Overexpression of the calcium sensor visinin-like protein-1 leads to a cAMP-mediated decrease of in vivo and in vitro growth and invasiveness of squamous cell carcinoma cells.

Visinin-like protein-1 (VILIP-1) is a member of the neuronal EF-hand Ca(2+)-sensor protein family. VILIP-1 is expressed in the central nervous system where it plays a crucial role in regulating cAMP levels, cell signaling, and differentiation. Screening of mouse skin tumor cell lines for differentially expressed genes showed high-level VILIP-1 expression in less aggressive squamous cell carcinoma (SCC) and papilloma cell lines. Conversely, expression was markedly decreased or lost in invasive SCC and spindle cell carcinoma cell lines. In addition, immunohistochemistry of normal skin and primary tumors showed that VILIP-1 is expressed in basal cells of the normal intrafollicular epidermis as well as in basal cells of papillomas. The expression was decreased in low-grade SCCs and disappeared in most high-grade SCCs. When two high-grade carcinoma cell lines were transfected with VILIP1-cDNA, the VILIP-1 transfectants had significantly higher cAMP levels than the respective vector alone-transfected lines. VILIP-1-transfected cells were less invasive (both in vivo and in vitro) than the control transfectants. Reduced invasiveness and elevation of cAMP levels were accompanied by decreased MMP-9, as well as decreased RhoA activity. These results indicate that VILIP-1 plays an important role in regulating tumor cell invasiveness and that its loss could aid in enhancing the advanced malignant phenotype.

Increased furin activity enhances the malignant phenotype of human head and neck cancer cells.

Many proteins are synthesized as inactive proforms requiring a proteolytic processing to render them active. A variety of proteases catalyze these cleavage reactions. Proprotein convertases are a family of serine proteases capable of activating substrates that will subsequently intervene in extracellular matrix (ECM) degradation, cell growth, differentiation and viral pathogenesis. Furin, the prototype of this family, has been implicated in many physiological and pathological processes. Some of its substrates such as TGF-beta, MT-MMP's, and IGFR-1 have been identified. Overexpression of furin has been observed in several human tumors. In this report we demonstrate that overexpression of furin causes a significant increase in the invasive potential of human tumor cells of low and moderate aggressive potential in vitro and in vivo. SCC12 and SCC15 were transfected with furin cDNA, resulting in efficient processing of furin substrates. An in vivo invasion assay showed enhancement of invasive ability. Inhibition of furin activity with the synthetic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl-ketone, CMK, showed a significant decrease in both processing and in vitro invasiveness. A moderate enhancement in proliferation rate was observed when cells were transfected with furin. CMK treatment resulted in a marked reduction of this effect. Tumors obtained after subcutaneous (s.c.) inoculation of furin-overexpressing cells were larger and developed earlier than the controls. Furin overexpression caused an imbalance in the activation of invasion and proliferation-related substrates leading to the acquisition of an advanced malignant phenotype. In addition, inhibition of furin activity decreases substrate activation, proliferation rate, and invasive potential of cancer cells, suggesting that furin is a potentially useful target for therapeutics.

Malignant conversion of non-tumorigenic murine skin keratinocytes overexpressing PACE4.

Proprotein convertases (PCs) have been implicated in tumor cell invasion by processing a variety of substrates including matrix metalloproteinases (MMPs). PACE4, a member of the family of PCs was shown to enhance mouse skin carcinoma progression by increasing tumor cell invasiveness. However, the effects of PACE4 on malignant conversion have not been investigated. In the present study we address the possible role of PACE4 as a trigger of malignant conversion by transfecting with a full-length PACE4 cDNA, three keratinocyte cell lines with no or little tumorigenic potential, i.e. non-tumorigenic BALB/MK-2 cells, tumorigenic non-invasive MT1/2 cells and tumorigenic moderately invasive p117 mouse skin keratinocytes. Overexpression of PACE4 led to a significant increase in the processing of stromelysin-3, a well-characterized substrate of this PC. When assayed for invasive ability, the PACE4-transfected cells were invasive both in vitro and in vivo, whereas their control counterparts were not. In addition, an enhanced processing ability of MT2-MMP a known substrate of PCs was detected in the PACE4-transfected cells. This was accompanied by MMP-2 and MMP-9 activation in PACE4 transfectants. Invasion and MMP processing were remarkably reduced when PACE4 was inhibited with a specific antibody. By triggering the processing of crucial invasion-related proteases, PACE4 is not only able to enhance the invasive ability of malignant cells as demonstrated previously, but also played a significant role in converting non-invasive keratinocytes into malignant cells.