Adriane S. Siqueira

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour

Siqueira A S, Carvalho M R D, Monteiro A C D, Freitas V M, Jaeger R G & Pinheiro J J V.
(2010) Histopathology 57, 128–137
Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and β1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin α1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin α1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73-induced colocalization of syndecan-1 and β1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin α1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and β1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and β1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion, and MMP secretion of ameloblastoma-derived cells.

Laminin-111 derived peptides AG73 and C16 regulate invadopodia activity of a human adenoid cystic carcinoma cell line

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and distant metastasis long time after treatment. Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are actin-rich membrane protrusions that localize enzymes required for ECM degradation. Breakdown of ECM macromolecules releases fragments and bioactive peptides. We have already demonstrated that laminin-111 and its derived peptides regulate migration, invasion and protease activity of adenocarcinoma cells. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells (CAC2) derived from human adenoid cystic carcinoma. CAC2 cells were treated by AG73 and C16, and subjected to fluorescent gelatin substrate degradation assay. In this assay invadopodia activity areas appear as black dots in a fluorescent background. Both peptides significantly increased invadopodia formation and activity compared to controls. We analyzed putative receptors and signaling pathways related to peptide effects. β1 integrin silencing by siRNA decreased AG73- and C16-induced invadopodia. Furthermore inhibition of Rac1 and ERK signaling pathways decreased both C16- and AG73-related invadopodia activities. We propose that laminin-111 peptides AG73 and C16 increase invadopodia activity in CAC2 cells through β1 integrin. Rac1 and ERK1/2 signaling pathways would transduce signals generated by both peptides.

AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility

Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment.

Invadopodia proteins, cortactin and membrane type I matrix metalloproteinase (MT1‐MMP) are expressed in ameloblastoma

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A Novel Cell Line Derived from Pleomorphic Adenoma Expresses MMP2, MMP9, TIMP1, TIMP2, and Shows Numeric Chromosomal Anomalies

Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1) derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1). Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG). Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

The laminin-derived peptide C16 regulates GPNMB expression and function in breast cancer

ABSTRACT Breast cancer is an important public health problem, and its progression may be related to the extracellular matrix (ECM), which acts as a structural scaffold and instruction source for neoplastic cells. Laminins are ECM proteins regulating tumor biology. The laminin‐derived peptide C16 regulates different properties of tumor cells. Here we analyzed C16‐induced differential gene expression in MDA‐MB‐231 breast cancer cells. MCF‐10A normal‐like breast cells served as control. Among different cancer‐related genes, C16 induced overexpression of GPNMB. This gene encodes a transmembrane protein GPNMB (glycoprotein non‐metastatic B), involved with malignant phenotype of breast cancer cells. Immunoblot validated microarray results. To correlate gene and protein expression with cellular function, we investigated whether C16 would regulate invasion in breast cancer cells. siRNA experiments strongly suggested that C16 and GPNMB cooperate to regulate invasion of highly aggressive MDA‐MB‐231 cancer cells. We addressed regulatory mechanisms involved in C16‐mediated increase of GPNMB protein levels in MDA‐MB‐231 cells, and observed that C16 stimulates &bgr;1 integrin and Src phosphorylation. Furthermore, Src inhibition decreases peptide‐induced GPNMB expression levels. To contextualize in vivo our results in vitro, we addressed GPNMB immunostaining in breast cancer human tissue microarrays. Quantitative immunohistochemistry showed that GPNMB is significantly more expressed in breast cancer compared to normal tissue. We concluded that laminin‐derived peptide C16 regulates gene and protein expression of GPNMB in breast cancer cells. C16 and GPNMB may cooperate to regulate invasion of highly aggressive MDA‐MB‐231 cells, probably through Src signaling. GPNMB presented increased expression in breast cancer in vivo compared to normal breast tissue. HIGHLIGHTSA laminin‐derived peptide C16 regulates GPNMB, a putative breast cancer biomarker.C16 and GPNMB interact to control the aggressive phenotype of breast cancer cells.The mechanism regulating this interaction relies on &bgr;1 integrin and Src.

Keratocystic odontogenic tumor overexpresses invadopodia-related proteins, suggesting invadopodia formation

OBJECTIVE Keratocystic odontogenic tumor (KOT) is an odontogenic neoplasm that shows aggressive clinical behavior and local invasiveness. Invadopodia are actin-rich cellular protrusions exhibiting proteolytic pericellular activity, thereby inducing focal invasion in neoplastic cells and increasing neoplasms aggressiveness. Thus, this study aimed to evaluate immunoexpression of invadopodia-related proteins, cortactin, MT1-MMP, Tks4, and Tks5, in KOT. STUDY DESIGN Immunohistochemistry of 16 cases of KOT, eight cases of calcifying cystic odontogenic tumor (CCOT), and eight samples of the oral mucosa (OM) was carried out to assess the expression of the above described invadopodia-related proteins in the basal and suprabasal layer. RESULTS KOT samples showed higher and significant immunoexpression of cortactin, MT1-MMP, TKs4, and TKs5 compared with the CCOT and OM samples. Significant expression of all these proteins was observed in the basal layer compared with the suprabasal layer in KOT. CONCLUSIONS Overexpression of cortactin, MT1-MMP, TKs4, and TKs5 was observed in KOT compared with samples of CCOT and OM. These proteins were also overexpressed in the basal over the suprabasal layer of KOT samples. Taken together, these results suggest the participation of invadopodia-related proteins on the pathogenesis of this lesion.