Open Access Austin Journal of Clinical Pathology

Original Article MMP13 and SERPINB2 as Novel Biomarkers for Hypopharyngeal Cancer

Lv F1, Huang W2 and Ji X3* 1Department of Oncology, Shengjing Hospital of China Abstract Medical University, China Matrix Metalloproteinase (MMP)13 and serine peptidase inhibitor, clade B, 2Department of Pathology, Shengjing Hospital of China member 2 (SERPINB2) are important components of the extracellular matrix Medical University, People’s Republic of China and play a regulatory role in tumor stromal remodeling. The purpose of the study 3Department of Otorhinolaryngology Head and Neck is to evaluate the expression of MMP13 and SERPINB2 in hypopharyngeal Surgery, the First Hospital of China Medical University, cancer. We utilized the public datasets to find the differentially expressed People’s Republic of China of hypopharyngeal cancer, which were verified by immunohistochemistry. At the *Corresponding author: Xu Ji, Department of same time, the role of differential expressed genes in prognosis was explored. Otorhinolaryngology Head and Neck Surgery, the First We found that MMP13 is upregulated and SERPINB2 is downregulated in Hospital of China Medical University, Shenyang 110001, public datasets. We confirmed this conclusion by immunohistochemistry Liaoning, People’s Republic of China of hypopharyngeal cancer tissues and found that MMP13 and SERPINB2 were related to some clinicopathological factors. Moreover, SERPINB2 is an Received: September 21, 2020; Accepted: October 13, independent prognostic factor for hypopharyngeal cancer patients. MMP13 and 2020; Published: October 20, 2020 SERPINB2 may predict the early recurrence of hypopharyngeal cancer. MMP13 combined with SERPINB2 may be potential prognostic biomarkers and drug targets in hypopharyngeal cancer.

Keywords: Hypopharyngeal neoplasms; Matrix metalloproteinase 13; Plasminogen activator inhibitor 2; Immunohistochemistry

Abbreviations wide range of proteolytic functions, and MMP13 participates in various physiological and pathological processes [3]. SERPINB2, AUC: Area Under ROC Curve; CI: Confidence Interval; DAB: also known as PAI-2, is a member of the superfamily of 3, 3’-Diaminobenzidine tetrahydrochloride; DEGs: Differentially serine protease inhibitors. SERPINB2 mainly inhibits uPA and tissue Expressed Genes; ECM: Extracellular Matrix; FC: Fold-Change; plasminogen activator [4]. Previous studies have shown that MMP13 GEO: Expression Omnibus; H&E: Hematoxylin and Eosin; [5,6] or SERPINB2 [7,8] are associated with the occurrence or H3K4me3: Histone H3 trimethylation at lysine 4; HR: Hazard Ratio; prognosis of different tumors. However, until now, there has been no ING: Inhibitor of Growth; MMPs: Matrix Metalloproteinases; PAI: report on the expression and regulation of MMP13 and SERPINB2 in Plasminogen Activator Inhibitor; TCGA: The Cancer Genome Atlas; hypopharyngeal cancer. As a rare tumor, hypopharyngeal cancer also uPA: urokinase type Plasminogen Activator; WHO: World Health lacks markers for diagnosis and prognosis. Organization This is the first study to investigate the expression and correlation Introduction of MMP13 and SERPINB2 in hypopharyngeal carcinoma. Through As an uncommon malignant tumor, hypopharyngeal cancer public datasets and immunohistochemistry, we showed the accounts for 3–5% of head and neck tumors [1]. Most pathological expression changes and correlations of MMP13 and SERPINB2 in types of hypopharyngeal cancer are squamous cell carcinoma. Due hypopharyngeal cancer. MMP13 and SERPINB2 are related to the to the occult anatomical location of hypopharyngeal cancer and poor survival of hypopharyngeal carcinoma patients and may predict surgical effect, local recurrence or distant metastasis often occurs in early recurrence. These findings suggest that MMP13 and SERPINB2 patients with hypopharyngeal cancer following surgery. Pharyngeal play an important role in hypopharyngeal cancer and may be a fistula also affects the quality of life of hypopharyngeal cancer potential prognostic marker and therapeutic targets for patients with patients. Therefore, it is necessary to search forhypopharyngeal hypopharyngeal cancer. cancer markers. Methods Tumor stroma is mainly composed of collagen fibers, vascular Public datasets searching vessels, and Extracellular Matrix (ECM) components. ECM From the National Center of Biotechnology Information components are over-deposited in tumor tissues and function in Omnibus (GEO) database (https://www.ncbi. nourishing tumor cells. The system composed of urokinase type nlm.nih.gov/gds/), we searched raw gene expression data of the Plasminogen Activator (uPA), plasmin, Plasminogen Activator hypopharyngeal cancer as the following key terms: (hypopharyngeal Inhibitor (PAI), and Matrix Metalloproteinases (MMPs) is one of or hypopharynx) and (cancer or tumor or carcinoma or neoplasm). the main ways to regulate ECM degradation. MMP13, also known as The publication time was not limited. Homo sapiens was selected in Collagenase 3, can degrade a variety of ECM components, including the Organism and Expression profiling by array in the Type. tenascin C, fibronectin, and type I–IV collagen [2]. MMP13 hasa

Austin J Clin Pathol - Volume 7 Issue 2 - 2020 Citation: Lv F, Huang W and Ji X. MMP13 and SERPINB2 as Novel Biomarkers for Hypopharyngeal Cancer. ISSN : 2381-9170 | www.austinpublishinggroup.com Austin J Clin Pathol. 2020; 7(2): 1065. Ji et al. © All rights are reserved Ji X Austin Publishing Group

GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) is an online obtained for uncolored, yellow and brown respectively. The final tool provided by GEO, which is based on the R language limma score was obtained by multiplying the scores of the two items and package [9]. GEO2R was used to screen DEGs (differentially expressed taking the average score of the five visual fields. The total score of 0-4 genes) between hypopharyngeal cancer and non-cancerous samples was defined as Low-No expression, and more than 4 was defined as in the GEO datasets and we further visualized DEGs by volcano plot Medium-High expression. [10]. A P-value <0.05 and absolute log Fold-Change (FC) greater than 2 for the DEGs were used as the cut-off criteria. We also used Statistical analysis the online tool Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/ Each experiment was repeated at least three times and data was index.html) to identify the overlapping DEGs that were up-/down- analyzed by SPSS 21.0 (IBM Corp.). The expression of MMP13 and regulated in the GEO datasets. SERPINB2 in cancer and non-cancerous groups was compared by The Cancer Genome Atlas (TCGA) is a public funded project Student’s t-test (public data: unpaired t-test; specimen data: paired 2 that aims to catalogue and discover major cancer-causing genomic t-test). Pearson’s X correlation was applied to test the correlations alterations to create a comprehensive “atlas” of cancer genomic between expression of MMP13 and SERPINB2. Univariate analysis profiles. We also obtained gene expression data in hypopharyngeal and (Kaplain-Meier test) was used to determine the clinicopathological non-cancerous tissues from TCGA database (https://cancergenome. characteristics related to survival, and multivariate analysis (Cox nih.gov/). Both GEO and TCGA datasets’ non-cancerous tissues came regression) was used to further analyze the covariates (P <0.05) in from keratinized epithelium of hypopharynx in healthy volunteers. univariate analysis. Finally, the diagnostic value was evaluated by area under ROC curve (R package ggplot2). Differences were considered Patients and tissue samples significant when p -values <0.05. The research was in consent by the First Affiliated Hospital of China Medical University Ethics Committee. We involved patients Results who signed informed consent and collected samples according Identification of Differentially Expressed Genes (DEGs) to the informed consent. We collected 40 hypopharyngeal cancer patients who underwent partial hypopharyngeal resection or total The gene expression profiles of GEO datasets, including GSE686 laryngectomy and collected their cancer and paracancerous tissues. (cancer samples: 9, non-cancerous samples: 3), GSE2379 (cancer Two independent pathologists examined all tissues. No distant samples: 14, non-cancerous samples: 4), and GSE10774 (cancer metastasis or preoperative chemoradiotherapy was observed. Besides, samples: 10, non-cancerous samples: 4) were downloaded from the we collected patients’ clinical information and followed up. Smokers public GEO database by searching the terms mentioned. DEGs in ware defined as those who smoke continuously or accumulatively the GSE686 dataset were screened and we obtained 231 DEGs (36 for six months or more in their lifetime [11], according to the World upregulated and 172 downregulated), as shown in (Figure 1A). Health Organization (WHO) guideline. In the GSE2379 and GSE10774 datasets, we identified 403 (204 Immunohistochemistry upregulated and 199 downregulated) and 99 (23 upregulated and 76 downregulated) DEGs, respectively (Figure 1B- C). Among the Formalin-fixed tissues were embedded in paraffin and cut into 5 DEGs, we found two overlapped DEGs, one upregulated (MMP13) μm–thick sections for Hematoxylin and Eosin (H&E) staining and immunostaining. The expression of MMP13 and SERPINB2 was and one downregulated (SERPINB2) (Figure 2A-B). assessed by immunohistochemical staining kit (Maixin, China). De- The Cancer Genome Atlas (TCGA) database contains normalized waxed sections were washed in PBS and exposed to 3% H2O2 for 15 RNA sequencing data of hypopharyngeal cancer patients using min at room temperature to quench endogenous peroxidase activity. the RNASeqV2 system. Thus, we verified our results in the TCGA Then the tissues were blocked with normal goat serum for 20 min at dataset. By analyzing MMP13 and SERPINB2 expression levels in ° room temperature. After incubation overnight at 4 C with primary unpaired hypopharyngeal (n = 5) and non-cancerous tissues (n = antibody (1:200) (MMP13: proteintech, 18165-1-AP; SERPINB2: 19) of the TCGA–HNSCC/hypopharyngeal cohort, we found that proteintech, 16035-1-AP), the tissues were incubated with the second MMP13 expression was significantly upregulated in hypopharyngeal antibody and biotin-labeled horseradish peroxidase. Subsequently, tissues (Figure 2C), while SERPINB2 was unchanged between the antibody binding was visualized with a 3,3’-Diaminobenzidine hypopharyngeal and non-cancerous tissues (Figure 2D). tetrahydrochloride (DAB) kit (Maixin, China) before brief counterstaining with hematoxylin. Eventually, tissues were gradually dehydrated, sealed with neutral gum, observed and photographed with an inverted phase contrast microscope. Immunoreactivity was semi-quantitatively evaluated and evaluated by two pathologists. Five representative regions were randomly selected from the 400-fold field of view of the microscope. The score of each field of view was determined by the proportion of positive cells and the color rendering intensity. According to the Figure 1: Volcano plot of gene expression profile data in hypopharyngeal proportion of positive cells: <5%, 5-25%, 25% - 50%, 50% - 75%, cancer and non-cancerous samples. Volcano plot of GSE686 (a), GSE2379 75% - 100%, the scores of 0, 1, 2, 3 and 4 were obtained respectively. (b), and GSE10774 (c). Red spots represent up-regulated genes, and blue According to the intensity of colouring, 0, 1 and 2 points were spots represent down-regulated genes.

Submit your Manuscript | www.austinpublishinggroup.com Austin J Clin Pathol 7(2): id1065 (2020) - Page - 02 Ji X Austin Publishing Group

ranged from 46 to 77 and most were male patients. Approximately one-third of patients had stage IV tumors, most of which were located in the piriform fossa. This is consistent with the epidemiological data of hypopharyngeal cancer reported in 2011 [12]. MMP13 expression was increased in 57.5% (23/40) of the patients and SERPINB2 expression was decreased in 62.5% (25/40) of the patients. MMP13 was expressed in the cytoplasm of hypopharyngeal cancer cells and negative in squamous epithelial cells, while expression of SERPINB2 was the opposite (Figure 3A-B). Expression of MMP13 in hypopharyngeal cancer was correlated with N stage (P = 0.017) and smoking history (P = 0.005); expression of SERPINB2 was correlated with N stage (P = 0.005), but not with other clinicopathological features (Tables 1 and 2). We also found a slight negative correlation between MMP13 and SERPINB2 expression, but the difference was not statistically significant (Pearson correlation coefficient = -0.274, P = 0.083, (Figure 4)). Prognostic significance of MMP13 and SERPINB2 Figure 2: Venn diagram of overlapping Differentially Expressed Genes (DEGs) and expression of DEGs in the TCGA dataset. (a,b) Venn diagram Hypopharyngeal cancer patients were followed up for 2–18 of upregulated overlapping DEGs (a) and downregulated DEGs (b). (c,d) Dot months, with a median follow-up of 12 months. At the end of follow- plot indicating expression level of MMP13 (c) and SERPINB2 (d) in TCGA– HNNC/hypopharyngeal cancer. P value was determined using Student’s up, 12 patients died as a result of disease progression and the common t-test. Error bars represent mean ± standard deviation. sites of progression included the lungs and cervical lymph nodes. Univariate analysis showed that MMP13 (hazard ratio [HR] = 3.691, 95% confidence interval [CI] = 1.157–11.78,P = 0.02, (Figure 5A) and SERPINB2 (HR = 0.2305, 95% CI = 0.072–0.739, P = 0.01, (Figure 5B)) expression along with N stage, and smoking and alcohol history were important factors for prognosis. Furthermore, multivariate analysis showed SERPINB2 expression was an independent factor for prognosis (HR = 0.120, 95% CI = 0.015–0.929, P = 0.04, (Table 3)). Diagnostic significance of MMP13 and SERPINB2 for early recurrence Recurrence within one year after radical resection is called early recurrence of hypopharyngeal cancer. To further detect whether MMP13 and SERPINB2 expression has a role in the prediagnosis of early recurrence of hypopharyngeal cancer, we calculated the area under ROC curve (AUC) in 27 patients who can be diagnosed with early recurrence or not. MMP13 and SERPINB2 levels were able to differentiate between early recurrence and non-early recurrence Figure 3: Immunohistochemistry of MMP13 and SERPINB2 Table 1: Correlation between expression levels of MMP13 and clinicopathological expression in HNSCC tissues. (a) Expression of MMP13 in hypopharyngeal parameters of hypopharyngeal cancer patients. cancer (upper) and adjacent normal squamous epithelium (lower). Upper Variable n MMP13 expression level P left: Hematoxylin and Eosin (H&E) staining of hypopharyngeal cancer. Upper middle and right: MMP13 of hypopharyngeal cancer (middle: 200×, Medium-High Low-No right: 400×). Lower left: H&E staining of squamous epithelium. Lower middle <60 19 13 6 Age 0.184 and right: MMP13 of squamous epithelium (middle: 200×, right: 400×). (b) >=60 21 10 11 Male 39 23 16 Expression of SERPINB2 in hypopharyngeal cancer (upper) and adjacent Gender 0.239 normal squamous epithelium (lower). Upper left: H&E of hypopharyngeal female 1 0 1 II III 25 17 8 cancer. Upper middle and right: SERPINB2 of hypopharyngeal cancer T stage 0.083 (middle: 200×, right: 400×). Lower left: H&E staining of squamous epithelium. IV 15 6 9 Negative 11 3 8 Lower middle and right: SERPINB2 of squamous epithelium (middle: 200×, N stage 0.017 positive 29 20 9 right: 400×). Yes 35 23 12 Smoke 0.005 no 5 0 5 Expression of MMP13 and SERPINB2 in hypopharyngeal Yes 33 21 12 Alcohol 0.088 cancer tissues no 7 2 5 >=4g/ml 21 15 6 Fibrinogen 0.061 To further test our conclusions predicted from public databases <4g/ml 19 8 11 Pyriform sinus 33 18 15 at the mRNA level, we performed immunohistochemistry to detect Position 0.412 others 7 5 2 the expression of MMP13 and SERPINB2 at the protein level. We Medium-High 15 6 9 SERPINB2 0.083 detected the expression of MMP13 and SERPINB2 in paraffin- Low-No 25 17 8 embedded tissues of 40 hypopharyngeal cancer patients. The ages all 40 23 (57.5%) 17 (42.5%)

Submit your Manuscript | www.austinpublishinggroup.com Austin J Clin Pathol 7(2): id1065 (2020) - Page - 03 Ji X Austin Publishing Group

Table 2: Correlation between expression levels of SERPINB2 and clinicopathological parameters of hypopharyngeal cancer patients. Variable n MMP13 expression level P Medium-High Low-No <60 19 6 13 Age 0.462 >=60 21 9 12 Male 39 14 25 Gender 0.191 female 1 1 0 II III 25 8 17 T stage 0.354 IV 15 7 8 Negative 11 8 3 N stage 0.005 positive 29 7 22 Yes 35 13 22 Smoke 0.902 no 5 2 3 Yes 33 11 22 Alcohol 0.237 no 7 4 3 >=4g/ml 21 10 11 Figure 4: Representative images of differences in the expression of MMP13 Fibrinogen 0.165 <4g/ml 19 5 14 and SERPINB2 in hypopharyngeal cancer tissues. Pyriform sinus 33 13 20 Position 0.591 others 7 2 5 Medium-High 23 6 17 SERPINB2 0.083 Low-No 17 9 8 all 40 15 (37.5%) 25 (62.5%)

Table 3: Univariate and multivariate analyses of factors associated with OS in hypopharyngeal cancer patients. Univariate analysis Multivariate analysis Variables HR (95% CI) P HR (95% CI) P Age 0.643 (0.205-1.974) 0.44 Gender 3.062 (0.199-47.10) 0.42 T stage 0.913 (0.271-3.071) 0.88 N stage 6.060 (1.112-11.83) 0.04 Figure 5: Relationship between MMP13 (a) and SERPINB2 (b) expression Smoke 4.181 (0.962-18.18) 0.05 and overall survival in hypopharyngeal cancer patients. Alcohol 3.982 (1.008-15.74) 0.04 Fibrinogen 0.830 (0.261-2.640) 0.75 Position 1.193 (0.278-5.125) 0.81 MMP13 3.691 (1.157-11.78) 0.02 SERPINB2 0.2305 (0.072-0.739) 0.01 0.120 (0.015-0.929) 0.04 with an AUC of 0.650 (95% CI = 0.500–0.800, (Figure 6A)) and 0.750 (95% CI = 0.500–1.000, (Figure 6B)). Diagnostic accuracy can be enhanced by combining biomarkers [13], so we explored whether the combined expression levels of MMP13 and SERPINB2 could improve the diagnosis of early recurrence. First, we defined increased MMP13 expression and decreased SERPINB2 expression as the high-risk group and the rest as the low-risk group. The results showed no significant change in AUC (0.744, 95% CI = 0.688–0.800, (Figure 6C)). Then, another classification method was adopted to test whether the diagnostic accuracy could be improved. We defined the high-risk group as increased MMP13 expression and decreased SERPINB2 expression, the low-risk group as decreased MMP13 expression and increased SERPINB2 expression, and the rest as a Figure 6: ROC analysis for MMP13 (a), SERPINB2 (b), and combined diagnostic markers (c, d) in early recurrence. medium risk group. The results showed that the AUC was 0.775 (95% CI = 0.688–0.800, (Figure 6D)). After the combined application of of hypopharyngeal cancer are few. According to bioinformatics markers, the diagnostic accuracy did not improve significantly and prediction and immunohistochemical results, this study reported the markers had only moderate predictive value for early recurrence. the expression and correlation of MMP13 and SERPINB2 in This may be due to the small sample size; in future studies, we will hypopharyngeal cancer for the first time. MMP13 and SERPINB2 can increase the sample size to look for additional markers suitable for be used to evaluate prognosis and early recurrence of hypopharyngeal predicting early recurrence. cancer. This is consistent with a previous study of MMP13 and Discussion SERPINB2 in osteosarcoma [14]. With the development of molecular biology and In the present study, MMP13 showed a weak negative immunohistochemistry, the prognostic indicators of head and correlation with SERPINB2, but no statistical significance. MMP13 neck tumors are increasing, but studies of molecular markers and SERPINB2 are important regulatory components of the ECM

Submit your Manuscript | www.austinpublishinggroup.com Austin J Clin Pathol 7(2): id1065 (2020) - Page - 04 Ji X Austin Publishing Group network and play an important role in tumor ECM remodeling. 5. Jiao XL, Chen D, Wang JG and Zhang KJ. Clinical significance of serum SERPINB2 is an inhibitor of urokinase and ultimately plasmin, the matrix metalloproteinase-13 levels in patients with Esophageal Squamous Cell Carcinoma (ESCC). Eur Rev Med Pharmacol Sci. 2014; 18: 509-515. latter is an activator of pro-MMP13. But there is a lack of research on the relationship between MMP13 and SERPINB2. As a member of 6. Vincent-Chong VK, Salahshourifar I, Karen-Ng LP, Siow MY, Kallarakkal TG and Ramanathan A, et al. Overexpression of MMP13 is associated with the serpin superfamily, SERPINB1 and SERPINB2 share 24% amino clinical outcomes and poor prognosis in oral squamous cell carcinoma. acid sequence identity [15]. Several previous studies have reported Scientific World Journal. 2014; 2014: 897523. a correlation between MMP13 and SERPINB1. In liver fibrosis, Hu 7. Becker M, Szarvas T, Wittschier M, Vom DF, Totsch M and Schmid KW, et et al found that by knocking down SERPINB1, MMP13 expression al. Prognostic impact of plasminogen activator inhibitor type 1 expression in was upregulated [16]. Ohkouchi et al found that Inhibitor of Growth bladder cancer. Cancer-AM Cancer SOC. 2010;116: 4502-4512. (ING)-2 combined with histone H3 trimethylation at lysine 4 8. Wei X, Li S, He J, Du H, Liu Y and Yu W, et al. Tumor-secreted PAI-1 (H3K4me3) participated in remodeling to activate promotes breast cancer metastasis via the induction of adipocyte-derived MMP13 and SERPINB1 was upregulated with overexpression of collagen remodeling. Cell Commun Signal. 2019; 17: 58. ING2 [17]. Transforming growth factor (TGF)-β is an important 9. Diboun I, Wernisch L, Orengo CA and Koltzenburg M. Microarray analysis cytokine in ECM remodeling [18]. Studies have shown that TGF-β after RNA amplification can detect pronounced differences in gene expression using limma. BMC Genomics. 2006; 7: 252. regulates both MMP13 and SERPINB2 [19,20]. Other studies [21] have shown that glucose stimulated mRNA expression of MMP13, 10. Kumar N, Hoque MA and Sugimoto M. Robust volcano plot: identification of differential metabolites in the presence of outliers. BMC Bioinformatics. while SERPINB1 suppressed expression of the mRNAs induced 2018; 19: 128. by glucose. Matrix components such as MMP13 and SERPINB2 11. Weishaar H, Collin J, Smith K, Gruning T, Mandal S and Gilmore A. Global have complex regulatory relationships, and their specific regulatory health governance and the commercial sector: a documentary analysis of mechanisms require further study. tobacco company strategies to influence the WHO framework convention on tobacco control. PLOS MED. 2012; 9: e1001249. In HNSCC, recurrence within 1 year following surgery is called early recurrence, and the prognosis of patients with early recurrence 12. Rose BS, Jeong JH, Nath SK, Lu SM and Mell LK. Population-based study of competing mortality in head and neck cancer. J Clin Oncol. 2011; 29: 3503- is very poor [22,23]. Currently, there are no markers to predict early 3509. recurrence of HNSCC. In this study, we found that MMP13 and 13. Masoy SE. Combined diagnosis and therapy. Ultraschall Med. 2010; 31: 537. SERPINB2 alone or in combination played a role in distinguishing early and non-early recurrence of hypopharyngeal cancer, although 14. Hirahata M, Osaki M, Kanda Y, Sugimoto Y, Yoshioka Y and Kosaka N, et al. PAI-1, a target gene of miR-143, regulates invasion and metastasis by these results need to be confirmed with a larger sample size. We upregulating MMP-13 expression of human osteosarcoma. Cancer Med. expect that additional markers could be used for early diagnosis of 2016; 5: 892-902. hypopharyngeal cancer. 15. Croucher DR, Saunders DN, Stillfried GE and Ranson M. A structural basis for differential cell signalling by PAI-1 and PAI-2 in breast cancer cells. This study has some limitations. First, this study was only based Biochem J. 2007; 408: 203-210. on public databases and patient tissues; the role of MMP13 and SERPINB2 needs to be further verified in vitro. Secondly, the sample 16. Hu PF, Chen H, Zhong W, Lin Y, Zhang X and Chen YX, et al. Adenovirus- mediated transfer of siRNA against PAI-1 mRNA ameliorates hepatic fibrosis size of the study is small and all of our patients are from North-East in rats. J Hepatol. 2009; 51: 102-113. of China. We will further confirm our experimental conclusion with 17. Ohkouchi C, Kumamoto K, Saito M, Ishigame T, Suzuki SI and Takenoshita a larger sample size. S, et al. ING2, a tumor associated gene, enhances PAI1 and HSPA1A expression with HDAC1 and mSin3A through the PHD domain and C-terminal. Conclusion Mol Med Rep. 2017; 16: 7367-7374. Our study assessed the role of MMP13 and SERPINB2 in 18. Chakravarthy A, Khan L, Bensler NP, Bose P and De Carvalho DD. TGF- hypopharyngeal cancer. The results showed that MMP13 may be β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure. Nat Commun. 2018; 9: 4692. an oncogene while SERPINB2 may be an anti-oncogene and could serve as a prognostic and diagnostic factor for hypopharyngeal cancer 19. Jimenez MJ, Balbin M, Lopez JM, Alvarez J, Komori T and Lopez-Otin C. Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene patients. We believe MMP13 and SERPINB2 could be promising family involved in bone formation. Mol Cell Biol. 1999; 19: 4431-4442. biomarkers and drug targets for HNSCC. 20. Oda T, Jung YO, Kim HS, Cai X, Lopez-Guisa JM and Ikeda Y, et al. PAI-1 References deficiency attenuates the fibrogenic response to ureteral obstruction. Kidney 1. Chung EJ, Kim GW, Cho BK, Park HS and Rho YS. Pattern of lymph node Int. 2001; 60: 587-596. metastasis in hypopharyngeal squamous cell carcinoma and indications for 21. Higuchi C, Tanihata Y, Nishimura H, Naito T and Sanaka T. Effects of level VI lymph node dissection. Head Neck. 2016; 38: 1969-1973. glucose and plasminogen activator inhibitor-1 on collagen metabolism in the peritoneum. Ther Apher Dial. 2005; 9: 173-1781. 2. Knauper V, Lopez-Otin C, Smith B, Knight G and Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem. 1996; 271: 1544- 22. Kibe Y, Nakamura N, Kuno H, Hiyama T, Hayashi R and Zenda S, et al. 1550. Frequency and predictors of detecting early locoregional recurrence/disease progression of oral squamous cell carcinoma with high-risk factors on imaging 3. Vincenti MP and Brinckerhoff CE. Transcriptional regulation of collagenase tests before postoperative adjuvant radiotherapy. Int J Clin Oncol. 2019; 24: (MMP-1, MMP-13) genes in arthritis: integration of complex signaling 1182-1189. pathways for the recruitment of gene-specific transcription factors. Arthritis Res. 2002; 4: 157-164. 23. Fakhry C, Krapcho M, Eisele DW and D’Souza G. Head and neck squamous cell cancers in the United States are rare and the risk now is higher among 4. Fortenberry Y. The role of in tumor cell migration. Biol Chem. 2015; white individuals compared with black individuals. Cancer-AM Cancer SOC. 396: 205-213. 2018; 124: 2125-2133.

Submit your Manuscript | www.austinpublishinggroup.com Austin J Clin Pathol 7(2): id1065 (2020) - Page - 05