Abstract
Background/Aim: p16 and PTEN are tumor suppressor genes. Loss of these molecules in oral squamous cell carcinoma (OSCC) has been studied worldwide. In this study, we explored whether p16 cooperates with inactive PTEN during the pathogenesis of OSCC, especially in regard to tumor aggressiveness and proliferation. Materials and Methods: Immunocytochemistry and western blot analysis were used to examine the levels of p16 and PTEN. Sequencing analysis was performed to identify mutations in the PTEN gene and HPV infection. Fluorescence in situ hybridization was used to examine the presence of the PTEN locus. Results: PTEN analysis showed high positivity in T4 samples. HPV-positive tumors correlated with tabagism, tumor size 3 and 4, disease stages 3 and 4, presence of lymph node metastasis (N1) and poor differentiation. Immunoexpression of p16 was strongly correlated with the presence of HPV. Conclusion: PTEN demonstrated a higher reactivity in advanced disease stages and p16 was strongly associated with HPV. Viral presence decreases tumor aggressiveness. Patients with advanced stage lesions demonstrated lower survival rate.
Oral squamous cell carcinoma (OSCC) is a subgroup of head and neck cancer squamous cell carcinoma (HNSCC), and the sixth most common malignancy in the world. Despite achievements in the prevention, diagnosis, and treatment management in the last three decades, it still has a weak 50% overall 5-year survival rate (1-3). Despite the well-described synergistic interaction between heavy tobacco and alcohol consumption as significant risk factors, in the last decades, epidemiological studies have shown an essential role for high-risk human papillomavirus (HPV) infections in a subset of HNSCCs, predominantly in oropharynx tumorigenesis (4-7).
HPV-positive head and neck tumors, mainly in the oropharynx, have inherently different epidemiological, clinical, and molecular characteristics compared to those caused by classic risk factors and their HPV-negative counterparts. Noteworthy, the global impact of HPV-associated OSCC cases and the distinct molecular mechanisms that play a significant role in HPV-positive and HPV-negative tumor progression are still unclear (8-10). Oral carcinogenesis is a complex process characterized by aberrant gene and protein expression. In this context, the investigation of p16 expression changes and the PTEN gene status has gained prominence (11, 12).
p16 overexpression has been recognized as a reliable surrogate marker for oncogenic HPV infection (13, 14). This protein binds to CDK4 or 6 to impair complex formation with cyclins, which in turn is responsible for the phosphorylation and consequent inactivation of pRB, resulting in cell cycle arrest at G1/S checkpoint, a way through which p16 protein can inhibit cancer cell proliferation (8, 15).
PTEN is a well-established tumor suppressor gene and a negative regulator of the PI3K/Akt/mTOR pathway; loss of PTEN results in Akt hyperactivation (16, 17). Importantly, loss of PTEN expression was found in 29% of tongue cancers, and loss of heterozygosity (LOH) was identified in 40% of HNSCCs, which has been related to worse outcomes after adjuvant radiotherapy (18-20).
Based on the above, this study aimed to provide an initial insight into p16INK2a expression as a potential prognostic marker, as well as the relevance of additional LOH of the PTEN gene and its protein expression levels in OSCC in a Northern Brazilian cohort.
Materials and Methods
Study design. This retrospective study included a cohort of 119 patients diagnosed with primary OSCC at the Oral Pathology Department of João de Barros Barreto University Hospital (Belém, Brazil) (Table I). The study was conducted following the principles of the Declaration of Helsinki, and the Ethics Committee approved the protocol of the University Hospital João de Barros Barreto (n° 1.426.757). Two oral pathologists and one head and neck surgeon analyzed the available samples to define the TNM classification according to the American Society of Clinical Oncology classification (21).
Immunohistochemistry (IHC). The reactions were carried out in 3 μm thick histological sections, dewaxed in two xylol baths, the first in an oven at 60°C and the second at room temperature. Then, the sections were hydrated in a descending series of ethanol (100%, 95% and 85%). To remove the formolic pigment, the sections were dipped in 10% ammonium hydroxide and 95% ethanol and washed with distilled water. To uncover antibody epitopes, the sections were immersed in 10 mM EDTA buffer solution (pH 8.0), incubated in a microwave for 15 min, and then washed in distilled water. To block endogenous peroxidase, the sections were dipped in a solution of 6% hydrogen peroxide and methanol (1: 1, v/v) twice for 15 min at room temperature and then, washed with Tris solution (pH 7, 4). Then, 3 μm thick histological sections were incubated with antibodies against PTEN (polyclonal anti-human PTEN, Invitrogen, Waltham, MA, USA) and p16 (monoclonal anti-human p16, Santa Cruz Biotechnology, Dallas, TX, USA). After rinsing with PBS, slides were incubated with secondary antibody followed by streptavidin-biotin-peroxidase complex, both for 30 min at room temperature with a PBS wash between each step (LSAB-Kit + HRP; Agilent Technologies, Santa Clara, CA, USA). After diaminobenzidine (DAB+; Agilent Technologies) visualization of peroxidase activity, sections were subsequently counterstained with Mayer's hematoxylin solution (Merck, Darmstadt, Germany). Two pathologists classified the results according to Tsurutani and colleagues (22).
Clinicopathological characteristics of the analyzed samples.
Fluorescence in situ hybridization (FISH). The FISH assay was carried out using 5 μl of Vysis LSI PTEN SpectrumOrange Probe (Abbott Molecular Inc. Chicago, IL, USA) in a humidified chamber at 37°C overnight and the slides were counterstained with DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride). For FISH evaluation, 200 randomly chosen cells stained with the probe in each allele of the PTEN gene per sample were counted. Signals of homozygous PTEN deletion were considered when a simultaneous lack of both PTEN locus signals in 25%-30% of the analyzed cells' scored nuclei was observed (23-25).
Western blotting analysis. Fifty protein samples from human mouth lining epithelium were extracted using RIPA lysis and extraction buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Thermo Fisher Scientific, Waltham, MA, USA), solubilized in 4X Laemmli sample buffer and denatured for 5 min at 95°C. The proteins were separated by electrophoresis on 12% or 15% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare Chicago, Illinois, USA). PVDF membranes (Millipore, Billerica, MA, USA) were then blocked at 4°C overnight with 5% non-fat dry milk in PBS containing 0.1% Tween 20. Then, the membranes were incubated with an antibody against p16 (dilution 1:1000; Thermo Fisher Scientific). The antigen-antibody complexes were visualized using an HRP-conjugated secondary antibody and an enhanced chemiluminescence system (Pierce™ ECL Western Blotting Substrate, Thermo Fisher Scientific). Acquired images were quantified by ImageQuant 350 digital image system (GE) and β-Actin (1:200, Abcam, Cambridge, UK) was used as an internal control of the experiments.
Immunohistochemical analysis of PTEN and p16 protein expression in OSCC. (A) Immunoreaction for PTEN protein was predominantly in the cytoplasm, less frequent in the nucleus (DAB, ×40). (B) Immunolabeling for p16 demonstrating a nucleus pattern (DAB, ×20).
Immunofluorescence for PTEN evaluating the loss of alleles. (A) Two signals, homozygosity. (B) Loss of homozygosity, one signal.
DNA isolation and molecular analysis of PTEN. Ten 5-μm thick histological sections were obtained for DNA extraction by the QIAamp DNA FFPE Tissue Kit (Qiagen, Venlo, the Netherlands), following the protocol of the manufacturer. PTEN gene exons 1-9 (26-28) were amplified by polymerase chain reaction (PCR), and samples were then purified with Sephadex® G-50 (Merck KGaA, Darmstadt, Germany). Subsequently, the sequencing reactions were performed using the BigDye Terminator v3.1 (Applied Biosystems 3500, Darmstadt, Germany). Sequencing of non-tumor DNA (from normal mucosa) was used to ascertain that PTEN mutations found in the tumors were somatic. Independent repeated analyses confirmed the mutations.
HPV analysis by direct sequencing. The efficacy of DNA extraction was evaluated by PCR amplification of the β-globin gene (29). For the HPV PCR detection, viral gene-specific reactions were performed using GP5+ and GP6+ primers, which amplify a 150 bp fragment of the L1 viral gene, corresponding to a conserved region of the virus genome (30). The viral identification was performed by direct sequencing of the PCR product using the 3500 Genetic Analyzer (Thermo Fisher Scientific).
Statistical analyses. Data were analyzed statistically using the GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) and PASW Statistics program 18.0 (Quarry Bay, Hong Kong). Associations between HPV and other clinicopathological features were analyzed using chi-square (χ2) and logistic regression. Overall survival rates were estimated by the Kaplan–Meier method and compared using a Log-rank test. The level of significance was based on a p-value ≤0.05, and the confidence interval was 95%.
Somatic origin of mutations.
Results
PTEN immunohistochemistry. PTEN was positive in 79 cases (66.4%), and negative in 40 cases (33.6%). The immunoreaction of PTEN protein was predominantly in the cytoplasm (72.3%), less frequent in the nucleus (5%) and concomitantly in the cytoplasm/nucleus (22.7%). Stage IV tumors showed a higher cytoplasmic/nuclear (51, 9%) immunolabeling (p=0.002) (Figure 1).
High positivity of PTEN was observed in T4 tumors (46.8%) when compared to T1 (22.8%), T2 (11.4%) and T3 (19%) tumors (p<0.01). Patients who presented lymph node metastasis showed a higher PTEN positive index when compared to those who did not present lymph node metastasis (55% vs. 44.6%, p=0.02). In contrast, there was no statistically significant correlation between PTEN positivity and age at diagnosis (p=0.98), gender (p=0.16), primary tumor site (p=0.21), disease stage (p=0.61), mutation (p=0.17), histological grade (p=0.57) and p16 expression (p=0.74).
Positive PTEN protein expression was significantly increased in smoking patients when compared to the non-smoking group. Smoking patients had 2.5 times higher expression of PTEN than non-smoking patients (95%CI=1.156-5.549; p=0.03). Our results demonstrated a significant result when the disease stage was associated with mutation, showing that stage IV (65.2%) presented a higher number of mutated cases when compared with stage II (21.7%) and stage III (13%) (p=0.043).
PTEN FISH and molecular analysis. FISH detected loss of alleles in 23 tumors (19.3%), and sequencing of exons 1 to 9 of PTEN gene revealed point mutations in 16 samples. We considered that the PTEN allele was deleted when 25% or more of the cells in the FISH presented only one fluorescent signal. We were able to observe one fluorescent signal in 49-98% of the interphasic nuclei analyzed, which indicated a deletion of the PTEN gene in our samples (Figure 2).
Even with this high index (19.3% of the samples with more than 25% of the cells had deleted PTEN gene), we could not observe a significant association (p>0.05) between the deletion of the PTEN gene and any of the clinical, pathological, or genetic variables analyzed in the present study. In the other 96 OSCC samples (81, 7%), no alteration of the PTEN gene was observed, and the alleles losses occurred in less than 25% of nuclei. No correlation was found between the presence of PTEN mutations and the overall survival of the patients studied (p>0.05). However, when we examined the number of mutations in different tumor stages, we could observe a statistically significant difference between stage IV (60% of them) and stages II (27% of them) and III (20% of them) (p=0.043). There is a seven times greater risk for advanced stages in the presence of mutation of PTEN. We did not detect mutations in stage I. The results of the molecular analyses and FISH of the PTEN gene are summarized in Table II.
Representative western blot of p16 (lower gel) and beta-actin (upper gel) that was used as an endogenous marker for normalization. Lanes are labeled on the top of the figure, and the analyzed patients' case numbers are shown at the bottom. Lane 1 L=ladder band pattern. Lane 2 NC=normal control (pool of p16 proteins extracted from 55 normal human mouth lining epithelium). Lanes 3, 5, and 7 correspond to cases negative for the HPV virus that do not overexpress p16 (p16 expression similar to the normal human mouth lining epithelium). Lanes 4, 6, 8, 9 and 10 correspond to HPV-positive cases [subtype 6 (lanes 4 and 6) 16 (line 8), 11 (line 9) and 18 (line 10)] with p16 overexpression. Lanes 3 and 5 correspond to samples with wild type p16 that are negative for HPV and do not overexpress p16. Lanes 4, 8, and 9 correspond to samples with wild type p16 that are positive for HPV and overexpress p16. Lanes 6 and 10 correspond to samples with mutated p16 that are positive for HPV and overexpress p16, and lane 7 corresponds to a sample with mutated p16 that is negative for HPV virus and does not overexpress p16. The relative quantification of OSCC tumor p16 protein in relation to the pool of p16 proteins extracted from 55 normal human mouth lining epithelium was: 0.98 (lane 3), 1.58 (lane 4), 0.96 (lane 5), 1.58 (lane 6), 1.17 (lane 7), 2.01 (lane 8), 1.59 (lane 9), 2.23 (lane 10).
Distribution of HPV status. In the analyzed samples, it was observed that 64 samples (53.8%) were positive for HPV, whereas 55 (46.2%) were negative for HPV. Among those positive, 15 samples demonstrated a positive reaction product for low-risk HPV-6 (23.5%), 9 for subtype 11 (14%), 25 subtype 16 (39%) and 15 subtype 18 (23.5%), totaling 40 cases with high risk for HPV(HR-HPV). HPV-positive tumors demonstrated a statistically significant result for tabagism (p<0.0001), evidencing an association between tobacco consumption and HPV positive samples. Furthermore, the results revealed a significant correlation between HPV-positive status, tumor size 3 and 4 (p<0.0001) and disease stage 3/4 (p<0.0001) when compared to HPV negative samples. Additionally, we observed a strong correlation between HPV positive status and lymph node metastasis (N1) (p<0.0001), as well as with poorly differentiated tumor samples (p<0.0001).
Distribution of p16 positive cases in all tumors. p16 immunoreaction demonstrated primarily a cytoplasmic pattern (82.3%), less frequently a nuclear pattern (7.8%) and concomitantly in the cytoplasm/nucleus (9.9%) (Figure 1B).
p16 expression was strongly associated with the presence of HPV (p<0.0001). However, there was no statistically significant correlation between the expression of p16 and some clinicopathological features, like age (p=0.10), gender (p=0.58), TNM, and mutational status of PTEN (p=0.52). It is worth emphasizing that fifty-eight percent of patients with p16-positive tumors were non-smokers, and 85.7% of those with p16-negative tumors had more than ten pack-years of exposure.
Survival analysis and its association with clinicopathological factors. (A) Smoking patients demonstrated 14 months less survival than non-smoking patients (p<0.001). (B) Individuals who presented tumor sizes 3 and 4 demonstrated a 1.94 times higher risk of death when compared with tumor sizes 1 and 2 (p<0.001). (C) Patients diagnosed with 2 or 3 metastatic lymph nodes were 2.23 times more likely to die compared to patients without lymph node metastasis or with only one lymph node metastasis (p=0.001). (D) Individuals who demonstrated disease stage 3 or 4 showed 2.03 times higher risk of death than stage 1 and 2. (E) The odds of death among those who presented poorly differentiated histological grade were 1.76 times (p<0.001) higher than those classified as well-differentiated tumors. (F) Individuals who had no HPV had a 2.22 times greater chance of death than that seen for HPV-positive patients (p=0.0001). the odds of death among patients who were HPV -negative was 2.22 times (p=0.0001) greater than that observed for HPV-positive patients. (G) Patients who were p16 negative showed 2.14 times longer survival time than those who were p16 positive (p<0.001).
Figure 3 is a representative western blot of p16 that lists OSCC HPV negative cases that do not overexpress p16 (p16 expression similar to the normal human mouth lining epithelium) and OSCC HPV-positive cases with p16 overexpression.
Survival outcomes. The follow-up time was 5 years (60 months). Overall, 102 patients (85.7%) deceased. Overall cumulative survival rates were 95% at one year, 33% at three years, and 14% at five years. Univariate analysis found prognosis to be significantly related to seven clinicopathological factors. These variables were: smoking, tumor size, lymph node metastasis, disease stage, histological grade, HPV status, and subtypes. Multivariate analysis of these six significant variables determined by univariate analysis identified that all six prognostic factors independently impacted on survival and one (p16 status) that did not independently impact on survival. The log-rank test showed that the odds ratios of death among smoking patients were 1.5 times higher, equivalent to 14 months less of survival (p<0.001), than non-smoking patients (Figure 4A). Patients who presented tumor sizes 3 and 4 demonstrated a 1.94 times higher risk of death (p<0.001) when compared with tumor sizes 1 and 2 (Figure 4B). The odds of death for patients who were diagnosed with lymph node metastasis 2 and 3 was 2.23 times. The risk of death was 2.23 times greater for patients with lymph node metastasis (N2 and N3) (p<0.001) than patients with no lymph node metastasis (0 and 1) (Figure 4C). Regarding the disease stage, our results demonstrated that the odds of death among those who presented disease stages 3 and 4 was 2.03 times (p<0.0001) greater than those staged as 1 and 2 (Figure 4D).
The odds of death among those who presented poorly differentiated histological grade were 1.76 times (p<0.001) higher than those classified as well-differentiated tumors (Figure 4E). Furthermore, the odds of death among patients who were HPV-negative was 2.22 times (p=0.0001) greater than those observed for HPV-positive patients (Figure 4F). We examined survival outcomes based on p16 status and observed that those patients who presented p16 positive immunostaining had 2.14 times longer survival time than those who were negative (p<0.001) (Figure 4G). There were also survival outcome differences between the p16-positive and p16-negative patients that had a smoking history. The patients who were smokers and had no p16 protein expression had two times fewer survival months compared with non-smokers that were p16 positive (p<0.001).
Discussion
OSCC is the most common malignant neoplasm of the oral cavity (31, 32). This pathological entity commonly affects male individuals, such as those in the present study, belonging to an age group above 40 years and is frequently diagnosed in the sixth decade of life, which is in accordance with our results (33-37). Furthermore, it has been shown that the tongue and the floor of the mouth are the lesion sites in the majority of cases (23, 31, 34, 38).
The tumor suppressor gene PTEN is also involved in oral tumorigenesis, as it has been demonstrated that PTEN inactivation or deletion mimics activation of the mTOR signaling pathway in the pathogenesis of OSCC. Moreover, PTEN LOH and reduced levels of the protein play an essential role in aggressive tumors and contribute to poor disease-free and overall survival (39, 40). In support, from 119 patients with OSCC analyzed in the present study, 40 samples were negative for PTEN immunoexpression.
Squarize et al. (38) have analyzed PTEN expression in OSCC by immunohistochemistry. They showed that protein expression was related to the degree of malignancy and that aggressive tumors with high malignancy score did not express PTEN, suggesting that it can be used as a prognostic marker. Conversely, our study demonstrated a statistically significant association between increased PTEN expression and T4 tumors, lymph node metastasis, and smoking. This unexpected result may be explained by the fact that increased PTEN levels occur to compensate for the increased levels of Akt, since they directly influence the molecular pathways of progression of malignant neoplasms (17, 23). Additionally, Troeltzsch and colleagues (41) have shown that poorly differentiated tumors are associated with the occurrence of lymph node metastasis, which corroborates with our results since an increased number of poorly differentiated tumors was observed.
Mutations in the PTEN gene were also evaluated in the lesions, and we observed 16 point mutations and allelic deletions in 19.3% of the analyzed tumors. The results showed a statistically significant association between stage IV tumors and the presence of a mutation.
Previous studies that evaluated gene expression and copy number alterations in OSCC demonstrated loss of PTEN in 59.4% of cases (42). Indeed, somatic mutations or deletions of PTEN have been previously described in a wide variety of premalignant and malignant lesions, including breast, prostate, endometrial, and oral squamous cell carcinomas, head and neck cancer, glioblastoma and melanoma (43). In head and neck squamous cell carcinoma, loss of the PTEN gene was found in 8.16% of cases, and somatic mutations in 5.45% (44). Interestingly, protein expression was not correlated with the presence of mutations, indicating that another mechanism can alter PTEN function.
Another gene with suppressor function and a significant role in carcinogenesis is p16, a cyclin-dependent kinase inhibitor that controls tumor progression by inhibiting transition through the G1 phase of the cell cycle (45). In this study, we examined the correlation between HR-HPV infection and P16 and PTEN LOH and observed that high expression of p16 and PTEN in OSCC was sometimes associated with clinicopathological data.
Recent studies have found that abnormal expression of the tumor suppressor gene P16 was closely associated with HR-HPV infection during carcinogenesis in a subset of HNSCC. Multiple studies have found that p16 is an important prognostic biomarker in oropharyngeal squamous cell carcinoma (OPSCC) (46). The prognostic role of p16 in non-oropharyngeal HNSCC, including OSCC, however, is unclear. Several studies have concluded that p16 has no prognostic significance in non-OPSCC (14, 47, 48), whereas others have concluded the opposite (49).
HPV DNA and expression of p16 has been suggested as a gold standard marker for the detection of HPV infection in many cases of cancers (49, 50). The frequency of HPV infection is significantly higher in patients with OSCC, as identified through the detection of HPV DNA and p16 expression (51). Our results revealed that 64 of the 119 cases were HPV positive, with a high expression of p16. Among these positive samples, 40 showed the presence of the virus subtypes 16 or 18. Interestingly, a study that evaluated the expression of p16 in correlation with an HPV infection in HNSCC observed that the protein expression was higher in the oropharynx and p16 positive patients had better survival, demonstrating that it is a useful prognostic marker in combination with the status of the p16 HPV (52).
A recent study analyzed 174 samples of OSCC for p16 levels and the presence of HPV DNA and its genotypes. Of the total, 24 samples were found to overexpress p16 and in 13, HPV DNA was detected. In agreement, the current study demonstrated that HR-HPV (16 or 18) genotypes were the most prevalent, enhancing the hypothesis about p16 immunoreactivity and HPV genotyping as a useful marker of virus infection in OSCC (53).
Several studies have examined the relationship between smoking status and p16 expression mechanisms and reported that smoking was significantly associated with promoting p16 methylation (54). Corroboratively, a recent study from China has reported that smoking is positively correlated with p16 hypermethylation in NSCLC (55). However, their meta-analysis was limited to promoter methylation, which is not the most common mechanism of inactivation for p16. Another meta-analysis confirmed that all inactivation mechanisms are genuinely associated with loss of protein expression. There is also a study that identified specific associations between p16 inactivation mechanisms and other genetic changes and smoking status (54).
Regarding patient's survival, we observed that smoking patients presented 14 months shorter survival compared to non-smoking patients. Kawakita et al. have explained that smoking induces p53 mutations and may trigger many harmful cascades in the human body, leading to a poor prognosis (56). Increased tumor size (3 and 4) demonstrated a 1.94 times higher risk of death when compared with smaller tumor sizes, consistent with other previous studies (41, 57). Additionally, we showed that the odds of death for patients who were diagnosed with lymph node metastasis N2 or N3 were higher (2.23 times higher) than those with N0 or N1. Furthermore, we demonstrated that the odds of death among those who presented with disease stages 3 and 4 were two times greater than those staged as 1 and 2. Therefore, cancer staging based on the TNM stage was a significant prognostic variable (58).
Positive HPV samples were also significantly correlated with increased tumor size, which can be explained by the inactivation of the Rb protein through the E7 HPV protein. In this scenario, p16 overexpression may be an attempt to recover Rb normal function, thus allowing normal cellular progression from the G1 phase to the S phase of the cell cycle. Furthermore, similar to our results, it has been shown that advanced tumors also showed a significant association with p16 overexpression (59).
In addition, the possibility of death of patients with poorly differentiated histological grades was 1.76 times greater than those with well-differentiated tumors, which is compatible with previous studies (60). The tumor's aggressiveness may explain, for instance, the association of the histological grade with lymph node metastasis, recurrence index, and distant metastasis (56, 58).
Besides that, p16 has been shown to sensitize HPV-positive cells to ionizing radiation by inhibiting homologous recombination-mediated DNA damage response and down-regulating cyclin D1 expression. The inhibition of the DNA damage response by p16 is thought to be independent of its cell-cycle regulation–inhibition CDK4/6 activity (61).
Conclusion
In conclusion, PTEN and p16 are suppressor genes that control tumor progression. In the current study, PTEN demonstrated a higher immunoreactivity in advanced disease stages (seven more times). p16 was strongly associated with HPV and did not demonstrate a significant association with any clinicopathological factor analyzed. Both proteins present great importance for the prognosis of patients. It was demonstrated that the presence of the virus decreases tumor aggressiveness. Also, advanced stages lesions demonstrated a lower survival rate. We observed that smoking and p16 absence were significantly associated with two times decreased survival rate.
Further understanding of the role of PTEN in the initiation and progression of cancer increases may be used for the design of novel therapeutic approaches, such as inhibition of Akt, which the protein involved in the most common signaling pathway associated with OSCC progression.
Acknowledgements
This work was supported by the Coordination of Improvement of Higher Education –Brazil (CAPES, Finance Code 001 to V.J.O.K); National Counsel of Technological and Scientific Development - Brazil (CNPq, grants #301350/2019-1 to R.M.R.B.) and Dean of Research and Graduate Studies of Federal University of Pará – Brazil (PROPESP for payment of the publication fees).
Footnotes
Authors' Contributions
Study concepts: RMRB, VJOK, MCA, HARP; Study design: RMRB, VJOK, MCA, HARP; Data acquisition: VJOK, MCA, LLS, ASK, AMBK; Quality control of data and algorithms: AGJ, ASK, CCS, LLS, MCA, RMRB; Data analysis and interpretation: VJOK, AMBK, AGJ, ASK, CCS, LLS, MCA, RMRB; Statistical analysis: ASK, MCA, LLS, RMRB; Manuscript preparation: VJOK, MCA, LLS, RMRB, FSCP, HARP; Manuscript editing: VJOK, MCA, AMBK, LLS, ASK, CCS, FSCP, AGJ, HARP, RMRB; Manuscript review: VJOK, MCA, AMBK, LLS, ASK, CCS, FSCP, AGJ, HARP, RMRB.
Conflicts of Interest
The Authors declare that they have no competing interests regarding this study.
- Received September 9, 2020.
- Revision received October 5, 2020.
- Accepted October 8, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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