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The 5-year survival rate for patients with oral cancer remains at 50%, in large part due the high rate of post-treatment recurrence. In this study, we transfected epithelial-specific integrin αvβ6 and Fyn-kinase, a member of the Src-family kinases, into embryonic murine fibroblasts. In oral cancer, expression of αvβ6 is neo-expressed. Using a variety of in vitro assays, including cell migration and multicellular spheroid formation, we determined that these embryonic fibroblasts expressing αvβ6 and Fyn-kinase were able to acquire an epithelial phenotype. This is in direct contrast to human oral SCC, where expression of αvβ6 with Fyn-kinase promotes epithelial to mesenchymal transition. This demonstrates that signaling pathways can be species-specific., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvβ6 integrin. Invasive oral SCC cells express the αvβ6 integrin, which contains an 11-amino-acid extension on its β-subunit unique to the integrin family. We determined that this β6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the β6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length β6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the β6 integrin cytoplasmic domain.

Squamous cell carcinoma (SCC) accounts for 96 percent of all intraoral malignancies. The five-year survival rate is 50 percent and has not improved in 60 years. During SCC progression, subsets of SCC cells undergo an epithelial-to-mesenchymal transition (EMT) to become highly invasive. The extracellular matrix metalloproteinase inducer (EMMPRIN) contributes to EMT by activating local matrix metalloproteinases (MMPs). In this study, we found that EMMPRIN modulates the invasive phenotype and may be a potential therapeutic target.

Oral cancer is the sixth most frequent cancer worldwide. Prognosis for these patients remains poor. Recently, the epidermal growth factor receptor has been targeted as an adjunct to radiotherapy and surgery with limited success. The authors now present data suggesting that the alphanubeta6 integrin, which is a marker for aggressive oral cancer, may regulate epidermal growth factor receptor expression. The authors suggest perhaps targeting both alphanubeta6 and EGFR may provide additional benefits.

Oral squamous cell carcinoma (SCC) is composed of a heterogeneous population of cells which range anywhere from epithelial to mesenchymal in phenotype. Several oral cancer specimens with antibodies to TRA160, a marker of pluripotent cells, were screened. Compared with the well differentiated lesions, pluripotent cells were more numerous in specimens from poorly differentiated tumors. In vitro, the expression of TRA160 was much greater in invasive oral SCC9beta6 cells compared with the poorly invasive SCC9SN or SCC9beta6D1 cells, which express a truncated beta6. In vitro, pluripotent cells were instrumental in aggressively closing an experimental wound assay. Lastly, TRA-1-60+/beta6+ tumor cells which formed vascular-like structures in vivo were identified. SCC9beta6 cells formed interconnecting channels, whereas SCC9SN cells did not in an in vitro Matrigel angiogenesis assay. The results of this study clearly demonstrated the differential distribution of pluripotent stem cells in oral SCC and that the beta6 integrin may be an important regulatory component of the pluripotent phenotype.

In this study, we evaluated whether the forced expression of beta6 integrin would modulate the epithelial to mesenchymal transition (EMT). When the full length beta6 integrin was expressed in poorly invasive squamous cell carcinoma SCC9 cells, the resulting SCC9/6 cells acquired a fibroblast-like morphology, increased expression of the mesenchymal marker vimentin and reduced expression of the epithelial markers keratin and E-cadherin. SCC9beta6D1 cells, which express a truncated form of beta6 subunit lacking the C-terminal 11 amino acids (AA), retained their epithelial morphology and did not alter vimentin or E-cadherin expression. This suggests that the full-length beta6 subunit can induce EMT in oral SCC cells. We previously showed that expression of beta6 increases both MMP-3 activation and tenascin-C expression and we now show that both molecules are MEK dependent. These results also demonstrate that the terminal 11 AA of beta6 contain information important for establishing an epithelial to mesenchymal transition.

The expression of the integrin alpha(v)beta6 has been correlated with oral SCC invasion. We evaluated its expression in three 4NQO transformed murine oral keratinocyte cell lines (B7E3, B7E11 and B4B8). The B7E3 cells were negative for beta6, whereas the B7E11 and the B4B8 cells were both positive. The beta6 negative B7E3 cells were fibroblast-like in appearance, whereas the B7E11 cells were more epithelial-like. The B4B8 cells were a mixture of the two cell types. Using immunofluorescent microscopy, we found that vimentin was highly expressed in the B7E3 cells, whereas the B7E11 cells keratin positive. The B4B8 cells expressed both filaments. The B7E3 cells formed large tumors when injected into nude mice, whereas the B4B8 cells formed small tumors and the B7E11 cells formed none. These results suggest that the expression of the alpha(v)beta6 integrin suppresses tumor formation and may promote the epithelial phenotype in 4NQO-transformed murine oral keratinocytes.

We have previously shown that the integrin beta6 is neo-expressed in invasive oral squamous cell carcinoma (SCC) and is correlated with oral tumor progression. However, the mechanism by which the integrin beta6 promotes oral tumor progression is not well understood. The purpose of the present study was to determine whether integrin beta6 signaling activates Fyn and thus promotes oral squamous cell carcinoma progression. We analyzed the integrin beta6 signaling complex and investigated the function of these signaling molecules in oral SCC cells. We found that, upon ligation of the integrin beta6 with fibronectin, beta6 complexed with Fyn and activated it. The activation of Fyn recruited and activated focal adhesion kinase to this complex. This complex was necessary to activate Shc and to couple beta6 signaling to the Raf-ERK/MAPK pathway. This pathway transcriptionally activated the matrix metalloproteinase-3 gene and promoted oral SCC cell proliferation and experimental metastasis in vivo. These findings indicate that integrin beta6 signaling activates Fyn and thus promotes oral cancer progression.

Floor of the mouth squamous cell carcinomas exhibit many characteristics that suggest they represent a distinct biological subset within head and neck tumors. The features of preinvasive lateral intraepithelial spread, high rate of conversion of intraepithelial neoplasia to invasive carcinoma, and high incidence of occult metastases, suggest the importance of motility-associated proteins in the pathogenesis of these lesions. Two such proteins, tenascin and beta 6 integrin, are generally overexpressed in squamous carcinomas, and may play a central role in the invasive process of floor of the mouth lesions. The purpose of this study was to evaluate in situ and invasive squamous cell carcinomas from the floor of the mouth for the expression of tenascin and beta 6 integrin. Twenty lesions each of floor of the mouth in situ carcinomas and squamous cell carcinomas, and 10 normal controls were stained for tenascin and beta 6 using a standard immunohistochemical protocol for formalin-fixed specimens. Sections were assessed for staining intensity, pattern, and co-localization. Tenascin was highly expressed at the keratinocyte-connective tissue interface of both in situ and invasive carcinomas. beta 6 was expressed in basal keratinocytes of all in situ and invasive lesions, but was not evident in any of the control epithelia. There was no significant difference in staining of in situ and invasive carcinomas, but there was a significant difference in staining between these lesions and controls. Staining was colocalized in serial sections, supporting a receptor-ligand relationship. Both tenascin and beta 6 were weakly expressed in dysplastic areas adjacent to carcinomas suggesting that changes in the expression of these proteins occurs prior to the invasive phenotype. We conclude that tenascin and beta 6 are overexpressed in in situ and invasive floor of the mouth carcinomas, but that transgression of the basement membrane by neoplastic epithelial cells requires additional changes to the keratinocyte molecular profile.

Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the alpha v beta 6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the beta 6 subunit into poorly invasive SCC9 cells to establish the SCC9 beta 6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of beta 6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to alpha v beta 6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-alpha 5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of beta 6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9 beta 6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-alpha v beta 6 antibodies. The expression of beta 6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9 beta 6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-alpha v beta 6 antibodies. These results demonstrate that expression of beta 6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin alpha v beta 6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.

Abstract: Cytoskeletal reorganization is partially mediated through cofilin, an actin assembly regulatory protein. Cofilin activity is modulated by reversible phosphorylation at Ser3. In this study, using K1735 murine melanoma cells, we examined the relationship between β3-integrin expression, phosphorylation of cofilin, and metalloproteinase production. The levels of phosphorylated cofilin were 10-fold higher in cells expressing αvβ3 than in αvβ3-negative cells when plated on vitronectin for 30 min. However, by 60 min, phosphorylation of cofilin was greater in the β3-negative cells. Expression of the wild type (WT) or non-phosphorylatable cofilin (A3 mutant) increased melanoma cell migration on vitronectin and invasion through a reconstituted basement membrane. Expression of a pseudophosphorylated, poorly active cofilin (E3 mutant) reduced cell motility. Expression of active cofilin accelerated the phosphorylation of FAK at Y397 and at Y576, strongly implicating cofilin as a mediator of cell signaling. The expression of MT1-MMP and MMP2 was also increased by expression of wild type or A3 cofilin. A 50% reduction of both enzymes was observed by the expression of the E3 cofilin. Overexpression of non-phosphorylatable cofilin was sufficient to induce the expression of MT1-MMP and MMP2 in the β3-negative M2Tβ3 cells. Interestingly, the invasion of M2Tβ3 cells could be sustained by overexpression of cofilin A3. These results suggest that the integrin αvβ3 and cofilin together regulate K1735 melanoma cell invasion. [Copyright &y& Elsevier]

Summary: β-6 Integrin, tenascin-C, and MMP-1 (matrix metalloproteinase-1) are invasion-related proteins that are frequently overexpressed in many human malignancies. The objective of this study was to determine whether there is overexpression of these molecules in three types of salivary neoplasms showing markedly different behavior. A total of 55 formalin-fixed, paraffin-embedded archived specimens comprising 19 adenoid cystic carcinomas (ACC), 18 polymorphous low-grade adenocarcinomas (PLGA) and 18 pleomorphic adenomas (PA) were utilized in this study. A standard immunohistochemical technique was used to determine the expression levels of β-6 integrin, tenascin-C, and matrix metalloproteinase-1 (MMP-1) proteins. Sections were assessed semiquantitatively, and tumors were divided into two groups, low-expressors (0–1+) and high-expressors (2–3+) for statistical analysis. Staining was graded as 0 (<1% positive tumor cells), 1+ (<25% positive tumor cells), 2+ (25–50% positive tumor cells), and 3+ (>50% positive cells). The results showed that the malignant tumors were higher expressors of β-6 than the benign tumors. ACCs showed significantly higher expression of β-6 than PAs (p=0.04). No significant difference was observed between ACCs and PLGAs. β-6 expression was rarely seen in normal salivary gland epithelium and was occasionally present in mucosa overlying the tumors. PAs were high-expressors of tenascin-C with a significant difference relative to ACCs (p=0.03). A majority of tumors in all three tumor types showed high expression of MMP1 with expression significantly greater in the PAs compared to ACCs (p=0.008). We conclude that ACCs and PLGAs express β-6, tenascin-C, and MMP-1, but that their expression patterns are not significantly different. β-6 appears to be more closely associated with the malignant tumors, and MMP-1 more closely associated with the benign tumors. We believe that β-6, tenascin-C, and MMP-1 proteins are part of the molecular repertoire used by salivary tumors for malignant invasion and benign tumor expansion. [Copyright &y& Elsevier]

In this study we now show that deposition of the mesenchymal matrix marker, tenascin-C (TN-C), is mediated through β3 expression and activation of Src. There was a striking upregulation of TN-C matrix organization in cell lines expressing β3 and activated Src when compared to cell lines with neither of these attributes. When β3 function was suppressed so was the deposition of TN-C. The same was true for function and activation of Src. When Src was inactive, the deposition of TN-C was low. We also determined that one of the downstream effectors of Src, MAPK, was also required to promote TN-C deposition. When MAPK activation was inhibited, TN-C deposition was also decreased. MMP activation is also implicated in TN-C deposition. The broad spectrum MMP inhibitor, GM6001, suppressed TN-C organization. These results indicate that β3 integrin ligand binding and the activation of the Src/MAPK/MMP pathway modulate deposition of TN-C. [Copyright &y& Elsevier]

The integrin β6 has been shown to promote invasion and experimental metastasis by oral squamous cell carcinoma (SCC). In this study, we demonstrate that the expression of β6 by oral SCC9 cells increased activation of the UPA → MMP3 → MMP9 pathway. We also demonstrate that the deposition of fibronectin and tenascin-C matrices by SCC9β6 cells and peritumor fibroblast cocultures is counter-regulated by the UPA → MMP3 → MMP9 pathway. Suppression of individual components of this pathway increased the deposition of fibronectin, but decreased tenascin-C matrix assembly by the cocultures. When the SCC9β6/PTF cocultures were incubated with TGFβ1, the deposition of fibronectin and tenascin-C as well as the activation of MMP3 and MMP9 was increased. These results indicate that MMP3, MMP9, and TGFβ1 are important for the modulation, composition, and maintenance of the ECM in oral SCC. [Copyright &y& Elsevier]

We have previously shown that the integrin β[sub 6] is neoexpressed in invasive oral squamous cell carcinoma (SCC) and is correlated with oral tumor progression. However, the mechanism by which the integrin β[sub 6] promotes oral tumor progression is not well understood. The purpose of the present study was to determine whether integrin β[sub 6] signaling activates Fyn and thus promotes oral squamous cell carcinoma progression. We analyzed the integrin β[sub 6] signaling complex and investigated the function of these signaling molecules in oral SCC cells. We found that, upon ligation of the integrin β[sub 6] with fibronectin, β[sub 6] complexed with Fyn and activated it. The activation of Fyn recruited and activated focal adhesion kinase to this complex. This complex was necessary to activate Shc and to couple β[sub 6] signaling to the Raf-ERK/MAPK pathway. This pathway transcriptionally activated the matrix metalloproteinase-3 gene and promoted oral SCC cell proliferation and experimental metastasis in vivo. These findings indicate that integrin β[sub 6] signaling activates Fyn and thus promotes oral cancer progression. [ABSTRACT FROM AUTHOR]

Expression of β3 integrins is increased in invasive melanoma. In this study we show that K1735 cell proliferation is enhanced by the expression of either β3 or a constitutively active Src. We investigated possible modulators of FN matrix assembly and found that matrix metalloproteinase 2 (MMP2) was activated by αvβ3. αvβ3 integrin was localized to focal contacts whereas αvβ5 was peripherally distributed. MMP2 was also activated by expression of CASrc. MMP2 activation inversely correlated with FN matrix assembly, in that it dramatically reduced the organization of a FN matrix. K1735 cell migration on VN and invasion through a reconstituted basement membrane were decreased in the presence of anti-MMP2 antibodies. These results demonstrate that the expression of the αvβ3 complex modulates melanoma cell behavior including activation of Src, organization of the cytoskeleton, assembly of the extracellular matrix, cell motility, and activation of MMP2. [Copyright &y& Elsevier]