- Email: [email protected]
Autophagy analysis in oral carcinogenesis
Accepted Manuscript Title: Autophagy Analysis in Oral Carcinogenesis Authors: T.B. de Lima, A.H.R. Paz, P.V. Rados, R. Leonardi, P. Bufo, M.C. Pedicillo, A. Santoro, S. Cagiano, G. Aquino, G. Botti, G. Pannone, F. Visioli PII: DOI: Reference:
S0344-0338(17)30331-X http://dx.doi.org/doi:10.1016/j.prp.2017.07.027 PRP 51864
To appear in: Received date: Revised date: Accepted date:
6-4-2017 4-7-2017 28-7-2017
Please cite this article as: T.B.de Lima, A.H.R.Paz, P.V.Rados, R.Leonardi, P.Bufo, M.C.Pedicillo, A.Santoro, S.Cagiano, G.Aquino, G.Botti, G.Pannone, F.Visioli, Autophagy Analysis in Oral Carcinogenesis, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.07.027 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.
1
Autophagy Analysis in Oral Carcinogenesis
TB de Lima1, AHR Paz2, PV Rados1, R Leonardi3, P Bufo4, MC Pedicillo4, A Santoro4, Cagiano S4, G Aquino5, G Botti5, G Pannone4,5, F Visioli1
1. Department of Oral Pathology – Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil. 2. Experimental Research Center, Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil. 3. Department of Medical and Surgical Science. II Dental Unit - University of Catania, Catania, Italy. 4. Department of Clinical and Experimental Medicine. Pathological Anatomy Unit – University of Foggia, Foggia, Italy. 5. Pathology Unit, Instituto Nazionale per lo Studio e la Cura dei Tumori, “Fondazione G. Pascale”, IRCCS, Naples, Italy.
Corresponding author: Fernanda Visioli Email: [email protected] Address: Rua Ramiro Barcelos 2492, room 503 Porto Alegre, RS, Brazil 90035-003 Phone: 55 51 33085011 Phax: 55 51 33085023
2
Abstract Objective: The aim of this study was to evaluate the levels of autophagy in oral leukoplakia and squamous cell carcinoma and to correlate with clinical pathological features, as well as, the evolution of these lesions. Methodology: 7 normal oral mucosa, 51 oral leukoplakias, and 120 oral squamous cell carcinomas (OSCC) were included in the study. Histological sections of the mucosa and leukoplakias were evaluated throughout their length, while the carcinomas were evaluated using Tissue Microarray. After the immunohistochemical technique, LC3-II positive cells were quantified in the different epithelial layers of the mucosa and leukoplakias and in the microarrays of the squamous cell carcinomas. The correlation between positive cells with the different clinical-pathological variables and with the evolution of the lesions was tested using the t test, ANOVA, and Kaplan-Meier survival analysis. Results: We observed increased levels of autophagy in the oral squamous cell carcinomas (p<0.001) in relation to the other groups, but without any association with poorer evolution or survival of these patients. Among the leukoplakias, we observed a higher percentage of positive cells in the intermediate layer of the dysplastic leukoplakias (p=0.0319) and in the basal layer of lesions with poorer evolution (p=0.0133). Conclusion: The levels of autophagy increased during the process of oral carcinogenesis and are correlated with poorer behavior of the leukoplakias. Key-words: Autophagy, LC3-II, leukoplakia, oral squamous cell carcinoma.
Introduction
Oral squamous cell carcinoma is considered to be the sixth most common type of cancer worldwide [1,2]. The five-year survival rate is about 50% when lymphonodal metastases are present and the survival of patients affected by this pathology has not improved in recent decades [3]. New
3
advances in understanding the biology of oral cancer are necessary to reduce its mortality rates.
Squamous cell carcinoma can be preceded by visible changes in the oral mucosa, known as potentially malignant disorders. The most common of these is leukoplakia and its prevalence is estimated at approximately 2% world-wide. The risk of malignant transformation is higher in dysplastic lesions, but some of these lesions can remain clinically unchanged or even regress completely [4]. Currently, the main risk factors considered for malignant transformation are: female gender, long duration of the lesion, size greater than 2 cm, leukoplakia in non-smokers, and the presence of epithelial dysplasia [5,6]. However, it is not yet possible to predict the behavior of these lesions, and the search for biomarkers to detect cellular alterations signaling the carcinogenetic process is necessary.
Therefore, we need to better understand the mechanisms related to the development and progression of tumors. In this context, the cellular autophagy may be an important mechanism for the occurrence of this outcome [7]. Autophagy is a catabolic process for the degradation and recycling of cytosol components and damaged cellular organelles. It plays a critical role in the maintenance of cellular homeostasis, and its purpose is to preserve cellular viability under conditions of stress [8]. The initiation process of autophagy involves several members of the Atg family of proteins [9]. The formation of autophagosomes is mediated by Atg8 and Atg12. Atg8 is modified by Atg4 to form LC3-I, which in turn, is modified by Atg3 and Atg7 into a smaller form, LC3II. LC3-II is recruited by Atg12 for the growth of the autophagic membrane. The
4
Atg5-Atg12 complex then binds to Atg16 to initiate the process of membrane prolongation through the recruitment of LC3-II. Once the autophagosome is formed, the Atg5-Atg12-Atg16 complex unbinds from the autophagic membrane and is recycled together with the LC3-II. The autophagosome containing the material to be degraded then fuses with a lysosome [10].
Autophagy is a process that may act in a dual way in carcinogenesis and during the progression of cancer [11,12]. It can act as a tumor suppressor mechanism, thus avoiding the accumulation of damaged organelles and proteins, reducing the levels of oxidative stress and consequently, genetic instability. On the other hand, autophagy may be a fundamental mechanism for the survival of cells in the inhospitable microenvironment that emerges during carcinogenesis, which is characterized by hypoxia, acidosis and nutrient deprivation [13]. Therefore, it is important to study how the levels of autophagy behave in the different stages of oral carcinogenesis. The objective of this study was to evaluate the levels of LC3-II, a marker of autophagy, in tissue samples from oral squamous cell carcinomas and oral leukoplakias, comparing them with oral normal mucosa, and correlating them with the clinical pathological features and the evolution of the lesions.
Methods
Sample
This is a multicenter study, the samples included in this analysis were selected from the Oral Pathology Archive of the Universidade Federal do Rio
5
Grande do Sul (Porto Alegre, Brazil) and from the National Cancer Institute "G.Pascale" Foundation (Napoli, Italy). Were included: 7 samples of normal oral mucosa (from lip frenectomies), 51 samples of oral leukoplakia (with clinical and microscopic confirmation) and 120 samples of oral squamous cell carcinomas (OSCC). OSCC samples were derived from tumor resection prior to any other treatment. All the hematoxylin-eosin stained slides were reviewed by two pathologists (FV, GP). Clinical and pathological information were obtained from the files of the laboratories and patient records. This study was approved by the Ethics Committee (Federal University of Rio Grande do Sul, number 1.394.505).
The criteria for histopathological analysis of oral leukoplakias in order to diagnose epithelial dysplasia were established by the World health Organization (WHO). In addition, the criteria for classification of oral squamous cell carcinomas in well, moderate and poor differentiated tumors were also obtained from WHO [14].
For the leukoplakia samples, the evolution was considered good, if the lesion did not recur after a total biopsy, if it remained unchanged after a partial biopsy, if it decreased in size or regressed, or if the clinical aspect improved over time. The evolution was considered poor, if the lesion worsened its clinical appearance or increased its size, if there was recurrence, if new lesions appeared or if malignant transformation occurred [4].
Alcohol consumption was considered positive when the patient consumed at least 30g of alcohol per week. Patients were considered former smoker or former drinker if they stopped consuming these substances for at least 1 year.
6
Tissue Microarray Immunohistochemistry
A tissue microarray (TMA) block was constructed from OSCC samples. After selecting representative areas, two cylinders with an area of 0.28 mm2 were collected of each paraffin block tand transferred to the receiving block using Galileo TMA CK 3500 (ISE TMA software, Systems Engineering Integrated). For each patient, two different areas were selected, to increase the area of tumor assessed and to improve the representativeness of the selected fields. From the paraffin blocks, 3μm thick sections were obtained, dewaxed and rehydrated in ethanol. After blocking the endogenous peroxidase with methanol and hydrogen peroxide, antigen retrieval was performed in a pressure cooker at 121°C, and then samples were incubated with primary antibody (LC3-II, 1: 100, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The detection system used was EnVisionTM + Dual Link (Dako, Carpinteria, CA, USA) and 3,3-diaminobenzidine (DAB, Dako, Carpinteria, CA, USA) was used for visualization. Slides were counterstained with Hematoxylin. As a positive control, a previously tested OSCC sample was used. For negative control, the same OSCC sample was used and the primary antibody was replaced with nonimmune goat serum.
Quantification of Immunopositive Cells
Images of the entire length of the histological section were obtained. The samples images were captured at a magnification of 400 times using the software cellSens™ (Olympus, Tokyo, Japan), in a binocular microscope model CX41RF (Olympus Latin AmericaInc., Miami, Coupled to a Qcolor 5 camera,
7
Coolet, RTV (Olympus Latin America Inc.). ImageJ software for Mac OS X version 1.5 (NIH, USA) was used to count 1000 cells per layer in order to determine the percentage of positive and negative autophagy tagged cells, totaling 1000 cells in the basal layer (BL), 1000 cells in the parabasal layer (PBL), 1000 cells in the intermediate layer (IL) and 1000 cells in the surface layer (SL). The basal layer was defined as having at least one point of contact with the basal membrane, the parabasal layer was composed of cell layers immediately above the basal layer (non-flattened), the spinous layer located between the parabasal and superficial layer, being the superficial layer characterized by flattened cells [15].
In OSCC TMA cores, LC3-II positive cells were counted using a binocular microscope model CX41RF (Olympus Latin AmericaInc., Miami, FL, USA). All the tumor cells were quantified in each cylinder and the percentage of cells positive was determined. At the end of the quantification, the mean between the two cores analyzed for each patient was performed.
Statistical Analysis
For statistical analysis, the software SPSS 19.0 (Statistical Package for the Social Sciences, IBM, New York, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, USA) were used. Data distribution was analyzed by the Kolmogorov-Sminorv test. The sample followed the parametric distribution; therefore, the data was assessed by the Student t test or ANOVA. Survival analysis was performed using the Kaplan-Meier curve.
8
Results
51 patients with leukoplakia were studied. The demographic profile of these patients is summarized in Table 1. The majority of the sample was composed of men (60%), smokers and alcohol consumers, the mean age was 55.86 years. The most affected sites were palate (27.45%), tongue (25.5%) and floor of mouth. Most of the lesions were non-homogeneous and non-dysplastic. The average duration of follow-up of these patients was 4.3 years, and for most of them, the course of the disease worsened (39.21%). 4 patients underwent malignant transformation, being the majority of these lesions located in the tongue border in female patients.
120 tumor samples of oral squamous cell carcinoma were analyzed, among the samples, the male gender predominated (70.8%) and the mean age of the patients constituting the sample was 67.3 years old. The majority of lesions were located on the tongue and floor of mouth, and lesions of grade 2 predominated. Most of the patients underwent radiotherapy associated with the surgical resection of the tumor (Table 1).
It was observed an increase in the percentage of LC3-II positive cells from the normal oral mucosa to the leukoplakia samples and from leukoplakia to the oral squamous cell carcinomas (Figure 1, A,B,C,D), this increase was statistically significant (p<0.0001) (Figure 2). An increase in LC3-II immunostaining was observed in oral leukoplakias compared to normal oral mucosa samples, especially in the upper layers of the epithelium (p=0.0013) (Table 2, Figure 1, A, B and C). No significant associations were found between LC3-II immunostaining and the clinical type of leukoplakia, nor with smoking and
9
alcohol consumption (Table 2). We observed an increase of LC3-II positive cells in dysplastic lesions compared to non-dysplastic lesions (Figure 1, B and C, Figure 2), in the intermediate layer of the epithelium this increase was statistically significant (p=0.0319). In the lesions of worse evolution, the expression of LC3-II increased (Figure 2), which was significant in the basal layer (p= 0.0133).
Increased LC3-II positive cells were detected in Grade 2, T3 and T4 tumors, with regional metastasis, with greater depth of invasion, and in patients who died because of the tumor. However, we did not find statistically significant associations (Table 3). To evaluate the association between LC3-II and patient survival, we performed a Kaplan-Meier survival curve, dividing the sample into >= 70% of LC3-II positive cells and <70% of LC3-II positive cells, based in the median cut-off. We observed greater survival for patients with a lower percentage of positive cells, but this data is not statistically significant (p = 0.21) (Figure 3).
Discussion
Our study sought to evaluate the levels of cellular autophagy during the oral carcinogenesis process. We observed a progressive increase of the percentage of positive cells according with progressive severity of alterations, being the number LC3-II cells greater in leukoplakic lesions than in normal oral mucosa, reaching the highest averages in oral squamous cell carcinomas.
Additionally, a greater number of cells performing autophagy were observed in dysplastic samples and in those with poorer evolution. Despite the dual role of autophagy, in leukoplakic lesions the increase in autophagic levels seems to signal the process of malignant transformation. There are no previous studies of autophagy detection in leukoplakias to establish comparisons. However, the study conducted by Li et al. [16] evaluated the autophagy process in oral submucosal fibroid lesions and, they also observed the increased expression of LC3-II in potentially malignant oral submucosal fibroid lesions when compared to control samples, using both immunohistochemistry and PCR in real time. A possible mechanism to explain the increase in autophagy in epithelial dysplasias and leukoplakias with poorer evolution is the increase in proliferative activity that usual occurs in these lesions [17]. Increased proliferation in the tissue can result in the emergence of areas of hypoxia and with decreased nutrition available, consequently activating the autophagy process [18,19].
In the normal oral mucosa, the autophagic cells were concentrated in the basal, parabasal and intermediate layers, which can be considered as the physiological
autophagy process in healthy tissue, since in these layers cells are still metabolic active. However, in leukoplakia we also observed a large number of cells positive for the LC3-II immunomarker in the superficial layer of the epithelium. Probably this may be related to increased proliferation of these lesions and consequently, higher metabolic status or, alternatively, by external stimuli, which act directly on the outermost layer of the oral epithelium inducing the autophagy process [20]. Regarding the association between the clinical type of leukoplakia and the levels of autophagy, we did not find association because, probably, what influences the autophagy process is the presence or absence of dysplasia, and the distribution of dysplastic lesions in our sample was similar among homogenous and non-homogenous leukoplakias.
In addition, we have not found association with smoking and drinking habits and LC3-II staining, although both substances are known to induce oxidative stress and consequently could led to cell autophagy. The literature reports that smoking can induce autophagy in lung tissues, however we have not found this evidence for oral leukoplakia [20]. Since, we have not found such association in our sample, this may indicate a specific response for the lung tissues. There is also evidence that ethanol can induce autophagy due to high level of oxidative stress in hepatocytes, however chronic intake of ethanol suppressed autophagy in the Lieber-DeCarli animal model (liquid diet for chronic ethanol-feeding), suggesting that autophagy modulation by these substances may be dose-dependent [21].
In malignant lesions, the percentage of LC3-II positive cells increases significantly, reaching high proportions (an average of 80.46% positive tumor cells). This
observation suggests that autophagy is highly activated in oral squamous cell carcinomas. In the literature, there are previous studies reporting low survival rates for patients with oral squamous cell carcinoma and high LC3-II expression [22,23]. The same was also found in other types off neoplasia, such as pancreatic cancer [24]. However, the association between autophagy and cancer prognosis seems to vary for tumors of different origins. In ovarian and hypopharyngeal cancers, the opposite was observed, a higher percentage of LC3-II positive cells was associated with tumors with better prognoses [25,26].
It has already been shown that LC3-II is a marker of autophagosomes, which is sensitive in the verification of autophagy in humans [27]. In the meantime, there are other markers of autophagy that have also been investigated in various solid tumors. In ovarian and hypopharyngeal cancers, low levels of Beclin-I were associated with poorer survival [25,26]. Another marker used for autophagy is P62. In studies conducted with oral squamous cell carcinomas, the increased expression of the cytoplasmic protein P62 is associated with a poorer prognosis for the disease [28,23].
In our study, observing the steady increase of LC3-II in the process of carcinogenesis from normal oral mucosa, up to invasive carcinomas, we can conclude that the autophagy marker could be correlated with neoplastic transformation and used in precancerous lesions as a predictive marker of transformation. Moreover, increased expression of LC3-II were detected in OSCCs characterized by a moderate grade of differentiation, an advanced stage (T3-T4), with regional metastasis, greater depth of invasion, and in patients who died because of the tumor; then, worst survival have been
noted in patients with higher percentage of positive cells. These results confirm the cytoprotective role of autophagy in oral carcinogenesis and also in cancer progression.
By well-orchestrated self-repair mechanisms, autophagy prevents cell death and is exploited by cells to become resistant to several therapeutic agents. Different studies have demonstrated that autophagy suppression may render the cancer cells more susceptible to treatments [29,30,31]. Different agents (3-MA, G15, Apicidin) have been employed to inhibit, directly or indirectly, cellular autophagy and, consequently, induce apoptosis in OSCC [31,32,33]. The identification of molecules involved in the autophagy process may aid the clinicians in the early diagnosis of oral lesions with a potential risk of malignant evolution and in the recognition of oral cancer with a more aggressive behavior and probably characterized by resistance to standardized therapeutic agents. New molecules able to directly or indirectly inducing apoptosis, in this way inhibiting the autophagy process, should be further investigated in order to offer an adequate and efficacious therapeutic chance to OSCC patients.
Acknowledgements We would like to thank CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil, process number 455496/2014-5) and Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas for supporting this study.
REFERÊNCIAS
[1] D.M. PARKIN, F. BRAY, J. FERLAY, P. PISANI. Global cancer statistics, 2002. CA Cancer J Clin (2005) 55: 74-108.
[2] R. SIEGEL, Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths, CA Cancer J. Clin. 61 (2011) 212-236. [3] R. TACHEZY, J. KLOZAR, L. RUBENSTEIN, E. SMITH, M. SALÁKOVÁ, J. SMAHELOVÁ, Demographic and risk factors in patients with head and neck tumors, J. Med. Virol. 5 (2009) 878-887.
[4] I. VAN DER WAAL, Potentially malignant disorders of the oral and oropharyngeal mucosa; terminology, classification, and present concepts of management, Oral Oncol. 45 (2009) 317–323.
[5] P. HOLMSTRUP, Long-term treatment outcome of oral premalignant lesions, Oral Oncol. 42 (2006) 461-474.
[6] J. S. LIMA, Oral leukoplakia manifests differently in smokers and non smokers. Braz. Oral. Res. 26 (2012) 543-549.
[7] H. WU, S. HUANG, D. ZHANG, Autophagic responses to hypoxia and anticancer therapy in head and neck cancer, Pathol. Res. Practice 211 (2015) 101–108.
[8] Z. J. YANG, The Role of Autophagy in Cancer: Therapeutic Implications, Mol. Cancer Ther. 10 (2011) 1533–1541.
[9] Z. YANG, D. J. KLIONSKY, Eaten Alive: A History Of Macroautophagy, Nat. Cell Biol. 12 (2010) 814-822.
[10] J. VAN LIMBERGEN, Autophagy: from basic science to clinical application. Mucosal Immunol. 2 (2009) 315-330. [11] M. M. HIPPERT, P. S. O’TOOLE, A. THORBURN, Autophagy in cancer: good, bad, or both? Cancer Res. 66 (2006) 9349-9351. [12] J. CUI, Z. GONG, H. SHEN, The role of autophagy in liver cancer: Molecular mechanisms and potential therapeutic targets. Biochim. Biophys. Acta. (2013) 15-26.
[13] X. ZHI, Q. ZHONG, Autophagy in cancer. F1000Prime Rep. 7 (2015) 18.
[14] A.K. EL-NAGGAR, J.K.C. CHAN, J.R. GRANDIS, T. TAKATA, P.J. SLOOTWEG, World Health Organization Classification of Tumours: Pathology and Genetics of Head and Neck Tumours. 4th ed. Lyon: IARCPress; 2017. [15] F. VISIOLI, I.S. LAUXEN, M. SANT’ANA FILHO, P. V. RADOS, Expression of the cell cycle regulation proteins p53 and p21
waf1
in different types of non-dysplastic
leukoplakias, J. Appl. Oral Sci. 20 (2012) 369-375.
[16] J. LI, T. T. ZHAO, P. ZHANG, Autophagy mediates oral submucous fibrosis, Exp. Ther. Med. 5 (2016) 1859-1864. [17] P. J. THOMSON, J. V. SOAMES, C. BOOTH, J.A. O’SHEA, Epithelial cell proliferative activity and oral cancer progression, Cell Prolif. 35 (2002) 110–120.
[18] W. M. TILAKARATNE, Z. IQBAL, M. T. THE, Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis, J. Oral Pathol. Med. 37 (2008) 372-377. [19] H. ZHANG, M. BOSCH-MARCE, L. A. SHIMODA, Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia, J. Biol. Chem. 283 (2008) 10892-10903. [20] M.K. SANNIGRAHI, V. SINGH, R. SHARMA, N.K. PANDA, M. KHULLAR. Role of autophagy in head and neck cancer and therapeutic resistance, Oral Diseases. 21 (2015) 283–291. [21] L.WANG, B. KHAMBU, H. ZHANG, X. YIN. Autophagy in alcoholic liver disease, self-eating triggered by drinking, Clinics and Research in Hepatology and Gastroenterology 39 (2015) S2—S6. [22] J. Y. TANG, High LC3 expression correlates with poor survival in patients with oral squamous cell carcinoma, Hum. Pathol. 44 (2013) 2558–2562.
[23] L. J. LIU, F. F. CHEN, J. LUNG, H. C. LO, H. F. LEE, C. Y. LU, H. C. HUNG, Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma, Br. J. Cancer. 26 (2014) 944-954. [24] S. FUJII, S. MITSUNAGA, M. YAMAZAKI, Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome, Cancer Sci. 99 (2008) 18131819. [25] J. WANG, X. PAN, L. DING, D. LIU, D. LEI, T. JIN, Aberrant Expression of
Beclin-1 and LC3 Correlates with Poor Prognosis of Human Hypopharyngeal Squamous Cell Carcinoma, Plos One. 7 (2013) e69038. [26] G. VALENTE, F. MORANI, G. NICOTRA, G. Expression and Clinical Significance of the Autophagy Proteins BECLIN 1 and LC3 in Ovarian Cancer, Biomed Res. Int. (2014) 462658. [27] S. J. CHANG, F. OU-YANG, H. P. TU, Decreased expression of autophagy protein LC3 and stemness (CD44+/CD24−/low) indicate poor prognosis in triplenegative breast cancer, Hum. Pathol. 48 (2016) 48–55. [28] T. INUI, T. CHANO, M. TAKIKITA-SUZUKI, M. NISHIKAWA, G. YAMAMOTO, H. OKABE, Association of p62/SQSTM1 excess and oral carcinogenesis, Plos One. 8 (2013) e74398.
[29] N. MIZUSHIMA, T. YOSHIMORI, B. LEVINE, Methods in mammalian autophagy research, Cell. 140 (2010) 313–326.
[30] J.D. RABINOWITZ, E. WHITE, Autophagy and metabolism, Science. 330 (2010) 1344–8. Korolchuk VI, Rubinsztein DC. Regulation of autophagy by lysosomal positioning. Autophagy. 2011;7(8):927–928.
[31] S. PATIL, R.S. RAO, A.T. RAJ, Dual Role of Autophagy in Oral Cancer, J. Int. Oral Health. 7 (2015) i–ii.
[32] L.Y. BAI, J.R. WENG, J.L. HU, D. WANG, A.M. SARGEANT, C.F. CHIU, G15, a GPR30 antagonist, induces apoptosis and autophagy in human oral squamous
carcinoma cells, Chem. Biol. Interact. 206 (2013) 375–384.
[33] M.Y. AHN, S.G. AHN, J.H. YOON. Apicidin, a histone deacetylase inhibitor, induces both apoptosis and autophagy in human oral squamous carcinoma cells, Oral Oncol. 47 (2011) 1032–1038.
Figure Captions:
Figure 1. LC3-II staining in the studied lesions in comparison to the normal oral mucosa. A, Normal oral mucosa. B, Non-dysplastic leukoplakia. C, Dysplastic leukoplakia. D, Oral squamous cell carcinoma. Magnification at 400x.
Figure 2. A, Distribution of positive cells to LC3-II according to the group studied. B, Distribution of LC3-II positive cells according to the epithelial layer and histopathological diagnosis. C, Distribution of LC3-II positive cells according to the epithelial layer and lesion evolution.
Figure 3. Kaplan-Meier survival analysis of oral squamous cell carcinomas according to LC3-II staining.
Table 1. Clinical-pathological characteristics of oral leukoplakias and OSCCs. Characteristics
N
%
3
6
LEUKOPLAKIA SAMPLE
Gender
Male
0
0.78 2
Female
1
3 9.22 55.86 ±
Age
Mean (±SD)
12.24
Range
26-79 1
Location
Palate
4
7.45 1
Tongue
3
8
4
Smoker
8
6
4
Alcohol Consumption
3
Drinker 8
3 5.29
0 5
4 5.09
1
Former Drinker
0 7.84
2 NI
1 1.76
0 Never smoked
3 5.29
0 Former Smoker
2 7.45
1 Smoking status
1 5.69
1 Others
2 5.52
0 Floor of mouth
2
0 9.80
Never drinker
0
0
5 NI
9.80 2
4
3 Histopathological
5.11
Dysplasic
Diagnosis
20 39.21
Non-Dysplastic
31 60.79
Clinical Type
Homogeneous
05 09.80
Non-Homogeneous
25 49.02
NI
21 41.18
Evolution
Good
11 21.57
Bad
20 39.21
NI
20 39.21
OSCC SAMPLE 8 Gender
Male
5
7 0.8
3 Female Age
5
Mean (±SD)
2 9.2
67.3 ± 11.0
Variation
31-92 5
Location
Tongue
9
4 9.2
1 Floor of mouth
4
1 1.7
1 Trigone and Gum
1
7
4.1 3
Others
2
0
5 4
Adjuvant Treatment
Radiotherapy
3
5.8 1.
2
Chemotherapy Radiotherapy
3
+
Chemotherapy
66
2
2
7
2.5 1
None
1
5
2.5 3
NI
3
Well differentiated –Grade 1 Differentiation Grade
7.5 2
1
3 Moderately differentiated –
Grade 2 Poorly
9.2 6
5
1 differentiated
Grade 3 NI
Tumor size
2
Mean (±SD)
–
0.8 2
2
9
4.2 5.
7
8
2.73 ± 1.26 cm
Variation Depht of invasion
Mean (±SD)
03-6.0 cm 11.17 ± 4.73 mm
Variation
SD, standard deviation. NI, Not informed
0.9-24mm
Table 2. LC3-II immunostaining comparison between normal oral mucosa and oral leukoplakia and distribution of LC3-II immunostaining according to the variables studied in leukoplakia. % LC3-II Positive Cells Basal Layer
Normal
Mucosa
Parabasal
Intermediat
Superficial
Layer
e Layer
Layer
Mean ±SD
Mean ±SD
Mean ±SD
Mean ±SD
50.837(±18.
46.467
55.712
22.344
x
Leukoplakia Oral Mucosa
Leukoplakia p (t Test)
99) 57.793 (±22.19)
(±16.88) 54.865 (±19.63)
(±11.62) 62.502 (±19.65)
(±19.22) 53.576 (±24.20)
0,8690
0,7501
0,7539
0,0013
63.64
60.76
69.26
56.66
Leukoplakia Sample Clinical Aspect Homogeneous
Non-Homogeneous
(±22.28)
(±17.32)
50.20 50.65
(±26.24)
(±14.34)
60.10 (±24.43)
(±30.00)
48.15 (±27.76)
(±28.63) p (t Test)
0.3529
0.4023
0.4313
0.5469
53.46
51.00
62.81
49.04
Smoking Status Smoker
Former Smoker
Never smoked p (ANOVA)
(±29.19) 46.33 (±30.08) 73.48 (±21.3) 0.3652
(±27.05) 43.78 (±27.82) 68.85 (±20.37) 0.3779
(±26.18) 48.23 (±24.67) 74.55 (±14.20) 0.3370
(±29.85) 48.68 (±30.09) 58.58 (±28.14) 0.8384
Alcohol Status Drinker
Former Drinker
55.85 (±30.13)
55.14 (±25.92)
37.53 38.30
(±30.13)
64.12 (±22.28)
46.67 (±30.70)
53.84 (±28.93)
44.03 (±36.07)
(±32.20)
Never Drinker
52.04 56.36
(±13.55)
65.54 (±12.20)
58.22 (±24.04)
(±14.26) p (ANOVA)
SD, standard deviation.
0.6012
0.5295
0.4253
0.7967
Table 3. Analysis of LC3-II immunostaining according to the clinical and histological variables of oral squamous cell carcinoma lesions.
% LC3-II Positive Cells Mean ±SD
P
Differentiation Grade 1
79.81 (±14.78)
Grade 2
78.83 (±12.84)
Grade 3
80.76 (±11.94)
0.9280
Tumor Size T1+T2
79.67 (±14.54)
T3+T4
81.72 (±10.43)
0.4544
Regional metastasis N0
79. 84 (±13.54)
N1
81.02 (±12.96)
0.6568
Evolution Alive Dead
80.52 (±13.50) (because
tumor)
of
the 82.74 (±12.12)
0.4587
Depht of invasion Up to 10mm
79.58 (±14.38)
>10mm
81.14 (±12.28)
SD, standard deviation.
0.5615
S0344-0338(17)30331-X http://dx.doi.org/doi:10.1016/j.prp.2017.07.027 PRP 51864
To appear in: Received date: Revised date: Accepted date:
6-4-2017 4-7-2017 28-7-2017
Please cite this article as: T.B.de Lima, A.H.R.Paz, P.V.Rados, R.Leonardi, P.Bufo, M.C.Pedicillo, A.Santoro, S.Cagiano, G.Aquino, G.Botti, G.Pannone, F.Visioli, Autophagy Analysis in Oral Carcinogenesis, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.07.027 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.
1
Autophagy Analysis in Oral Carcinogenesis
TB de Lima1, AHR Paz2, PV Rados1, R Leonardi3, P Bufo4, MC Pedicillo4, A Santoro4, Cagiano S4, G Aquino5, G Botti5, G Pannone4,5, F Visioli1
1. Department of Oral Pathology – Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil. 2. Experimental Research Center, Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil. 3. Department of Medical and Surgical Science. II Dental Unit - University of Catania, Catania, Italy. 4. Department of Clinical and Experimental Medicine. Pathological Anatomy Unit – University of Foggia, Foggia, Italy. 5. Pathology Unit, Instituto Nazionale per lo Studio e la Cura dei Tumori, “Fondazione G. Pascale”, IRCCS, Naples, Italy.
Corresponding author: Fernanda Visioli Email: [email protected] Address: Rua Ramiro Barcelos 2492, room 503 Porto Alegre, RS, Brazil 90035-003 Phone: 55 51 33085011 Phax: 55 51 33085023
2
Abstract Objective: The aim of this study was to evaluate the levels of autophagy in oral leukoplakia and squamous cell carcinoma and to correlate with clinical pathological features, as well as, the evolution of these lesions. Methodology: 7 normal oral mucosa, 51 oral leukoplakias, and 120 oral squamous cell carcinomas (OSCC) were included in the study. Histological sections of the mucosa and leukoplakias were evaluated throughout their length, while the carcinomas were evaluated using Tissue Microarray. After the immunohistochemical technique, LC3-II positive cells were quantified in the different epithelial layers of the mucosa and leukoplakias and in the microarrays of the squamous cell carcinomas. The correlation between positive cells with the different clinical-pathological variables and with the evolution of the lesions was tested using the t test, ANOVA, and Kaplan-Meier survival analysis. Results: We observed increased levels of autophagy in the oral squamous cell carcinomas (p<0.001) in relation to the other groups, but without any association with poorer evolution or survival of these patients. Among the leukoplakias, we observed a higher percentage of positive cells in the intermediate layer of the dysplastic leukoplakias (p=0.0319) and in the basal layer of lesions with poorer evolution (p=0.0133). Conclusion: The levels of autophagy increased during the process of oral carcinogenesis and are correlated with poorer behavior of the leukoplakias. Key-words: Autophagy, LC3-II, leukoplakia, oral squamous cell carcinoma.
Introduction
Oral squamous cell carcinoma is considered to be the sixth most common type of cancer worldwide [1,2]. The five-year survival rate is about 50% when lymphonodal metastases are present and the survival of patients affected by this pathology has not improved in recent decades [3]. New
3
advances in understanding the biology of oral cancer are necessary to reduce its mortality rates.
Squamous cell carcinoma can be preceded by visible changes in the oral mucosa, known as potentially malignant disorders. The most common of these is leukoplakia and its prevalence is estimated at approximately 2% world-wide. The risk of malignant transformation is higher in dysplastic lesions, but some of these lesions can remain clinically unchanged or even regress completely [4]. Currently, the main risk factors considered for malignant transformation are: female gender, long duration of the lesion, size greater than 2 cm, leukoplakia in non-smokers, and the presence of epithelial dysplasia [5,6]. However, it is not yet possible to predict the behavior of these lesions, and the search for biomarkers to detect cellular alterations signaling the carcinogenetic process is necessary.
Therefore, we need to better understand the mechanisms related to the development and progression of tumors. In this context, the cellular autophagy may be an important mechanism for the occurrence of this outcome [7]. Autophagy is a catabolic process for the degradation and recycling of cytosol components and damaged cellular organelles. It plays a critical role in the maintenance of cellular homeostasis, and its purpose is to preserve cellular viability under conditions of stress [8]. The initiation process of autophagy involves several members of the Atg family of proteins [9]. The formation of autophagosomes is mediated by Atg8 and Atg12. Atg8 is modified by Atg4 to form LC3-I, which in turn, is modified by Atg3 and Atg7 into a smaller form, LC3II. LC3-II is recruited by Atg12 for the growth of the autophagic membrane. The
4
Atg5-Atg12 complex then binds to Atg16 to initiate the process of membrane prolongation through the recruitment of LC3-II. Once the autophagosome is formed, the Atg5-Atg12-Atg16 complex unbinds from the autophagic membrane and is recycled together with the LC3-II. The autophagosome containing the material to be degraded then fuses with a lysosome [10].
Autophagy is a process that may act in a dual way in carcinogenesis and during the progression of cancer [11,12]. It can act as a tumor suppressor mechanism, thus avoiding the accumulation of damaged organelles and proteins, reducing the levels of oxidative stress and consequently, genetic instability. On the other hand, autophagy may be a fundamental mechanism for the survival of cells in the inhospitable microenvironment that emerges during carcinogenesis, which is characterized by hypoxia, acidosis and nutrient deprivation [13]. Therefore, it is important to study how the levels of autophagy behave in the different stages of oral carcinogenesis. The objective of this study was to evaluate the levels of LC3-II, a marker of autophagy, in tissue samples from oral squamous cell carcinomas and oral leukoplakias, comparing them with oral normal mucosa, and correlating them with the clinical pathological features and the evolution of the lesions.
Methods
Sample
This is a multicenter study, the samples included in this analysis were selected from the Oral Pathology Archive of the Universidade Federal do Rio
5
Grande do Sul (Porto Alegre, Brazil) and from the National Cancer Institute "G.Pascale" Foundation (Napoli, Italy). Were included: 7 samples of normal oral mucosa (from lip frenectomies), 51 samples of oral leukoplakia (with clinical and microscopic confirmation) and 120 samples of oral squamous cell carcinomas (OSCC). OSCC samples were derived from tumor resection prior to any other treatment. All the hematoxylin-eosin stained slides were reviewed by two pathologists (FV, GP). Clinical and pathological information were obtained from the files of the laboratories and patient records. This study was approved by the Ethics Committee (Federal University of Rio Grande do Sul, number 1.394.505).
The criteria for histopathological analysis of oral leukoplakias in order to diagnose epithelial dysplasia were established by the World health Organization (WHO). In addition, the criteria for classification of oral squamous cell carcinomas in well, moderate and poor differentiated tumors were also obtained from WHO [14].
For the leukoplakia samples, the evolution was considered good, if the lesion did not recur after a total biopsy, if it remained unchanged after a partial biopsy, if it decreased in size or regressed, or if the clinical aspect improved over time. The evolution was considered poor, if the lesion worsened its clinical appearance or increased its size, if there was recurrence, if new lesions appeared or if malignant transformation occurred [4].
Alcohol consumption was considered positive when the patient consumed at least 30g of alcohol per week. Patients were considered former smoker or former drinker if they stopped consuming these substances for at least 1 year.
6
Tissue Microarray Immunohistochemistry
A tissue microarray (TMA) block was constructed from OSCC samples. After selecting representative areas, two cylinders with an area of 0.28 mm2 were collected of each paraffin block tand transferred to the receiving block using Galileo TMA CK 3500 (ISE TMA software, Systems Engineering Integrated). For each patient, two different areas were selected, to increase the area of tumor assessed and to improve the representativeness of the selected fields. From the paraffin blocks, 3μm thick sections were obtained, dewaxed and rehydrated in ethanol. After blocking the endogenous peroxidase with methanol and hydrogen peroxide, antigen retrieval was performed in a pressure cooker at 121°C, and then samples were incubated with primary antibody (LC3-II, 1: 100, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The detection system used was EnVisionTM + Dual Link (Dako, Carpinteria, CA, USA) and 3,3-diaminobenzidine (DAB, Dako, Carpinteria, CA, USA) was used for visualization. Slides were counterstained with Hematoxylin. As a positive control, a previously tested OSCC sample was used. For negative control, the same OSCC sample was used and the primary antibody was replaced with nonimmune goat serum.
Quantification of Immunopositive Cells
Images of the entire length of the histological section were obtained. The samples images were captured at a magnification of 400 times using the software cellSens™ (Olympus, Tokyo, Japan), in a binocular microscope model CX41RF (Olympus Latin AmericaInc., Miami, Coupled to a Qcolor 5 camera,
7
Coolet, RTV (Olympus Latin America Inc.). ImageJ software for Mac OS X version 1.5 (NIH, USA) was used to count 1000 cells per layer in order to determine the percentage of positive and negative autophagy tagged cells, totaling 1000 cells in the basal layer (BL), 1000 cells in the parabasal layer (PBL), 1000 cells in the intermediate layer (IL) and 1000 cells in the surface layer (SL). The basal layer was defined as having at least one point of contact with the basal membrane, the parabasal layer was composed of cell layers immediately above the basal layer (non-flattened), the spinous layer located between the parabasal and superficial layer, being the superficial layer characterized by flattened cells [15].
In OSCC TMA cores, LC3-II positive cells were counted using a binocular microscope model CX41RF (Olympus Latin AmericaInc., Miami, FL, USA). All the tumor cells were quantified in each cylinder and the percentage of cells positive was determined. At the end of the quantification, the mean between the two cores analyzed for each patient was performed.
Statistical Analysis
For statistical analysis, the software SPSS 19.0 (Statistical Package for the Social Sciences, IBM, New York, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, USA) were used. Data distribution was analyzed by the Kolmogorov-Sminorv test. The sample followed the parametric distribution; therefore, the data was assessed by the Student t test or ANOVA. Survival analysis was performed using the Kaplan-Meier curve.
8
Results
51 patients with leukoplakia were studied. The demographic profile of these patients is summarized in Table 1. The majority of the sample was composed of men (60%), smokers and alcohol consumers, the mean age was 55.86 years. The most affected sites were palate (27.45%), tongue (25.5%) and floor of mouth. Most of the lesions were non-homogeneous and non-dysplastic. The average duration of follow-up of these patients was 4.3 years, and for most of them, the course of the disease worsened (39.21%). 4 patients underwent malignant transformation, being the majority of these lesions located in the tongue border in female patients.
120 tumor samples of oral squamous cell carcinoma were analyzed, among the samples, the male gender predominated (70.8%) and the mean age of the patients constituting the sample was 67.3 years old. The majority of lesions were located on the tongue and floor of mouth, and lesions of grade 2 predominated. Most of the patients underwent radiotherapy associated with the surgical resection of the tumor (Table 1).
It was observed an increase in the percentage of LC3-II positive cells from the normal oral mucosa to the leukoplakia samples and from leukoplakia to the oral squamous cell carcinomas (Figure 1, A,B,C,D), this increase was statistically significant (p<0.0001) (Figure 2). An increase in LC3-II immunostaining was observed in oral leukoplakias compared to normal oral mucosa samples, especially in the upper layers of the epithelium (p=0.0013) (Table 2, Figure 1, A, B and C). No significant associations were found between LC3-II immunostaining and the clinical type of leukoplakia, nor with smoking and
9
alcohol consumption (Table 2). We observed an increase of LC3-II positive cells in dysplastic lesions compared to non-dysplastic lesions (Figure 1, B and C, Figure 2), in the intermediate layer of the epithelium this increase was statistically significant (p=0.0319). In the lesions of worse evolution, the expression of LC3-II increased (Figure 2), which was significant in the basal layer (p= 0.0133).
Increased LC3-II positive cells were detected in Grade 2, T3 and T4 tumors, with regional metastasis, with greater depth of invasion, and in patients who died because of the tumor. However, we did not find statistically significant associations (Table 3). To evaluate the association between LC3-II and patient survival, we performed a Kaplan-Meier survival curve, dividing the sample into >= 70% of LC3-II positive cells and <70% of LC3-II positive cells, based in the median cut-off. We observed greater survival for patients with a lower percentage of positive cells, but this data is not statistically significant (p = 0.21) (Figure 3).
Discussion
Our study sought to evaluate the levels of cellular autophagy during the oral carcinogenesis process. We observed a progressive increase of the percentage of positive cells according with progressive severity of alterations, being the number LC3-II cells greater in leukoplakic lesions than in normal oral mucosa, reaching the highest averages in oral squamous cell carcinomas.
Additionally, a greater number of cells performing autophagy were observed in dysplastic samples and in those with poorer evolution. Despite the dual role of autophagy, in leukoplakic lesions the increase in autophagic levels seems to signal the process of malignant transformation. There are no previous studies of autophagy detection in leukoplakias to establish comparisons. However, the study conducted by Li et al. [16] evaluated the autophagy process in oral submucosal fibroid lesions and, they also observed the increased expression of LC3-II in potentially malignant oral submucosal fibroid lesions when compared to control samples, using both immunohistochemistry and PCR in real time. A possible mechanism to explain the increase in autophagy in epithelial dysplasias and leukoplakias with poorer evolution is the increase in proliferative activity that usual occurs in these lesions [17]. Increased proliferation in the tissue can result in the emergence of areas of hypoxia and with decreased nutrition available, consequently activating the autophagy process [18,19].
In the normal oral mucosa, the autophagic cells were concentrated in the basal, parabasal and intermediate layers, which can be considered as the physiological
autophagy process in healthy tissue, since in these layers cells are still metabolic active. However, in leukoplakia we also observed a large number of cells positive for the LC3-II immunomarker in the superficial layer of the epithelium. Probably this may be related to increased proliferation of these lesions and consequently, higher metabolic status or, alternatively, by external stimuli, which act directly on the outermost layer of the oral epithelium inducing the autophagy process [20]. Regarding the association between the clinical type of leukoplakia and the levels of autophagy, we did not find association because, probably, what influences the autophagy process is the presence or absence of dysplasia, and the distribution of dysplastic lesions in our sample was similar among homogenous and non-homogenous leukoplakias.
In addition, we have not found association with smoking and drinking habits and LC3-II staining, although both substances are known to induce oxidative stress and consequently could led to cell autophagy. The literature reports that smoking can induce autophagy in lung tissues, however we have not found this evidence for oral leukoplakia [20]. Since, we have not found such association in our sample, this may indicate a specific response for the lung tissues. There is also evidence that ethanol can induce autophagy due to high level of oxidative stress in hepatocytes, however chronic intake of ethanol suppressed autophagy in the Lieber-DeCarli animal model (liquid diet for chronic ethanol-feeding), suggesting that autophagy modulation by these substances may be dose-dependent [21].
In malignant lesions, the percentage of LC3-II positive cells increases significantly, reaching high proportions (an average of 80.46% positive tumor cells). This
observation suggests that autophagy is highly activated in oral squamous cell carcinomas. In the literature, there are previous studies reporting low survival rates for patients with oral squamous cell carcinoma and high LC3-II expression [22,23]. The same was also found in other types off neoplasia, such as pancreatic cancer [24]. However, the association between autophagy and cancer prognosis seems to vary for tumors of different origins. In ovarian and hypopharyngeal cancers, the opposite was observed, a higher percentage of LC3-II positive cells was associated with tumors with better prognoses [25,26].
It has already been shown that LC3-II is a marker of autophagosomes, which is sensitive in the verification of autophagy in humans [27]. In the meantime, there are other markers of autophagy that have also been investigated in various solid tumors. In ovarian and hypopharyngeal cancers, low levels of Beclin-I were associated with poorer survival [25,26]. Another marker used for autophagy is P62. In studies conducted with oral squamous cell carcinomas, the increased expression of the cytoplasmic protein P62 is associated with a poorer prognosis for the disease [28,23].
In our study, observing the steady increase of LC3-II in the process of carcinogenesis from normal oral mucosa, up to invasive carcinomas, we can conclude that the autophagy marker could be correlated with neoplastic transformation and used in precancerous lesions as a predictive marker of transformation. Moreover, increased expression of LC3-II were detected in OSCCs characterized by a moderate grade of differentiation, an advanced stage (T3-T4), with regional metastasis, greater depth of invasion, and in patients who died because of the tumor; then, worst survival have been
noted in patients with higher percentage of positive cells. These results confirm the cytoprotective role of autophagy in oral carcinogenesis and also in cancer progression.
By well-orchestrated self-repair mechanisms, autophagy prevents cell death and is exploited by cells to become resistant to several therapeutic agents. Different studies have demonstrated that autophagy suppression may render the cancer cells more susceptible to treatments [29,30,31]. Different agents (3-MA, G15, Apicidin) have been employed to inhibit, directly or indirectly, cellular autophagy and, consequently, induce apoptosis in OSCC [31,32,33]. The identification of molecules involved in the autophagy process may aid the clinicians in the early diagnosis of oral lesions with a potential risk of malignant evolution and in the recognition of oral cancer with a more aggressive behavior and probably characterized by resistance to standardized therapeutic agents. New molecules able to directly or indirectly inducing apoptosis, in this way inhibiting the autophagy process, should be further investigated in order to offer an adequate and efficacious therapeutic chance to OSCC patients.
Acknowledgements We would like to thank CNPQ (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil, process number 455496/2014-5) and Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas for supporting this study.
REFERÊNCIAS
[1] D.M. PARKIN, F. BRAY, J. FERLAY, P. PISANI. Global cancer statistics, 2002. CA Cancer J Clin (2005) 55: 74-108.
[2] R. SIEGEL, Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths, CA Cancer J. Clin. 61 (2011) 212-236. [3] R. TACHEZY, J. KLOZAR, L. RUBENSTEIN, E. SMITH, M. SALÁKOVÁ, J. SMAHELOVÁ, Demographic and risk factors in patients with head and neck tumors, J. Med. Virol. 5 (2009) 878-887.
[4] I. VAN DER WAAL, Potentially malignant disorders of the oral and oropharyngeal mucosa; terminology, classification, and present concepts of management, Oral Oncol. 45 (2009) 317–323.
[5] P. HOLMSTRUP, Long-term treatment outcome of oral premalignant lesions, Oral Oncol. 42 (2006) 461-474.
[6] J. S. LIMA, Oral leukoplakia manifests differently in smokers and non smokers. Braz. Oral. Res. 26 (2012) 543-549.
[7] H. WU, S. HUANG, D. ZHANG, Autophagic responses to hypoxia and anticancer therapy in head and neck cancer, Pathol. Res. Practice 211 (2015) 101–108.
[8] Z. J. YANG, The Role of Autophagy in Cancer: Therapeutic Implications, Mol. Cancer Ther. 10 (2011) 1533–1541.
[9] Z. YANG, D. J. KLIONSKY, Eaten Alive: A History Of Macroautophagy, Nat. Cell Biol. 12 (2010) 814-822.
[10] J. VAN LIMBERGEN, Autophagy: from basic science to clinical application. Mucosal Immunol. 2 (2009) 315-330. [11] M. M. HIPPERT, P. S. O’TOOLE, A. THORBURN, Autophagy in cancer: good, bad, or both? Cancer Res. 66 (2006) 9349-9351. [12] J. CUI, Z. GONG, H. SHEN, The role of autophagy in liver cancer: Molecular mechanisms and potential therapeutic targets. Biochim. Biophys. Acta. (2013) 15-26.
[13] X. ZHI, Q. ZHONG, Autophagy in cancer. F1000Prime Rep. 7 (2015) 18.
[14] A.K. EL-NAGGAR, J.K.C. CHAN, J.R. GRANDIS, T. TAKATA, P.J. SLOOTWEG, World Health Organization Classification of Tumours: Pathology and Genetics of Head and Neck Tumours. 4th ed. Lyon: IARCPress; 2017. [15] F. VISIOLI, I.S. LAUXEN, M. SANT’ANA FILHO, P. V. RADOS, Expression of the cell cycle regulation proteins p53 and p21
waf1
in different types of non-dysplastic
leukoplakias, J. Appl. Oral Sci. 20 (2012) 369-375.
[16] J. LI, T. T. ZHAO, P. ZHANG, Autophagy mediates oral submucous fibrosis, Exp. Ther. Med. 5 (2016) 1859-1864. [17] P. J. THOMSON, J. V. SOAMES, C. BOOTH, J.A. O’SHEA, Epithelial cell proliferative activity and oral cancer progression, Cell Prolif. 35 (2002) 110–120.
[18] W. M. TILAKARATNE, Z. IQBAL, M. T. THE, Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis, J. Oral Pathol. Med. 37 (2008) 372-377. [19] H. ZHANG, M. BOSCH-MARCE, L. A. SHIMODA, Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia, J. Biol. Chem. 283 (2008) 10892-10903. [20] M.K. SANNIGRAHI, V. SINGH, R. SHARMA, N.K. PANDA, M. KHULLAR. Role of autophagy in head and neck cancer and therapeutic resistance, Oral Diseases. 21 (2015) 283–291. [21] L.WANG, B. KHAMBU, H. ZHANG, X. YIN. Autophagy in alcoholic liver disease, self-eating triggered by drinking, Clinics and Research in Hepatology and Gastroenterology 39 (2015) S2—S6. [22] J. Y. TANG, High LC3 expression correlates with poor survival in patients with oral squamous cell carcinoma, Hum. Pathol. 44 (2013) 2558–2562.
[23] L. J. LIU, F. F. CHEN, J. LUNG, H. C. LO, H. F. LEE, C. Y. LU, H. C. HUNG, Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma, Br. J. Cancer. 26 (2014) 944-954. [24] S. FUJII, S. MITSUNAGA, M. YAMAZAKI, Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome, Cancer Sci. 99 (2008) 18131819. [25] J. WANG, X. PAN, L. DING, D. LIU, D. LEI, T. JIN, Aberrant Expression of
Beclin-1 and LC3 Correlates with Poor Prognosis of Human Hypopharyngeal Squamous Cell Carcinoma, Plos One. 7 (2013) e69038. [26] G. VALENTE, F. MORANI, G. NICOTRA, G. Expression and Clinical Significance of the Autophagy Proteins BECLIN 1 and LC3 in Ovarian Cancer, Biomed Res. Int. (2014) 462658. [27] S. J. CHANG, F. OU-YANG, H. P. TU, Decreased expression of autophagy protein LC3 and stemness (CD44+/CD24−/low) indicate poor prognosis in triplenegative breast cancer, Hum. Pathol. 48 (2016) 48–55. [28] T. INUI, T. CHANO, M. TAKIKITA-SUZUKI, M. NISHIKAWA, G. YAMAMOTO, H. OKABE, Association of p62/SQSTM1 excess and oral carcinogenesis, Plos One. 8 (2013) e74398.
[29] N. MIZUSHIMA, T. YOSHIMORI, B. LEVINE, Methods in mammalian autophagy research, Cell. 140 (2010) 313–326.
[30] J.D. RABINOWITZ, E. WHITE, Autophagy and metabolism, Science. 330 (2010) 1344–8. Korolchuk VI, Rubinsztein DC. Regulation of autophagy by lysosomal positioning. Autophagy. 2011;7(8):927–928.
[31] S. PATIL, R.S. RAO, A.T. RAJ, Dual Role of Autophagy in Oral Cancer, J. Int. Oral Health. 7 (2015) i–ii.
[32] L.Y. BAI, J.R. WENG, J.L. HU, D. WANG, A.M. SARGEANT, C.F. CHIU, G15, a GPR30 antagonist, induces apoptosis and autophagy in human oral squamous
carcinoma cells, Chem. Biol. Interact. 206 (2013) 375–384.
[33] M.Y. AHN, S.G. AHN, J.H. YOON. Apicidin, a histone deacetylase inhibitor, induces both apoptosis and autophagy in human oral squamous carcinoma cells, Oral Oncol. 47 (2011) 1032–1038.
Figure Captions:
Figure 1. LC3-II staining in the studied lesions in comparison to the normal oral mucosa. A, Normal oral mucosa. B, Non-dysplastic leukoplakia. C, Dysplastic leukoplakia. D, Oral squamous cell carcinoma. Magnification at 400x.
Figure 2. A, Distribution of positive cells to LC3-II according to the group studied. B, Distribution of LC3-II positive cells according to the epithelial layer and histopathological diagnosis. C, Distribution of LC3-II positive cells according to the epithelial layer and lesion evolution.
Figure 3. Kaplan-Meier survival analysis of oral squamous cell carcinomas according to LC3-II staining.
Table 1. Clinical-pathological characteristics of oral leukoplakias and OSCCs. Characteristics
N
%
3
6
LEUKOPLAKIA SAMPLE
Gender
Male
0
0.78 2
Female
1
3 9.22 55.86 ±
Age
Mean (±SD)
12.24
Range
26-79 1
Location
Palate
4
7.45 1
Tongue
3
8
4
Smoker
8
6
4
Alcohol Consumption
3
Drinker 8
3 5.29
0 5
4 5.09
1
Former Drinker
0 7.84
2 NI
1 1.76
0 Never smoked
3 5.29
0 Former Smoker
2 7.45
1 Smoking status
1 5.69
1 Others
2 5.52
0 Floor of mouth
2
0 9.80
Never drinker
0
0
5 NI
9.80 2
4
3 Histopathological
5.11
Dysplasic
Diagnosis
20 39.21
Non-Dysplastic
31 60.79
Clinical Type
Homogeneous
05 09.80
Non-Homogeneous
25 49.02
NI
21 41.18
Evolution
Good
11 21.57
Bad
20 39.21
NI
20 39.21
OSCC SAMPLE 8 Gender
Male
5
7 0.8
3 Female Age
5
Mean (±SD)
2 9.2
67.3 ± 11.0
Variation
31-92 5
Location
Tongue
9
4 9.2
1 Floor of mouth
4
1 1.7
1 Trigone and Gum
1
7
4.1 3
Others
2
0
5 4
Adjuvant Treatment
Radiotherapy
3
5.8 1.
2
Chemotherapy Radiotherapy
3
+
Chemotherapy
66
2
2
7
2.5 1
None
1
5
2.5 3
NI
3
Well differentiated –Grade 1 Differentiation Grade
7.5 2
1
3 Moderately differentiated –
Grade 2 Poorly
9.2 6
5
1 differentiated
Grade 3 NI
Tumor size
2
Mean (±SD)
–
0.8 2
2
9
4.2 5.
7
8
2.73 ± 1.26 cm
Variation Depht of invasion
Mean (±SD)
03-6.0 cm 11.17 ± 4.73 mm
Variation
SD, standard deviation. NI, Not informed
0.9-24mm
Table 2. LC3-II immunostaining comparison between normal oral mucosa and oral leukoplakia and distribution of LC3-II immunostaining according to the variables studied in leukoplakia. % LC3-II Positive Cells Basal Layer
Normal
Mucosa
Parabasal
Intermediat
Superficial
Layer
e Layer
Layer
Mean ±SD
Mean ±SD
Mean ±SD
Mean ±SD
50.837(±18.
46.467
55.712
22.344
x
Leukoplakia Oral Mucosa
Leukoplakia p (t Test)
99) 57.793 (±22.19)
(±16.88) 54.865 (±19.63)
(±11.62) 62.502 (±19.65)
(±19.22) 53.576 (±24.20)
0,8690
0,7501
0,7539
0,0013
63.64
60.76
69.26
56.66
Leukoplakia Sample Clinical Aspect Homogeneous
Non-Homogeneous
(±22.28)
(±17.32)
50.20 50.65
(±26.24)
(±14.34)
60.10 (±24.43)
(±30.00)
48.15 (±27.76)
(±28.63) p (t Test)
0.3529
0.4023
0.4313
0.5469
53.46
51.00
62.81
49.04
Smoking Status Smoker
Former Smoker
Never smoked p (ANOVA)
(±29.19) 46.33 (±30.08) 73.48 (±21.3) 0.3652
(±27.05) 43.78 (±27.82) 68.85 (±20.37) 0.3779
(±26.18) 48.23 (±24.67) 74.55 (±14.20) 0.3370
(±29.85) 48.68 (±30.09) 58.58 (±28.14) 0.8384
Alcohol Status Drinker
Former Drinker
55.85 (±30.13)
55.14 (±25.92)
37.53 38.30
(±30.13)
64.12 (±22.28)
46.67 (±30.70)
53.84 (±28.93)
44.03 (±36.07)
(±32.20)
Never Drinker
52.04 56.36
(±13.55)
65.54 (±12.20)
58.22 (±24.04)
(±14.26) p (ANOVA)
SD, standard deviation.
0.6012
0.5295
0.4253
0.7967
Table 3. Analysis of LC3-II immunostaining according to the clinical and histological variables of oral squamous cell carcinoma lesions.
% LC3-II Positive Cells Mean ±SD
P
Differentiation Grade 1
79.81 (±14.78)
Grade 2
78.83 (±12.84)
Grade 3
80.76 (±11.94)
0.9280
Tumor Size T1+T2
79.67 (±14.54)
T3+T4
81.72 (±10.43)
0.4544
Regional metastasis N0
79. 84 (±13.54)
N1
81.02 (±12.96)
0.6568
Evolution Alive Dead
80.52 (±13.50) (because
tumor)
of
the 82.74 (±12.12)
0.4587
Depht of invasion Up to 10mm
79.58 (±14.38)
>10mm
81.14 (±12.28)
SD, standard deviation.
0.5615