Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

The onset of p53 loss of heterozygosity is differentially induced in various stem cell types and may involve the loss of either allele

p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.Heterozygosity, caused by a mutation in a single allele of a tumor suppressor gene (TSG), is one of the first steps in malignant transformation.¹ Often, TSGs undergo loss of the wild-type (WT) allele, designated as loss of heterozygosity (LOH).^{2, 3, 4} Patients with the rare cancer predisposition Li-Fraumeni syndrome (LFS), carrying germ-line heterozygous p53 mutations,⁵ apparently exhibit normal development yet later in adult life develop a wide spectrum of tumors; predominantly sarcomas,^{6, 7, 8} where 40–60% of tumors exhibit WT p53 loss of heterozygosity (p53LOH).⁸Giving that cancer development could be associated with stemness deregulation challenges, the notion that the occurrence of p53LOH in stem cells (SCs) may contribute to the emergence of cancer SCs. Genomic fidelity is a hallmark of SCs.⁹ The genome of embryonic stem cells (ESCs) is extremely stable, whereas adult stem cells (ASCs) exhibit a less stable genome.¹⁰ Genetic deregulation in ASCs was shown to be associated with tumor development.^{11, 12, 13} Mesenchymal stem cells (MSCs) that acquire mutations in oncogenes/TSGs such as p53 may function as tumor-initiating cells leading to de-novo sarcomagensis.^{14, 15, 16, 17} Furthermore, MSCs isolated from young mice, aged in culture acquired clinically relevant p53 mutations.¹⁸ In all, these findings suggest a link between p53 inactivation in SCs and tumorigenesis.Although induced pluripotent stem cells (iPSCs) seemed to represent ESCs,^{19, 20} several studies questioned the assumption that iPSCs are as genomically stable as ESCs.^{21, 22, 23, 24} p53 was found to have a major role in the generation of iPSCs both in attenuating reprogramming and controlling the quality of the reprogrammed cells.^{25, 26} An additional role of p53 during reprogramming may be an indirect effect on cell proliferation²⁷ and on the restriction of mesenchymal–epithelial transition during the early phases of reprogramming.²⁸ Importantly, Mutp53 cells exhibiting a fully reprogrammed iPSC phenotype in vitro, form malignant tumors in vivo, instead of the benign teratomas induced by the WTp53-iPSCs.²⁵ As p53 is the guardian of the genome, it is important to investigate how p53LOH would affect genome stability and tumorigenicity of iPSCs.The availability of in vitro SC p53LOH models (iPSCs, MSCs) can help decipher the role of p53LOH in cancer initiation. Indeed, the incidence of p53LOH was found to be extremely different between these SCs. Surprisingly, we found that reprograming of heterozygous p53 (HZp53) fibroblasts, which frequently undergo p53LOH, gave rise to iPSC clones, most of which retained their HZp53 status and exhibited features of normal WTp53-iPSCs. However, p53LOH process is robust in MSCs. Interestingly, single-cell sub-cloning of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that, in addition to the loss of the WTp53, loss of the Mutp53 allele also takes place. Of note, this bi-directional p53LOH occurred in an age-dependent manner linking LOH to aging and tumorigenesis. Surprisingly, most of the p53LOH events in BM progenitors preferred the loss of the Mutp53 allele. Taken together, our results of a bi-directional p53LOH process, accompanied by a burst of DNA repair pathways, may suggest that p53LOH can be regarded as a DNA repair event. In the case of a DNA repair-orientated productive LOH process, where the Mutp53 allele is lost, cells are rescued of tumorigenesis. However, when the WTp53 allele is lost, cells become prone to tumor initiation.