SPINK7 Expression Changes Accompanied by HER2, P53 and RB1 may be Potential Biomarkers to Predict Oral Squamous Cell Carcinoma at Molecular Level

Background. The oral squamous cell carcinoma (OSCC) affects more than 300,000 patients annually worldwide with a high morbidity rate (37.8%). Several tumor biomarkers have been suggested to anticipate outcome but results were poor. Changes of SPINK7 and associated proteins in precancerous oral lesions could lead to genomic instability and promote oncogenesis. Our aim was to evaluate SPINK7as apotential molecular biomarkerpredictive of OSCC stages, compared with well-known molecules altered in cancer: HER2,TP53, RB1, NFKB and CYP4B1. Methods.Oral biopsies from patientswith dysplasia (n=33), less invasive(n=28) andhighly invasiveOSCC (n=18) were collected. 20 cases with a clinical suspicion but normal mucosa conrmedwere included ascontrol. Gene expression of SPINK7, P53,RB, NFKBand CYP4B1 were quantied by qPCR.SPINK7 levels were correlated with a cohort of 330 patients from the TCGA. Also,SPINK7, HER2, TP53, and RB1, were evaluated by immunohistouorescence. One-way Kruskal-Wallis test and Dunn's post-hocwith a p<0.05 signicance were used to data analyze. Results.In OSCC, SPINK7wasdown regulated andP53, RB, NFKB and CYP4B1were up regulatedrespect tothe others groups (p<0.001). Also,SPINK7 expressionwasdiminished in patients of TCGA(p=2.10e-6). In less invasive OSCC,SPINK7 and HER2 proteinswere decreasedandTP53 and RB1 signicantly increasedrespect todysplasia and highly invasivegroups (p<0.05). Conclusion. Our results suggest that SPINK7changes accompanied of HER2, P53 and RB1 can be used to classify the molecular stage of epithelial oral lesion inthe OSCC, allowing a more accuratediagnosis to molecular and histopathological level. One μg of RNA was reverse transcribed for 60 min at 42 °C using 200 U M-MLV reverse transcriptase (Invitrogen) and 0.5 μM oligo-dT primers (Invitrogen). Real time PCR was performed in a nal volume of 10 μL containing 50 ng of cDNA, Power SYBR Green PCR master mix (Life Technologies, Grand Island, NY) and 0.5 μM of each specic primer, using the Step One Plus PCR system (Life Technologies).


Introduction
Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck, with a high morbidity rate (37.8%) ve years after diagnosis (1). Despite great improvement in treatment and therapy,prognosis remains poor (2).Furthermore, OSCC often causes dysfunctions and aesthetic disorders, and have a high incidence of cervical lymph node metastasis, worsen patients quality of life (3). Several tumor biomarkers have been suggested as predictive for OSCC prognosis with poor outcome (4), however, speci c molecular prognostic factors have only been partially identi ed (5).The pattern of invasion (POI) presented by Brandwein-Gensler et al., classi es the POI in ve types (6), and has been validated as an independent prognostic factor in oral cancer (7). However, it is necessary to identify changes in proteins and genes, to improve diagnostic strategies in precancerous, invasive and metastatic stages (8).
One of the cancer hallmarks is the alteration of molecules related to cell adhesion and migration (9). Adhesion molecules play a central role in pathogenesis and progression of malignant tumors (10). Serine Peptidase Inhibitor, Kazal Type 7 (SPINK7, ECRG2) belongs to a family of 13 members (1-13) of proteins with inhibitory Serine Peptidase activity identi ed in 1998 in esophageal tissue (11). This novel tumorsuppressor genewas identi ed as a tumor suppressor gene by comparing normal esophageal epithelia and primary squamous cell carcinomas tissues (12,13). It has been reported that SPINK7 inhibits tumor cells growth, promotes cell apoptosis, and inhibits cancer cell migration, invasion and metastasisin vitro (14,15).
The HER2, P53, RB1, NFKB and CYP4B1 genes and their proteins have been described altered inOSCC carcinogenesis (16).The human epidermal growth factor receptors (HER/EGFR)are a family oftrans membrane tyrosine kinase receptors comprising 1 to 4 (HER1-4) (17). The overexpression of HER isinvolved in the development of oncogenesis, including OSCC, through regulating different cellular pathways. HER2(also known as C-erbB-2/ERBB2/ErbB2) plays a critical role in the cell proliferation, survival, migration, angiogenesis, and metastasis through a variety of intracellular signaling cascades such as MAPK/ERK1/2 and Pi3K/Akt (18,19).An imbalance in these pathways can lead to permanent activation (20,21). Studies have established a marked correlation between HER2 expression and the poor survival of OSCC patients (22).It has been reported that the SPINKs protein family share 50% of homology to EGF moleculeand can interact by binding to EGFR, activating EGFR downstream AKT signaling pathway, inducing epithelial mesenchymal transition (23). The SPINK6 protein is secreted and act as a functional regulator of nasopharyngeal carcinoma cells metastasis through the bound to EGFR extracellular domain (23).
In cancer cells, tumor-suppressor genes like Protein 53 (TP53) and Retinoblastoma (RB1) are inactivated by mutation, deletion and methylation (24). It is well established that TP53 is a genome guardian and plays a pivotal role in regulating the cell cycle, cellular differentiation, DNA repair, and apoptosis (25,26).
Somatic mutations in TP53 are detected in >60% of OSCC and in 10% of oral dysplasia (27). Recently, GenomeWide Association Study data has shown that TP53 is usually mutated in papillomavirus-negative OSCC patients (28). TheTP53 mutations in OSCC(classi ed in low-and high-risk missense mutations) are associated with resistance to Cisplatin, distant metastasis and poor prognosis (29)(30)(31). The overall survival of TP53-mutant OSCC patients is also markedly worse than patients with TP53 wildtype (32).Previous studies reported that SPINK7 also participates in centrosome ampli cation in TP53dependent manner and has a role in maintaining chromosome stability (33). Other gene altered is RB1 that plays a key role in the regulation of cell cycle and differentiation. Its active form is phosphorylated (pRB1), acts a regulator at the G1-S restriction point arresting the cell cycle (34). Mutations lead to functional pRB1 inactivation and failure of growth and tumor suppression control (35).
Another molecule altered in carcinogenesis is the Nuclear factor-κB (NFKB), this is a proin ammatory transcription factor that plays a pivotal role in initiation and progression of the cancer (36). NFKBis constitutively activated in OSCCs and is involved in promoting the invasive characteristics (37).
Regarding the cell detoxi cation machinery,Cytochrome P450 enzyme family (CYP450) is one of the most important (38,39). Their activity consists in catalyzing reactions that participate in both biosynthesis and degradation of drug metabolism and xenobiotic biotransformation pathways (40). These enzymes can participate indirectly in the OSCC carcinogenesis through activation and detoxi cation of these compounds (38,40).
The Cancer Genome Atlas (TCGA) is an important tool to provide expression pro les from cancer patient samples and the associated clinical-pathological data for > 30 human cancer types (41). However, there are few studies on genome-wide pro ling of OSCC tumors.
The aim of this work was to determine ifSPINK7 gene expressionis associatedto molecules altered in cancer p53, RB1, NFKB, CYP4B1 and HER2as a good biomarker candidate of premalignant epithelial oral lesions and OSCC stages, havingpotential therapeutic applications (early detection and targeted therapies).

Study population
Patients with suspected oral lesions of OSCC were enrolled. After signing the informed consent, the subjects were interviewed using a standard questionnaire that requested information about sociodemographic, medical, and lifestyle factors. The patients from Department of Head and Neck surgery of The National Cancer Institute,Dental school of Universidad de Valparaíso,Dental school of Universidad del Desarrollo (Chile), The Hospital Lencinas and the Servicio de Estomatología y Medicina Bucal Dental school, Universidad Nacional de Cuyo (Argentina); received a routine intraoral examination and oral mucosal biopsies were taken and classi ed according to thediagnosis and POI in three groups:oral epithelial dysplasia, less invasive OSCC (POI type 1 and 2) and invasive OSCC (POI type 3, 4 and 5)group. Seventy-one cases of primary OSCC diagnosed over a period of 2 years (2017-2019) were included in the study. None of the patientshad received any tumor speci c therapy (chemotherapy or radiotherapy) before the resection.Twenty cases diagnosed as in ammatory lesions and histologically con rmed with normal mucosal margins from the resection specimens were included as control group in the qPCR analysis. The Ethics Committee of the School of Medicine of Universidad del Desarrollo (FM-UDD CAS), National Cancer Institute of Chile and Medicine School of Universidad Nacional de Cuyo (FCM-UNCuyo) approved this study according to Declaration of Helsinki to experimentation with human subjects.

Histopathologicalanalysis
TheOral biopsieswere xed in 10% buffered formalin (Merck, USA), embedded in para n (Merck), and sectioned. Tissue sections of 4 μm were depara nized with Neoclear (Merck), rehydrated with graded alcohols, stained with hematoxylin-eosin (H&E, Merck), and visualized with a light microscope (DM2000; Leica, Germany). Images were captured with a digital camera (DFC295; Leica). Samples were classi ed according to the revised criteria given by the World Health Organization (2005). Three independent observers performed histological analyses blind; one of them is a pathologist expert in oral diseases (42,43).

Confocal microscopy analysis
A Gaussian lter of 1 was applied anda constant background value of 150 wassubtracted for each image. The same threshold value was set for each channel including the structures of interest and the corresponding masks were obtained. The yellow pixels (red and green pixels overlap) versus the total pixels were quanti ed and the colocalization was measured with Coloc2 plugin(FijiImageJ) (44).

Gene expression analysis
Total RNA was isolated from theoral biopsies. The mRNA was puri ed using RNEasy PlusMini Kit (Qiagen, Germany). Contaminating genomic DNA was degraded with 1 U of DNAse RQ1 (Promega). One μg of RNA was reverse transcribed for 60 min at 42 °C using 200 U M-MLV reverse transcriptase (Invitrogen) and 0.5 μM oligo-dT primers (Invitrogen). Real time PCR was performed in a nal volume of 10 μL containing 50 ng of cDNA, Power SYBR Green PCR master mix (Life Technologies, Grand Island, NY) and 0.5 μM of each speci c primer, using the Step One Plus PCR system (Life Technologies).
Controls without reverse transcriptase were included. Amplicons were analyzed according to their size and melting temperature (Supplementary Table 1, S1). To normalize data, 18S RNA and β-actin were used as reference genes. The RNA level of a target gene was calculated using the 2ΔCt method and graphed as fold change (45).

Geneexpressions TCGA pro le
The data studied was programmatically extracted from the publicly available data set of OSCC from The Cancer Genome Atlas Project (TCGA) on May, 2019 using the recount2 platform (https://jhubiostatistics.shinyapps.io/recount/). Non-standardized RNASeq gene expression levels from tumor samples and 32 non-tumoral tissue samples. RNA expression levels were evaluated for 6 genes (NFKB1, RB1, TP53, ERBB2, CYP4B1, SPINK7). Crude counts were scaled by the total coverage of the sample (area under the curve, 'AUC') and differential gene expression analysis (DGE) was performed using the generalized linear model method of the EdgeR R package comparing non-tumor versus tumor samples (46). Log2 Fold change values were obtained associated with exact p-values and False Discovery Rate values (FDR). To evaluate gene expression correlation, data was transformed using Voom conversion from the R limma package, allowing normal linear modeling of the RNA counts. Afterwards, pairwise Pearson's product-moment correlation analysis was performed for the aforementioned genes and p-values were calculated (47).

Gene mutationsTCGA pro le
The mutational analysis of OSCC, data was programmatically downloaded using the TCGAbiolinks package of Bioconductor (48). Mutation Annotation Format (MAF) les with aggregated mutation information generated from whole-exome sequencing were downloaded.From 546 samples of Head and Neck cancer, 329 samples of OSCC were obtained. The maftools Bioconductor package was used to analyze and visualize the MAF les (49). An Oncoplot was drawn showing the variants (SNP) of the 15 most mutated genes in OSCC, followed by 5 genes of interest (RB1, ERBB2, NFKB1, CYP4B1 and SPINK7) (47).

Statistical analysis
The population distribution of the samples from our patientswas non-parametric.Comparisonsof gene and protein expression among the groups were performed using One-way Kruskal-Wallis test and Dunn's test as post-test. Stat Graph Prism 5.0 software was used for statistical analysis. Data are presented as median ± SEM, andp<0.05 was considered statistically signi cative.

Study population data
Of a total of 71Caucasian patients with oral dysplasia or OSCC were consent and enrolled in the present study.The average age was 52 years. The predominant gender was male. Of the total of patients 30% had no smoking habits, 20% were light smokers (less than 10 cigarettes daily), 40% heavy smokers (more than 10 cigarettes daily) and a 10% did not specify if they hadsmoking habits. Being the most frequent tumor location the tongue ride (80%)( Table 1). Table 1 Data of the study population The oral epithelium changesamong OSCCstages To analyze epithelial changes in OSCC progression, the oral biopsies were evaluated by H&E and classi ed indysplasia, less invasive and highly invasive OSCC. We found 33 cases of dysplasia, 28 cases of less invasive OSCC and 18 cases of highly invasive OSCC. In all dysplasia cases, the tissue did not show loss of basement membrane continuity or presence of epithelial cells invading the stroma, although an increase in epithelial cell layers and presence of mild leukocyte in ltrate was observed ( Figure 1A-D). In the OSCC groups both (less invasive and highly invasive), showed more than 5 layers of epithelial cells, hyperchromatism, cellular atypias and presence of keratin pearls, loss of continuity of the basement membrane and severe leukocyte in ltrate ( Figure 1B-C-E-F). In the highly invasive OSCC group, epithelial cell nests were observed in the stroma, total epithelial disorganization, keratin pearls and leucocyte in ltration severe.These results were correlated with poor prognosis( Figure 1C). The inserts (black square) shownat high magni cation the oral epithelium changes among the OSCC stages( Figure 1D-F SPINK7 generate a distinctive molecular signature among the OSCC stages To evaluate the molecular status of the biopsies among the OSCC stages, we assessed the gene expression of SPINK7with reported altered genes in carcinogenesis: TP53, RB1, NFKBandCYP4B1in the groups.We found SPINK7progressively down-regulated in oral dysplasia and OSCC groupsrespect tocontrol (p<0.001). RegardingTP53, RB1, NFKBandCYP4B1 all were upregulated in OSCC groups with respect todysplasia and control groups (p<0.001). Additionally,with exception of SPINK7, we observed differential expression levels of the rest of the genes between less invasive OSCC and highlyinvasive OSCC groups (TP53, RB1 and NFKB(p<0.05); CYP4B1p<0.001))( Figure 2A).The differences observed regarding the gene expression among the groups were correlated with a poor prognosis.

SPINK7 was down regulated inpatients from TCGA
To evaluate if theSPINK7gene expression pro le observed in our study populationwas reproducible with other cohort of OSCC patients,we analyzedthegene expression levels in 581 patients with OSCC from the TCGA (primary tissue) and the results were compared with normal subjects data (normal tissue)throughin silico analysis (50). The results were graphed as box plot comparing the gene expression of normal group (box plot blue) versus primary tumors group (box plot yellow) showing signi cant downregulation of SPINK7 p=2.10e-06and this result was correlated with our results respect to SPINK7 gene expression ( Figure 3A).

SPINK7 does not show mutationsaccording toTCGAmutation gene pro le
To understand if the differential expression of SPINK7among the OSCC stages is related to a mutational pro le, we analyzed in silicomutations described to date of a cohort of 329 patients fromTCGA database.We identi ed 15 genes with differential mutations rate associated to OSCC, being gene of interest SPINK7 included in the analysis. We found thatTP53 was the gene with the highest number of mutations in the OSCC cohort, being mutated in 69% of patients( Figure 3B). The most frequent type of variant was missense mutations followed by nonsense mutations, frameshift deletions, and multi hits mutations. TTN gene showed a mutation rate of 34%, FAT1 (26%), CDKN2A (22%), NOTCH1 (18%), PIK3CA and MUC16 (16%), CASP8 (15%), SYNE1 (14%), CSMD3 and PCL2 (13%) and nally, KMT2D, LRP1B, DNAH5 and FLG genes were mutated in 12% of the patients.On the other hand, with a more stable mutational pro le, we found RB1 (mutated in 2% of the samples), HER2, NFKB1and CYP4B1(1%) of the cohort. Finally we found that theSPINK7 is a genomic stable gene, which showed no mutations in none of the cases analyzed ( Figure 3B).

SPINK7 and HER2change differentially among the OSCC stages
It has been reported previously that SPINKs proteins can interact with HER2 receptor (51). We evaluatedthe presence and abundance of SPINK7 among the different groups and its correlation with HER2 protein by confocal microscopy. We found that SPINK7 and HER2 were signi cantly decreased in less invasive OSCC group compared with dysplasia and highly invasive OSCC groups (p<0.05). On another hand, the highly invasive OSCC group showed SPINK7 protein levels similar to dysplasiaand no signi cant differences were found. Regarding HER2, it was foundsigni cantly reduced in less invasive OSCCcompared to the other groups. Meanwhile, the highly invasive OSCC group showed a signi cant increase of HER2 compare toless invasive OSCC (p<0.001), being similar to thedysplasia group( Figure   4A).

SPINK7 and HER2 were colocalized
Due to the overlappingof signals between SPINK7 and HER2 in confocal microscopy we evaluated at high magni cation images (120X digital zoom) through colocalizationanalysis.The intensity variability of both channels was statistically evaluated using Pearson's coe cient of 1 as positive result (r=0,99). Yellow versus red and green pixels were quanti ed yielding a co-occurrence value of 56.58% ( Figure 4B).

TP53 and pRB1 change differentially among the OSCC stages
To evaluate cell cycle regulators inthe OSCC, we analyzed TP53 and pRB1 protein levels among the groups by confocal microscopy. The less invasive OSCC groupshowed a signi cant increase of TP53 and pRB1compared with dysplasia and highly invasive OSCC groups (p<0.05). Regarding the highly invasive OSCC group, both proteins (TP53 and pRB1) signi cantly decreased respect to the other groups (p<0.001) ( gure 5).

Discussion
The oral squamous cell carcinoma has a high morbidity rate in the world (1). Despite the progress in research and therapy, survival has not improved signi cantly in the last decades (52). The biomarkers study aims to understand the role of genetic and lifestyle factors of the tumor biology included the OSCC (53).We studied changes in proteins related with some of the cancer hallmarks (cell survival, cell cycle, in ammation, metastasis and metabolism) to stratify molecularly oral precancerous and cancerous lesions (54). Currently, the gold standard of OSCC diagnosis is the biopsy, however, the results are observer-dependent and subjective (52,55).
The current study is reporting the SPINK7 expression changes among the OSCC stages andwe propose this protein as a "new biomarker" associated with the natural progression of the OSCC. We found differences among the oral epithelial organization indysplasia and less or highly invasive OSCC groups, and these results were correlated with the literature (56,57), showingadifferential gene expression pro le by qPCR analysiswith a distinctive "molecular signature" in each stage. We found SPINK7signi cantly downregulated at dysplasia and OSCC comparedwith control group. MeanwhileTP53,RB1, NFKB and CYP4B1 were signi cantly upregulated at OSCC stages compare with dysplasia and control groups.The results obtained by SPINK7 in our study population were compared with a cohort of 541 patients from the TGCA database (47).The comparative analysis showed that SPINK7 was signi cantly down regulated in patients with OSCC compare to normal tissue and this could be related with the advance grade of the malignant lesion (44). Additionally, we analyzed the mutation pro le of genes described in TCGA altered in OSCC included SPINK7, TP53, RB1, NFKB and CYP4B1. We found that TP53 showed a high mutation rate in OSCC meanwhile SPINK7 was the most stable without any mutation described.These results suggest that the downregulation of the gene would be related to other mechanisms, not associated to TP53 gene, andneed to be explored in a future.
Due the SPINK's protein family is related with extracellular matrix remodeling and cell migration regulation (51). We evaluatedthe abundance of SPINK7 and HER2 and if there correlation between them,because has been reported that SPINK7 shares 50% of homology with EGF(51). We found SPINK7 up regulated in the highly invasive OSCC group, these results were similar to previous studies describing that SPINK6 was up regulated in highly metastatic tumors (51). SPINK6 regulate the metastasis via EGFR signaling and their expression levels change during the carcinogenesis (58). Interestingly SPINK7 and HER2 were overexpressed in the highlyinvasive OSCC compared with lessinvasive OSCC.Additionally the SPINK7 and HER2 colocalization analysis showed that both proteins are close suggesting interaction, however subsequent tests with a larger sample size are necessary to evaluate and understand its interaction or co-compartmentalization (44).The differential expression of the studied proteins among the OSCC stages could be related with disorganization of the oral epithelium and to a non-functional protein or absence of their ligands, but it needs to be explored in more detail in the future(59).The differential proteins expression among the stagesallowedstratifyingthegroups to molecular and histological levels correlated with prognosis.It has been reported in esophageal cancer that cells treated with a siRNA for SPINK1 were resistant to the antitumoral drug Cisplatin (60). This could be interesting in order to stratify the patients who respond, or not, to the standard chemotherapy.
Regarding the studied cell cycle factors, TP53 and pRB1, we found through confocal microscopy analysis that both proteins were upregulated in theless invasive OSCCrespect to dysplasia; meanwhile in thehighly invasive OSCC both were downregulated, which consistent with previous studies (61). These could be explained according to the TCGAin silico analysis with the high rate of mutation pro le that both genes showed in OSCC. These results suggest that in OSCC,TP53 and pRB1 are present but non-functional and this could be related with a more aggressive tumor(61).

Conclusions
Our results suggest that the changes in the expression of SPINK7 can be used to predict the molecular stage of the OSCC lesions. Thismolecule could be a new "potential" biomarker. Futures studies are needed to validate this noveltumor suppressor genethatcould be applied as a possible early diagnostic method to precancerous oral lesions and OSCC.   Figure 1 OSCC stages H&E analysis. Oral biopsies of patients where analyzed by H&E and classi ed into dysplasia, less invasive OSCC and highly invasive OSCC according to the changes in the epithelium. In all dysplasia cases, the tissue did not show loss of basal membrane continuity or presence of epithelial cells invading the stroma, although an increase in epithelial cell layers and presence of mild leukocyte in ltrate was observed. On the contrary, in the biopsies of patients with OSCC both less and highly invasive cases, more than 5 layers of epithelial cells, hyperchromatism, presence of keratin pearls, loss of continuity of the basement membrane and severe leukocyte in ltrate were seen. In the highly invasive OSCC group, epithelial cell nests were observed in the stroma and severe epithelium disorganization, accompanied of leucocyte in ltration of high grade ( Figure 1A-F).

Figure 2
SPINK7, TP53, RB1, NFKB and CYP4B1 gene expression change among the OSCC stages. The graph bar showed the gene expression evaluated by qPCR of each group (normal, dysplasia, less invasive and highly invasive OSCC) and the results were expressed as arbitrary units. The differences were considered statistically signi cance with P values of (*P<0.05, **P<0.01 and ***P<0.001).   pRB1 and TP53 proteins analysis among the OSCC stages. A) Immunohisto uorescence of both proteins evaluated in biopsies of dysplasia, less invasive and highly invasive OSCC. Graphs bar show quantitative analysis of pixels intensity (green=pRB and red=p53) assessed by ImageJ. Representative images by group, n=6/group. White bar = 50 μm. The differences were considered statistically signi cance with P values of (*P<0.05and ***P<0.001).