ID3 INDUCES AN ELK-1- AND CASPASE-8-DEPENDENT APOPTOTIC PATHWAY IN SQUAMOUS CARCINOMA CELLS

Abstract

Inhibitors of differentiation/DNA binding (Id) proteins are helix-loop-helix (HLH) transcription factors. The Id protein family (Id1-Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Previously, we found that Id3 induced apoptosis in immortalized human keratinocytes (Simbulan-Rosenthal et al., 2006), consistent with its role as a tumor suppressor (Richter et al., 2012; Schmitz et al., 2012). To investigate the role of Id3 in malignant SCC cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an ETS-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescence analysis, we determined that the tumor size decrease was also mediated through apoptosis. Further, we show that Id3 synergizes with 5-FU and cisplatin therapies for non-melanoma skin cancer cells. Our studies have shown that Id3 induces apoptosis in SCC cells via an Elk-1- and caspase-8-dependent pathway, and this information can provide some strategic insights for new SCC treatments.