Lysosomes are single-membraned organelles that mediate the intracellular degradation of macromolecules. Various stress can induce lysosomal membrane permeabilization (LMP), translocating intralysosomal components, such as cathepsins, to the cytoplasm, which induces lysosomal-dependent cell death (LDCD). This study reports that p53 regulates LMP in response to DNA-damaging drugs. Treating wild-type TP53 A549 cells with DNA-damaging drugs (namely, doxorubicin, carboplatin, and etoposide) induced LMP and accelerated cell death more rapidly than treating TP53-knockout (KO) A549 cells. This suggested p53-dependent LMP and LDCD induction in response to DNA damage. LMP was induced by p53-dependent BID upregulation and activation, followed by trans of truncated BID to lysosomes. Simultaneously, autophagy for damaged lysosome elimination (lysophagy) was activated via the p53-mTOR-TEFB/TFE3 pathways in response to DNA damage. These data suggested the dichotomous nature of p53 for LMP regulation; LMP induction and repression via the p53-BID axis and p53-mTOR-TFEB/TFE3 pathway, respectively. Blocking autophagy with hydroxychloroquine or azithromycin as well as ATG5 KO enhanced LMP and LDCD induction after exposure to DNA-damaging drugs. Furthermore, lysosomal membrane stabilization using U18666A, a cholesterol transporter Niemann-Pick disease C1 (NPC1) inhibitor, suppressed LMP as well as LDCD in wild-type TP53, but not in TP53-KO, A549 cells. Thus, LMP is finely regulated by TP53 after exposure to DNA-damaging drugs.

Cancer cells use autophagy for growth, survival, and cytoprotection from chemotherapy. Therefore, autophagy inhibitors appear to be good candidates for cancer treatment. Our group previously reported that macrolide antibiotics, especially azithromycin (AZM), have potent autophagy inhibitory effects, and combination treatment with tyrosine kinase inhibitors or proteasome inhibitors enhances their anti-cancer activity. In this study, we evaluated the effect of combination therapy with DNA-damaging drugs and AZM in non-small-cell lung cancer (NSCLC) cells. We found that the cytotoxic activities of DNA-damaging drugs, such as doxorubicin (DOX), etoposide, and carboplatin, were enhanced in the presence of AZM in NSCLC cell lines, whereas AZM alone exhibited almost no cytotoxicity. This enhanced cell death was dependent on wild-type-p53 status and autophagosome-forming ability because TP53 knockout (KO) and ATG5-KO cells attenuated AZM-enhanced cytotoxicity. DOX treatment upregulated lysosomal biogenesis by activating TFEB and led to lysosomal membrane damage as assessed by galectin 3 puncta assay and cytoplasmic leakage of lysosomal enzymes. In contrast, AZM treatment blocked autophagy, which resulted in the accumulation of lysosomes/autolysosomes. Thus, the effects of DOX and AZM were integrated into the marked increase in damaged lysosomes/autolysosomes, leading to prominent lysosomal membrane permeabilization (LMP) for apoptosis induction. Our data suggest that concomitant treatment with DNA-damaging drugs and AZM is a promising strategy for NSCLC treatment via pronounced LMP induction.

Although recent reports have revealed the importance of the inactivation of both RB1 and TP53 in the transformation from lung adenocarcinoma into neuroendocrine carcinoma (NEC), the requirements for complete transformation into NEC have not been elucidated. To investigate alterations in the characteristics associated with the inactivation of RB1/TP53 and define the requirements for transformation into NEC cells, RB1/TP53 double-knockout A549 lung adenocarcinoma cells were established, and additional knockout of REST and transfection of ASCL1 and POU class 3 homeobox transcription factors (TFs) was conducted. More than 60 genes that are abundantly expressed in neural cells and several genes associated with epithelial-to-mesenchymal transition were up-regulated in RB1/TP53 double-knockout A549 cells. Although the expression of chromogranin A and synaptophysin was induced by additional knockout of REST (which mimics the status of most NECs), the expression of another neuroendocrine marker, CD56, and proneural TFs was not induced. However, coexpression of ASCL1 and POU3F4 in RB1/TP53/REST triple-knockout A549 cells induced the expression of not only CD56 but also other proneural TFs (NEUROD1 and insulinoma-associated 1) and induced NEC-like morphology. These findings suggest that the inactivation of RB1 and TP53 induces a state necessary for the transformation of lung adenocarcinoma into NEC and that further inactivation of REST and coexpression of ASCL1 and POU3F4 are the triggers for complete transformation into NEC.

The p53 protein is crucial for regulating cell survival and apoptosis in response to DNA damage. However, its influence on therapy effectiveness is controversial: when DNA damage is high p53 directs cells toward apoptosis, while under moderate genotoxic stress it saves the cells from death and promote DNA repair. Furthermore, these processes are influenced by the metabolism of transition metals, particularly copper since they serve as cofactors for critical enzymes. The metallochaperone Atox1 is under intensive study in this context because it serves as transcription factor allegedly mediating described effects of copper. Investigating the interaction between p53 and Atox1 could provide insights into tumor cell survival and potential therapeutic applications in oncology. This study explores the relationship between p53 and Atox1 in HCT116 and A549 cell lines with wild type and knockout TP53. The study found an inverse correlation between Atox1 and p53 at the transcriptional and translational levels in response to genotoxic stress. Atox1 expression decreased with increased p53 activity, while cells with inactive p53 had significantly higher levels of Atox1. Suppression of both genes increased apoptosis, while suppression of the ATOX1 gene prevented apoptosis even under the treatment with chemotherapeutic drugs. The findings suggest that Atox1 may act as one of key elements in promotion of cell cycle under DNA-damaging conditions, while p53 works as an antagonist by inhibiting Atox1. Understanding of this relationship could help identify potential targets in cell signaling pathways to enhance the effectiveness of combined antitumor therapy, especially in tumors with mutant or inactive p53.