LAMP3 (CD208) Expression in Squamous Cell Carcinoma and Epithelial Dysplasia of the Oral Cavity and Clinicopathological Characteristics of Unfavorable Prognosis

Background: This study aimed to evaluate LAMP3 (CD208) gene expression in oral squamous cell carcinoma (OSCC) and dysplastic oral epithelium by quantitative real-time polymerase chain reaction (qPCR) and compare LAMP3 expression in different disease grades and stages. Methods: In this study, 60 OSCC and dysplastic oral epithelium samples were obtained from the Mashhad University of Medical Sciences together with their demographic and clinicopathological documents. LAMP3 expression was measured by qPCR. Results: LAMP3 expression was significantly greater in OSCC than in dysplasia samples (P=0.001), in grade III OSCC than in grades I and II, and also greater in advanced than in early OSCC disease stage (P=0.001). Conclusions: The significantly greater LAMP3 expression in OSCC than in dysplastic epithelium indicates a role for LAMP3 in carcinogenesis in oral mucosa. Our results suggest LAMP3 may be useful as an anticancer target and/or to predict disease pathogenesis in OSCC patient’s cells.


Introduction
Oral squamous cell carcinoma (OSCC) is a major subcategory of head and neck squamous cell carcinomas and accounts for more than 90% of oral cavity cancers (1). Although some investigations found OSCC related to tobacco and alcohol consumption, it has also been found in patients who used neither (2). Recently, the number of new OSCC cases has increased annually worldwide (3), and its high incidence rate concurs with metastasis, high recurrence, and poor prognosis in advanced stages (4,5). Utilizing the novel diagnostic methods related to molecular markers could provide earlier or faster OSCC patient prognoses. Lysosome-associated membrane protein 3 (LAMP3, CD208, DC-LAMP), a member of the LAMP protein family, is considered a molecular marker of mature dendritic cells (MDCs) (6). Increased LAMP3 expression is associated with unfavorable prognosis in many cancers including esophageal squamous cell carcinoma (ESCC) (7), gastrointestinal stromal tumor (GIST) (8), breast cancers (9, 10), cervical cancer (11), and head and neck squamous cell carcinomas (6). This marker is associated with induction and promotion of tumor migration and invasion (12,13). A significant relationship was seen between LAMP3 expression and lymph node metastasis (6,7). Consequently, LAMP3 expression may be a predictor of cancer patient survival (6). Investigations of LAMP3 expression and its association with OSCC patients' clinical features and pathological characteristics are lacking; hence, we aimed to analyze LAMP3 expression in OSCC tissue samples and dysplastic epithelium by quantitative polymerase chain reaction (qPCR). Furthermore, the relationship between LAMP3 expression and OSCC patient demographic features and histological characteristics were assessed. Our results suggest LAMP3 expression may be a treatment target in for OSCC patients.

Study participants and sample preparation
This study was performed in the Oral and Maxillofacial Pathology Department of Mashhad Dental School, Mashhad University of Medical Science, from November 2016 to November 2019. Sixty specimens from 60 patients were obtained as paraffin blocks from the dentistry pathology archive. We recorded the patients' demographic characteristics including age, sex, and alcohol, smoking, and drug consumption. In addition, the tumor grades and stages were recorded.
Patients with OSCC who had received no treatment until the time of sampling, who had no previous radiotherapy or chemotherapy, and currently had no other skin or oral diseases were included. In addition, we included all paraffin block specimens with sufficient lesion volume for testing. All RNA samples to be used for qRT-PCR were first analyzed for quality on a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, Thermo, USA). Thereafter, cDNA was synthesized by standard procedure (Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit, Thermo Scientific, USA). cDNA was synthesized in 20 µL reactions containing 4 µL of 5x reaction buffer, 1 µL of RiboLock RNase inhibitor, 2 µL of 10 mM dNTP mix and 1 µL of reverse transcriptase on an ABI thermocycler (One Step, USA). Only cDNA samples of sufficient quality for testing and samples that met required storage conditions were included in the study. Five-micron-thick tumor and dysplastic tissue sections were obtained, sterilized in microtubules, and transferred to the Mashhad Medical School Pathology Laboratory for LAMP3 expression analyses.

Quantitative Real-time PCR (qPCR)
For qPCR, tumor and dysplastic tissue samples were placed in sterile micro-tubes containing RNAlater and stored at -80 o C. RNA was extracted using an RNeasy mini kit following the manufacturer's protocol (High Pure RNA Tissue Kit, Roche, Germany). After RNA extraction, LAMP3 cDNA was synthesized (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit, Scientific Thermo, USA) in 20 µL reactions containing 4 µL of 5x reaction buffer, 1 µL of RiboLock RNase inhibitor, 2 µL of 10 mM dNTP mix and 1 µL of reverse transcriptase on an ABI thermocycler (One Step, USA). The quality and concentration of the cDNAs were determined on a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
qPCR was performed to quantitate the relative expression of LAMP3 using a SYBR Green master mix kit (Thermos Scientific, Germany) on an ABI thermocycler (One Step, USA) with appropriate primers (Table 1). All reactions were performed in duplicate in 20 µL volumes in separate wells. Each reaction contained 0.5 µL of each primer (10 pM), 10 µL of SYBR Green master mix, 7 µL of DEPC water, and 2 µL (4 ng) of cDNA extract. The PCR program started with 1 cycle at 94 ºC for 10 min followed by denaturation, annealing, and replication processes including amplification cycles, respectively, at 94 ºC for 30 sec, 60 ºC for 30 sec, and 72 ºC for 30 sec. The final amplification followed at 72 ºC for 35 sec. Differential expression was analyzed by the ΔΔCT method. The housekeeping GAPDH gene was used as the reference gene (Figs. 1 and 2). Fold change above 2 was regarded as over expression, and below 2 as low or no expression (14-16).

Statistical Analyses
Data were analyzed by SPSS software version 19.9 (SPSS Inc., Chicago, IL, USA). Descriptive data including age and LAMP3 expression were summarized as means with the standard errors of means (SEMs). LAMP3 expression was compared in the tumoral and dysplastic tissues by the Mann-Whitney Test. LAMP3 expression in tumor and dysplastic tissues and its relationship with clinicopathological parameters, including tumor stage and histological grade, were evaluated by one-way ANOVA. The association between LAMP3 expression and demographic features between the dysplastic and OSCC groups were compared by independent sample T test. P values less than 0.05 were considered statistically significant.

Study participant characteristics, and demographic and histologic features
The 60 patient samples included 15 Table 3). LAMP3 expression was significantly greater in dysplasia tissue specimens from patients under 60 years of age than in those 60 years and older (P=0.01; Table 4); however, LAMP3 expression was not significantly different in OSCC samples from the two age groups (P=0.09). No significant difference in LAMP3 expression was seen between males and females in either the OSCC or dysplasia samples (P>0.05 for both, Table 4).   LAMP3 expression was significantly greater in OSCC grades 1, 2, and 3 than in the dysplasia group, and increased as the OSCC grade severity increased. The expression difference between OSCC grades 1 and 2 was not significant, however, the differences between grade 3 and grades 1 and 2 were significant. (P<0.05). Also, LAMP3 expression was significantly greater in both early and advanced OCSS stages than in dysplasia, and significantly greater in advanced than in early OCSS. (P=0.01) ( Table 5).

Discussion
The role of lysosomes in homeostasis, cancer biology, and macromolecular degradation has been well studied (17). Throughout cancer progression or cell transformation, lysosomes move to different cellular locations and arrangement by release of enzymes (18). LAMPs are a family of glycosylated proteins that primarily associate with the lysosome membrane and are expressed at different levels in different tissues. An anticancer therapy role for LAMPs has been suggested for five members of this family including LAMP1, LAMP2, LAMP3, CD68/macrosialin/LAMP4, and BAD-LAMP/LAMP5. It has been shown that cancer aggressiveness is increased by lysosomal release of LAMPs (19). LAMP3, located on a chromosome 3q segment, was initially isolated as a gene specifically expressed in lung. A role for LAMP3e was characterized in some human cancers including SCC and uterine and cervical cancers (11).
A previous study found a gain of 3q in 90% of tumor tissue and only 7% in severe dysplasia of invasive cervical carcinomas. The authors suggested that a functionally important gene for cervical carcinogenesis might exist at 3q24-27. According to in cancers, this could be a suitable candidate gene in previous investigation on altered LAMP3 expression dysplastic and tumor tissues (20).
This present study investigated the expression of LAMP3 mRNA in grades 1-3 of oral SCC and in dysplasia. Due to limited studies and the importance of this marker in dysplasia and carcinomas, we compared dysplastic mucosa and OSCC in this study. Our results indicate a role for LAMP3 and its increased expression in the process of oral mucosal carcinogenesis.
LAMP3 expression and patient age were inversely related in the dysplasia group; however, no significant difference in LAMP3 expression was seen between age groups with OSCC. Similar to our result,, Jun Lu et al. found no correlation between LAMP3 expression in OSCC and patient age or gender (6,21).
Our results indicate that LAMP3 may be a relevant oncogene candidate potential prognosis factor in OSCC, and due to the many similarities, possibly other human cancers as well.