Abstract 2025: SIX1 overexpression extends the lifespan of normal human keratinocytes and promotes epithelial-mesenchymal transition

SIX1, a homeodomain-containing transcription factor, has a critical role in the expansion of progenitor cells during embryogenesis. The overexpression of SIX1 contributes to tumorigenesis, promoting malignant transformation and metastasis. Six1 promotes tumor progression by induction of epithelial-to-mesenchymal transition (EMT). In HPV16-transformed human keratinocytes (HKc), SIX1 overexpression produces EMT and a differentiation-resistant phenotype at early stages of in-vitro progression, and transforms cells to malignancy at late stages. We transfected normal HKc with a plasmid encoding human-SIX1 in order to determine to what extent SIX1 overexpression may induce growth, differentiation and EMT changes in normal cells. We also studied the effects of expressing the HRas-V12 oncogene while downregulating the p53 tumor suppressor gene in normal HKc expressing SIX1, in order to push these keratinocytes to a precancerous state. Normal HKc overexpressing SIX1 (HKc/SIX1) or a combination of SIX1, HRas-V12 and p53i (HKc/ALL3) grow continuously while their vector-transfected controls senesced. Continuous growth past the end of the normal lifespan was also observed in normal human fibroblasts overexpressing SIX1. HKc/SIX1 and HKc/ALL3 exhibit a spindle-shape and fibroblastic appearance rather than the cuboidal morphology of epithelial cells. They are also morphologically distinct from SIX1-overexpressing human fibroblasts. HKc /SIX1 and HKc/ALL3 grow best in KSFM supplemented with 10% FBS and 1mM CaCl2, while normal HKc grow best in KSFM with 0.1 mM CaCl2. RT-PCR confirmed that mRNA levels of SIX1 were significantly increased and p53 levels were significantly decreased, and that HRasV12 was expressed in HKc/ALL3. The growth rates of HKc/SIX1 and HKc/ALL3 are comparable to those of early-stage HPV16-immoratlized cells (HKc/HPV16). Invasion assays demonstrated that HKc/SIX1 are more invasive than normal HKc, and their invasiveness is comparable to that of HKc/HPV16. The invasiveness of HKc/ALL3 is greater than that of late-stage, differentiation-resistant HPV-immortalized cells (HKc/DR), which are about 2.5-fold more invasive than HKc/HPV16. In conclusion, SIX1 promotes continued proliferation and invasiveness, signs indicative of cellular transformation, even in the background of normal HKc. Currently, we are exploring the gene expression changes induced by SIX1 in normal cells. Additionally, we plan to investigate whether these cells express stem cell markers. T