- Email: [email protected]
PKC-δ attenuates the cancer stem cell among squamous cell carcinoma cells through down-regulating p63
Accepted Manuscript Title: PKC-␦ attenuates the cancer stem cell among squamous cell carcinoma cells through down-regulating p63 Authors: Dongmei Zhang, Mingjing Fu, Lingyan Li, Huan Ye, Zhiqi Song, Yongjun Piao PII: DOI: Reference:
S0344-0338(17)30055-9 http://dx.doi.org/doi:10.1016/j.prp.2017.07.013 PRP 51850
To appear in: Received date: Revised date: Accepted date:
18-1-2017 15-6-2017 12-7-2017
Please cite this article as: Dongmei Zhang, Mingjing Fu, Lingyan Li, Huan Ye, Zhiqi Song, Yongjun Piao, PKC-␦ attenuates the cancer stem cell among squamous cell carcinoma cells through down-regulating p63, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page PKC-δ attenuates the cancer stem cell among squamous cell carcinoma cells through down-regulating p63
Running title: PKC-δ attenuates the cancer stem cell through p63
Dongmei Zhang1, Mingjing Fu2, Lingyan Li2, Huan Ye2, Zhiqi Song2, Yongjun Piao2
1 Departments of Physiology, Dalian Medical University, Dalian, China 2 Department of Dermatology, The First Affiliated Hospital of Dalian Medical University Correspondence: Yongjun Piao Department of Dermatology, The First Affiliated Hospital of Dalian Medical University. No. 222, zhongshan road, Xigang District, Dalian 116011, China. E-mail address: [email protected]
Tel: +86 18098870608
Abstract Protein kinase C delta (PKC-δ) has been identified as a tumor suppressor. However, the effects of PKC-δ on the cancer stem cells in squamous cell carcinomas (SCC) have not been clarified. The purpose of this study was to detect the regulation of PKC-δ on cancer stem cell among SSC cells and the role of p63 during the regulation. Immunohistochemistry of human cutaneous SCC tissues was performed to detect the expression of PKC-δ. After the human SCC13 cells infected by recombinant adenoviruses, the cell proliferation were determined. The correlation of PKC-δ and p63 was detected by western blot. The colony forming activity and the number of cancer stem cells (CSCs) in SCC identified by double-staining with anti-integrin 6 and anti-CD71 antibodies were detected. The expression of PKC-δ was obviously decreased in SCC tissues compared with that in
normal skin tissues. The higher protein level of p63 in SCC was attenuated by the transfection of PKC-δ. The higher proliferation capacity of SCC13 cells, the higher activity and expression of CSCs in SCC13 cells induced by p63 were significantly suppressed by the transfection of PKC-δ. In conclusion, PKC-δ played as a protective role in SCC partly by down-regulating p63, leading to the suppression of SCC cell proliferation, attenuation of the activity and expression of CSCs in SCC cells.
Key words: PKC-δ; squamous cell carcinomas; cancer stem cells; p63
1. Introduction Squamous cell carcinoma (SCC), as one of the most common malignancies, is potentially aggressive, metastatic, and lethal. During recent years, efforts have been made on various aspects of SCC, including detection techniques, treatment strategies, and mechanisms involved in cancer initiation and progression. Despite advancements in oncology researches and therapies in the last decade, SCC with high morbidity and resistance was still being observed in large numbers of patients, leading to reduced life quality. A novel effective clinic treatment of SCC is imperative. Recent studies have demonstrated that a variety of human malignant tumor comprises a rare subpopulation of cells, which are referred to as cancer stem cells (CSCs)[1]. They possess self-renewal, tumor-initiating capabilities and sustain the tumor growth. It is believed that the existence of CSCs may be one reason for the lack of effectiveness and resistance of the cancers to the conventional therapies, and be considered as a major cause of the tumor recurrence after the conventional chemo- or radio-treatment[2, 3, 14]. Targeted elimination of CSCs has drawn attentions these years and believed to provide a framework for treatment of SCC. Protein kinase C (PKC) is a Ser/Thr kinase family including at least 12 isoforms. The
expression and activation patterns of PKC isoform vary in tissues. Activation of PKC is involved in a wide range of cellular functions, notably proliferation, differentiation, and cell survival[10, 15, 18]. It is demonstrated that PKC-δ (a subtype of PKC) functions as a critical mediator in macrophage-colony stimulating factor induced differentiation signaling[11, 15]. PKC-δ is also displayed with the involvement in differentiation of embryonic stem cells as well as H9c2 cells[5, 6]. However, whether PKC-δ affects the CSCs in SCC has not been identified. p63 is well recognized as a master squamous regulator and is the primary marker used to clinically diagnose SCCs[19]. It is confirmed that p63 is indispensable to maintain the high proliferative potential of CSCs[17]. Our aim of the study is to investigate the relationships among PKC-δ, p63 and CSCs in SCC. 2. Methods and materials Immortalized human SCC13 cells and 293A cells were provided by Professor Tae-Jin Yoon (Gyeongsang National University, Jinju, South Korea). Skin samples were obtained from patients who underwent surgical resection of squamous cell carcinoma in Chungnam National University Hospital. The written informed consent was obtained from all the donators. All the experiments were created in accordance with the ethical committee approval process of Chungnam National University Hospital. 2.1 Immunohistochemistry Skin samples were prepared for this experiment. Paraformaldehyde-fixed and paraffin embedded specimens were deparaffinized, rehydrated and washed by phosphate-buffered saline (PBS). After treatment with proteinase K (1 mg/ml) at 37 ºC for 5 min, sections were treated with H2O2 for 10 min at room temperature, blocked in 1% bovine serum albumin (BSA) in PBS for 20 min, followed by reaction with purified goat anti-PKC-δ antibody (1:100, Santa Cruz Biotechnologies, CA, USA) for 1h. Sections were incubated sequentially with peroxidase-conjugated secondary antibodies (Upstate, Lake Placid, NY) and visualized with ChemMate Envision Detection kit (Dako, Carpinteria, CA). 2.2 Cell culture and adenovirus infection experiments Immortalized human SCC13 cells were cultured in Dulbecco’s modified Eagle’s
medium(DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Rockville, MD) and penicillin/streptomycin antibiotics (1:100). The cells were maintained at 37 °C in humidified air with 5% CO2. The PKC-δ full-length cDNA was amplified by polymerase chain reaction (PCR) and subcloned into pENT/CMV vector that has attL sites for site-specific recombination with a Gateway destination vector (Invitrogen, Carlsbad, CA). All plasmid constructs were sequence-verified. The recombinant adenoviruses containing PKC-δ (Ad-PKC-δ) or p63 (Ad-p63) were created using Virapower adenovirus expression system (Invitrogen, USA) according to the method previously described[12]. Briefly, site-specific recombination between entry vector and adenoviral destination vector was achieved by LR clonase (Invitrogen, USA). The resulting adenoviral expression vector was transduced into 293A cells using Lipofectamine 2000 (Invitrogen, USA). The control recombinant adenovirus containing galactosidase gene (Ad-lacZ) was constructed in the same way. Cells were grown until 80% cytopathic effect (CPE) was seen, then harvested. For adenovirus infection, SCC13 cells were seeded in 60-mm culture dish, and infected by different recombinant adenoviruses overnight. 2.3 Western blot analysis Cells were harvested and lysed after trypsinization. After vigorous pipetting, extracts were centrifuged for 15 min at 13,000 rpm. Total protein concentration was determined with BCA Protein Assay Kit (Pierce, U.S.A). Proteins (50 μg) were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene fluoride membrane. After blocking with 5% fat-free dry milk overnight at 4 °C, the membrane was incubated with primary antibodies directed against PKC-δ or p63 (Santa Cruz Biotechnologies, CA, USA) overnight at 4 °C with gentle agitation. After incubating with peroxidase-conjugated secondary antibodies for 30 min at room temperature, target protein was visualized by enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA) and the density was determined using Image J software (US National Institutes of Health, Bethesda, MD, USA). β-Actin (1 : 5000) was used as a loading control. 2.4 Cell proliferation assay
For determination of cell growth, [3H] thymidine uptake assay was performed. Cells were replenished with fresh medium and treated with 1 mL of [3H] thymidine (Amersham, Buckinghamshire, UK). After incubated for the indicated time points (12h, 24h, 48h, 72h), cells were washed twice with PBS and incubated with 0.1 N NaOH at room temperature. Radioactivity of cell lysate was measured by liquid scintillation counter. 2.5 Colony formation assay Transfected cells were plated with solidified DMEM containing 40% FBS, 0.66% agar (2 ml) and incubated at 37 °C for 2 weeks. Plates were incubated with medium containing G418 (0.5 μg/μL) for 2 weeks. The colonies were then fixed by methanol and stained with crystal violet (0.005%; Sigma, USA). 2.6 Flow cytometry analysis All the antibodies used in this experiment were purchased from BD Biosciences, San Diego, CA. Cells were gently trypsinized to prepare single cell suspension. After washed with PBS (pH 7.4) and examined cell viability with trypan blue exclusion assay (to make sure cell viability≥95%), cells (1×106 cells/tube) were incubated with antibody directed against integrin α6 (1:100) or CD71 (1:100) for 1 h at 4 °C. Incubation of cells with IgG1 antibody (1:100) was used as the isotype controls. Unbound antibodies were removed by washing with PBS (pH 7.4) at 4 °C. Fluorescein isothiocyanate (FITC)-conjugated anti-IgG (1:100) for integrin α6 or alkaline phosphatase (AP)-conjugated anti-IgG (1:100) for CD71 antibody was then applied to incubate the cells for 45 min at 4 °C. The unbound secondary antibody was washed off, and the cell mixture was adjusted to 250 μL with PBS (pH 7.4) before assessed in a FACScan flow cytometer (BD Biosiences, San Diego, CA). Experiments were performed in duplicate and repeated for three times. 2.7 Statistical Analysis All the statistical tests were performed with statistical analysis software (SPSS version 17.0, Chicago, IL, USA). A P value < 0.05 was considered to be statistically significant. All the data were expressed as mean±SD. Multiple comparisons were conducted with normal linear model by Duncan test.
3. Results 3.1 PKC-δ expression decreased in cutaneous squamous cancer tissues The PKC-δ expressions detected by immunohistochemistry were shown in Figure 1. PKC-δ expression was identified in the normal skin tissue including surrounding normal epidermis and dermal fibroblasts (Figure 1A). In contrast, the expression of PKC-δ was hardly found in the SCC tissues (Figure 1B). 3.2 Overexpression of PKC-δ inhibited the proliferation of SCC13 cells To investigate the role of PKC-δ in the carcinogenesis of SCC, we examined the effect of PKC-δ on SCC13 cell proliferation after PKC-δ cDNA was transfected into the SCC13 cells using adenovirus system. As shown in Figure 2, the proliferation of the SCC13 cells were significantly inhibited in Ad-PKC-δ group compared with the Ad-LacZ group from the time points of 12 hours to 72 hours (P<0.05). 3.3 Overexpression of PKC-δ suppressed the protein expression of p63 and inhibited the SCC13 cell proliferation induced by p63 As shown in Figure 3A, overexpression of PKC-δ significantly inhibited the protein expression of p63. While overexpression of p63 had no markedly influence on the expression of PKC-δ. The [3H] thymidine uptake results was displayed that p63 obviously promoted the proliferation of SCC13 cells (P<0.01), while proliferation was significantly attenuated by PKC-δ when PKC-δ was concurrent with p63 (P<0.05), suggesting that PKC-δ inhibits SCC13 cell growth partly by regulating p63 (Figure 3B). 3.4 Overexpression of PKC-δ inhibited the activity and expression of the CSCs in SCC13 cells As shown in Figure 4, the colony-forming number was the lowest in Ad-PKC-δ group, and was the highest in Ad-p63 group. Transfection of the PKC-δ significantly suppressed the colony-formation number caused by the transfection of p63. To explore the the number of cancer stem cells, we examined the expression of cell surface markers with integrin 6 and CD71. As shown in Figure 5, the transfection of PKC-δ significantly decreased the expression of 6bri/CD71dim, and the overexpression of p63 significantly elevated the expression of
6bri/CD71dim. However, increased expression of PKC-δ obviously suppressed the elevation of 6bri/CD71dim induced by p63. All the data suggested that PKC-δ expression inhibited the number of CSCs by suppressing the expression of p63. 4. Discussion Cutaneous SCC is the second most frequent skin cancer and affects more than 500,000 new patients per year throughout the world. PKC-δ, ubiquitously expressed in a tissue-specific manner, plays a vital role in cellular processes including cell proliferation[22]. Down-regulation of PKC-δ gene in the human cutaneous SCC was clarified[21]. In our study, the less expression of PKC-δ could be found in cutaneous SCC samples, and the overexpression of PKC-δ obviously inhibited the proliferation of SCC13 cells, consistently proving the critical role of PKC-δ in SCC. Recently, abundant studies have identified the existence of CSCs in SCC[4, 7, 13]. Given that CSCs are the origins of the cancers and play vital roles in cancer cell proliferation, invasion, and metastasis, the regulation of CSCs has been a promising framework for the treatment of SCC[9]. p63, as a homologue of p53, plays a fundamental role in the regulation of epithelial development and carcinogenesis[20]. Overexpression of p63 has been verified with involvement in the formation and development of squamous cell tumors[8]. Consistently, we found that p63 could promote the SCC cell growth, the activity and expression of the CSCs in SCC13 cells, indicating that p63 played a critical role in regulating CSCs in SCC and promoting the process of SCC. Recent study of MicroRNA 203 has confirmed the positive regulation of PKC on p63 during human papillomaviruses (HPV) infection[16]. In our study, we found that PKC-δ was also a regulator on p63. It could attenuate the protein level of p63 in SCC13 cells, suppress the higher activity and expression of CSCs in SCC13 cells induced by p63, demonstrating that the favourable performance of PKC-δ on SCC cells was partly relying on the downreguation of p63 expression, illuminating the relationship among PKC-δ, p63, and SCC. Our study has some limitations. The mechanism between PKC-δ and p63 should be investigated to prove our findings. And the reason for increased expression of p63 in SCC has
not been elucidated. More investigations should be included in further studies to facilitate the application of PKC-δ in clinical treatment of SCC. 5. Conclusion PKC-δ played as a protective role in SCC partly by down-regulating p63, leading to the suppression of SCC cell proliferation, attenuation of the activity and expression of CSCs in SCC cells.
Conflict of Interest Statement None
Acknowledgments None
References [1] A. Allegra, A. Alonci, G. Penna, V. Innao, D. Gerace, F. Rotondo, C. Musolino, The cancer stem cell hypothesis: a guide to potential molecular targets. Cancer Invest 32 (2014) 470-495. [2] D.F. Altomare, G. Guanti, J. Hoch, M. Vician, Z. Krivokapic, R. Bergamaschi, G. Colorectal Micrometastases Study, Noncolonic cancer stem cells in bone marrow of colorectal cancer patients. Colorectal Dis 12 (2010) 206-212. [3] X. Cheng, H.C. O'Neill, Oncogenesis and cancer stem cells: current opinions and future directions. J Cell Mol Med 13 (2009) 4377-4384. [4] T. Featherston, H.H. Yu, J.C. Dunne, A.M. Chibnall, H.D. Brasch, P.F. Davis, S.T. Tan, T. Itinteang, Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System. Front Surg 3 (2016) 52. [5] X. Feng, J. Zhang, K. Smuga-Otto, S. Tian, J. Yu, R. Stewart, J.A. Thomson, Protein kinase C mediated extraembryonic endoderm differentiation of human embryonic stem cells. Stem Cells 30 (2012) 461-470. [6] J. Holgersson, P.A. Jovall, B.E. Samuelsson, M.E. Breimer, Structural characterization of non-acid glycosphingolipids in kidneys of single blood group O and A pigs. J Biochem 108 (1990) 766-777. [7] Z. Jian, A. Strait, A. Jimeno, X.J. Wang, Cancer Stem Cells in Squamous Cell Carcinoma. J Invest Dermatol (2016). [8] T. Kakuki, M. Kurose, K. Takano, A. Kondoh, K. Obata, K. Nomura, R. Miyata, Y. Kaneko, T. Konno, S. Takahashi, T. Hatakeyama, T. Kohno, T. Himi, T. Kojima, Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma. Oncotarget 7 (2016) 33887-33900. [9] S. Kerk, K. Finkel, A.T. Pearson, K. Warner, F. Nor, Z. Zhang, V.P. Wagner, P.A. Vargas, M.S. Wicha, E. Hurt, R.E. Hollingsworth, D.A. Tice, J.E. Nor, 5T4-targeted therapy ablates cancer stem cells and prevents recurrence of head and neck squamous cell carcinoma. Clin Cancer Res (2016). [10] F.R. Khuri, Y. Cho, D.A. Talmage, Retinoic acid-induced transition from protein kinase C beta to protein kinase C alpha in differentiated F9 cells: correlation with altered regulation
of proto-oncogene expression by phorbol esters. Cell Growth Differ 7 (1996) 595-602. [11] J.H. Kim, K.C. Sohn, T.Y. Choi, M.Y. Kim, H. Ando, S.J. Choi, S. Kim, Y.H. Lee, J.H. Lee, C.D. Kim, T.J. Yoon, Beta-catenin regulates melanocyte dendricity through the modulation of PKCzeta and PKCdelta. Pigment Cell Melanoma Res 23 (2010) 385-393. [12] J. Li, H. Li, L. Zhu, W. Song, R. Li, D. Wang, K. Dou, The adenovirus-mediated linamarase/linamarin suicide system: a potential strategy for the treatment of hepatocellular carcinoma. Cancer Lett 289 (2010) 217-227. [13] X.M. Li, Y.J. Piao, K.C. Sohn, J.M. Ha, M. Im, Y.J. Seo, K.U. Whang, J.H. Lee, Y. Lee, C.D. Kim, Sox9 is a beta-catenin-regulated transcription factor that enhances the colony-forming activity of squamous cell carcinoma cells. Mol Med Rep 14 (2016) 337-342. [14] Y.C. Lim, S.Y. Oh, Y.Y. Cha, S.H. Kim, X. Jin, H. Kim, Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas. Oral Oncol 47 (2011) 83-91. [15] M. Marchisio, V. Bertagnolo, C. Celeghini, M. Vitale, S. Capitani, G. Zauli, Selective modulation of specific protein kinase C (PKC) isoforms in primary human megakaryocytic vs. erythroid cells. Anat Rec 255 (1999) 7-14. [16] M. Melar-New, L.A. Laimins, Human papillomaviruses modulate expression of microRNA 203 upon epithelial differentiation to control levels of p63 proteins. J Virol 84 (2010) 5212-5221. [17] E.M. Memmi, A.G. Sanarico, A. Giacobbe, A. Peschiaroli, V. Frezza, A. Cicalese, F. Pisati, D. Tosoni, H. Zhou, G. Tonon, A. Antonov, G. Melino, P.G. Pelicci, F. Bernassola, p63 Sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling. Proceedings of the National Academy of Sciences of the United States of America 112 (2015) 3499-3504. [18] H. Mischak, W. Kolch, J. Goodnight, W.F. Davidson, U. Rapp, S. Rose-John, J.F. Mushinski, Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific. J Immunol 147 (1991) 3981-3987. [19] B.Z. Ring, R.S. Seitz, R.A. Beck, W.J. Shasteen, A. Soltermann, S. Arbogast, F. Robert, M.T. Schreeder, D.T. Ross, A novel five-antibody immunohistochemical test for subclassification of lung carcinoma. Mod Pathol 22 (2009) 1032-1043.
[20] A. Sinha, S. Chandra, V. Raj, I. Zaidi, S. Saxena, R. Dwivedi, Expression of p63 in potentially malignant and malignant oral lesions. J Oral Biol Craniofac Res 5 (2015) 165-172. [21] V. Yadav, N.C. Yanez, S.E. Fenton, M.F. Denning, Loss of protein kinase C delta gene expression in human squamous cell carcinomas: a laser capture microdissection study. Am J Pathol 176 (2010) 1091-1096. [22] H. Zhang, M. Okamoto, E. Panzhinskiy, W.M. Zawada, M. Das, PKCdelta/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells. Am J Physiol Cell Physiol 306 (2014) C648-658.
Figure Legends Figure 1 Expression of PKC-δ: A) the normal skin tissue; B) squamous cancer tissues. Figure 2 Effect of overexpression of PKC-δ on cell proliferation. *,** vs Ad-LacZ group, *P < 0.05 and ** P < 0.01. Figure 3 Effect of overexpression of PKC-δ on the p63 expression and SCC13 cell growth: A) results of western blot; B) results of [3H] thymidine uptake assay. Ad-LacZ group : Ad-LacZ +, Ad-PKC-δ -, Ad-p63 -; Ad-PKC-δ group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 -; Ad-p63 group: Ad-LacZ -, Ad-PKC-δ -, Ad-p63 +; Ad-PKC-δ and p63 group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 +. #
*,**
vs Ad-LacZ group, *P < 0.05 and ** P < 0.01.# vs Ad-p63 group,
P < 0.05.
Figure 4 Effect of overexpression of PKC-δ on the activity of cancer stem cells among the SCC13 cells: A) a: Ad-LacZ group; b: Ad-PKC-δ group; c: Ad-p63 group; d: Ad-PKC-δ and p63 group. Ad-LacZ group : Ad-LacZ +, Ad-PKC-δ -, Ad-p63 -; Ad-PKC-δ group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 -; Ad-p63 group: Ad-LacZ -, Ad-PKC-δ -, Ad-p63 +; Ad-PKC-δ and p63 group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 +. *P < 0.05 and
**
P < 0.01;
#,##
vs Ad-p63
group, #P < 0.05 and ##P < 0.01. Figure 5 Effect of overexpression of PKC-δ on the expression of cancer stem cells among the SCC13 cells by flow cytometry analysis. A) a: The ratio of 6bri/CD71dim in Ad-LacZ group; b: The ratio of 6bri/CD71dim in Ad-PKC-δ group; c: The ratio of 6bri/CD71dim in Ad-p63 group; d: The ratio of 6bri/CD71dim in Ad-PKC-δ and p63 group. Ad-LacZ group : Ad-LacZ +, Ad-PKC-δ -, Ad-p63 -; Ad-PKC-δ group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 -; Ad-p63 group: Ad-LacZ -, Ad-PKC-δ -, Ad-p63 +; Ad-PKC-δ and p63 group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 +. *,** vs Ad-LacZ group, *P < 0.05 and and ##P < 0.01.
**
P < 0.01;
#,##
vs Ad-p63 group, #P < 0.05
S0344-0338(17)30055-9 http://dx.doi.org/doi:10.1016/j.prp.2017.07.013 PRP 51850
To appear in: Received date: Revised date: Accepted date:
18-1-2017 15-6-2017 12-7-2017
Please cite this article as: Dongmei Zhang, Mingjing Fu, Lingyan Li, Huan Ye, Zhiqi Song, Yongjun Piao, PKC-␦ attenuates the cancer stem cell among squamous cell carcinoma cells through down-regulating p63, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page PKC-δ attenuates the cancer stem cell among squamous cell carcinoma cells through down-regulating p63
Running title: PKC-δ attenuates the cancer stem cell through p63
Dongmei Zhang1, Mingjing Fu2, Lingyan Li2, Huan Ye2, Zhiqi Song2, Yongjun Piao2
1 Departments of Physiology, Dalian Medical University, Dalian, China 2 Department of Dermatology, The First Affiliated Hospital of Dalian Medical University Correspondence: Yongjun Piao Department of Dermatology, The First Affiliated Hospital of Dalian Medical University. No. 222, zhongshan road, Xigang District, Dalian 116011, China. E-mail address: [email protected]
Tel: +86 18098870608
Abstract Protein kinase C delta (PKC-δ) has been identified as a tumor suppressor. However, the effects of PKC-δ on the cancer stem cells in squamous cell carcinomas (SCC) have not been clarified. The purpose of this study was to detect the regulation of PKC-δ on cancer stem cell among SSC cells and the role of p63 during the regulation. Immunohistochemistry of human cutaneous SCC tissues was performed to detect the expression of PKC-δ. After the human SCC13 cells infected by recombinant adenoviruses, the cell proliferation were determined. The correlation of PKC-δ and p63 was detected by western blot. The colony forming activity and the number of cancer stem cells (CSCs) in SCC identified by double-staining with anti-integrin 6 and anti-CD71 antibodies were detected. The expression of PKC-δ was obviously decreased in SCC tissues compared with that in
normal skin tissues. The higher protein level of p63 in SCC was attenuated by the transfection of PKC-δ. The higher proliferation capacity of SCC13 cells, the higher activity and expression of CSCs in SCC13 cells induced by p63 were significantly suppressed by the transfection of PKC-δ. In conclusion, PKC-δ played as a protective role in SCC partly by down-regulating p63, leading to the suppression of SCC cell proliferation, attenuation of the activity and expression of CSCs in SCC cells.
Key words: PKC-δ; squamous cell carcinomas; cancer stem cells; p63
1. Introduction Squamous cell carcinoma (SCC), as one of the most common malignancies, is potentially aggressive, metastatic, and lethal. During recent years, efforts have been made on various aspects of SCC, including detection techniques, treatment strategies, and mechanisms involved in cancer initiation and progression. Despite advancements in oncology researches and therapies in the last decade, SCC with high morbidity and resistance was still being observed in large numbers of patients, leading to reduced life quality. A novel effective clinic treatment of SCC is imperative. Recent studies have demonstrated that a variety of human malignant tumor comprises a rare subpopulation of cells, which are referred to as cancer stem cells (CSCs)[1]. They possess self-renewal, tumor-initiating capabilities and sustain the tumor growth. It is believed that the existence of CSCs may be one reason for the lack of effectiveness and resistance of the cancers to the conventional therapies, and be considered as a major cause of the tumor recurrence after the conventional chemo- or radio-treatment[2, 3, 14]. Targeted elimination of CSCs has drawn attentions these years and believed to provide a framework for treatment of SCC. Protein kinase C (PKC) is a Ser/Thr kinase family including at least 12 isoforms. The
expression and activation patterns of PKC isoform vary in tissues. Activation of PKC is involved in a wide range of cellular functions, notably proliferation, differentiation, and cell survival[10, 15, 18]. It is demonstrated that PKC-δ (a subtype of PKC) functions as a critical mediator in macrophage-colony stimulating factor induced differentiation signaling[11, 15]. PKC-δ is also displayed with the involvement in differentiation of embryonic stem cells as well as H9c2 cells[5, 6]. However, whether PKC-δ affects the CSCs in SCC has not been identified. p63 is well recognized as a master squamous regulator and is the primary marker used to clinically diagnose SCCs[19]. It is confirmed that p63 is indispensable to maintain the high proliferative potential of CSCs[17]. Our aim of the study is to investigate the relationships among PKC-δ, p63 and CSCs in SCC. 2. Methods and materials Immortalized human SCC13 cells and 293A cells were provided by Professor Tae-Jin Yoon (Gyeongsang National University, Jinju, South Korea). Skin samples were obtained from patients who underwent surgical resection of squamous cell carcinoma in Chungnam National University Hospital. The written informed consent was obtained from all the donators. All the experiments were created in accordance with the ethical committee approval process of Chungnam National University Hospital. 2.1 Immunohistochemistry Skin samples were prepared for this experiment. Paraformaldehyde-fixed and paraffin embedded specimens were deparaffinized, rehydrated and washed by phosphate-buffered saline (PBS). After treatment with proteinase K (1 mg/ml) at 37 ºC for 5 min, sections were treated with H2O2 for 10 min at room temperature, blocked in 1% bovine serum albumin (BSA) in PBS for 20 min, followed by reaction with purified goat anti-PKC-δ antibody (1:100, Santa Cruz Biotechnologies, CA, USA) for 1h. Sections were incubated sequentially with peroxidase-conjugated secondary antibodies (Upstate, Lake Placid, NY) and visualized with ChemMate Envision Detection kit (Dako, Carpinteria, CA). 2.2 Cell culture and adenovirus infection experiments Immortalized human SCC13 cells were cultured in Dulbecco’s modified Eagle’s
medium(DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Rockville, MD) and penicillin/streptomycin antibiotics (1:100). The cells were maintained at 37 °C in humidified air with 5% CO2. The PKC-δ full-length cDNA was amplified by polymerase chain reaction (PCR) and subcloned into pENT/CMV vector that has attL sites for site-specific recombination with a Gateway destination vector (Invitrogen, Carlsbad, CA). All plasmid constructs were sequence-verified. The recombinant adenoviruses containing PKC-δ (Ad-PKC-δ) or p63 (Ad-p63) were created using Virapower adenovirus expression system (Invitrogen, USA) according to the method previously described[12]. Briefly, site-specific recombination between entry vector and adenoviral destination vector was achieved by LR clonase (Invitrogen, USA). The resulting adenoviral expression vector was transduced into 293A cells using Lipofectamine 2000 (Invitrogen, USA). The control recombinant adenovirus containing galactosidase gene (Ad-lacZ) was constructed in the same way. Cells were grown until 80% cytopathic effect (CPE) was seen, then harvested. For adenovirus infection, SCC13 cells were seeded in 60-mm culture dish, and infected by different recombinant adenoviruses overnight. 2.3 Western blot analysis Cells were harvested and lysed after trypsinization. After vigorous pipetting, extracts were centrifuged for 15 min at 13,000 rpm. Total protein concentration was determined with BCA Protein Assay Kit (Pierce, U.S.A). Proteins (50 μg) were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene fluoride membrane. After blocking with 5% fat-free dry milk overnight at 4 °C, the membrane was incubated with primary antibodies directed against PKC-δ or p63 (Santa Cruz Biotechnologies, CA, USA) overnight at 4 °C with gentle agitation. After incubating with peroxidase-conjugated secondary antibodies for 30 min at room temperature, target protein was visualized by enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA) and the density was determined using Image J software (US National Institutes of Health, Bethesda, MD, USA). β-Actin (1 : 5000) was used as a loading control. 2.4 Cell proliferation assay
For determination of cell growth, [3H] thymidine uptake assay was performed. Cells were replenished with fresh medium and treated with 1 mL of [3H] thymidine (Amersham, Buckinghamshire, UK). After incubated for the indicated time points (12h, 24h, 48h, 72h), cells were washed twice with PBS and incubated with 0.1 N NaOH at room temperature. Radioactivity of cell lysate was measured by liquid scintillation counter. 2.5 Colony formation assay Transfected cells were plated with solidified DMEM containing 40% FBS, 0.66% agar (2 ml) and incubated at 37 °C for 2 weeks. Plates were incubated with medium containing G418 (0.5 μg/μL) for 2 weeks. The colonies were then fixed by methanol and stained with crystal violet (0.005%; Sigma, USA). 2.6 Flow cytometry analysis All the antibodies used in this experiment were purchased from BD Biosciences, San Diego, CA. Cells were gently trypsinized to prepare single cell suspension. After washed with PBS (pH 7.4) and examined cell viability with trypan blue exclusion assay (to make sure cell viability≥95%), cells (1×106 cells/tube) were incubated with antibody directed against integrin α6 (1:100) or CD71 (1:100) for 1 h at 4 °C. Incubation of cells with IgG1 antibody (1:100) was used as the isotype controls. Unbound antibodies were removed by washing with PBS (pH 7.4) at 4 °C. Fluorescein isothiocyanate (FITC)-conjugated anti-IgG (1:100) for integrin α6 or alkaline phosphatase (AP)-conjugated anti-IgG (1:100) for CD71 antibody was then applied to incubate the cells for 45 min at 4 °C. The unbound secondary antibody was washed off, and the cell mixture was adjusted to 250 μL with PBS (pH 7.4) before assessed in a FACScan flow cytometer (BD Biosiences, San Diego, CA). Experiments were performed in duplicate and repeated for three times. 2.7 Statistical Analysis All the statistical tests were performed with statistical analysis software (SPSS version 17.0, Chicago, IL, USA). A P value < 0.05 was considered to be statistically significant. All the data were expressed as mean±SD. Multiple comparisons were conducted with normal linear model by Duncan test.
3. Results 3.1 PKC-δ expression decreased in cutaneous squamous cancer tissues The PKC-δ expressions detected by immunohistochemistry were shown in Figure 1. PKC-δ expression was identified in the normal skin tissue including surrounding normal epidermis and dermal fibroblasts (Figure 1A). In contrast, the expression of PKC-δ was hardly found in the SCC tissues (Figure 1B). 3.2 Overexpression of PKC-δ inhibited the proliferation of SCC13 cells To investigate the role of PKC-δ in the carcinogenesis of SCC, we examined the effect of PKC-δ on SCC13 cell proliferation after PKC-δ cDNA was transfected into the SCC13 cells using adenovirus system. As shown in Figure 2, the proliferation of the SCC13 cells were significantly inhibited in Ad-PKC-δ group compared with the Ad-LacZ group from the time points of 12 hours to 72 hours (P<0.05). 3.3 Overexpression of PKC-δ suppressed the protein expression of p63 and inhibited the SCC13 cell proliferation induced by p63 As shown in Figure 3A, overexpression of PKC-δ significantly inhibited the protein expression of p63. While overexpression of p63 had no markedly influence on the expression of PKC-δ. The [3H] thymidine uptake results was displayed that p63 obviously promoted the proliferation of SCC13 cells (P<0.01), while proliferation was significantly attenuated by PKC-δ when PKC-δ was concurrent with p63 (P<0.05), suggesting that PKC-δ inhibits SCC13 cell growth partly by regulating p63 (Figure 3B). 3.4 Overexpression of PKC-δ inhibited the activity and expression of the CSCs in SCC13 cells As shown in Figure 4, the colony-forming number was the lowest in Ad-PKC-δ group, and was the highest in Ad-p63 group. Transfection of the PKC-δ significantly suppressed the colony-formation number caused by the transfection of p63. To explore the the number of cancer stem cells, we examined the expression of cell surface markers with integrin 6 and CD71. As shown in Figure 5, the transfection of PKC-δ significantly decreased the expression of 6bri/CD71dim, and the overexpression of p63 significantly elevated the expression of
6bri/CD71dim. However, increased expression of PKC-δ obviously suppressed the elevation of 6bri/CD71dim induced by p63. All the data suggested that PKC-δ expression inhibited the number of CSCs by suppressing the expression of p63. 4. Discussion Cutaneous SCC is the second most frequent skin cancer and affects more than 500,000 new patients per year throughout the world. PKC-δ, ubiquitously expressed in a tissue-specific manner, plays a vital role in cellular processes including cell proliferation[22]. Down-regulation of PKC-δ gene in the human cutaneous SCC was clarified[21]. In our study, the less expression of PKC-δ could be found in cutaneous SCC samples, and the overexpression of PKC-δ obviously inhibited the proliferation of SCC13 cells, consistently proving the critical role of PKC-δ in SCC. Recently, abundant studies have identified the existence of CSCs in SCC[4, 7, 13]. Given that CSCs are the origins of the cancers and play vital roles in cancer cell proliferation, invasion, and metastasis, the regulation of CSCs has been a promising framework for the treatment of SCC[9]. p63, as a homologue of p53, plays a fundamental role in the regulation of epithelial development and carcinogenesis[20]. Overexpression of p63 has been verified with involvement in the formation and development of squamous cell tumors[8]. Consistently, we found that p63 could promote the SCC cell growth, the activity and expression of the CSCs in SCC13 cells, indicating that p63 played a critical role in regulating CSCs in SCC and promoting the process of SCC. Recent study of MicroRNA 203 has confirmed the positive regulation of PKC on p63 during human papillomaviruses (HPV) infection[16]. In our study, we found that PKC-δ was also a regulator on p63. It could attenuate the protein level of p63 in SCC13 cells, suppress the higher activity and expression of CSCs in SCC13 cells induced by p63, demonstrating that the favourable performance of PKC-δ on SCC cells was partly relying on the downreguation of p63 expression, illuminating the relationship among PKC-δ, p63, and SCC. Our study has some limitations. The mechanism between PKC-δ and p63 should be investigated to prove our findings. And the reason for increased expression of p63 in SCC has
not been elucidated. More investigations should be included in further studies to facilitate the application of PKC-δ in clinical treatment of SCC. 5. Conclusion PKC-δ played as a protective role in SCC partly by down-regulating p63, leading to the suppression of SCC cell proliferation, attenuation of the activity and expression of CSCs in SCC cells.
Conflict of Interest Statement None
Acknowledgments None
References [1] A. Allegra, A. Alonci, G. Penna, V. Innao, D. Gerace, F. Rotondo, C. Musolino, The cancer stem cell hypothesis: a guide to potential molecular targets. Cancer Invest 32 (2014) 470-495. [2] D.F. Altomare, G. Guanti, J. Hoch, M. Vician, Z. Krivokapic, R. Bergamaschi, G. Colorectal Micrometastases Study, Noncolonic cancer stem cells in bone marrow of colorectal cancer patients. Colorectal Dis 12 (2010) 206-212. [3] X. Cheng, H.C. O'Neill, Oncogenesis and cancer stem cells: current opinions and future directions. J Cell Mol Med 13 (2009) 4377-4384. [4] T. Featherston, H.H. Yu, J.C. Dunne, A.M. Chibnall, H.D. Brasch, P.F. Davis, S.T. Tan, T. Itinteang, Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System. Front Surg 3 (2016) 52. [5] X. Feng, J. Zhang, K. Smuga-Otto, S. Tian, J. Yu, R. Stewart, J.A. Thomson, Protein kinase C mediated extraembryonic endoderm differentiation of human embryonic stem cells. Stem Cells 30 (2012) 461-470. [6] J. Holgersson, P.A. Jovall, B.E. Samuelsson, M.E. Breimer, Structural characterization of non-acid glycosphingolipids in kidneys of single blood group O and A pigs. J Biochem 108 (1990) 766-777. [7] Z. Jian, A. Strait, A. Jimeno, X.J. Wang, Cancer Stem Cells in Squamous Cell Carcinoma. J Invest Dermatol (2016). [8] T. Kakuki, M. Kurose, K. Takano, A. Kondoh, K. Obata, K. Nomura, R. Miyata, Y. Kaneko, T. Konno, S. Takahashi, T. Hatakeyama, T. Kohno, T. Himi, T. Kojima, Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma. Oncotarget 7 (2016) 33887-33900. [9] S. Kerk, K. Finkel, A.T. Pearson, K. Warner, F. Nor, Z. Zhang, V.P. Wagner, P.A. Vargas, M.S. Wicha, E. Hurt, R.E. Hollingsworth, D.A. Tice, J.E. Nor, 5T4-targeted therapy ablates cancer stem cells and prevents recurrence of head and neck squamous cell carcinoma. Clin Cancer Res (2016). [10] F.R. Khuri, Y. Cho, D.A. Talmage, Retinoic acid-induced transition from protein kinase C beta to protein kinase C alpha in differentiated F9 cells: correlation with altered regulation
of proto-oncogene expression by phorbol esters. Cell Growth Differ 7 (1996) 595-602. [11] J.H. Kim, K.C. Sohn, T.Y. Choi, M.Y. Kim, H. Ando, S.J. Choi, S. Kim, Y.H. Lee, J.H. Lee, C.D. Kim, T.J. Yoon, Beta-catenin regulates melanocyte dendricity through the modulation of PKCzeta and PKCdelta. Pigment Cell Melanoma Res 23 (2010) 385-393. [12] J. Li, H. Li, L. Zhu, W. Song, R. Li, D. Wang, K. Dou, The adenovirus-mediated linamarase/linamarin suicide system: a potential strategy for the treatment of hepatocellular carcinoma. Cancer Lett 289 (2010) 217-227. [13] X.M. Li, Y.J. Piao, K.C. Sohn, J.M. Ha, M. Im, Y.J. Seo, K.U. Whang, J.H. Lee, Y. Lee, C.D. Kim, Sox9 is a beta-catenin-regulated transcription factor that enhances the colony-forming activity of squamous cell carcinoma cells. Mol Med Rep 14 (2016) 337-342. [14] Y.C. Lim, S.Y. Oh, Y.Y. Cha, S.H. Kim, X. Jin, H. Kim, Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas. Oral Oncol 47 (2011) 83-91. [15] M. Marchisio, V. Bertagnolo, C. Celeghini, M. Vitale, S. Capitani, G. Zauli, Selective modulation of specific protein kinase C (PKC) isoforms in primary human megakaryocytic vs. erythroid cells. Anat Rec 255 (1999) 7-14. [16] M. Melar-New, L.A. Laimins, Human papillomaviruses modulate expression of microRNA 203 upon epithelial differentiation to control levels of p63 proteins. J Virol 84 (2010) 5212-5221. [17] E.M. Memmi, A.G. Sanarico, A. Giacobbe, A. Peschiaroli, V. Frezza, A. Cicalese, F. Pisati, D. Tosoni, H. Zhou, G. Tonon, A. Antonov, G. Melino, P.G. Pelicci, F. Bernassola, p63 Sustains self-renewal of mammary cancer stem cells through regulation of Sonic Hedgehog signaling. Proceedings of the National Academy of Sciences of the United States of America 112 (2015) 3499-3504. [18] H. Mischak, W. Kolch, J. Goodnight, W.F. Davidson, U. Rapp, S. Rose-John, J.F. Mushinski, Expression of protein kinase C genes in hemopoietic cells is cell-type- and B cell-differentiation stage specific. J Immunol 147 (1991) 3981-3987. [19] B.Z. Ring, R.S. Seitz, R.A. Beck, W.J. Shasteen, A. Soltermann, S. Arbogast, F. Robert, M.T. Schreeder, D.T. Ross, A novel five-antibody immunohistochemical test for subclassification of lung carcinoma. Mod Pathol 22 (2009) 1032-1043.
[20] A. Sinha, S. Chandra, V. Raj, I. Zaidi, S. Saxena, R. Dwivedi, Expression of p63 in potentially malignant and malignant oral lesions. J Oral Biol Craniofac Res 5 (2015) 165-172. [21] V. Yadav, N.C. Yanez, S.E. Fenton, M.F. Denning, Loss of protein kinase C delta gene expression in human squamous cell carcinomas: a laser capture microdissection study. Am J Pathol 176 (2010) 1091-1096. [22] H. Zhang, M. Okamoto, E. Panzhinskiy, W.M. Zawada, M. Das, PKCdelta/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells. Am J Physiol Cell Physiol 306 (2014) C648-658.
Figure Legends Figure 1 Expression of PKC-δ: A) the normal skin tissue; B) squamous cancer tissues. Figure 2 Effect of overexpression of PKC-δ on cell proliferation. *,** vs Ad-LacZ group, *P < 0.05 and ** P < 0.01. Figure 3 Effect of overexpression of PKC-δ on the p63 expression and SCC13 cell growth: A) results of western blot; B) results of [3H] thymidine uptake assay. Ad-LacZ group : Ad-LacZ +, Ad-PKC-δ -, Ad-p63 -; Ad-PKC-δ group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 -; Ad-p63 group: Ad-LacZ -, Ad-PKC-δ -, Ad-p63 +; Ad-PKC-δ and p63 group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 +. #
*,**
vs Ad-LacZ group, *P < 0.05 and ** P < 0.01.# vs Ad-p63 group,
P < 0.05.
Figure 4 Effect of overexpression of PKC-δ on the activity of cancer stem cells among the SCC13 cells: A) a: Ad-LacZ group; b: Ad-PKC-δ group; c: Ad-p63 group; d: Ad-PKC-δ and p63 group. Ad-LacZ group : Ad-LacZ +, Ad-PKC-δ -, Ad-p63 -; Ad-PKC-δ group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 -; Ad-p63 group: Ad-LacZ -, Ad-PKC-δ -, Ad-p63 +; Ad-PKC-δ and p63 group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 +. *P < 0.05 and
**
P < 0.01;
#,##
vs Ad-p63
group, #P < 0.05 and ##P < 0.01. Figure 5 Effect of overexpression of PKC-δ on the expression of cancer stem cells among the SCC13 cells by flow cytometry analysis. A) a: The ratio of 6bri/CD71dim in Ad-LacZ group; b: The ratio of 6bri/CD71dim in Ad-PKC-δ group; c: The ratio of 6bri/CD71dim in Ad-p63 group; d: The ratio of 6bri/CD71dim in Ad-PKC-δ and p63 group. Ad-LacZ group : Ad-LacZ +, Ad-PKC-δ -, Ad-p63 -; Ad-PKC-δ group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 -; Ad-p63 group: Ad-LacZ -, Ad-PKC-δ -, Ad-p63 +; Ad-PKC-δ and p63 group: Ad-LacZ -, Ad-PKC-δ +, Ad-p63 +. *,** vs Ad-LacZ group, *P < 0.05 and and ##P < 0.01.
**
P < 0.01;
#,##
vs Ad-p63 group, #P < 0.05