Carolina Cavalcante Bitu

A simple novel prognostic model for early stage oral tongue cancer.

The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low <5; high ≥5) and for depth of invasion at 4mm (low <4mm; high ≥4mm). The scores of B and D were combined into one model: the BD predictive model. On multivariate analysis, a high risk score (BD score 2) correlated significantly with loco-regional recurrence (P=0.033) and death due to OTSCC (P<0.001) in early stage OTSCC. The new BD model is a promising prognostic tool to identify those patients with aggressive cases of early stage OTSCC who might benefit from multimodality treatment.

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.

Cathepsin K Is Present in Invasive Oral Tongue Squamous Cell Carcinoma In Vivo and In Vitro

Objectives Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens. Materials and Methods OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated. Results Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively). Conclusions Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.

Human Bone Marrow Mesenchymal Stem Cells Induce Collagen Production and Tongue Cancer Invasion

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.

Anti-Heparanase Aptamers as Potential Diagnostic and Therapeutic Agents for Oral Cancer

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.

Cathepsin K expression is increased in oral lichen planus

BACKGROUND Oral lichen planus (OLP) is an idiopathic T-cell-mediated mucosal inflammatory disease. Cathepsin K (Cat K) is one of the lysosomal cysteine proteases. It is involved in many pathological conditions, including osteoporosis and cancer. The expression and role of Cat K in OLP are unknown. METHODS Twenty-five oral mucosal specimens diagnosed histopathologically as OLP and fourteen healthy controls (HC) were used to study the immunohistochemical (IHC) expression of Cat K. Colocalization of Cat K with CD1a, Melan-A, CD68, CD45, mast cell tryptase (MCT), and Toll-like receptors (TLRs) 4 and 9 were studied using double IHC and/or immunofluorescence (IF) staining. Expression of Cat K was also evaluated in OLP tissue samples before and after topical tacrolimus treatment. RESULTS Cat K was expressed in a higher percentage of cells in the epithelial zone, and the staining intensity was stronger in the stroma in OLP compared to controls (P < 0.001). In OLP, Cat K was present mostly in melanocytes and macrophages and sporadically in basal keratinocytes, endothelial cells, and extracellularly. Cat K was found also in some fibroblasts in HC and OLP samples. Coexpression of Cat K and TLRs 4 and 9 was seen in some dendritic cells (presumably melanocytes) and macrophages. In OLP, tacrolimus treatment reduced the expression of Cat K in the epithelium but increased it in the stroma. CONCLUSIONS These results suggest that Cat K is involved in the pathogenesis of OLP. Cat K possibly takes part in the modulation of matrix molecules and cellular receptors.

IL-1α induces cathepsin K in oral cancer cells – the invasion is unaffected

Background To study cathepsin K location in oral tongue squamous cell carcinoma (OTSCC), its traffic and expression levels in cultured oral cell lines; and to analyze the effect of interleukin (IL)-1α on OTSCC invasion. Methods Cathepsin K expression in OTSCC tissue samples was analyzed with immunostaining; its intracellular traffic was followed in HSC-3 cells after PMA treatment. HSC-3 and two oral keratinocyte cell lines, oral carcinoma associated fibroblasts (CAFs) and primary gingival fibroblasts (GF) were treated with IL-1α. Cathepsin K expression was measured using PCR and ELISA. Lastly, the effects of IL-1α on HSC-3 invasiveness, alone and in co-cultures with fibroblasts in the 3D myoma invasion model, were determined. Results Cathepsin K in OTSCC cells was found in vesicles close to cell membrane and within exosomes. While cathepsin K was expressed at the basal level in both epithelial cells and fibroblasts, basal IL-1α levels were higher in epithelial cells compared with GFs and CAFs. Cathepsin K expression was slightly induced by IL-1α in all cell lines, but it did not affect HSC-3 invasiveness. Conclusion In OTSCC, cathepsin K remains mostly intracellular and it is slightly secreted within exosomes. IL-1α treatment has no effect on HSC-3 invasiveness in 3D myoma model.

Abstract 4998: In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oral squamous cell carcinoma (OSCC) is the eighth most prevalent malignant neoplasm and accounts for 2% of all deaths by cancer worldwide. OSCCs have a highly variable clinical course, and because it is often diagnosed only after it has reached an advanced stage, the overall survival rate is less than 50% in 5 years. A better knowledge of the mechanisms that influence OSCC development and its progression is still needed. Particularly, most of the attention has been driven to mechanisms associated with tumor cell invasion and how the surrounding tumoral stroma may influence it. The myoma organotypic model is a novel fully human organotypic invasion model that mimics the tumor microenvironment (TME) (Nurmenniemi et al. 2009). Aiming to investigate the molecular mechanisms related to OSCC development and search for prognostic markers for this tumor, we used the myoma organotypic assay, followed by MS-ESI technique of laser microdissected invaded tissue, to analyze several features related to OSCC invasive vs. non-invasive cells. Our results showed that the HSC-3, an aggressive and invasive oral squamous cell carcinoma cell line, invaded as small islands and cell chords and penetrated deeply in the tissue. Unlike, the less aggressive LN2 cells, formed a well structured epithelium layer, but they invaded less in myoma. The proteomic analysis was able to characterize the differential expressed proteins of these, and revealed a group of proteins exclusively expressed in the cells that deeply invade the myoma, such as HMGA2 (high mobility group), JUP (junction plakoglobin), PLEC (plectin), HIST1H2B (Histone H2B), IQGAP1 (Ras GTPase-activating-like protein), EEF1A1 (elongation factor 1-alpha), HSPA9 (Stress-70 protein, mitochondrial), HADHA (Trifunctional enzyme subunit alpha). Additionally, the TME is a unique ambient created and shaped by the tumor, which orchestrates molecular and cellular events taking place in surrounding tissues. Interestingly, we found a group of high abundant proteins expressed in the TME surrounding the invasive island. Among them are PALLD (palladin), TNC (tenascin C), LMNA (laminin), VIM (vimentin), and COL1A (collagen-type I-alpha I), which have been described to be involved in cell migration and proliferation. Our preliminary data suggests that the myoma organotypic tumor invasion assay is successful in determining new molecules and potential pathways associated with OSCC tumor invasion. We also present here a new insight for head and neck cancer biomarkers research that can, ultimately, contribute to more individualized treatment of patients affected by OSCC. However, validation of such results in clinical sample cohorts is important for a better understanding of the biological events associate with tumor invasion and disease spread (metastasis) in those patients. Citation Format: Nilva K. Cervigne, Carolina Bitu, Bianca A. Pauletti, Adriana F. Paes Leme, Tuula Salo, Ricardo Della Coletta. In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2014-4998