PARP1 Inhibition Retards Tumor Growth By Inducing Oxidative Stress In Tumor Cells And By Impeding M2 Polarization In Tumor-associated Macrophages

PARP is a multifunctional posttranslational modification protein enzyme present in most eukaryotic cells.PARP1 accounts for most of the enzymatic activities of the PARP family memebers.PARP1 functions in DNA damage repair,chromatin remodeling,DNA methylation,apoptosis and the transcriptional regulation of many proinflammatory cytokines.It is activated by DNA breaks and is considered to be a sensor of DNA damage.PARP1 can ADP-ribosylate the histone to facilitate the repair of DNA damage.Therefore,PARP inhibitors alone or in combination with other chemotherapeutic drugs have been used to treat tumors,especially breast and ovarian cancer.PARP1 inhibitors can disrupt the repair of DNA single-strand breaks(SSBs),which will be converted to double-strand breaks(DSBs)during S phase and the latter can be repaired by homologous recombination(HR).It is generally believed that because tumor cells carrying BRCA1 or BRCA2 mutations cannot effectively repair DSBs by homologous recombination,inhibition of SSBs Repair by PARP1 inhibitors will cause synthetic lethality.However,PARP inhibitors are also effective against HR-proficient cancer.Therefore,mechanisms other than disrupting SSBs repair may also operate to confer the antitumor effect.In fact,PARP1 inhibitors were shown to modulate NF-κB activity and regulate HSP70 expression.Thus,the anti-tumor mechanisms of PARP1 inhibition remain to be further clarified.Many cancer drugs exert their therapeutic effect by inducing oxidative stress in the cancer cells.Oxidative stress compromises cell survival by inflicting lesions in macromolecules like DNA.Cancer cells rely on enhanced antioxidant metabolism and increased DNA repair function to survive oxidative assault.Because PARP1 senses DNA-strand breaks and orchestrates their repair,it is reasonable to assume that PARP1 may play an important role in the repair of oxidative DNA damage and inhibition of PARP1 may sensitize cancer cells to oxidative stress-inducing agents.We speculated that in addition to the direct killing effect on tumor cells,PARP inhibition may have an effect on the tumor microenvironment.We therefore studied the therapeutic effect of PARP inhibition from two angles:tumor cells per se and the tumor microenvironment.We found that PARP inhibition can not only reduce cancer cell proliferation by inducing oxidative stress,it can also compromise the tumor-permissive microenvironment by impairing the M2 polarization of tumor-associated macrophages.PART ONEIncreased oxidative stress mediates the antitumor effect of PARP inhibition in ovarian cancerOvarian cancer is one of the most common causes of death among women with gynecologic malignancies.Because of lack of symptoms and biomarkers at early stage,70%of the ovarian cancer cases are already progressed to advanced stages when diagnosed.Debulking surgery followed by platinum-taxane based chemotherapy is the standard care for patients with advanced stage ovarian cancer and overall 5-year survival is abou 47%.PARP inhibitors were shown to be highly successful against ovarian cancer in clinical trials.To understand the role of PARP1 in ovarian cancer,we first examined the expression of PARP1 in in 94 specimens of high grade serous ovarian cancer(HGSOC)and 26 matched fallopian tube(FT)tissues and found that the PARP1 levels are significantly higher in HGSOC when compared to FT tissues,indicating that PARP1 is generally overexpressed in ovarian cancer.To study the biological significance of PARP1 overexpression in the ovarian cancer,we determined the cell cycle distribution and clone formation after inhibiting the PARP1 activity in ovarian cancer cells with PJ-34 and found that PARP1 inhibition resulted in G2 cell cycle arrest and reduced clonogenic ability.To determine how PARP1 inhibition inhibits the proliferation of ovarian cancer cells,we treated different ovarian cancer cell lines with PARP inhibitor PJ-34 and found that inhibition of PARP1 leads to not only an increase in DNA damage,but also an elevation in the levels of reactive oxygen species(ROS).Two additional PARP1 inhibitors,niraparib and oliparib,exhibited similar effects.Importantly,antioxidant N-acetylcysteine(NAC)significantly attenuated the induction of DNA damage and the perturbation of proliferation by PARP inhibition or depletion,suggesting that elevation of ROS mediates the antitumor effect of PARP inhibitors.We further showed that NADPH oxidases(NOX)1 and 4 were significantly upregulated by PARP1 inhibition and were partially responsible for the induction of oxidative stress.Inhibition of NOX1 and NOX4 by GKT137831 greatly improved the proliferation of cancer cells in tumor xenografts in mice treated with PARP inhibitor PJ-34,which was concomitant with a reduction in oxidative base damage.Similarly,depletion of NOX1 and NOX4 attenuated oxidative DNA damage and rescued the growth inhibition of tumors formed by A2780-shPARP1 cells.Our findings suggest that in addition to compromising the repair of DNA damage,PARP inhibition or depletion may exert extra antitumor effect by elevating oxidative stress in ovarian cancer cells.PART TWOReduced M2 polarization of tumor-associated macrophages mediates the antitumor effect of PARP1 inhibitionTumor progression depends on a permissive microenvironment.In the tumor microenvironment,the stromal cells(such as myeloid derived suppressor cells,macrophages,endothelial cells,mesenchymal cells)and secreted inflammatory factors can promote the vascularization of tumor tissue,so that the tumor can evade the immune monitoring of the body.Many early studies suggest that PARP1 is involved in inflammatory response.We speculated that PARP1 inhibition may also affect tumor progression by altering the tumor microenvironment.To this end,we determined the growth of tumor xenografts formed by EL4 lymphoma cells in Parp1 knockout mice.We observed that tumor xenografts were retarded in their growth and had a lower accumulation of tumor-associated macrophages(TAMs)in Parp1 knockout mice when compared to Parp1+/+ mice.We examined the polarization of TAMs in tumor xenografts of Parp1+/+ mice and Parp1 knockout mice by PT-PCR and found that M2 polarization of TAMs was impaired in tumor xenografts of Parp1 knockout mice.These results indicate that PARP1 is required for the accumulation of TAMs and for their M2 polarization.To gain further insight into the function of PARP1 in M2 polarization of macrophages,we conducted RNA-seq of peritoneal macrophages from Parp1+/+ and Parpl knockout mice.KEGG enrichment analysis of the RNA-seq data revealed that multiple signaling pathways that play a role in tumorigenesis have been down-regulated in Parp1 knockout mice,such as the NF-kappa B signaling pathway,Jak-STAT signaling pathway,cytokine-cytokine receptor interaction,and chemokine signaling pathway.Basal expression levels of M2-markers were found to be downregulated in peritoneal macrophages of Parp1 deficient mice.Lactic acid(LA)was previously shown to promote M2 polarization of macrophages.We therefore next tested whether PARP1 is also required for lactic acid-induced M2 polarization.We found that PARP was activated in LA-treated macrophages and the LA-induced M2-polarization was impaired in macrophages derived from Parpl deficient mice.Furthermore,PARP inhibitor PJ-34 also inhibited lactic acid-induced M2 polarization of macrophage.Importantly,Parp1+/+ mice-derived peritonea macrophages resulted in significantly larger tumors than those from Parp1 knockout mice when co-injected with tumor cells.Meanwhile,lactic-acid-stimulated peritonea macrophages resulted in significantly larger tumors than control peritonea macrophages when co-injected with tumor cells.Finally,we tested whether the positive regulation of M2 polarization by PARP1 is mediated by HIF-1a and found that HIF-1a was downregulated when PARP1 is inhibited.ChIP experiment showed that PARP1 can bind to the promoter of HIF-1a.These results suggest that PARP1 may play a tumor-promoting role by promoting M2 polarization of tumor-associated macrophages.The results summarized above reveal two new mechanisms that underlie the anti-tumor effect of PARP inhibitors:One is by elevating oxidative stress in tumor cells;the other is by blocking M2 polarization of the tumor-associated macrophage in the tumor microenvironment.Our findings suggest that the PARP inhibitors may have broad applications as a cancer therapeutic agent.