Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

BACKGROUND: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. METHODS: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. RESULTS: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. CONCLUSION: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

Trophoblast cell surface antigen 2 expression predicts outcome in oral squamous cell carcinomas.

BACKGROUND: Trophoblast cell surface antigen 2 (TROP2) has unclear clinical role in oral squamous cell carcinomas (OSCC). Here, we investigated the association of TROP2 immunoexpression with clinicopathological parameters and survival of OSCC patients. SUBJECTS AND METHODS: Cancer-specific survival (CSS) and disease-free survival (DFS) were assessed in a cohort composed of 266 OSCC. An independent cohort with 88 OSCC samples matched with the normal oral tissue, as well as 17 metastatic lymph nodes, was used for validation. RESULTS: Multivariate analysis showed TROP2 as an independent marker of favorable prognosis for both CSS (HR: 0.60, 95% CI: 0.40-0.90, p = .01) and DFS (HR: 0.57, 95% CI: 0.36-0.89, p = .01). Furthermore, TROP2 protein expression was significantly higher in morphologically normal tissues compared to primary tumors (p < .0001) and lymph node metastases (p = .001), and it was significantly associated with CSS (HR: 0.26, 95% CI: 0.09-0.74, p = .008) in the validation cohort. A pooled mRNA analysis performed on the Oncomine™ database confirmed the underexpression in OSCC compared with normal tissues (p = .014). CONCLUSIONS: In summary, our results point to a favorable prognostic significance of TROP2 overexpression in a large cohort of oral cancer patients, suggesting it as an attractive clinical marker.

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

The Impact of Histopathological Features on the Prognosis of Oral Squamous Cell Carcinoma: A Comprehensive Review and Meta-Analysis.

OBJECTIVE: Over many decades, studies on histopathological features have not only presented high-level evidence of contribution for treatment directions and prognosis of oral squamous cell carcinoma (OSCC) but also provided inconsistencies, making clinical application difficult. The 8th TNM staging system of OSCC has acknowledged the importance of some histopathological features, by incorporating depth of invasion (DOI) to T category and extranodal extension (ENE) to N category. The aim of this systematic review with meta-analysis is to determine the most clinically relevant histopathological features for risk assessment and treatment planning of OSCC and to elucidate gaps in the literature. METHODS: A systematic review was conducted using PRISMA guidelines, and the eligibility criteria were based on population, exposure, comparison, outcome, and study type (PECOS). PubMed, Cochrane, Scopus, and Web of Science were searched for articles exploring the impact of histopathological features on OSCC outcomes with Cox multivariate analysis. Pooled data were subjected to an inverse variance method with random effects or fixed effect model, and the risk of bias was evaluated using quality in prognosis studies (QUIPS). Quality of evidence was assessed with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. RESULTS: The study included 172 articles published from 1999 to 2021. Meta-analyses confirmed the prognostic potential of DOI, ENE, perineural invasion, lymphovascular invasion, and involvement of the surgical margins and brought promising results for the association of bone invasion, tumor thickness, and pattern of invasion with increased risk for poor survival. Although with a small number of studies, the results also revealed a clinical significance of tumor budding and tumor-stroma ratio on predicted survival of patients with OSCC. Most of the studies were considered with low or moderate risk of bias, and the certainty in evidence varied from very low to high. CONCLUSION: Our results confirm the potential prognostic usefulness of many histopathological features and highlight the promising results of others; however, further studies are advised to apply consistent designs, filling in the literature gaps to the pertinence of histopathological markers for OSCC prognosis. SYSTEMATIC REVIEW REGISTRATION: International Prospective Register of Systematic Reviews (PROSPERO), identifier CRD42020219630.

Stanniocalcin 2 contributes to aggressiveness and is a prognostic marker for oral squamous cell carcinoma.

Stanniocalcin 2 (STC2), a glycoprotein that regulates calcium and phosphate homeostasis during mineral metabolism, appears to display multiple roles in tumorigenesis and cancer progression. This study aimed to access the prognostic value of STC2 in oral squamous cell carcinoma (OSCC) and its implications in oral tumorigenesis. STC2 expression was examined in 2 independent cohorts of OSCC tissues by immunohistochemistry. A loss-of-function strategy using shRNA targeting STC2 was employed to investigate STC2 in vitro effects on proliferation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT) and possible activation of signaling pathways. Moreover, STC2 effects were assessed in vivo in a xenograft mouse cancer model. High expression of STC2 was significantly associated with poor disease-specific survival (HR: 2.67, 95% CI: 1.37-5.21, p = 0.001) and high rate of recurrence with a hazard ratio of 2.80 (95% CI: 1.07-5.71, p = 0.03). In vitro downregulation of STC2 expression in OSCC cells attenuated proliferation, migration and invasiveness while increased apoptotic rates. In addition, the STC2 downregulation controlled EMT phenotype of OSCC cells, with regulation on E-cadherin, vimentin, Snail1, Twist and Zeb2. The reactivation of STC2 was observed in the STC2 knockdown cells in the in vivo xenograft model, and no influence on tumor growth was observed. Modulation of STC2 expression levels did not alter consistently the phosphorylation status of CREB, ERK, JNK, p38, p70 S6K, STAT3, STAT5A/B and AKT. Our findings suggest that STC2 overexpression is an independent marker of OSCC outcome and may contribute to tumor progression via regulation of proliferation, survival and invasiveness of OSCC cells.

Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma.

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain­of­function (treatment with recombinant activin A) or loss­of­function [treatment with activin A­antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA­mediated depletion of activin A was also tested. The profile of pro­ and anti­angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription­qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30­4.71; P=0.006) for disease­specific survival and 2.09 (95% CI, 1.07­4.08l: P=0.03) for disease­free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A­depleted OSCC cells. Activin A­knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro­angiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

Interleukin-17F Has Anti-Tumor Effects in Oral Tongue Cancer.

We recently showed that extracellular interleukin-17F (IL-17F) correlates with better disease-specific survival in oral tongue squamous cell carcinoma (OTSCC) patients. However, the underlying mechanisms of such effect remain obscure. Here, we used qRT-PCR to assess the expression of IL-17F and its receptors (IL-17RA and IL-17RC) in two OTSCC cell lines (HSC-3 and SCC-25) and in normal human oral keratinocytes (HOKs). IL-17F effects on cancer cell proliferation, migration, and invasion were studied using a live-imaging IncuCyte system, and a Caspase-3/7 reagent was used for testing apoptosis. 3D tumor spheroids were utilized to assess the impact of IL-17F on invasion with or without cancer-associated fibroblasts (CAFs). Tube-formation assays were used to examine the effects of IL-17F on angiogenesis using human umbilical vein endothelial cells (HUVEC). OTSCC cells express low levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with HOKs. IL-17F inhibited cell proliferation and random migration of highly invasive HSC-3 cells. CAFs promoted OTSCC invasion in tumor spheroids, whereas IL-17F eliminated such effect. IL-17F suppressed HUVEC tube formation in a dose-dependent manner. Collectively, we suggest that IL-17F counteracts the pro-tumorigenic activity in OTSCC. Due to its downregulation in tumor cells and inhibitory activity in in vitro cancer models, targeting IL-17F or its regulatory pathways could lead to promising immunotherapeutic strategies against OTSCC.

Extracellular vesicles derived from cancer-associated fibroblasts induce the migration and invasion of oral squamous cell carcinoma.

As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.

Enamel renal syndrome: A novel homozygous FAM20A founder mutation in 5 new Brazilian families.

Enamel renal syndrome (ERS) is a rare autosomal recessive disorder not fully characterized. Here we investigated ERS characteristics in 11 patients from 5 Brazilian families through clinical examination, imaging, renal ultrasonography, laboratory tests and DNA sequencing. The patients' age ranged from 6 to 25 years-old, and the presence of hypoplastic amelogenesis imperfecta, microdontia, intra-pulpal calcification, impacted posterior teeth with hyperplastic pericoronal follicles, gingival fibromatosis, ectopic calcifications on gingival and pericoronal tissues, and nephrocalcinosis were common findings to all patients. Only 4 patients showed abnormal laboratory tests (vitamin D, parathyroid hormone, phosphate, calcium). Intellectual disability and renal cysts were present in 2 patients each. Biallelic loss of function mutation in FAM20A gene, characterized by one base pair deletion in exon 11, resulting in a frameshift replacing a glutamine at codon 483 for a lysine and terminating at position 24 [NG_029809.1: c.1447delG; p.(Glu483Lysfs*24)], was detected in all patients, strongly suggesting a founder effect. Our results reinforce the distinct orofacial features of ERS, which are the clue for kidney examination and genetic testing. Early diagnosis is essential to minimize the deleterious effects related to ERS. Here we report the largest series of patients with ERS in a same population, and describe, for the first time, a founder mutation for FAM20A.

Prognostic value of the immunohistochemical detection of cancer-associated fibroblasts in oral cancer: A systematic review and meta-analysis.

AIM: To perform a meta-analysis to assess whether the presence of cancer-associated fibroblasts (CAF) is a prognostic marker of oral squamous cell carcinomas (OSCC). METHODS: Immunohistochemical studies assessing the prognostic relevance of CAF (alpha smooth muscle actin (α-SMA)-positive fibroblasts) in patients with OSCC were systematically reviewed using Cochrane, Lilacs, PubMed, Scopus, and Web of Science databases. The outcomes assessed were overall survival (OS) and disease-free survival (DFS). The meta-analysis was performed using the random- and fixed-effects model with adjusted hazard ratio (HR) and 95% confidence intervals (95% CI) as effect measures. The methodological quality of the included studies was assessed using the Meta-Analysis of Statistics Assessment and Review Instrument (MAStARI) tool, and the evidence quality was assessed by the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) system. RESULTS: The presence of high levels of CAF in the stroma of OSCC predicted shortened time to DFS (HR = 3.32, 95% CI: 2.09-5.26, P < .00001) and an overall decrease in survival (HR: 2.16, 95% CI: 1.60-2.92, P < .00001). Moreover, high presence of CAF was frequently reported in association with parameters that worsen the prognosis in OSCC, including advanced disease stage (TNM classification), recurrence, tumor grade, depth of invasion, vascular, lymphatic and neural invasion, and extranodal metastatic spread. CONCLUSION: The presence of CAF, as assessed by α-SMA-positive fibroblasts in the stroma, indicates poor prognosis in patients with OSCC.

Organotypic three-dimensional assays based on human leiomyoma-derived matrices.

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel® However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel® Additionally, Myogel can replace Matrigel® in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in ex vivo OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate in vitro and in vivo the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.

Enamel Renal Syndrome: A Case History Report.

Enamel renal syndrome (ERS) is a rare, commonly misdiagnosed condition that results in amelogenesis imperfecta and nephrocalcinosis and can lead to renal impairment in adulthood. This case history report describes a multidisciplinary dental management approach in a young adult patient with ERS.