-actin and antiLC3B (Sigma-Aldrich); anti-ATG5 (Novus Biologicals, LLC, Littleton, CO); anti-p62 (Medical & Biological Laboratories, Nagoya, Japan); anti-p53, anti-phospho-p53 (Ser15), anti-Rb, anti-phospho-Rb (Ser807/811), anti-p21, anti-phospho-mTOR (Ser2448), anti-phospho-p70S6K (Thr389), and anti-Beclin1 (all from Cell Signaling Technology, Inc., Danvers, MA); and anti-ATF3 (Santa Cruz Biotechnology).
Cell culturing The SCC13 human squamous cell carcinoma (SCC) cell line and ATF3-overexpressing SCC13 cells were provided by Dr. G. Paolo Dotto (Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA, USA). SCC13 cells originated from a human SCC of the facial epidermis with a moderate degree of mitosis and proliferation (23), with a wild-type ras gene and harbouring single p53 missense mutations (24). These cells were cultured in DMEM (Welgene, Deagu, Korea) containing 10% foetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% antibiotic–antimycotic solution (100 9 ) (Gibco). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. Serum starvation was performed in 5% and 1% serum for 72 h.
Transient siRNA transfection Cells were placed 6-well plates, seeded at a density of 1 9 105 cells and transfected with siRNA against p53, ATF3 or negative control siRNA using LipofectamineTM RNAiMAX (Invitrogen, Merelbeke, Belgium) according to the manufacturer’s instructions.
Transmission electron microscopy To further verify autophagy in SCC13 and ATF3-SCC13 cells, we directly examined the cellular autophagosomes using transmission electron microscopy (TEM). Briefly, the cells were fixed at 4°C overnight using a 2.5% glutaraldehyde and 0.1 mol/L phosphate buffer (pH 7.2). After 90 min of postfixation in 1% osmium tetroxide and 0.1 mol/L phosphate buffer at 4°C, the samples were dehydrated in a graded series of alcohol and embedded in Epon media. Ultrathin sections were cut to 50–60 nm, stained with ura-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Western blot analysis Total proteins were extracted using PRO-PREP extraction solution (Intron, Deajeon, Korea). Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Equally loaded proteins from each sample were resolved in SDSPAGE gels (Invitrogen, Merelbeke, Belgium) and transferred to nitrocellulose membranes (WhatmanTM, GE Healthcare Life Sciences, Buckinghamshire, UK). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween20 and 5% non-fat dry milk. Membranes were then incubated overnight at 4°C with primary antibodies that were diluted in 5% non-fat dry milk or 5% BSA in TBS-T with Tween-20. Membranes were then washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). After further washing with TBS-T, the protein bands were visualized using a SuperSignal West Pico Trial kit (Thermo Scientific, Rockford, IL, USA). Primary antibodies against the following proteins were used: b-actin (SigmaAldrich), LC3B (Sigma-Aldrich), ATG5 (Novus Biologicals), p62 (Medical & Biological Laboratories), Beclin1, p53, phospho-p53 (Ser15), p21, Rb, phospho-Rb (Ser807/811), phospho-mTOR (Ser2448), phospho-p70S6K (Thr389) (all from Cell Signaling) and ATF3 (Santa Cruz Biotechnology). All experiments were performed at least three times.
Senescence-associated beta-galactosidase (SA-b-Gal) staining Cells were seeded at the same density in 6-well plates and stained with SA-b-Gal using a senescence b-galactosidase staining kit (Cell Signaling Technology). SA-b-gal is a commonly used senescence biomarker that can be detected at pH 6.0 (21). This is because senescent cells express a high level of lysosomal b-gal (25,26).
Statistical analysis Statistically significant differences between groups were assessed using analysis of variance (ANOVA) and the Student t-test. In this study, P < 0.05 was considered statistically significant.
Results Metastatic cutaneous SCC and ATF3-SCC13 cells demonstrate significantly increased ATF3 expression The clinicopathologic data of patients who provided skin biopsy tissues were examined (Table 1). Differences in ATF3 expression in cutaneous SCC in situ, invasive cutaneous SCC and metastatic
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cutaneous SCC were compared using immunohistochemical analysis (Fig. 1a). ATF3 was not expressed in the cutaneous SCC in situ samples. Metastatic cutaneous SCC samples demonstrated stronger ATF3 immunoreactivity than cutaneous SCC in situ and invasive cutaneous SCC samples. Mouse IgG1/kappa did not demonstrate positive signals in metastatic cutaneous SCC samples. ATF3 showed a more distinct nuclear staining in metastatic SCC cells compared with SCC in situ or invasive SCC cells. This finding is consistent with the fact that ATF3 is a transcription factor with robust nuclear expression. Also, in cell culture experiments, ATF3 expression was higher in ATF3-SCC13 than SCC13 cells (Fig. 1b) and there were two ATF3 bands apparent on a Western blot. The mobility of ATF3 was observed to differ between SCC13 and ATF3-SCC13 cells (Fig. 1b), implying that ATF3 expression may be subject to post-transcriptional regulation. Based on these results, we choose ATF3-SCC13 and control SCC13 cells as in vitro representative of metastatic cutaneous SCC and localized cutaneous SCC, respectively.
ATF3-SCC13 cells demonstrate high proliferative capacity, low p53 expression and low basal autophagy compared with SCC13 cells ATF3-SCC13 cells demonstrated better growth than SCC13 cells (Fig. 2a). Because p53 is a well-known senescence marker that is counteracted by ATF3 in cutaneous SCC samples (12), we knocked down ATF3 and p53 in SCC13 cells using siRNA. When we targeted ATF3, p53 expression was higher than the negative control siRNA (siControl) in both SCC13 and ATF3-SCC13 cells (Fig. 2b). In addition, ATF3 expression increased in SCC13 cells following p53 knock-down (Fig. 2b). These results indicate that ATF3 is inversely correlated with p53 in SCC cells. There were two ATF3 bands on a Western blot, and the upper of these bands (ATF3-SCC13 cells) in Fig. 2b was stronger than that in Fig. 1. We then evaluated basal autophagy in each cell because p53 may be associated with autophagy (27). The expression levels of autophagy-related proteins, including ATG5, Beclin1 and the microtubule-associated protein 1 light chain 3 (LC3B)-II/LC3B-I ratio, were lower, and p62 was higher in ATF3-SCC13 cells than in SCC13 cells (Fig. 2c). The phosphorylation of mTOR and p70S6K in ATF3-SCC13 cells was higher than that in SCC13 cells (Fig.
(a)
(b)
Figure 1. ATF3 expression in dermal tissues and cutaneous SCC and SCC cell lines. (a) Immunohistochemical analysis of cutaneous squamous cell carcinoma (SCC) in situ, invasive cutaneous SCC and metastatic cutaneous SCC lesions. ATF3 expression in metastatic cutaneous SCC was higher than cutaneous SCC in situ or invasive cutaneous SCC. ATF3 showed a more distinct nuclear staining in metastatic SCC cells than in SCC in situ or invasive SCC cells. Representative images of one of five experiments are shown. Scale bar, 50 lm. (b) Western blot analysis of ATF3 expression in SCC cells. ATF3 expression was higher in ATF3SCC13 cells than SCC13 cells.
S1). By TEM, the number of autophagosomes was lower in ATF3SCC13 cells than in SCC13 cells (Fig. 2d). These results provide evidence that ATF3-SCC13 cells demonstrate lower basal autophagy levels, as well as lower cellular senescence, than SCC13 control cells.
Table 1. Summary of clinical and pathologic data Age (years)
Sex
Diagnosis
Primary site
84 82 89 69 58 49 84 88 66 76 64 74 75 49 74
F F F F F M F M F M M M M M M
SCC in situ SCC in situ SCC in situ SCC in situ SCC in situ SCC SCC SCC SCC SCC Metastatic SCC Metastatic SCC Metastatic SCC Metastatic SCC Metastatic SCC
Lt cheek Rt temporal area Nose Rt cheek Rt infraorbital area Nose Rt cheek Lt cheek Neck Rt temporal scalp Scalp Chest Lt clavicular area Scalp Lt axilla
State of differentiation
Metastasis (location)
ATF3 staining
p62 staining
Moderately Well Well Well Well Poorly Poorly Moderately Well Poorly
– – – – – – – – – – + + + + +
+ + + + + +++ ++ ++ +++ ++ ++++ ++++ +++ ++++ ++++
+ + + + + +++ +++ +++ ++ ++ ++++ ++++ +++ ++++ ++
(Lung) (LN) (LN) (Lung) (LN)
0, negative staining; +, focal or < 10% positive of SCC cells; ++, 10–30% positive of SCC cells; +++, 30–50% positive of SCC cells; and ++++, > 50% positive of SCC cells. M, male; F, female; Lt, left; Rt, right; LN, lymph node; SCC, squamous cell carcinoma.
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(a)
(b)
(c)
(a)
(c)
(d)
(b)
Figure 2. Cell proliferation, ATF3 and p53 expression, and basal autophagy in SCC cells. (a) ATF3-SCC13 cells demonstrate a higher growth level than SCC13 cells. Cell growth levels were analysed using a cell viability assay that measured the absorbance at 450 nm. Data are presented as the mean SD (n = 3). **P < 0.01 and ***P < 0.001 according to the Student t-test. (b) Western blot analysis validating ATF3 and p53 knock-down in SCC cells that were transfected with ATF3, p53 or negative control siRNA (siControl) for 48 h. When ATF3 was targeted, p53 expression was higher compared with negative control siRNA (siControl) in both SCC13 and ATF3-SCC13 cells. In addition, ATF3 expression increased in SCC13 cells following p53 knock-down. b-actin expression was used as the loading control. These results are representative of three independent experiments. (c) Western blot analysis of autophagy-related proteins, including ATG5, Beclin1, LC3B and p62. ATG5 and the LC3B-II/LC3B-I ratio were significantly decreased, and the p62 level was slightly increased in ATF3-SCC13 cells compared with SCC13 cells. b-actin expression was used as the loading control. These results are representative of three independent experiments. (d) Examination of autophagosomes in SCC13 and ATF3-SCC13 cells using transmission electron microscopy. The number of autophagosomes was lower in ATF3-SCC13 cells in comparison with SCC13 cells. (AP) autophagosome; (N) nuclei; (M) mitochondria. Scale bars, 50 lm.
Activating autophagy inhibits cellular growth and induces cellular senescence
Figure 3. Activation of autophagy in SCC cells by serum starvation induces cellular senescence more potently if ATF3 is overexpressed. SCC cells were cultured in complete medium for 24 h before being cultured in medium with 1% or 5% FBS for 72 h. (a) Western blot analysis of p53, phosphorylated p53 (Ser 15), p21, Rb, phosphorylated Rb (Ser807/811) and autophagy-related proteins, including ATG5, LC3B and p62. Autophagy activation (ATG5 and LC3B-II/LC3B-I ratio) in ATF3SCC13 cells was more potent than that in SCC13 cells. b-actin expression was used as the loading control. The results are representative of three independent experiments. (b) Effects of serum starvation on cellular senescence detected by SA-b-gal staining. Under serum starvation, SA-b-gal activity in ATF3-SCC13 cells increased compared with that in SCC13 cells. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells are expressed as a percentage of the total cells. Scale bar, 50 lm. (c) Effects of serum starvation on cellular apoptosis were detected using annexin V/PI staining. Apoptosis was not induced in either SCC13 or ATF3-SCC13 cells by serum starvation.
To determine the effects of autophagy activation by serum starvation in SCC cells, we subjected these cells to serum starvation for 72 h and measured ATF3 expression and autophagy level. Because cell viability decreased to about 60% after 72 h of serum-free condition (Fig S2a), the results of serum-free condition might not have been accurate compared with serum 1% and 5% conditions. Therefore, we conducted further serum starvation experiments at serum 1% and 5%. Unexpectedly, autophagy activation (according to ATG5 expression, the LC3B-II/LC3B-I ratio) in ATF3-SCC13 cells was more potent than that in SCC13 cells (Fig. 3a), while the basal autophagy level was far lower in ATF3-SCC13 cells (Fig 2c). The phosphorylation of mTOR and p70S6K was suppressed in both SCC13 and ATF3-SCC13 cells by serum starvation compared with the control, and the suppressed degrees were higher in ATF3-SCC13 cells (Fig S2b). Furthermore, the cellular growth of both cells was significantly suppressed by serum starvation in comparison with the 10% serum-containing control. ATF3-SCC13 cells showed further inhibition of cell growth, while the basal proliferation capacity was higher in ATF3-SCC13 cells (Fig S2c). To confirm whether an autophagic pathway is activated by serum starvation in SCC cells, we treated the ATF3-SCC13 and SCC13 cells with an autophagy inhibitor bafilomycin A1 (BFA1) (2.5 nM) for 48 h in the presence or absence of serum starvation. We first assessed the cytotoxic effects of BFA1 on SCC cells
(Fig.S3a). According to the cytotoxicity results, we chose 2.5 nM as the maximal concentration of this inhibitor. Serum starvation in conjunction with BFA1 treatment decreased the LC3B-II/LC3BI ratio and ATG5 levels and increased p62 expression in both SCC13 and ATF3-SCC13 cells (Fig.S3b). We determined the protein levels of p53, phosphorylated p53 (Ser 15) and p21 as other markers of senescence. While p21 demonstrated no change, phosphorylated p53 levels were higher in ATF3-SCC13 cells than SCC13 control cells following serum starvation (Fig. 3a). Apoptosis was not induced, but senescence-associated b-galactosidase activity (SA-b-gal) increased in both cells following serum starvation (Figs 3b, c and S2c). These findings imply that autophagy activation inhibits growth and induces senescence in ATF3-SCC13 cells to a greater degree. We also evaluated the effects of rapamycin, as another autophagy activator, on cellular proliferation versus senescence in the SCC13 and ATF3-SCC13 cells. The results of rapamycin treatment on SCC cells showed similar results to serum starvation. Autophagy activation (ATG5, LC3B-II/LC3B-I ratio and p62) was similarly induced in both SCC13 and ATF3-SCC13 cells by rapamycin (Fig. S4a). Senescence was also induced, but apoptosis was not, in
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
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both cell types following rapamycin treatment. Activation of autophagy by rapamycin induces senescence more potently in ATF3SCC13 cells (Fig.S4b–d). We conducted immunohistochemical analysis of SCC samples from patients using anti-p62 antibodies to assess autophagy in vivo. By immunohistochemical comparison, the expression of p62 in metastatic cutaneous SCC and invasive cutaneous SCC was higher than that in cutaneous SCC in situ. In metastatic cutaneous SCC, p62 staining was mostly positive in both the nucleus and cytoplasm of the SCC cells. In invasive cutaneous SCC, p62 staining was positive in the cytoplasm of the SCC cells (Fig. S5). These results are consistent with increased ATF3 expression in metastatic SCC with more distinct staining in the nucleus, which suggests a relationship between the expression of p62 and ATF3. Both SCC13 and ATF3-SCC13 cells are susceptible to autophagic stimuli by serum starvation, leading to autophagy activation, growth inhibition and senescence. Interestingly, highly proliferative ATF3-SCC13 cells, which demonstrated low basal autophagy and senescence, were more susceptible to autophagic stimulation.
Discussion Autophagy is an evolutionarily conserved catabolic process whereby cells degrade and recycle aggregated protein complexes, poorly functioning organelles and pathogens allowing cells to survive starvation and other stresses (28). Conventional transmission electron microscopy (TEM) is one method for detecting the accumulation of autophagic compartments in mammalian cells. In TEM, autophagosomes have double or sometimes multiple limiting membranes and their cytoplasmic contents begin to show dark granular/amorphous clumps with age (29). Microtubule-associated protein 1 light chain 3 (LC3) is a specific marker protein for autophagy that exists in two forms, LC3-I and its proteolytic derivative LC3-II (18 and 16 kDa, respectively), which are localized in the cytosol (LC3-I) or in autophagosomal membranes (LC3-II). In addition to LC3, Atg5, Beclin-1, p62 and mTORC1 protein can be used as markers of autophagy. Atg5 is involved in the elongation of the isolation membrane during autophagosome formation (30). Beclin-1 localizes to the trans-Golgi network, belongs to the class III phosphatidylinositol 3-kinase complex and is involved in autophagosome formation (31). The p62 protein acts as an axillary autophagy factor by directly binding ubiquitinated proteins and LC3 to facilitate their degradation by autophagy (32). The accumulation of p62 has been used to indicate abnormalities in the autophagic degradation (33) and also for measuring the flux of autophagosomes to demonstrate inhibition of autophagy by bafilomycin A1 (34). The mTORC1 is a cellular sensor of nutritional conditions that negatively regulates autophagy (35). Inhibition of mTORC1 activity induces autophagy in many types of cells (19). We used the above-mentioned multiple methods to monitor autophagy in our current study. The involvement of autophagy in tumorigenesis is, at present, incompletely understood, and the opposing effects of autophagy on cell survival and death have been documented (36,37). Whether it is autophagy activation or inhibition that demonstrates anticancer effects is controversial. Many tumors contain poorly vascularized regions that lack both nutrients and oxygen, which is also associated with high levels of autophagosomes (38). In these settings, we believe that autophagy is a cytoprotective mechanism for tumor cells and can therefore be considered oncogenic. How-
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ever, some autophagy-deficient murine models are reportedly prone to lung carcinoma (39), and it has been demonstrated that key autophagy-related genes are inactivated in the major sporadic forms of human cancer (39–41). This suggests that autophagy contributes to tumor suppression, at least in some tumor types and at some point during tumor development. In cutaneous SCC, the role of autophagy has only been sporadically analysed. Recent studies show that inhibiting autophagy may lead to increased apoptosis in cutaneous SCC, thereby benefiting antitumor therapies (18,19). To explain contrary results, it is has been suggested that the proper level of autophagy promotes cell survival, while higher levels result in autophagic cell death. Many anticancer agents induce autophagy to restrain growth in various cancers, and studying autophagy is a new direction in the development of anticancer drugs. Here, for the first time, we investigated autophagy activation and induction as a tumor-suppressing mechanism in cutaneous SCC. Autophagy is commonly induced by in vitro serum starvation or rapamycin treatment. Serum starvation is the most common and reliable tool in experiments on autophagy activation, while mTOR-inhibiting rapamycin induces apoptosis, cytotoxicity and autophagy (42). The target of rapamycin (TOR) is a highly conserved serine/threonine protein kinase that acts as a central sensor for growth factors, nutrient signals, cellular energy status and a master regulator of autophagy (43). Rapamycin also increases apoptosis, the p53/MDM2 protein ratio and p53-dependent apoptosis via the translational inhibition of MDM2 in cancer cells (44). Other previous studies performed on various murine models also report that the effects of rapamycin include reduction in tumor number, tumor size and the progression of benign lesions to SCCs in UVB- or cyclosporine-induced carcinogenesis and in solid organ transplant recipients (45–48). Human clinical trials on using rapamycin to treat metastatic cutaneous SCC are still ongoing (18). ATF3 belongs to the ATF/cAMP-responsive element-binding protein family of transcription factors and can bind to DNA (via the ATF/CREB consensus sequence, 50 -TGACGTCA-30 ) (49) and other proteins (e.g. p53 and E6) (50), modifying gene expression and cellular functions. ATF3 is a stress-inducible and/or adaptive response gene, as its mRNA and protein levels rapidly are increased by stress signals, such as cytokines, genotoxic agents, nerve injury and tissue damage (49). Recent studies have implicated ATF3 in many cancers (51), but its exact role remains unclear. There has been much evidence characterizing ATF3 as an oncogene. Its overexpression has been found in human breast cancer, prostate cancer and Hodgkin lymphomas. (11). A recent study showed that an elevated level of ATF3 expression suppresses p53dependent senescence and increases tumorigenic potential in squamous skin cancer (15). However, some evidence suggests that ATF3 may be able to inhibit tumorigenesis. ATF3 expression decreased in human colorectal cancer, and ATF3 overexpression resulted in apoptosis of human prostate cancer cells (52). ATF3 plays the context-dependent role in cancer possibly due to the complex protein–protein interaction networks. In addition to transcriptional regulation, it has been reported that ATF3 interacts with a number of critical cellular proteins while regulating their functions. The wild-type p53 is one of the ATF3-binding proteins that have been well documented (50). The major p53 suppressor
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Role of autophagy in cutaneous squamous cell carcinoma
MDM2 binds to and ubiquitinates ATF3 by accelerating its degradation (53). ATF3 binds to and stabilizes p53 by blocking its ubiquitination, thereby activating p53 as a countermeasure to DNA damage (50). ATF3 has also been known to be a negative regulator of TP53 gene expression (51). Based on these results, it can be deduced that ATF3 may serve as a p53 activator that stabilizes p53 protein or as a p53 inhibitor that downregulates the transcriptional activity of TP53. This study confirms higher ATF3 expression in metastatic cutaneous SCC tissues in comparison with SCC in situ and localized cutaneous SCC lesions. In addition, we confirmed that ATF3SCC13 cells were more proliferative at lower p53 levels, which is consistent with a previous study (12). High ATF3 expression suppresses p53-dependent senescence and enhances tumorigenic potential (15). p53 is a representative tumor suppressor gene and marker of senescence. Cellular senescence is a permanent feature of cell cycle arrest that is believed to play a role in tumor growth inhibition and suppression (54,55). Consistent with this, using reciprocal knock-down experiments, our data show an interconnected relationship between ATF3 and p53 expression in SCC cells. p53 expression increases when ATF3 is antagonized by siRNA, and ATF3 expression increases in SCC cells when p53 is antagonized. Overall, ATF3-SCC13 represents a more advanced type of SCC that is treatment resistant. Here, we report the repression of cell proliferation and increase in cellular senescence in SCC cells following serum starvation. ATF3-SCC13 cells exhibited significantly less proliferation than SCC13 cells and demonstrated increased senescence following serum starvation. Rapamycin treatment produced similar results to serum starvation. It is interesting that ATF3-SCC13 cells, in other words, clinically metastatic or advanced cutaneous SCC, in which p53 is suppressed, demonstrated more obviously inhibited growth and increased senescence when p53 and autophagy-related proteins increased following serum starvation compared with SCC13 cells. SCC cells seemed to show different responses to autophagy activation, at least in terms of ATF3 expression and basal autophagy. As expected, we found that ATF3-SCC13 cells demonstrated low basal autophagy in comparison with SCC13 cells. Serum starvation increased the expression of autophagy-associated proteins and decreased cell growth in ATF3-SCC13 and SCC13 cells. We also found that serum starvation increased p53 activation and induced senescence in ATF3-SCC13 and SCC13 cells. In particular, reactions to serum starvation were more obvious in ATF3-SCC13 cells. ATF3 is known to be the regulator of p53 in response to a wide range of environmental insults (50,51). Autophagy has been suggested to be regulated by several oncogenes and tumor suppressors. p53 is one of the most well-known
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tumor suppressors that may play a dual role in autophagy regulation, that is positively or negatively (56,57). Taken together, ATF3 regulates p53, which in turn regulates and affects autophagy. One of the possible causes behind the various reactions to autophagy among SCC cells at different levels of ATF3 expression could be the difference in p53 regulation by ATF3. However, the relationships among ATF3, p53 and autophagy have yet to be clarified. Therefore, further research is required to elucidate the exact mechanism. In our results, there were two ATF bands evident by Western blotting and the upper of these two bands in Fig. 2b (ATFSCC13) was stronger than that in Fig. 1b. There could be two major causes of this, including post-transcriptional modification of the protein or as ATF3 is increased by stress signals, a degree of basal stress in the cells. The mobility of ATF3 was also different between SCC13 and ATF3-SCC13 cells in Fig. 1b. This difference of mobility may be caused by post-translational modifications (PTMs) of ATF3. There is evidence that ATF3 is regulated by a PTM; the protein activity level of ATF3 has been shown to be modulated by ubiquitination and/or small ubiquitin-related modifier (SUMO)ylation. Among PTMs, the mechanisms of SUMOylation are very similar to those of ubiquitination, but the biological functions of these two processes are very different. Recent studies have reported that ATF3 can be SUMOylated at lysine 42 as a major SUMO site and that the de-SUMOylation of ATF3 augments the ATF3-p53 physical interaction and the trans-activation of p53 responsive promoters (53). In conclusion, autophagic activation results in growth inhibition and senescence in both SCC13 and ATF3-SCC13 cells, which is stronger in ATF3-SCC13 cells. These findings could potentially advance the practical induction of autophagy and its future use as a therapeutic intervention for advanced cutaneous SCC, which is notorious for chemotherapeutic resistance. Agents that activate autophagy may be promising cytostatic treatments for metastatic cutaneous SCC.
Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science, and Technology (2011-0409) and Asan Life Science Institute (2011-415). We thank Dr. Dotto for generously providing the SCC13 and ATF3-SCC13 cells.
Author contributions SRC performed the research; BYC and SRC wrote the manuscript; WJY and SWK designed the study; MWL, JHC and CDK analysed the data; and SEC supervised the study and the manuscript.
Conflicts of interest The authors have no conflicts of interest to declare.
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Supporting Information Additional supporting data may be found in the supplementary information of this article: Figure S1. Western blot analysis of phospho-mTOR (Ser2448) and phospho-p70S6K (Thr389) in SCC13 and ATF3-SCC13 cells. The phosphorylation of mTOR (Ser2448) and p70S6K (Thr389) in ATF3-SCC13 cells increased compared with that in SCC13 cells. b-actin expression was used as the loading control. These results are representative of three independent experiments. Figure S2. (a) Cell viability of SCC cells on serumfree condition (FBS 0%). When SCC cells were cultured with serum-free medium, cell viability decreased to about 60% after 72 hr in both SCC13 and ATF3SCC13 cells. Cell viability was evaluated using EZ-Cytox cell viability assay that measured the absorbance at 450 nm. Data are presented as the mean SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. (b) Activation of autophagy in SCC cells by serum starvation. The phosphorylation of mTOR and p70S6K was suppressed in both SCC13 and ATF3SCC13 cells by serum starvation compared with the 10% serum-containing control, and the suppressed degrees were higher in ATF3-SCC13 cells. b-actin expression was used as the loading control. These results are representative of three independent experiments. (c) Left, the inhibitory effects of serum starvation on cell growth. Cell growth of SCC cells was significantly suppressed by serum starvation compared with a 10% serum-containing control. ATF3-SCC13 cells showed further inhibition of cell growth, while the basal proliferation capacity was higher in ATF3-SCC13 cells. Cell growth levels were analysed using EZ-Cytox cell growth assay that measured the absorbance at 450 nm. Right, effects of serum starvation on cellular senescence as detected by SA-b-gal staining. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells were expressed as percentage of the total cells. Data are presented as the mean sd (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. Figure S3. (a) Effects of bafilomycin A1 (BFA1) on cell viability in SCC cells. The cells were seeded in 96well plates for 1 day and then treated with BFA1 (0, 1,
2.5, 5 nM) for 48 h. Cell viability was evaluated using EZ-Cytox cell viability assay and expressed as a percentage of the control. Data are presented as the mean SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. (b) Inhibitory effects of autophagy in SCC cells by bafilomycin A1 (BFA1; 2.5 nM) in the presence and absence of serum starvation. Autophagy-related proteins, including ATG5, p62 and LC3B were determined using Western blotting. Autophagy was suppressed by BFA1 in both SCC13 and ATF3SCC13 cells. b-actin expression was detected as a loading control. Results are representative of three independent experiments. Figure S4. Activation of autophagy by rapamycin in SCC cells induces senescence more potently if ATF3 is overexpressed. Rapamycin at 20 nM or 200 nM was added to SCC cultures for 48 h. (a) Western blotting analysis of p53, phosphorylated p53 (Ser 15), p21, Rb, phosphorylated Rb (Ser807/811) and autophagy-related proteins, including ATG5, p62 and LC3B. Autophagy activation (ATG5 and LC3B-II/LC3B-I ratio) was similarly induced in both SCC13 and ATF3-SCC13 cells. bactin expression was detected as a loading control. Results are representative of three independent experiments. (b) Effects of rapamycin on cellular senescence detected by SA-b-gal staining. SA-b-gal activity increased in both SCC cells but more potently in ATF3-SCC13 cells by rapamycin. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells were expressed as percentage of the number of total cells. Data are presented as the mean sd (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. Scale bar, 50 lm. (c) Effects of rapamycin on cellular apoptosis detected by annexin V/PI staining. Apoptosis was not induced in either SCC13 or ATF3-SCC13 cells by rapamycin. (d) Left, the inhibitory effects of rapamycin on cell growth. Cell growth of both SCC13 and ATF3-SCC13 cells was suppressed by rapamycin in a dose-dependent manner. Cell growth levels were analysed using EZ-Cytox cell growth assay that measured the absorbance at 450 nm. Right, effects of rapamycin on cellular senescence as detected by SAb-gal staining. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells were expressed as percentage of the total cell number. Data are presented as the mean SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. Figure S5. Immunohistochemical analysis of p62 in cutaneous SCC in situ, invasive cutaneous SCC and metastatic cutaneous SCC lesions. The expression of p62 in metastatic cutaneous SCC and invasive cutaneous SCC was higher than that in cutaneous SCC in situ. In metastatic cutaneous SCC, p62 staining was mostly positive in both the nucleus and cytoplasm of the SCC cells. In invasive cutaneous SCC, p62 staining was positive in the cytoplasm of SCC cells. Representative images from one of five experiments are shown. Scale bars, 50 lm.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Original Article
www.wileyonlinelibrary.com/journal/EXD
Activation of autophagic pathways is related to growth inhibition and senescence in cutaneous squamous cell carcinoma So Ra Choi1*, Bo Young Chung1*, Seong Who Kim2, Chang Deok Kim3, Woo Jin Yun1, Mi Woo Lee1, Jee Ho Choi1 and Sung Eun Chang1 1 Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 2Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; 3Department of Dermatology and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, Korea Correspondence: Sung Eun Chang, Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, 86 Asanbyeongwon-gil, Songpa-gu, Seoul 138-736, Korea, Tel.: +82-2-3010-3467, Fax: +82-2-486-7831; e-mail: [email protected] *These authors contributed equally to this work.
Abstract: Cutaneous squamous cell carcinoma (SCC) is a very common resectable cancer; however, cutaneous SCC is highly resistant to chemotherapy if metastasis develops. Activating transcription factor 3 (ATF3) has been suggested as a marker of advanced or metastatic cutaneous SCC. Autophagy is one of the most important mechanisms in cancer biology and commonly induced by in vitro serum starvation. To investigate the role of autophagy activation in cutaneous SCC, we activated autophagic pathways by serum starvation in SCC13 and ATF3-overexpressing SCC13 (ATF3-SCC13) cell lines. ATF3-SCC13 cells demonstrated high proliferative capacity and low p53 and autophagy levels in
comparison with control SCC13 cells under basal conditions. Intriguingly, autophagic stimulation via serum starvation resulted in growth inhibition and senescence in both cells, while ATF3SCC13 cells further demonstrated growth inhibition and senescence. Apoptosis was not significantly induced by autophagy activation. Taken together, autophagy activation may be a promising antitumor approach for advanced cutaneous SCC.
Introduction
ATF3 expression is elevated in human breast cancer (11) and prostate cancer (13) and significantly elevated in cutaneous SCCs that are metastatic or arise in organ transplant recipients (14). ATF3 is a well-known suppressor of p53-dependent senescence and enhances tumorigenic potential in cutaneous SCC (15). In a previous study, ATF3-overexpressing SCC13 cells (ATF3-SCC13) were far more advanced and metastatic than control SCC13 cells (15). The role of autophagy remains unknown in skin cancers. Recent studies have reported that autophagic activation was correlated with the disease progression and prognosis of cutaneous SCC and malignant melanoma (16,17). Autophagy has been described as a process that induces non-apoptotic cell death and as an antitumor mechanism (18–21). Autophagy, originally known as a survival mechanism, is a lysosomal-associated catabolic process that degrades and recycles cellular constituents, such as longlife proteins and organelles (22). Experimental tools in vitro for autophagy activation include serum starvation and/or rapamycin treatment, a well-known mammalian target of rapamycin (mTOR) inhibitor. To investigate the role of autophagy activation in cutaneous SCC, we activated autophagic pathways in SCC13 and ATF3-overexpressing SCC13 (ATF3-SCC13) cell lines. Our current study was aimed to elucidate how autophagy activation in localized cutaneous and advanced cutaneous SCCs functions as an antitumor mechanism.
Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers worldwide, and SCC is the second most common skin cancer (1). Early-stage cutaneous SCC is highly curable and demonstrates a relatively low rate of metastasis (3–5%) (2). However, cutaneous SCC with lymph node or distant metastasis demonstrates poor prognosis regardless of treatment (3). Standard first-line treatments for advanced cutaneous SCC currently include surgical resection, radiation therapy or both. Chemotherapy has been integrated into standard treatment regimens for advanced cutaneous SCC in an attempt to improve survival outcomes. However, current therapies for advanced cutaneous SCC demonstrate poor response and high recurrence rates, demonstrating the need for new therapeutic targets (2,4). The incidence of cutaneous SCC is increasing, partly due to new therapeutic regimens involving for example BRAF inhibitors (5,6). Patients treated with BRAF inhibitors, targeted therapeutics for advanced melanomas harbouring certain BRAF mutations, frequently develop non-melanoma skin cancers including cutaneous SCC. The cause of the keratinocyte proliferation underlying these SCC lesions has been suggested to be paradoxical hyperactivation of the mitogen-activated protein kinase (MAPK) pathway (7). As another mechanism of cutaneous SCC development and progression, a recent study has reported the involvement of p53 family members and their signalling via Notch (8). Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding family of transcription factors (9). ATF3 is significantly increased by a variety of stress signals, including anoxia, DNA damage and carcinogenesis (10–12).
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Key words: activating transcription factor 3 – autophagy – cutaneous squamous cell carcinoma – growth inhibition – senescence
Accepted for publication 17 July 2014
Materials and methods Ethics statement All patient biopsies were obtained with written informed consent and in accordance with the Helsinki declaration. The study was
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Role of autophagy in cutaneous squamous cell carcinoma
approved by the institutional review board of the Asan Medical Center (file reference: 2014-0403).
nyl acetate and lead citrate and examined at 50 kV using a JEM 1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan).
Immunohistochemistry
Cell growth analysis and apoptosis assay
Five sets of biopsy tissues were obtained from each patient, fixed in 10% buffered neutral formalin and embedded in paraffin. These specimens were cut to 4-lm-thick sections, and serial sections were prepared for immunohistochemistry. For antigen retrieval, sections were autoclaved in antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) and immunostained using Vector Elite ABC Kit (Vector Laboratories) according to the manufacturer’s instructions. Sections were treated with diaminobenzidine (0.5 mg/ml) and hydrogen peroxide. The primary antibodies included an ATF3 antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p62 antibody (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA). Staining positivity was classified into five grades: 0, negative; +, 50%. All slides were evaluated independently by two dermatologists with no knowledge of the identity of the patient or clinical outcome.
Following treatment with rapamycin, BFA1 or serum starvation, cell growth was determined using the EZ-Cytox cell growth assay kit (ITSBIO, Seoul, Korea) according to the manufacturer’s guidelines. Absorbance was measured at 450 nm using an enzyme immunosorbent assay (ELISA) reader (Molecular Devices Co., Sunnyvale, CA, USA). Apoptosis was measured by flow cytometry using the annexin V-FITC apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Annexin V-FITC and propidium iodide (PI) were added according to the manufacturer’s instructions. After 15 min of incubation at room temperature, cells were acquired by flow cytometry (BD Biosciences) and the presence of viable cells (annexin V- and PI-negative), early apoptotic cells (annexin V-positive and PI-negative) and late apoptotic cells (annexin V- and PI-positive) were assessed using Cell Quest software (BD Biosciences).
Reagents Rapamycin and bafilomycin A1 (BFA1) were purchased from Sigma-Aldrich (St Louis, MO, USA). The following primary antibodies against human proteins were used: anti-b-actin and antiLC3B (Sigma-Aldrich); anti-ATG5 (Novus Biologicals, LLC, Littleton, CO); anti-p62 (Medical & Biological Laboratories, Nagoya, Japan); anti-p53, anti-phospho-p53 (Ser15), anti-Rb, anti-phospho-Rb (Ser807/811), anti-p21, anti-phospho-mTOR (Ser2448), anti-phospho-p70S6K (Thr389), and anti-Beclin1 (all from Cell Signaling Technology, Inc., Danvers, MA); and anti-ATF3 (Santa Cruz Biotechnology).
Cell culturing The SCC13 human squamous cell carcinoma (SCC) cell line and ATF3-overexpressing SCC13 cells were provided by Dr. G. Paolo Dotto (Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA, USA). SCC13 cells originated from a human SCC of the facial epidermis with a moderate degree of mitosis and proliferation (23), with a wild-type ras gene and harbouring single p53 missense mutations (24). These cells were cultured in DMEM (Welgene, Deagu, Korea) containing 10% foetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% antibiotic–antimycotic solution (100 9 ) (Gibco). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. Serum starvation was performed in 5% and 1% serum for 72 h.
Transient siRNA transfection Cells were placed 6-well plates, seeded at a density of 1 9 105 cells and transfected with siRNA against p53, ATF3 or negative control siRNA using LipofectamineTM RNAiMAX (Invitrogen, Merelbeke, Belgium) according to the manufacturer’s instructions.
Transmission electron microscopy To further verify autophagy in SCC13 and ATF3-SCC13 cells, we directly examined the cellular autophagosomes using transmission electron microscopy (TEM). Briefly, the cells were fixed at 4°C overnight using a 2.5% glutaraldehyde and 0.1 mol/L phosphate buffer (pH 7.2). After 90 min of postfixation in 1% osmium tetroxide and 0.1 mol/L phosphate buffer at 4°C, the samples were dehydrated in a graded series of alcohol and embedded in Epon media. Ultrathin sections were cut to 50–60 nm, stained with ura-
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Western blot analysis Total proteins were extracted using PRO-PREP extraction solution (Intron, Deajeon, Korea). Protein concentrations were determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Equally loaded proteins from each sample were resolved in SDSPAGE gels (Invitrogen, Merelbeke, Belgium) and transferred to nitrocellulose membranes (WhatmanTM, GE Healthcare Life Sciences, Buckinghamshire, UK). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween20 and 5% non-fat dry milk. Membranes were then incubated overnight at 4°C with primary antibodies that were diluted in 5% non-fat dry milk or 5% BSA in TBS-T with Tween-20. Membranes were then washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). After further washing with TBS-T, the protein bands were visualized using a SuperSignal West Pico Trial kit (Thermo Scientific, Rockford, IL, USA). Primary antibodies against the following proteins were used: b-actin (SigmaAldrich), LC3B (Sigma-Aldrich), ATG5 (Novus Biologicals), p62 (Medical & Biological Laboratories), Beclin1, p53, phospho-p53 (Ser15), p21, Rb, phospho-Rb (Ser807/811), phospho-mTOR (Ser2448), phospho-p70S6K (Thr389) (all from Cell Signaling) and ATF3 (Santa Cruz Biotechnology). All experiments were performed at least three times.
Senescence-associated beta-galactosidase (SA-b-Gal) staining Cells were seeded at the same density in 6-well plates and stained with SA-b-Gal using a senescence b-galactosidase staining kit (Cell Signaling Technology). SA-b-gal is a commonly used senescence biomarker that can be detected at pH 6.0 (21). This is because senescent cells express a high level of lysosomal b-gal (25,26).
Statistical analysis Statistically significant differences between groups were assessed using analysis of variance (ANOVA) and the Student t-test. In this study, P < 0.05 was considered statistically significant.
Results Metastatic cutaneous SCC and ATF3-SCC13 cells demonstrate significantly increased ATF3 expression The clinicopathologic data of patients who provided skin biopsy tissues were examined (Table 1). Differences in ATF3 expression in cutaneous SCC in situ, invasive cutaneous SCC and metastatic
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cutaneous SCC were compared using immunohistochemical analysis (Fig. 1a). ATF3 was not expressed in the cutaneous SCC in situ samples. Metastatic cutaneous SCC samples demonstrated stronger ATF3 immunoreactivity than cutaneous SCC in situ and invasive cutaneous SCC samples. Mouse IgG1/kappa did not demonstrate positive signals in metastatic cutaneous SCC samples. ATF3 showed a more distinct nuclear staining in metastatic SCC cells compared with SCC in situ or invasive SCC cells. This finding is consistent with the fact that ATF3 is a transcription factor with robust nuclear expression. Also, in cell culture experiments, ATF3 expression was higher in ATF3-SCC13 than SCC13 cells (Fig. 1b) and there were two ATF3 bands apparent on a Western blot. The mobility of ATF3 was observed to differ between SCC13 and ATF3-SCC13 cells (Fig. 1b), implying that ATF3 expression may be subject to post-transcriptional regulation. Based on these results, we choose ATF3-SCC13 and control SCC13 cells as in vitro representative of metastatic cutaneous SCC and localized cutaneous SCC, respectively.
ATF3-SCC13 cells demonstrate high proliferative capacity, low p53 expression and low basal autophagy compared with SCC13 cells ATF3-SCC13 cells demonstrated better growth than SCC13 cells (Fig. 2a). Because p53 is a well-known senescence marker that is counteracted by ATF3 in cutaneous SCC samples (12), we knocked down ATF3 and p53 in SCC13 cells using siRNA. When we targeted ATF3, p53 expression was higher than the negative control siRNA (siControl) in both SCC13 and ATF3-SCC13 cells (Fig. 2b). In addition, ATF3 expression increased in SCC13 cells following p53 knock-down (Fig. 2b). These results indicate that ATF3 is inversely correlated with p53 in SCC cells. There were two ATF3 bands on a Western blot, and the upper of these bands (ATF3-SCC13 cells) in Fig. 2b was stronger than that in Fig. 1. We then evaluated basal autophagy in each cell because p53 may be associated with autophagy (27). The expression levels of autophagy-related proteins, including ATG5, Beclin1 and the microtubule-associated protein 1 light chain 3 (LC3B)-II/LC3B-I ratio, were lower, and p62 was higher in ATF3-SCC13 cells than in SCC13 cells (Fig. 2c). The phosphorylation of mTOR and p70S6K in ATF3-SCC13 cells was higher than that in SCC13 cells (Fig.
(a)
(b)
Figure 1. ATF3 expression in dermal tissues and cutaneous SCC and SCC cell lines. (a) Immunohistochemical analysis of cutaneous squamous cell carcinoma (SCC) in situ, invasive cutaneous SCC and metastatic cutaneous SCC lesions. ATF3 expression in metastatic cutaneous SCC was higher than cutaneous SCC in situ or invasive cutaneous SCC. ATF3 showed a more distinct nuclear staining in metastatic SCC cells than in SCC in situ or invasive SCC cells. Representative images of one of five experiments are shown. Scale bar, 50 lm. (b) Western blot analysis of ATF3 expression in SCC cells. ATF3 expression was higher in ATF3SCC13 cells than SCC13 cells.
S1). By TEM, the number of autophagosomes was lower in ATF3SCC13 cells than in SCC13 cells (Fig. 2d). These results provide evidence that ATF3-SCC13 cells demonstrate lower basal autophagy levels, as well as lower cellular senescence, than SCC13 control cells.
Table 1. Summary of clinical and pathologic data Age (years)
Sex
Diagnosis
Primary site
84 82 89 69 58 49 84 88 66 76 64 74 75 49 74
F F F F F M F M F M M M M M M
SCC in situ SCC in situ SCC in situ SCC in situ SCC in situ SCC SCC SCC SCC SCC Metastatic SCC Metastatic SCC Metastatic SCC Metastatic SCC Metastatic SCC
Lt cheek Rt temporal area Nose Rt cheek Rt infraorbital area Nose Rt cheek Lt cheek Neck Rt temporal scalp Scalp Chest Lt clavicular area Scalp Lt axilla
State of differentiation
Metastasis (location)
ATF3 staining
p62 staining
Moderately Well Well Well Well Poorly Poorly Moderately Well Poorly
– – – – – – – – – – + + + + +
+ + + + + +++ ++ ++ +++ ++ ++++ ++++ +++ ++++ ++++
+ + + + + +++ +++ +++ ++ ++ ++++ ++++ +++ ++++ ++
(Lung) (LN) (LN) (Lung) (LN)
0, negative staining; +, focal or < 10% positive of SCC cells; ++, 10–30% positive of SCC cells; +++, 30–50% positive of SCC cells; and ++++, > 50% positive of SCC cells. M, male; F, female; Lt, left; Rt, right; LN, lymph node; SCC, squamous cell carcinoma.
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Role of autophagy in cutaneous squamous cell carcinoma
(a)
(b)
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Figure 2. Cell proliferation, ATF3 and p53 expression, and basal autophagy in SCC cells. (a) ATF3-SCC13 cells demonstrate a higher growth level than SCC13 cells. Cell growth levels were analysed using a cell viability assay that measured the absorbance at 450 nm. Data are presented as the mean SD (n = 3). **P < 0.01 and ***P < 0.001 according to the Student t-test. (b) Western blot analysis validating ATF3 and p53 knock-down in SCC cells that were transfected with ATF3, p53 or negative control siRNA (siControl) for 48 h. When ATF3 was targeted, p53 expression was higher compared with negative control siRNA (siControl) in both SCC13 and ATF3-SCC13 cells. In addition, ATF3 expression increased in SCC13 cells following p53 knock-down. b-actin expression was used as the loading control. These results are representative of three independent experiments. (c) Western blot analysis of autophagy-related proteins, including ATG5, Beclin1, LC3B and p62. ATG5 and the LC3B-II/LC3B-I ratio were significantly decreased, and the p62 level was slightly increased in ATF3-SCC13 cells compared with SCC13 cells. b-actin expression was used as the loading control. These results are representative of three independent experiments. (d) Examination of autophagosomes in SCC13 and ATF3-SCC13 cells using transmission electron microscopy. The number of autophagosomes was lower in ATF3-SCC13 cells in comparison with SCC13 cells. (AP) autophagosome; (N) nuclei; (M) mitochondria. Scale bars, 50 lm.
Activating autophagy inhibits cellular growth and induces cellular senescence
Figure 3. Activation of autophagy in SCC cells by serum starvation induces cellular senescence more potently if ATF3 is overexpressed. SCC cells were cultured in complete medium for 24 h before being cultured in medium with 1% or 5% FBS for 72 h. (a) Western blot analysis of p53, phosphorylated p53 (Ser 15), p21, Rb, phosphorylated Rb (Ser807/811) and autophagy-related proteins, including ATG5, LC3B and p62. Autophagy activation (ATG5 and LC3B-II/LC3B-I ratio) in ATF3SCC13 cells was more potent than that in SCC13 cells. b-actin expression was used as the loading control. The results are representative of three independent experiments. (b) Effects of serum starvation on cellular senescence detected by SA-b-gal staining. Under serum starvation, SA-b-gal activity in ATF3-SCC13 cells increased compared with that in SCC13 cells. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells are expressed as a percentage of the total cells. Scale bar, 50 lm. (c) Effects of serum starvation on cellular apoptosis were detected using annexin V/PI staining. Apoptosis was not induced in either SCC13 or ATF3-SCC13 cells by serum starvation.
To determine the effects of autophagy activation by serum starvation in SCC cells, we subjected these cells to serum starvation for 72 h and measured ATF3 expression and autophagy level. Because cell viability decreased to about 60% after 72 h of serum-free condition (Fig S2a), the results of serum-free condition might not have been accurate compared with serum 1% and 5% conditions. Therefore, we conducted further serum starvation experiments at serum 1% and 5%. Unexpectedly, autophagy activation (according to ATG5 expression, the LC3B-II/LC3B-I ratio) in ATF3-SCC13 cells was more potent than that in SCC13 cells (Fig. 3a), while the basal autophagy level was far lower in ATF3-SCC13 cells (Fig 2c). The phosphorylation of mTOR and p70S6K was suppressed in both SCC13 and ATF3-SCC13 cells by serum starvation compared with the control, and the suppressed degrees were higher in ATF3-SCC13 cells (Fig S2b). Furthermore, the cellular growth of both cells was significantly suppressed by serum starvation in comparison with the 10% serum-containing control. ATF3-SCC13 cells showed further inhibition of cell growth, while the basal proliferation capacity was higher in ATF3-SCC13 cells (Fig S2c). To confirm whether an autophagic pathway is activated by serum starvation in SCC cells, we treated the ATF3-SCC13 and SCC13 cells with an autophagy inhibitor bafilomycin A1 (BFA1) (2.5 nM) for 48 h in the presence or absence of serum starvation. We first assessed the cytotoxic effects of BFA1 on SCC cells
(Fig.S3a). According to the cytotoxicity results, we chose 2.5 nM as the maximal concentration of this inhibitor. Serum starvation in conjunction with BFA1 treatment decreased the LC3B-II/LC3BI ratio and ATG5 levels and increased p62 expression in both SCC13 and ATF3-SCC13 cells (Fig.S3b). We determined the protein levels of p53, phosphorylated p53 (Ser 15) and p21 as other markers of senescence. While p21 demonstrated no change, phosphorylated p53 levels were higher in ATF3-SCC13 cells than SCC13 control cells following serum starvation (Fig. 3a). Apoptosis was not induced, but senescence-associated b-galactosidase activity (SA-b-gal) increased in both cells following serum starvation (Figs 3b, c and S2c). These findings imply that autophagy activation inhibits growth and induces senescence in ATF3-SCC13 cells to a greater degree. We also evaluated the effects of rapamycin, as another autophagy activator, on cellular proliferation versus senescence in the SCC13 and ATF3-SCC13 cells. The results of rapamycin treatment on SCC cells showed similar results to serum starvation. Autophagy activation (ATG5, LC3B-II/LC3B-I ratio and p62) was similarly induced in both SCC13 and ATF3-SCC13 cells by rapamycin (Fig. S4a). Senescence was also induced, but apoptosis was not, in
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both cell types following rapamycin treatment. Activation of autophagy by rapamycin induces senescence more potently in ATF3SCC13 cells (Fig.S4b–d). We conducted immunohistochemical analysis of SCC samples from patients using anti-p62 antibodies to assess autophagy in vivo. By immunohistochemical comparison, the expression of p62 in metastatic cutaneous SCC and invasive cutaneous SCC was higher than that in cutaneous SCC in situ. In metastatic cutaneous SCC, p62 staining was mostly positive in both the nucleus and cytoplasm of the SCC cells. In invasive cutaneous SCC, p62 staining was positive in the cytoplasm of the SCC cells (Fig. S5). These results are consistent with increased ATF3 expression in metastatic SCC with more distinct staining in the nucleus, which suggests a relationship between the expression of p62 and ATF3. Both SCC13 and ATF3-SCC13 cells are susceptible to autophagic stimuli by serum starvation, leading to autophagy activation, growth inhibition and senescence. Interestingly, highly proliferative ATF3-SCC13 cells, which demonstrated low basal autophagy and senescence, were more susceptible to autophagic stimulation.
Discussion Autophagy is an evolutionarily conserved catabolic process whereby cells degrade and recycle aggregated protein complexes, poorly functioning organelles and pathogens allowing cells to survive starvation and other stresses (28). Conventional transmission electron microscopy (TEM) is one method for detecting the accumulation of autophagic compartments in mammalian cells. In TEM, autophagosomes have double or sometimes multiple limiting membranes and their cytoplasmic contents begin to show dark granular/amorphous clumps with age (29). Microtubule-associated protein 1 light chain 3 (LC3) is a specific marker protein for autophagy that exists in two forms, LC3-I and its proteolytic derivative LC3-II (18 and 16 kDa, respectively), which are localized in the cytosol (LC3-I) or in autophagosomal membranes (LC3-II). In addition to LC3, Atg5, Beclin-1, p62 and mTORC1 protein can be used as markers of autophagy. Atg5 is involved in the elongation of the isolation membrane during autophagosome formation (30). Beclin-1 localizes to the trans-Golgi network, belongs to the class III phosphatidylinositol 3-kinase complex and is involved in autophagosome formation (31). The p62 protein acts as an axillary autophagy factor by directly binding ubiquitinated proteins and LC3 to facilitate their degradation by autophagy (32). The accumulation of p62 has been used to indicate abnormalities in the autophagic degradation (33) and also for measuring the flux of autophagosomes to demonstrate inhibition of autophagy by bafilomycin A1 (34). The mTORC1 is a cellular sensor of nutritional conditions that negatively regulates autophagy (35). Inhibition of mTORC1 activity induces autophagy in many types of cells (19). We used the above-mentioned multiple methods to monitor autophagy in our current study. The involvement of autophagy in tumorigenesis is, at present, incompletely understood, and the opposing effects of autophagy on cell survival and death have been documented (36,37). Whether it is autophagy activation or inhibition that demonstrates anticancer effects is controversial. Many tumors contain poorly vascularized regions that lack both nutrients and oxygen, which is also associated with high levels of autophagosomes (38). In these settings, we believe that autophagy is a cytoprotective mechanism for tumor cells and can therefore be considered oncogenic. How-
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ever, some autophagy-deficient murine models are reportedly prone to lung carcinoma (39), and it has been demonstrated that key autophagy-related genes are inactivated in the major sporadic forms of human cancer (39–41). This suggests that autophagy contributes to tumor suppression, at least in some tumor types and at some point during tumor development. In cutaneous SCC, the role of autophagy has only been sporadically analysed. Recent studies show that inhibiting autophagy may lead to increased apoptosis in cutaneous SCC, thereby benefiting antitumor therapies (18,19). To explain contrary results, it is has been suggested that the proper level of autophagy promotes cell survival, while higher levels result in autophagic cell death. Many anticancer agents induce autophagy to restrain growth in various cancers, and studying autophagy is a new direction in the development of anticancer drugs. Here, for the first time, we investigated autophagy activation and induction as a tumor-suppressing mechanism in cutaneous SCC. Autophagy is commonly induced by in vitro serum starvation or rapamycin treatment. Serum starvation is the most common and reliable tool in experiments on autophagy activation, while mTOR-inhibiting rapamycin induces apoptosis, cytotoxicity and autophagy (42). The target of rapamycin (TOR) is a highly conserved serine/threonine protein kinase that acts as a central sensor for growth factors, nutrient signals, cellular energy status and a master regulator of autophagy (43). Rapamycin also increases apoptosis, the p53/MDM2 protein ratio and p53-dependent apoptosis via the translational inhibition of MDM2 in cancer cells (44). Other previous studies performed on various murine models also report that the effects of rapamycin include reduction in tumor number, tumor size and the progression of benign lesions to SCCs in UVB- or cyclosporine-induced carcinogenesis and in solid organ transplant recipients (45–48). Human clinical trials on using rapamycin to treat metastatic cutaneous SCC are still ongoing (18). ATF3 belongs to the ATF/cAMP-responsive element-binding protein family of transcription factors and can bind to DNA (via the ATF/CREB consensus sequence, 50 -TGACGTCA-30 ) (49) and other proteins (e.g. p53 and E6) (50), modifying gene expression and cellular functions. ATF3 is a stress-inducible and/or adaptive response gene, as its mRNA and protein levels rapidly are increased by stress signals, such as cytokines, genotoxic agents, nerve injury and tissue damage (49). Recent studies have implicated ATF3 in many cancers (51), but its exact role remains unclear. There has been much evidence characterizing ATF3 as an oncogene. Its overexpression has been found in human breast cancer, prostate cancer and Hodgkin lymphomas. (11). A recent study showed that an elevated level of ATF3 expression suppresses p53dependent senescence and increases tumorigenic potential in squamous skin cancer (15). However, some evidence suggests that ATF3 may be able to inhibit tumorigenesis. ATF3 expression decreased in human colorectal cancer, and ATF3 overexpression resulted in apoptosis of human prostate cancer cells (52). ATF3 plays the context-dependent role in cancer possibly due to the complex protein–protein interaction networks. In addition to transcriptional regulation, it has been reported that ATF3 interacts with a number of critical cellular proteins while regulating their functions. The wild-type p53 is one of the ATF3-binding proteins that have been well documented (50). The major p53 suppressor
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
Role of autophagy in cutaneous squamous cell carcinoma
MDM2 binds to and ubiquitinates ATF3 by accelerating its degradation (53). ATF3 binds to and stabilizes p53 by blocking its ubiquitination, thereby activating p53 as a countermeasure to DNA damage (50). ATF3 has also been known to be a negative regulator of TP53 gene expression (51). Based on these results, it can be deduced that ATF3 may serve as a p53 activator that stabilizes p53 protein or as a p53 inhibitor that downregulates the transcriptional activity of TP53. This study confirms higher ATF3 expression in metastatic cutaneous SCC tissues in comparison with SCC in situ and localized cutaneous SCC lesions. In addition, we confirmed that ATF3SCC13 cells were more proliferative at lower p53 levels, which is consistent with a previous study (12). High ATF3 expression suppresses p53-dependent senescence and enhances tumorigenic potential (15). p53 is a representative tumor suppressor gene and marker of senescence. Cellular senescence is a permanent feature of cell cycle arrest that is believed to play a role in tumor growth inhibition and suppression (54,55). Consistent with this, using reciprocal knock-down experiments, our data show an interconnected relationship between ATF3 and p53 expression in SCC cells. p53 expression increases when ATF3 is antagonized by siRNA, and ATF3 expression increases in SCC cells when p53 is antagonized. Overall, ATF3-SCC13 represents a more advanced type of SCC that is treatment resistant. Here, we report the repression of cell proliferation and increase in cellular senescence in SCC cells following serum starvation. ATF3-SCC13 cells exhibited significantly less proliferation than SCC13 cells and demonstrated increased senescence following serum starvation. Rapamycin treatment produced similar results to serum starvation. It is interesting that ATF3-SCC13 cells, in other words, clinically metastatic or advanced cutaneous SCC, in which p53 is suppressed, demonstrated more obviously inhibited growth and increased senescence when p53 and autophagy-related proteins increased following serum starvation compared with SCC13 cells. SCC cells seemed to show different responses to autophagy activation, at least in terms of ATF3 expression and basal autophagy. As expected, we found that ATF3-SCC13 cells demonstrated low basal autophagy in comparison with SCC13 cells. Serum starvation increased the expression of autophagy-associated proteins and decreased cell growth in ATF3-SCC13 and SCC13 cells. We also found that serum starvation increased p53 activation and induced senescence in ATF3-SCC13 and SCC13 cells. In particular, reactions to serum starvation were more obvious in ATF3-SCC13 cells. ATF3 is known to be the regulator of p53 in response to a wide range of environmental insults (50,51). Autophagy has been suggested to be regulated by several oncogenes and tumor suppressors. p53 is one of the most well-known
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tumor suppressors that may play a dual role in autophagy regulation, that is positively or negatively (56,57). Taken together, ATF3 regulates p53, which in turn regulates and affects autophagy. One of the possible causes behind the various reactions to autophagy among SCC cells at different levels of ATF3 expression could be the difference in p53 regulation by ATF3. However, the relationships among ATF3, p53 and autophagy have yet to be clarified. Therefore, further research is required to elucidate the exact mechanism. In our results, there were two ATF bands evident by Western blotting and the upper of these two bands in Fig. 2b (ATFSCC13) was stronger than that in Fig. 1b. There could be two major causes of this, including post-transcriptional modification of the protein or as ATF3 is increased by stress signals, a degree of basal stress in the cells. The mobility of ATF3 was also different between SCC13 and ATF3-SCC13 cells in Fig. 1b. This difference of mobility may be caused by post-translational modifications (PTMs) of ATF3. There is evidence that ATF3 is regulated by a PTM; the protein activity level of ATF3 has been shown to be modulated by ubiquitination and/or small ubiquitin-related modifier (SUMO)ylation. Among PTMs, the mechanisms of SUMOylation are very similar to those of ubiquitination, but the biological functions of these two processes are very different. Recent studies have reported that ATF3 can be SUMOylated at lysine 42 as a major SUMO site and that the de-SUMOylation of ATF3 augments the ATF3-p53 physical interaction and the trans-activation of p53 responsive promoters (53). In conclusion, autophagic activation results in growth inhibition and senescence in both SCC13 and ATF3-SCC13 cells, which is stronger in ATF3-SCC13 cells. These findings could potentially advance the practical induction of autophagy and its future use as a therapeutic intervention for advanced cutaneous SCC, which is notorious for chemotherapeutic resistance. Agents that activate autophagy may be promising cytostatic treatments for metastatic cutaneous SCC.
Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science, and Technology (2011-0409) and Asan Life Science Institute (2011-415). We thank Dr. Dotto for generously providing the SCC13 and ATF3-SCC13 cells.
Author contributions SRC performed the research; BYC and SRC wrote the manuscript; WJY and SWK designed the study; MWL, JHC and CDK analysed the data; and SEC supervised the study and the manuscript.
Conflicts of interest The authors have no conflicts of interest to declare.
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Supporting Information Additional supporting data may be found in the supplementary information of this article: Figure S1. Western blot analysis of phospho-mTOR (Ser2448) and phospho-p70S6K (Thr389) in SCC13 and ATF3-SCC13 cells. The phosphorylation of mTOR (Ser2448) and p70S6K (Thr389) in ATF3-SCC13 cells increased compared with that in SCC13 cells. b-actin expression was used as the loading control. These results are representative of three independent experiments. Figure S2. (a) Cell viability of SCC cells on serumfree condition (FBS 0%). When SCC cells were cultured with serum-free medium, cell viability decreased to about 60% after 72 hr in both SCC13 and ATF3SCC13 cells. Cell viability was evaluated using EZ-Cytox cell viability assay that measured the absorbance at 450 nm. Data are presented as the mean SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. (b) Activation of autophagy in SCC cells by serum starvation. The phosphorylation of mTOR and p70S6K was suppressed in both SCC13 and ATF3SCC13 cells by serum starvation compared with the 10% serum-containing control, and the suppressed degrees were higher in ATF3-SCC13 cells. b-actin expression was used as the loading control. These results are representative of three independent experiments. (c) Left, the inhibitory effects of serum starvation on cell growth. Cell growth of SCC cells was significantly suppressed by serum starvation compared with a 10% serum-containing control. ATF3-SCC13 cells showed further inhibition of cell growth, while the basal proliferation capacity was higher in ATF3-SCC13 cells. Cell growth levels were analysed using EZ-Cytox cell growth assay that measured the absorbance at 450 nm. Right, effects of serum starvation on cellular senescence as detected by SA-b-gal staining. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells were expressed as percentage of the total cells. Data are presented as the mean sd (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. Figure S3. (a) Effects of bafilomycin A1 (BFA1) on cell viability in SCC cells. The cells were seeded in 96well plates for 1 day and then treated with BFA1 (0, 1,
2.5, 5 nM) for 48 h. Cell viability was evaluated using EZ-Cytox cell viability assay and expressed as a percentage of the control. Data are presented as the mean SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. (b) Inhibitory effects of autophagy in SCC cells by bafilomycin A1 (BFA1; 2.5 nM) in the presence and absence of serum starvation. Autophagy-related proteins, including ATG5, p62 and LC3B were determined using Western blotting. Autophagy was suppressed by BFA1 in both SCC13 and ATF3SCC13 cells. b-actin expression was detected as a loading control. Results are representative of three independent experiments. Figure S4. Activation of autophagy by rapamycin in SCC cells induces senescence more potently if ATF3 is overexpressed. Rapamycin at 20 nM or 200 nM was added to SCC cultures for 48 h. (a) Western blotting analysis of p53, phosphorylated p53 (Ser 15), p21, Rb, phosphorylated Rb (Ser807/811) and autophagy-related proteins, including ATG5, p62 and LC3B. Autophagy activation (ATG5 and LC3B-II/LC3B-I ratio) was similarly induced in both SCC13 and ATF3-SCC13 cells. bactin expression was detected as a loading control. Results are representative of three independent experiments. (b) Effects of rapamycin on cellular senescence detected by SA-b-gal staining. SA-b-gal activity increased in both SCC cells but more potently in ATF3-SCC13 cells by rapamycin. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells were expressed as percentage of the number of total cells. Data are presented as the mean sd (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. Scale bar, 50 lm. (c) Effects of rapamycin on cellular apoptosis detected by annexin V/PI staining. Apoptosis was not induced in either SCC13 or ATF3-SCC13 cells by rapamycin. (d) Left, the inhibitory effects of rapamycin on cell growth. Cell growth of both SCC13 and ATF3-SCC13 cells was suppressed by rapamycin in a dose-dependent manner. Cell growth levels were analysed using EZ-Cytox cell growth assay that measured the absorbance at 450 nm. Right, effects of rapamycin on cellular senescence as detected by SAb-gal staining. Three fields were selected to calculate the cell numbers in each group, and the average was taken as the final cell count. Senescent cells were expressed as percentage of the total cell number. Data are presented as the mean SD (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 by the Student t-test. Figure S5. Immunohistochemical analysis of p62 in cutaneous SCC in situ, invasive cutaneous SCC and metastatic cutaneous SCC lesions. The expression of p62 in metastatic cutaneous SCC and invasive cutaneous SCC was higher than that in cutaneous SCC in situ. In metastatic cutaneous SCC, p62 staining was mostly positive in both the nucleus and cytoplasm of the SCC cells. In invasive cutaneous SCC, p62 staining was positive in the cytoplasm of SCC cells. Representative images from one of five experiments are shown. Scale bars, 50 lm.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 718–724
