Knockdown of SMARCA4 leads to ferroptosis of HT1080 cells through inhibition of cholesterol synthesis / 中国病理生理杂志

AIM:To investigate the role and molecular mechanisms of SMARCA4(SWI/SNF-related,matrix-associated,actin-dependent regulator of chromatin,subfamily A,member 4)in ferroptosis.METHODS:(1)Human fi-brosarcoma HT1080 cells were treated with dimethyl sulfoxide(DMSO)and different concentrations(31.25,62.5 and 125 nmol/L)of Ras-selective lethal small molecule 3(RSL3;ferroptosis inducer).Each treatment had 3 replicate wells of cells.The protein levels of SMARCA4 were detected by Western blot.(2)Two small interfering RNAs(siSMARCA4-1 and siSMARCA4-2)were constructed according to the SMARCA4 gene sequence.After SMARCA4 knockdown,each treat-ment had 3 replicate wells of cells,and the protein levels of SMARCA4 were determined by Western blot.Effects of DMSO,necrostatin 2 racemate(Nec-1s;necroptosis inhibitor),Z-VAD(OMe)-FMK(Z-VAD,pan-caspase inhibitor/apoptosis inhibitor)and ferrostatin-1(Fer-1,ferroptosis inhibitor)on cell viability were assessed using high-content analy-sis.The levels of ferroptosis indicators,including prostaglandin-endoperoxide synthase 2(PTGS2)transcription,lipid peroxidation,reactive oxygen species(ROS),labile iron pool(LIP)and glutathione,were determined by RT-qPCR and flow cytometry.The mRNA expression levels of pivotal iron metabolism genes,ferroptosis-related ROS regulatory genes,and cholesterol synthesis-related genes were measured using RT-qPCR.Impact of cholesterol on the cell viability were as-sessed using high-content analysis.(3)Common differential gene analysis and gene ontology(GO)enrichment analysis were performed on published online data.RESULTS:(1)Treatment with RSL3 significantly reduced the protein level of SMARCA4(P＜0.05).(2)Knockdown of SMARCA4 resulted in ferroptosis.(3)Knockdown of SMARCA4 did not induce ferroptosis by modulating the LIP and the transcription levels of ROS-related genes.(4)Knockdown of SMARCA4 affected the pathways associated with the cell membrane,lipid raft,and cholesterol synthesis.(5)Addition of cholesterol to cell culture medium rescued the ferroptosis induced by SMARCA4 knockdown(P＜0.01).CONCLUSION:Treatment with RSL3 reduces the protein level of SMARCA4 in human fibrosarcoma HT1080 cells,and inhibition of cholesterol synthesis by SMARCA4 knockdown leads to the ferroptosis of HT1080 cells.

Inhibition of p53 and/or AKT as a new therapeutic approach specifically targeting ALT cancers

While the majority of all human cancers counteract telomere shortening by expressing telomerase, ~15% of all cancers maintain telomere length by a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). Here, we show that high load of intrinsic DNA damage is present in ALT cancer cells, leading to apoptosis stress by activating p53-independent, but JNK/c-Myc-dependent apoptotic pathway. Notably, ALT cells expressing wild-type p53 show much lower apoptosis than p53-deficient ALT cells. Mechanistically, we find that intrinsic DNA damage in ALT cells induces low level of p53 that is insufficient to initiate the transcription of apoptosis-related genes, but is sufficient to stimulate the expression of key components of mTORC2 (mTOR and Rictor), which in turn leads to phosphorylation of AKT. Activated AKT (p-AKT) thereby stimulates downstream anti-apoptotic events. Therefore, p53 and AKT are the key factors that suppress spontaneous apoptosis in ALT cells. Indeed, inhibition of p53 or AKT selectively induces rapid death of ALT cells in vitro, and p53 inhibitor severely suppresses the growth of ALT-cell xenograft tumors in mice. These findings reveal a previously unrecognized function of p53 in anti-apoptosis and identify that the inhibition of p53 or AKT has a potential as therapeutics for specifically targeting ALT cancers.