Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells.

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Endostatin induces proliferation of oral carcinoma cells but its effect on invasion is modified by the tumor microenvironment.

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen-fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment.

Phe27Cys polymorphism alters the maturation and subcellular localization of the human delta opioid receptor.

The human delta opioid receptor (hdeltaOR) is a G-protein-coupled receptor that is mainly involved in the modulation of pain and mood. Only one nonsynonymous single nucleotide polymorphism (T80G) has been described, causing Phe27Cys substitution in the receptor N-terminus and showing association with substance dependence. In this study, we expressed the two hdeltaOR variants in a heterologous expression system with an identical genetic background. They differed greatly during early steps of biosynthesis, displaying a significant difference in the maturation efficiency (50% and 85% for the Cys27 and Phe27 variants, respectively). The Cys27 variant also showed accumulation in pre-Golgi compartments of the secretory pathway and impaired targeting to endoplasmic reticulum (ER)-associated degradation following long-term expression. In addition, the cell surface receptors of the Cys27 variant internalized constitutively. Replacement of phenylalanine with other amino acids revealed that cysteine at position 27 decreased the mature receptor/precursor ratio most extensively, suggesting a thiol-mediated retention of precursors in the ER. However, cysteine did not cause a major folding defect because pharmacological characteristics and the maturation kinetics of the variants were identical, and an opioid antagonist was able to enhance the maturation of both variants. We conclude that, instead of causing loss of function, Phe27Cys polymorphism of the hdeltaOR causes a gain-of-function phenotype, which may have implications for the regulation of receptor expression at the cell surface and possibly also for the susceptibility to pathophysiological states.

A novel organotypic model mimics the tumor microenvironment.

Carcinoma cell invasion is traditionally studied in three-dimensional organotypic models composed of type I collagen and fibroblasts. However, carcinoma cell behavior is affected by the various cell types and the extracellular matrix (ECM) in the tumor microenvironment. In this study, a novel organotypic model based on human uterine leiomyoma tissue was established and characterized to create a more authentic environment for carcinoma cells. Human tongue squamous cell carcinoma cells (HSC-3) were cultured on top of either collagen or myoma. Organotypic sections were examined by immunohistochemistry and in situ hybridization. The maximal invasion depth of HSC-3 cells was markedly increased in myomas compared with collagen. In myomas, various cell types and ECM components were present, and the HSC-3 cells only expressed ECM molecules in the myoma model. Organotypic media were analyzed by radioimmunoassay, zymography, or Western blotting. During carcinoma cell invasion, matrix metalloprotease-9 production and collagen degradation were enhanced particularly in the myoma model. To evaluate the general applicability of the myoma model, several oral carcinoma, breast carcinoma, and melanoma cell lines were cultured on myomas and found to invade in highly distinct patterns. We conclude that myoma tissue mimics the native tumor microenvironment better than previous organotypic models and possibly enhances epithelial-to-mesenchymal transition. Thus, the myoma model provides a promising tool for analyzing the behavior of carcinoma cells.

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'.

Arresten, a collagen-derived angiogenesis inhibitor, suppresses invasion of squamous cell carcinoma.

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement membrane collagen IV α1 chain. As the mere prevention of tumor angiogenesis leads to hypoxia that can result in selection of more aggressive cell types and reduces the efficacy of chemotherapy, we aimed here to elucidate how arresten influences the aggressive human carcinoma cells. Arresten efficiently inhibited migration and invasion of HSC-3 tongue carcinoma cells in culture and in an organotypic model. Subcutaneous Arr-HSC xenografts grew markedly more slowly in nude mice and showed reduced tumor cell proliferation, vessel density and local invasiveness. In the organotypic assay, HSC-3 cells overproducing arresten (Arr-HSC) showed induction of cell death. In monolayer culture the Arr-HSC cells grew in aggregated cobblestone-like clusters and, relative to the control cells, showed increased expression and localization of epithelial marker E-cadherin in cell-cell contacts. Application of electric cell-substrate impedance sensing (ECIS) further supported our observations on altered morphology and motility of the Arr-HSC cells. Administration of a function-blocking α1 integrin antibody abolished the impedance difference between the Arr-HSC and control cells suggesting that the effect of arresten on promotion of HSC-3 cell-cell contacts and cell spreading is at least partly mediated by α1ß1 integrin. Collectively, our data suggest novel roles for arresten in the regulation of oral squamous carcinoma cell proliferation, survival, motility and invasion through the modulation of cell differentiation state and integrin signaling.