Key proteins of invadopodia are overexpressed in oral squamous cell carcinoma suggesting an important role of MT1-MMP in the tumoral progression

Invadopodia is one of the major mechanisms that metastatic neoplastic cells with high invasion capacity use the to spread to other tissues [5, 13, 23]. Our results showed overexpression of invadopodia proteins in OSCC in comparison with OM, suggesting the invadopodia is active in OSCC and may be behind of the unfavourable prognosis observed in this tumour [24]. Immunostaining of MT1-MMP, the most important protease involved in ECM in invadopodia front, increased according to tumour progresses to less favourable clinical-stages and histological grade.

The neoplastic progression that triggers metastasis is a complex process that has been attributed, in part to invadopodia, due to their capacity of focal invasion of the ECM [20, 25]. Epithelial cancer cells are subject to several stimuli that can activate the EGFr signalling, resulting in phosphorylation of TKs5. TKs5 then recruits and activates cortactin [26]; therefore, initiating actin rearrangement and polymerisation [5]. Cortactin [27] and Tks4 [17] are related to the recruitment of proteases to invadopodia front; but TKs4 plays a leading role in the localisation and stabilisation of MT1-MMP, resulting in activation of this and other MMPs. Because of this crosstalk between TKs5, cortactin, Tks4 and MT1-MMP, these proteins are recognised as key proteins for invadopodia formation and function.

Stylli et al. [28] verified the participation of TKs5 in the formation of invadopodia as an important component in the promotion of actin projections in melanoma. Another study has reported the expressive presence of TKs5 in breast and colon cancer, especially in cases of worse prognosis [29]. In prostate cancer, high expression of TKs5 was observed in high-grade tumours [13], suggesting that overexpression of TKs5 was associated with higher aggressiveness of this lesion. In our samples, the expression of TKs5 was uniform independent of cell differentiation, suggesting the overexpression of this protein was independent of the histological grade. However, in stage I, TKs5 expression was higher than in stage III, showing a decreasing tendency in advanced tumours.

Increased cortactin expression has been observed in OSCC and associated with spreading to lymph nodes. Eleven of 39 cases where cortactin was overexpressed showed lymph nodes invasion, suggesting this protein as a prognostic marker for invasive and metastatic OSCC [30]. Hofman et al. [31] evaluated 77 cases of OSCC and found a correlation between advanced clinical staging, histological grade and lower survival with higher expression of cortactin. Indeed, cortactin was overexpressed in OSCC compared to OM, however, we found no correlation between clinical stage and a higher expression in well differentiated tumours (GI) than in GII and GIII. These results suggest that more investigations are needed to clarify the role of cortactin as a determinant of prognosis marker. For instance, in oesophageal SCC, higher scores on cortactin immunolabelling were significantly associated with a higher histological grade and a worse prognosis; however, when a multivariate analysis was carried out, higher scores of cortactin weren´t an independent risk factor for lower survival rate [32].

There are few studies that have evaluated the expression of TKs4 in oral lesions. Ribeiro Ribeiro et al. [33] observed the expression of TKs4, cortactin, TKs5 and MT1-MMP in odontogenic keratocyst and found that these proteins were overexpressed in this aggressive odontogenic benign lesion, suggesting the formation and participation of invadopodia in the progression of this lesion. In melanoma, the expression of TKs4 was classified as high in both, metastatic and non-metastatic tumours, with no statistical difference between them [34].

In malignant epithelial tumours, neoplastic invasion has in the basal lamina its first physical barrier to transpose, so the initial steps for tumour dissemination requires localized proteolytic remodelling [25]. The basal lamina is composed mainly of type IV collagen and laminin [35], which protease degradation is a fundamental step for tumour cells to reach the adjacent tissues [36]. MT1-MMP was the first membrane-anchored metalloproteinase to be discovered and currently it stands out for its overexpression in epithelial malignancies [36, 37].

The physiological expression of MT1-MMP on the cell surface is generally low since it is rapidly internalised by endocytosis. However, its increased concentration in the plasma membrane is correlated with malignancy [38]. Elevated levels of MT1-MMP that cause ECM remodelling are associated increased trafficking of proteases in microtubule of regions were invadopodia is formed. The interaction of MT1-MMP-containing endosomes and the actin network are responsible for the recycling and stabilisation of this protein [25].

In this study, we found MT1-MMP overexpressed in OSCC, which is a common finding in malignancies [39]. The role of MT1-MMP has been studied in vitro using breast cancer cells and melanoma, which demonstrated that the ECM degradation mediated by invadopodia is MT1-MMP-dependent [40]. MT1-MMP accumulation and activity is observed in regions forming invadopodia in breast cancer cells, which reinforces the participation of this protease in invadopodia physiopathology and tumour progression [25].

Indeed, invadopodia is present not only in established SCC, but its expression is also seen in premalignant nondysplastic and dysplastic lesions. Ali et al. showed that progressing premalignant lesions that evolved to SCC have higher invadopodia scores, which were based on invadopodia markers, than non-progressing lesions [41]. This finding places invadopodia as an important marker for predicting malignant transformation and a potential determinant of treatment, where high-risk cases could require further aggressive treatment.

It is well-known that chronic inflammation creates an environment that helps the development of many cancers. A TNF-driven oral inflammation promotes a pro-inflammatory environment and proinvasion phenotype leading to the recruitment and activation of inflammatory cells and invadopodia formation [42]. An increase in local neutrophil infiltration is seen in aggressive cases with decreased survival rate [42] and also in progressing cases that undergo malignant transformation [41]. The identification of invadopodia promotors and regulators are quite important and can be a target for specific drugs development and anticancer therapy [43].

Thus, our results showed MT1-MMP expression as an important marker in OSCC. MT1-MMP immuoexpression gradually increased according tumour progresses from GI to GIII and from stage I to stage III. While clinical-stage and histological grade are so far the most used parameters to determine prognosis, our results have demonstrated a correlation between MT1-MMP expression and cancer progression, which can be used as a prognostic marker in OSCC.