Mp19-01 functional analysis of cancer stem-like cells by a novel hsp40 family member protein

INTRODUCTION AND OBJECTIVES: Cancer stem-like cells (CSCs) are small population of cancer cells with superior tumor initating, seilf-renewel, and differentiation properties. In the previous study, we found that HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma(RCC). HSF1 has been described to induce heat shock proteins by heat shock stress and be related to initiation and progression of cancer. In this study, we investigated the relations of HSF1 and CSC phenotype including DNAJB8. METHODS: RCC cell lines ACHN was given heat shock by incubating in 45 degrees for 60 minutes. HSF1 and DNAJB8 expression was evaluated by RT-PCR and Western blot analysis. CSCs were isolated by side population method by flowcytometry. SP(side population) cells mean CSCs, MP(main population) cells mean non-CSCs. As an inhibitor of HSF1, temsirolimus (mTOR inhibitor) was used. The cell viability was assessed by MTT assay. RESULTS: Heat shock stress enhanced the expression of DNAJB8 as well as SOX2. The expression of these molecules reached maximum 24 hours after heat shock, and the SP population increased at the same time. When MP cells sorted by flowcytometry were stimulated by heat shock, SP population could be newly detected. This fact implies that heat shock stress could induce the reversion from MP cells to SP cells. Concerning the mechanism of the reversion from MP cells to SP cells by heat shock, HSF1 seems to play an important role. We found that the expression of phosphorylated HSF1 increased by heat shock, although the expression level of HSF1 itself did not change. Interestingly, when HSF1 was knocked down by siRNA, DNAJB8 could not be induced even after heat shock stress. This suggests that the stress-inducible expression of DNAJB8 is regulated by HSF1, which bind to the heat shock element(HSE) in the promoter of DNAJB8 gene and stimulate its transcription. Treatment with temsirolimus to ACHN cells could inhibit phosphorylation of HSF1, followed by down regulation of DNAJB8, resulting in the decrease of SP population. SP cells were more susceptible by temsilolimus treatment than MP cells. CONCLUSIONS: Stress including heat shock such as hyperthermia might induce DNAJB8 expression, which is related to CSC phenotype. Phosphorylation of HSF1 is required in this process. mTOR inhibitor could inhibit CSC specifically by inhibiting phosphorylation of HSF1.