Synthetic Lethality Induced By Co-targeting Of PARP1 And Src In Hepatocellular Carcinoma And Its Molecular Mechanism

PurposesPrimary liver cancer is the second leading cause of cancer death worldwide,and hepatocellular carcinoma(HCC)accounts for nearly 90%of all cases.HCC is a highly heterogeneous malignant tumor with complex canceration,and patients with similar clinical symptoms and stages have very different response to drug treatment.Although in recent years,with the development of targeted drugs and immunotherapy,the treatment effect of various cancers has been improved,but the treatment efficiency in HCC is far lower than other cancers.Meanwhile,due to the early concealment of HCC and the difficulty of diagnosis,the majority of patients are diagnosed at an advanced stage,missing the chance for potential treatment such as surgery that could lead to long-term survival.Therefore,exploring new and effective treatment for liver cancer is a key problem that researchers and clinicians need to solve urgently.For liver cancer,a tumor with high heterogeneity and universal resistance to multiple therapies,combination therapy could be a necessary measure to maximize the efficacy.In the study of tumor therapy,combination therapy strategies based on synthetic lethality refer to simultaneously target two different genes or proteins to cause cell death,and have achieved good therapeutic effects in a variety of tumors.During the progression of HCC,the chronic inflammatory environment and persistent replication stress can lead to massive DNA damage in cells,which leads to the continuous activation of poly(adipose diphosphate[ADP]-ribose)polymerase 1(PARP1),a key molecule that senses DNA damage repair.Recent studies have shown that many cancers that are insensitive to immune checkpoint inhibitor therapy could be better treated with synthetic lethal therapy targeting PARP1.Current studies have confirmed that PARP1 plays a key role in the occurrence and progression of HCC,but the effect of targeting PARP1 in clinical trials of HCC was not satisfactory.Therefore,exploring a synthetic lethal partner with PARP1 is a research field with great scientific potential and clinical application prospects.MethodsIn this study,we identified the combination of PARP1 and Src has synthetic lethal potential in HCC through clinical tissue sample collection,experimental testing,and database analysis.It was further confirmed by the malignant phenotype detection of HCC cells such as cell proliferation,clone formation and migration ability,as well as zebrafish and nude mouse HCC tumor-bearing models.In the HCC cell model,the molecular mechanism of the combination therapy was further explored through Western Blot,immunofluorescence,flow cytometry and in vitro kinase experiments.The CRISPR/Cas9 technology was used to construct a PARP1 knockout cell line(PARP1-/1 Huh7),and the molecular mechanism of the synthetic lethality induced by the combined targeting of PARP1 and Src was further explored through cell rescue experiments.Results1.PARP1 and Src were a synthetic lethal combination of HCC with therapeutic potential.Analysis of the collected clinical patient tissue samples and the TCGA database showed that the high expression of PARP1 in HCC was significantly positively correlated with poor prognosis of HCC patients,which provided further evidence for the feasibility of PARP1 inhibitors in the treatment of HCC.The correlation analysis between protein and inhibitor resistance based on CTRP database showed that the non-receptor tyrosine kinase Src may be a synthetic lethal partner with PARP1 in the treatment of HCC.And we further constructed the PARP1-/-Huh7 cells by CRISPR/Cas9 to verify the synergistic effect of targeting Src and PARP1.2.The detection of malignant behavior of HCC cells showed that the combined targeting of PARP1 and Src has a synthetic lethal effect.HCC cells were treated with different concentration gradients of PARP1 or Src inhibitors alone or in combination,and the cell viability was determined by CCK-8 assay and the combination index(CI)was calculated.The results showed that the combined targeting PARP1 and Src had a synthetic lethal effect(CI<1).The results of cell proliferation,clone formation and cell migration experiments showed that this combined targeting strategy could significantly suppress the malignant phenotype of HCC cells.3.HCC tumor-bearing animal experiments verified the synthetic lethality of combined targeting of PARP1 and Src on HCCThe HCC tumor-bearing models of zebrafish and mice were constructed,and the combined treatment of PARP1 and Src inhibitor was given.The results showed that compared with the single-administered group,the combined targeting strategy could induce a synergistic effect,significantly inhibit tumor growth without causing tumor-bearing animals obvious side effects.4.Combined targeting of PARP1 and Src achieves synthetic lethality by inhibiting mTORC1 signaling.The results of Western Blot experiments showed that targeting PARP1 and Src simultaneously could inhibit the proliferation-related signaling pathways such as mTORC1 in HCC cells.At the same time,PARP1 inhibitor and Src inhibitor arrest HCC cells in G2/M and G1/S phases,respectively.Combination therapy have a pleiotropic effect on cell cycle arrest,resulting in DNA damage accumulation in cells,and eventually induce apoptosis.5.PARP1 and Src can interact with each other in HCC cells.In order to further clarify the molecular mechanism of PARP1 and Src as a synthetic lethal combination,we performed Western Blot and immunohistochemical experiments on clinical liver cancer tissue samples,and the results showed that the expression of PARP1 was significantly positively correlated with the activation of Src.The results of database analysis showed that the overactivation of PARP1 and Src was involved in the progression of liver cancer,and was significantly negatively correlated with the survival of patients,suggesting a mutual regulatory effect in the progression of HCC.The interaction between PARP1 and Src was further analyzed by co-immunoprecipitation(Co-IP)and confocal experiments.The results showed that PARP1 and Src were able to interact and co-localize in HCC cells,and the kinase domain of Src and the DNA-binding domain of PARP1 were the molecular basis for their binding.6.Tyrosine phosphorylation of Src on PARP1-Y992 leads to resistance of HCC cells to PARP1 inhibitors.The results of Co-IP and in vitro kinase assays showed that Src could tyrosine phosphorylation modifies PARP1,and further analysis showed that the Y992 of PARP1 was the modification residue of Src.The PARP1-Y992A mutant was transfected into PARP1-/Huh7 cells,and the results showed that phosphorylation at the PARP1-Y992 residue could induce HCC resistance to PARP1 inhibitors.Furthermore,it provides a molecular basis for the combined targeting of PARP1 and Src to achieve synthetic lethality.Conclusions and innovation1.Co-targeting of Src and PARP1 is a novel target combination that induces synthetic lethality in HCC.2.Src-mediated tyrosine phosphorylation at the PARP1-Y992 residue can induce HCC resistance to PARP1 inhibitor therapy.It is suggested that the phosphorylation state of PARP1-Y992 residue has the potential to become a new clinical marker of HCC,and provides a molecular typing strategy for the treatment of HCC patients.3.The combined treatment targeting PARP1 and Src can induce cell cycle arrest by affecting mTORC1 pathway and cell cycle checkpoint proteins in HCC cells,resulting in accumulation of DNA damage to further induce apoptosis and induce synthetic lethality.