Versican G3 Promotes Mouse Mammary Tumor Cell Growth, Migration, and Metastasis by Influencing EGF Receptor Signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis

The Motile Breast Cancer Phenotype Roles of Proteoglycans/Glycosaminoglycans.

The consecutive stages of cancer growth and dissemination are obligatorily perpetrated through specific interactions of the tumor cells with their microenvironment. Importantly, cell-associated and tumor microenvironment glycosaminoglycans (GAGs)/proteoglycan (PG) content and distribution are markedly altered during tumor pathogenesis and progression. GAGs and PGs perform multiple functions in specific stages of the metastatic cascade due to their defined structure and ability to interact with both ligands and receptors regulating cancer pathogenesis. Thus, GAGs/PGs may modulate downstream signaling of key cellular mediators including insulin growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), estrogen receptors (ERs), or Wnt members. In the present review we will focus on breast cancer motility in correlation with their GAG/PG content and critically discuss mechanisms involved. Furthermore, new approaches involving GAGs/PGs as potential prognostic/diagnostic markers or as therapeutic agents for cancer-related pathologies are being proposed

HA metabolism in skin homeostasis and inflammatory disease

Hyaluronan (HA), an unsulfated glycosaminoglycan, is an important component of the complex extracellular matrix network which surrounds and supports cells in tissues. HA is detected in all vertebrate tissues, but the bulk of HA is produced and deposited in the skin. In this review we focus on the role of HA in skin-associated inflammatory disease and wound healing. Properties of HA are directly dependent on its molecular weight. Thus, high molecular weight HA (HMWHA) is deposited in normal tissues during homeostasis and promotes their stability whereas low molecular weight HA fragments (LMWHA), on the other hand, may arise from enzymatic or chemical activities. The degradation of HMWHA to LMWHA fragments, often leads to the generation of biologically active oligosaccharides with different properties and postulated functions in wound scar formation and inflammation. More detailed studies of HA involvement in skin-associated inflammatory disease may result in novel treatment modalities

Contact allergen (PPD and DNCB)-induced keratinocyte sensitization is partly mediated through a low molecular weight hyaluronan (LMWHA)/TLR4/NF-κB signaling axis

Allergic contact dermatitis (ACD) is caused by topical exposure to chemical allergens. Keratinocytes play a key role in innate immunity, as well as in ACD progression. The transmembrane Toll-like receptor 4 (TLR4), strongly implicated in skin inflammation, has the ability to bind Damage Associated Molecular Patterns (DAMPs), like Low Molecular Weight Hyaluronan (LMWHA). Previously, we had determined that p-phenylenediamine (PPD) and 2,4-dinitrochlorobenzene (DNCB) modulate keratinocyte HA deposition in a manner correlated to their sensitization. In the present study, we aimed to investigate putative co-operation of HA and TLR4 in the process of PPD and DNCB-induced keratinocyte activation. Contact sensitizers were shown to significantly increase the expression of Hyaluronan Synthases (HAS) and TLR4 in NCTC2544 human keratinocytes, as demonstrated by western blot and Real-Time PCR. These data, in correlation to earlier shown enhanced HA degradation suggest that the contact sensitizers facilitate HA turnover of keratinocytes and increase the release of pro-inflammatory, LMWHA fragments. Treatment with exogenous LMWHA enhanced TLR4, HAS levels and Nuclear factor-kappa beta (NF-\u3ba\u392) activation. PPD, DNCB and LMWHA-effects were shown to be partly executed through TLR4 downstream signaling as shown by Real-Time, western blot, siRNA and confocal microscopy approaches. Specifically, PPD and DNCB stimulated the activation of the TLR4 downstream mediator NF-\u3baB. Therefore, the shown upregulation of TLR4 expression is suggested to further facilitate the release of endogenous, bioactive HA fragments and sustain keratinocyte activation. In conclusion, keratinocyte contact allergen-dependent sensitization is partly mediated through a LMWHA/TLR4/ NF-\u3baB signaling axis

Hyaluronan regulates chemical allergen-induced IL-18 production in human keratinocytes

Interleukin-18 (IL-18) has been shown to play a key proximal role in the induction of allergic contact dermatitis. Low molecular weight hyaluronan (LMWHA), an endogenous molecule and a member of the so-called damage associated molecular patterns (DAMPs), has been suggested to elicit immune-stimulatory effects. The purpose of this study was to examine the role of hyaluronan (HA) degradation in IL-18 production in human keratinocytes following stimulation with the contact sensitizers 2,4-dinitrochlorobenzene (DNCB) and PPD. IL-18 production in the human keratinocyte cell line NCTC2544 was measured by ELISA, whereas changes in HA metabolism were determined by Real-time PCR and immunofluorescence. Both contact allergens were able to enhance hyaluronidase (HYAL) 1 and 2 expression inducing HA degradation. Modulation of HA production, by HYAL or aristolochic acid pre-treatment, resulted in a significant reduction of contact allergen-induced IL-18 production. Oxidative stress appears to be the initial step in KC activation, as all the sequels of events can be blocked using antioxidants. This is the first indication that LMWHA can act as a DAMP in keratinocytes. In conclusion LMWHA fragments are important mediators in the process of contact sensitisation leading to IL-18 dependent responses