Matrix metalloproteinase-7 activates heparin-binding epidermal growth factor-like growth factor in cutaneous squamous cell carcinoma.

BACKGROUND: Tumour-specific expression of matrix metalloproteinase (MMP)-7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB). OBJECTIVES: To examine the potential role of MMP-7 in shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in RDEB-associated and sporadic SCCs. METHODS: Tissue microarrays of RDEB-associated SCC (n = 20), non-EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP-7, CD44 variant 3 (CD44v3) and HB-EGF. Shedding of HB-EGF was studied in vitro using two cutaneous SCC cell lines. RESULTS: Immunohistochemical analysis showed that HB-EGF was absent in tumour cells when MMP-7 and CD44v3 colocalized, and that the absence of HB-EGF was more pronounced in RDEB-associated SCCs than in non-EB SCCs. The loss of HB-EGF in MMP-7-CD44v3 double-positive areas was interpreted to indicate shedding and activation of HB-EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP-7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB-EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. CONCLUSIONS: These findings provide evidence for the role of MMP-7 in promoting the growth of cutaneous SCCs by shedding HB-EGF, and identify EGFR signalling as a potential therapeutic target in RDEB-associated SCC and unresectable sporadic cutaneous SCC.

Extended release of adenovirus from silica implants in vitro and in vivo.

Despite promising preclinical results, the clinical benefits of cancer gene therapy have been modest heretofore. The main obstacle continues to be the level and persistence of gene delivery to sufficiently large areas of the tumor. One approach for overcoming this might entail extended local virus release. We studied the utility of silica gel monoliths for delivery of adenovirus to advanced orthotopic gastric and pancreatic cancer tumors. Initially, the biochemical properties of the silica-virus matrix were studied and nearly linear release as a function of time was detected. Virus stayed infective for weeks at +37 degrees C and months at +4 degrees C, which may facilitate storage and distribution. In vivo, extended release of functional replication deficient and also replication-competent, capsid-modified oncolytic viruses was seen. Treatment of mice with pancreatic cancer doubled their survival (P<0.001). Also, silica gel-based delivery slowed the development of antiadenovirus antibodies.

p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells.

Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.

Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells.

Squamous cell carcinoma (SCC) cells of the head and neck specifically express collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the expression of which correlates with their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 and collagenase-1 (MMP-1) expression and invasion of SCC cells via p38 mitogen-activated protein kinase. Here, we have examined the role of Smad signaling in regulating MMP-13 expression and in invasion of head and neck SCC cells. Treatment with TGF-beta resulted in activation of Smad2 and Smad3 in SCC cells, but had no effect on their proliferation or viability. Basal activation of Smad3 and p38 was noted in SCC cells without exogenous TGF-beta stimulation, and adenoviral delivery of Smad7 and dominant-negative Smad3 inhibited p38 activation in these cells. Adenoviral overexpression of Smad3 augmented the upregulatory effect of TGF-beta on MMP-13 expression by SCC cells. Disruption of Smad signaling by adenoviral expression of kinase-defective TGF-beta type I receptor (activin-receptor-like kinase-5), Smad7, and dominant-negative Smad3 potently suppressed the basal and TGF-beta-induced expression of MMP-13 and MMP-1 in SCC cells, and inhibited their basal and TGF-beta-induced invasion through Matrigel and type I collagen. Adenoviral overexpression of Smad7 in cutaneous and oral SCC cells significantly inhibited their implantation in skin of SCID mice and growth of xenografts in vivo, as compared to LacZ adenovirus-transduced control cells. Together, these results show that Smad signaling plays an important role in promoting the invasive phenotype of human head and neck SCC cells by upregulating their collagenase expression.

Adenovirus mediated intra-articular expression of collagenase-3 (MMP-13) induces inflammatory arthritis in mice.

OBJECTIVES: To better understand the role of collagenase-3 (MMP-13) in joint inflammation by investigating the consequences of transient overexpression of human collagenase-3 (matrix metalloproteinase-13 (MMP-13)), introduced by adenoviral gene delivery, in the mouse knee joint. METHODS: A single dose (5x10(7) pfu) of recombinant adenovirus coding either for beta-galactosidase (RAdLacZ) or human MMP-13 (RAdMMP-13) was injected intra-articularly into the knee joint of adult mice. The joints were analysed at frequent intervals up to 4 weeks by histology, immunohistochemistry, and RNA analysis. RESULTS: When RAdLacZ reporter virus was used, adenoviruses efficiently infected synovial cells, chondrocytes of articular cartilage, and hypertrophic chondrocytes of the growth plate. The infection was transient as no reporter gene activity was detected 3 weeks after the injection. After RAdMMP-13 injection into the knee joints, expression of human MMP-13 in joint tissues resulted in an arthritis characterised by recruitment of inflammatory cells and increased production of cytokines and chemokines, synovial hyperplasia, and pannus formation. After the loss of MMP-13 transgene expression at 3 weeks, these inflammatory changes began to diminish. CONCLUSIONS: MMP-13 has a role in the onset of inflammatory reaction in synovium. However, damage to articular cartilage was only rarely detected after the short term overexpression of MMP-13.

Differential patterns of stromelysin-2 (MMP-10) and MT1-MMP (MMP-14) expression in epithelial skin cancers.

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumours. To investigate the role of stromelysin-2 (MMP-10) in growth and invasion of skin tumours, we studied cutaneous carcinomas with high metastatic capacity (squamous cell carcinomas, SCCs), only locally destructive tumours (basal cell carcinomas, BCCs) and pre-malignant lesions (Bowen's disease and actinic keratosis) using in situ hybridization. Expression of MMP-10 was compared with that of stromelysin-1 (MMP-3) and of MT1-MMP, the expression of which has been shown to correlate with tumour invasiveness. MMP-10 was expressed in 13/21 SSCs and 11/19 BCCs only in epithelial laminin-5 positive cancer cells, while premalignant lesions were entirely negative. MT1-MMP mRNA was detected in 19/21 SCCs both in epithelial cancer cells and stromal fibroblasts and in 14/18 BCCs only in fibroblasts. The level of MMP-10 was upregulated in a cutaneous SCC cell line (UT-SCC-7) by transforming growth factor-alpha and keratinocyte growth factor, and by interferon-gamma in combination with transforming growth factor-beta1 and tumour necrosis factor-alpha both in UT-SCC-7 and HaCaT cells. Our results show that MMP-10 expression does not correlate with the invasive behaviour of tumours as assessed by their histology and MT1-MMP expression, but may be induced by the wound healing and inflammatory matrix remodelling events associated with skin tumours.

Expression of human macrophage metalloelastase (MMP-12) by tumor cells in skin cancer.

Matrix metalloproteinases play an essential role in tumor growth and invasion. Different matrix metalloproteinases are often expressed in cancers with distinct patterns. To investigate the role of human macrophage metalloelastase (MMP-12) in epidermal tumors, we studied human macrophage metalloelastase mRNA and protein expression in malignant squamous cell and basal cell carcinomas, and in premalignant Bowen's disease. Human macrophage metalloelastase was detected in 11 of 17 squamous cell carcinomas in epithelial cancer cells, whereas macrophages were positive in 15 of 17 samples. In basal cell carcinomas, human macrophage metalloelastase was more often found in macrophages (seven of 19) than in cancer cells (four of 19). Human macrophage metalloelastase mRNA was also detected in three cell lines derived from squamous cell carcinomas of the head and neck and in transformed HaCaT cells, whereas premalignant tumors and primary keratinocytes were negative for human macrophage metalloelastase mRNA. Western analysis revealed human macrophage metalloelastase protein in squamous cell carcinoma cells. Our results show that human macrophage metalloelastase can be expressed in vivo and in vitro by transformed epithelial cells and indicate that the level of human macrophage metalloelastase expression correlates with epithelial dedifferentiation and histologic aggressiveness.

Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase.

Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by transformed squamous epithelial cells, i.e. squamous cell carcinoma (SCC) cells in culture and in vivo. Here, we have elucidated the signaling pathways regulating MMP-13 expression in transformed human epidermal keratinocytes, i.e. ras-transformed HaCaT cell line A-5 and cutaneous SCC cell line (UT-SCC-7). Treatment with tumor necrosis factor-(alpha) (TNF-(alpha) resulted in activation of extracellular signal-regulated kinase (ERK)1,2, Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) in both cell lines. In addition, transforming growth factor-(beta) (TGF-(beta) activated p38 MAPK in both cell lines, and ERK2 in A-5 cells. Selective inhibition of p38 activity with SB 203580 abolished the enhancement of MMP-13, as well as collagenase-1 (MMP-1) and 92-kDa gelatinase (MMP-9) expression by TNF-(alpha) and TGF-(beta). Blocking the ERK1, 2 pathway by PD 98059 had no effect on the induction of MMP-13 expression by TNF-(alpha) or TGF-(beta), but potently suppressed MMP-1 and MMP-9 production. Inhibition of p38 activity by SB 203580 also suppressed collagenolytic activity produced by both cell lines and inhibited invasion of TNF-(alpha) or TGF-(beta) stimulated A-5 cells through type I collagen and reconstituted basement membrane (Matrigel). These results show that activation of p38 MAPK pathway plays a crucial role in the invasive phenotype of transformed squamous epithelial cells, suggesting p38 MAPK as a target to specifically inhibit their invasion.

Inhibition of collagenase-3 (MMP-13) expression in transformed human keratinocytes by interferon-gamma is associated with activation of extracellular signal-regulated kinase-1,2 and STAT1.

Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate specificity and restricted expression. MMP-13 is specifically expressed by transformed human keratinocytes in squamous cell carcinomas in vivo and its expression correlates with their invasion capacity. Here, we show, that interferon-gamma (IFN-gamma) markedly inhibits expression of MMP-13 by human cutaneous SCC cells (UT-SCC-7) and by ras-transformed human epidermal keratinocytes (A-5 cells) at the transcriptional level. In addition, IFN-gamma inhibits collagenase-1 (MMP-1) expression in these cells. IFN-gamma abolished the enhancement of MMP-13 and MMP-1 expression by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), and inhibited invasion of A-5 cells through type I collagen. IFN-gamma also rapidly and transiently activates extracellular signal-regulated kinase 1,2 (ERK1,2) and blocking ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by specific MEK1,2 inhibitor PD98059 partially (by 50%) prevents Ser-727 phosphorylation of STAT1 and suppression of MMP-13 expression by IFN-gamma. Furthermore, Ser-727 phosphorylation of STAT1 by ERK1,2, or independently of ERK1,2 activation is associated with marked reduction in MMP-13 expression. These observations identify a novel role for IFN-gamma as a potent inhibitor of collagenolytic activity and invasion of transformed squamous epithelial cells, and show that inhibition of MMP-13 expression by IFN-gamma involves activation of ERK1,2 and STAT1.