PARP1基因敲除HCT116细胞

BACKGROUND & AIMS:Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. METHODS:Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. RESULTS:We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. CONCLUSIONS:Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3' flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3' nuclease, excises flaps at the immediate 3' end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.

Although it has been established that cannabidiol (CBD), the major non-psychoactive constituent of cannabis, exerts antitumoral activities, the exact mechanism(s) via which tumor cells are killed by CBD are not well understood. This study provides new insights into the potential mechanisms of CBD-induced mutual antagonism of apoptosis and macroautophagy using wild type (HCT116 p53wt, LS174T p53wt), knockout (HCT116 p53) and mutant (SW480 p53mut) human colorectal cancer cells (CRC). CBD causes a more pronounced loss in the viability of p53wt cells than p53 and p53mut cells, and a 5-week treatment with CBD reduced the volume of HCT116 p53wt xenografts in mice, but had no effect on the volume of HCT116 p53 tumors. Mechanistically, we demonstrate that CBD only significantly elevates ROS production in cells harboring wild-type p53 (HCT116, LS174T) and that this is associated with an accumulation of PARP1. CBD-induced elevated ROS levels trigger G0/G1 cell cycle arrest, a reduction in CDK2, a p53-dependent caspase-8/9/3 activation and macroautophagy in p53wt cells. The ROS-induced macroautophagy which promotes the activation of keap1/Nrf2 pathway might be positively regulated by p53wt, since inhibition of p53 by pifithrin-α further attenuates autophagy after CBD treatment. Interestingly, an inhibition of heat shock protein 70 (Hsp70) expression significantly enhances caspase-3 mediated programmed cell death in p53wt cells, whereas autophagy-which is associated with a nuclear translocation of Nrf2-was blocked. Taken together, our results demonstrate an intricate interplay between apoptosis and macroautophagy in CBD-treated colorectal cancer cells, which is regulated by the complex interactions of p53wt and Hsp70.

PURPOSE:Proton beam radiation therapy (PBT) offers superior accuracy of dose deposition, reducing the risk of adverse effects to surrounding healthy tissues. However, despite high medical need, molecular and cellular determinants of radiosensitivity to PBT remain underexplored, and prognostic biomarkers and therapeutic targets informing precision medicine strategies for PBT are mostly missing. This study aimed to investigate the role of DNA double-strand break (DSB) repair pathways in shaping tumor response to proton versus photon radiation. METHODS AND MATERIALS:The study employed genetic and pharmacologic methods to impair DSB repair, including CRISPR-Cas9 gene editing to generate DSB repair deficient (ATM, PARP1, and BRCA2 knockout) A549 and HCT116 cell lines, and pharmacologic inhibitors of ATM and PARP using KU55933 or AZD1390 and olaparib, respectively. Cellular responses to photon (x-rays) and proton irradiation were evaluated through clonogenic survival assays, crystal violet proliferation, and annexin V/7AAD apoptosis assays. To investigate DNA repair mechanisms, U2OS reporter systems were employed, complemented by chromosomal aberration analysis, and pulsed-field gel electrophoresis. Finally, the translational relevance of the findings was validated using the chorioallantoic membrane assay closer representing an in vivo situation. RESULTS:PBT triggered a stronger activation of resection-dependent DNA repair pathways, primarily homologous recombination and alternative end-joining (alt-EJ), compared with photon irradiation. This increased activation was further supported by classical cytogenetics results. Tumor cells deficient in BRCA2, ATM, or PARP1 showed significantly increased sensitivity to PBT, highlighting enhanced relative biological effectiveness in both, in vitro and in the chorioallantoic membrane model. Importantly, combining PBT with olaparib, AZD1390 or KU55933 potentiated tumor cell killing, even in repair-proficient models, showing synergy not observed with photons. CONCLUSIONS:The observed genotype-specific or drug-induced increase in radiosensitivity toward PBT highlights the promise of genetic profiling of DSB repair defects for biology-driven patient stratification and the use of PARP inhibitors in guiding personalized proton radiation therapy strategies.