Jun N-terminal kinase 1 mediates transcriptional induction of matrix metalloproteinase 9 expression.

Tumor cell invasion and metastasis require precise coordination of adherence to extracellular matrix (ECM) and controlled degradation of its components. Invasive cells secrete proteolytic enzymes known as matrix metalloproteinases (MMPs) which degrade specific basement membrane molecules. Expression of these enzymes is regulated by multiple signaling mechanisms, including the mitogen-activated protein kinase (MAPK) pathway. One of the terminal effectors of this signaling cascade is jun N-terminal kinase 1 (JNK1) which phosphorylates the transcription factor c-jun, a component of the AP-1 complex. MMP-9 expression is regulated by two well-characterized AP-1 sites in the promoter of this gene. To determine how JNK1 activity regulated MMP-9 expression in human squamous cell carcinoma lines, we overexpressed this kinase in SCC25 cells. JNK1 overexpression induced MMP-9 protein levels and activity in this cell line. Elevated MMP-9 expression correlated with increased invasion of reconstituted basement membranes by JNK1-overexpressing clones. Site-directed mutagenesis of the MMP-9 promoter revealed that JNK1 cooperated with its transcription factor target c-jun to increase MMP-9 expression at the transcriptional level via the proximal AP-1 site. These results suggest that elevated JNK1 expression may contribute to increased MMP-9 activity and ECM invasion by tumor cells.

The transcription factor ATF-2 inhibits extracellular signal regulated kinase expression and proliferation of human cancer cells.

The mitogen activated protein kinase (MAPK) pathway is a paradigm for regulation of growth factor signaling and cellular proliferation. The MAPK pathway is a major target for signaling by growth factor receptor kinases. The MAPK pathway consists of a series of protein kinases which is activated by phosphorylation of specific amino acid residues in their regulatory domains. The MAPK family can be divided into three subgroups: the extracellular signal regulated kinases (ERKs), the stress activated protein kinase/jun N terminal kinase (SAPK/JNK), and the p38 MAPK. These kinase cascades phosphorylate transcription factor targets such as ets, c-jun, and ATF-2. Of these, little is known about the role of ATF-2 in regulation of MAPK signaling and cellular proliferation. To begin to understand this role, we overexpressed ATF-2 in a human cancer cell line. ATF-2 inhibited the G1/S phase transition of the cell cycle and decreased the proliferation rate of these cells. Decreased proliferation correlated with cell cycle independent inhibition of ERK1 expression in ATF-2 clones. Genetic and pharmacologic inhibition of ERK1 activity was sufficient to reproduce the effects of ATF-2 on cell cycle progression and proliferation. These results indicate a novel role for ATF-2 in cancer cell proliferation and suggest a potential feedback mechanism that regulates MAPK signaling.

Head and neck squamous cell carcinoma lines produce biologically active angiogenic factors.

Angiogenesis is the formation of capillaries from pre-existing blood vessels. Angiogenesis occurs in many developmental, physiologic, and pathologic processes including tumor growth. Previous studies have shown that angiogenesis is required for growth and metastasis of solid tumors. Fibroblast growth factors (FGF-1, -2) and vascular endothelial growth factor (VEGF) are extremely potent angiogenesis inducers by stimulating the proliferation and migration of capillary endothelial cells. Expression of these factors is upregulated in many solid tumors and correlates with high vascularity, lymph node metastasis, and poor clinical prognosis. Few studies have examined whether established head and neck squamous cell carcinoma (SCC) lines produce biologically active angiogenic factors. By immunoprecipitation, we detected FGF and VEGF proteins in cellular lysates of SCC lines. We also detected FGF-1 and -2 proteins in serum-free-conditioned medium from these lines. Conditioned medium from SCC lines significantly increased proliferation of human umbilical vein endothelial cells (HUVEC). This increased proliferation was abrogated by pre-incubation of conditioned medium with neutralizing antibodies to FGFs and VEGF. Conditioned medium from SCC lines also significantly stimulated HUVEC invasion across a reconstituted basement membrane. We concluded that head and neck SCC lines secrete biologically active angiogenic factors.