A small-molecule inhibitor of TopBP1 exerts anti-MYC activity and synergy with PARP inhibitors.

We have previously identified TopBP1 (topoisomerase IIß-binding protein 1) as a promising target for cancer therapy, given its role in the convergence of Rb, PI(3)K/Akt, and p53 pathways. Based on this, we conducted a large-scale molecular docking screening to identify a small-molecule inhibitor that specifically targets the BRCT7/8 domains of TopBP1, which we have named 5D4. Our studies show that 5D4 inhibits TopBP1 interactions with E2F1, mutant p53, and Cancerous Inhibitor of Protein Phosphatase 2A. This leads to the activation of E2F1-mediated apoptosis and the inhibition of mutant p53 gain of function. In addition, 5D4 disrupts the interaction of TopBP1 with MIZ1, which in turn allows MIZ1 to bind to its target gene promoters and repress MYC activity. Moreover, 5D4 inhibits the association of the TopBP1-PLK1 complex and prevents the formation of Rad51 foci. When combined with inhibitors of PARP1/2 or PARP14, 5D4 synergizes to effectively block cancer cell proliferation. Our animal studies have demonstrated the antitumor activity of 5D4 in breast and ovarian cancer xenograft models. Moreover, the effectiveness of 5D4 is further enhanced when combined with a PARP1/2 inhibitor talazoparib. Taken together, our findings strongly support the potential use of TopBP1-BRCT7/8 inhibitors as a targeted cancer therapy.

ACTL6A promotes repair of cisplatin-induced DNA damage, a new mechanism of platinum resistance in cancer.

Cisplatin is a mainstay of systemic therapy for a variety of cancers, such as lung cancer, head and neck cancer, and ovarian cancer. However, resistance to cisplatin represents one of the most significant barriers for patient outcome improvement. Actin-like 6A (ACTL6A) is a component of several chromatin remodeling complexes, including SWI/SNF, NuA4/TIP60 histone acetylase, and INO80. Amplification of ACTL6A gene is often seen in lung squamous cell carcinoma, ovarian cancer, and esophageal cancer, but its significance remains to be fully determined. Here we identify ACTL6A overexpression as a novel cause for platinum resistance. High levels of ACTL6A are associated with chemoresistance in several types of human cancer. We show that overexpression of ACTL6A leads to increased repair of cisplatin-DNA adducts and resistance to cisplatin treatment. In contrast, depletion of ACTL6A inhibits the repair of cisplatin-induced DNA lesions, and increases cisplatin sensitivity in cisplatin-resistant ovarian cancer cells. The regulation of repair by ACTL6A is mediated through the SWI/SNF chromatin remodeling complex. Treatment with a histone deacetylase inhibitor can reverse the effect of ACTL6A overexpression on the repair of cisplatin-induced DNA damage and render cancer cells more sensitive to cisplatin treatment in a xenograft mouse model. Taken together, our study uncovers a novel role for ACTL6A in platinum resistance, and provides evidence supporting the feasibility of using HDAC inhibitors for platinum resistant tumors.

E2F1 sumoylation as a protective cellular mechanism in oxidative stress response.

Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.

Overexpression of TopBP1, a canonical ATR/Chk1 activator, paradoxically hinders ATR/Chk1 activation in cancer.

Topoisomerase IIß-binding protein 1 (TopBP1) is involved in cellular replication among other functions and is known to activate ATR/Chk1 during replicative stress. TopBP1 is also expressed at high levels in many cancers. However, the impact of TopBP1 overexpression on ATR/Chk1 activation and cancer development has not been investigated. Here we demonstrate that the degree of ATR/Chk1 activation is regulated by TopBP1 in a biphasic, concentration-dependent manner in a nontransformed MCF10A cell line and several cancer cell lines, including H1299, MDA-MB468, and U2OS. At low levels, TopBP1 activates ATR/Chk1, but once TopBP1 protein accumulates above an optimal level, it paradoxically leads to lower activation of ATR/Chk1. This is due to the perturbation of ATR-TopBP1 interaction and ATR chromatin loading by excessive TopBP1. Overexpression of TopBP1 thus hinders the ATR/Chk1 checkpoint response, leading to the impairment of genome integrity as demonstrated by the cytokinesis-block micronucleus assay. In contrast, moderate depletion of TopBP1 by shRNA in TopBP1-overexpressing cancer cells enhanced ATR/Chk1 activation and S-phase checkpoint response after replicative stress. The clinical significance of these findings is supported by an association between TopBP1 overexpression and genome instability in many types of human cancer. Taken together, our study illustrates an unexpected relationship between the levels of TopBP1 and the final functional outcome and suggests TopBP1 overexpression as a new mechanism directly contributing to genomic instability during tumorigenesis.

Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin.

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: (i) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and (ii) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

Role of the autotaxin-lysophosphatidate axis in cancer resistance to chemotherapy and radiotherapy.

High expression of autotaxin in cancers is often associated with increased tumor progression, angiogenesis and metastasis. This is explained mainly since autotaxin produces the lipid growth factor, lysophosphatidate (LPA), which stimulates cell division, survival and migration. It has recently become evident that these signaling effects of LPA also produce resistance to chemotherapy and radiation-induced cell death. This results especially from the stimulation of LPA(2) receptors, which depletes the cell of Siva-1, a pro-apoptotic signaling protein and stimulates prosurvival kinase pathways through a mechanism mediated via TRIP-6. LPA signaling also increases the formation of sphingosine 1-phosphate, a pro-survival lipid. At the same time, LPA decreases the accumulation of ceramides, which are used in radiation therapy and by many chemotherapeutic agents to stimulate apoptosis. The signaling actions of extracellular LPA are terminated by its dephosphorylation by a family of lipid phosphate phosphatases (LPP) that act as ecto-enzymes. In addition, lipid phosphate phoshatase-1 attenuates signaling downstream of the activation of both LPA receptors and receptor tyrosine kinases. This makes many cancer cells hypersensitive to the action of various growth factors since they often express low LPP1/3 activity. Increasing our understanding of the complicated signaling pathways that are used by LPA to stimulate cell survival should identify new therapeutic targets that can be exploited to increase the efficacy of chemo- and radio-therapy. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway.

JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

DNA2 Nuclease Inhibition Confers Synthetic Lethality in Cancers with Mutant p53 and Synergizes with PARP Inhibitors.

The tumor suppressor p53 promotes tumor-suppressive activities including cell-cycle inhibition, apoptosis, senescence, autophagy, and DNA repair. However, somatic mutations in the TP53 gene are one of the most common alterations in human cancers. We previously showed that mutant p53 (mutp53) can bind TopBP1, an ATR activator, to attenuate its ATR-activating function. A partially defective ATR function caused by mutp53 makes cancer cells more vulnerable to inhibitors of other TopBP1-independent ATR activators, such as DNA2. DNA2 plays a role in homologous recombination (HR) repair by resecting DNA ends in double-strand breaks and preparing them for invasion of homologous duplex. Here we identify a new DNA2 inhibitor, namely d16, and show that d16 exhibits anticancer activities and overcomes chemotherapy resistance in mutp53-bearing cancers. Similar to DNA2 depletion, d16 treatment results in cell-cycle arrest mainly at S-phase. Moreover, reexpression of mutp53 in a p53-null cancer cell line makes cells more vulnerable to d16-mediated inhibition of ATR activity. As d16 also inhibits HR, a combination of d16 and PARP inhibitors displays synergistic induction of cell death. DNA2 is often overexpressed in cancer, particularly in cancer cells harboring mutp53. Overexpression of DNA2 is associated with poor outcome in ovarian cancer. Overall, our results provide a rationale to target DNA2 as a new synthetic lethality approach in mutp53-bearing cancers, and further extend the benefit of PARP inhibitors beyond BRCA-mutated cancers. SIGNIFICANCE: This study identifies a new DNA2 inhibitor as a synthetic lethal targeted therapy for mutp53-harboring cancers, and provides a new therapeutic strategy by combining DNA2 inhibitors with PARP inhibitors for these cancers.

Virtual screening for LPA2-specific agonists identifies a nonlipid compound with antiapoptotic actions.

Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA2 G protein-coupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L5547924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA2 receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro. Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA(1&2) double-knockout mice reconstituted with the LPA2 receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA2, Na⁺ - H⁺ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA2-specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.

Associations of Warfarin Use with Risks of Ischemic Cerebrovascular Events and Major Bleeding in Patients with Hyperthyroidism-Related Atrial Fibrillation.

The use of oral anticoagulants for patients with new-onset hyperthyroidism-related atrial fibrillation (AF) is controversial. We aimed to evaluate the clinical benefits of warfarin therapy in this population. This retrospective cohort study used a data-cut of Taiwan Health and Welfare Database between 2000 and 2016. We compared warfarin users and nonusers among AF patients with hyperthyroidism. We used 1:2 propensity score matching to balance covariates and Cox regression model to calculate hazard ratios (HRs). The primary outcome was risk of ischemic stroke/transient ischemic attack (TIA), and the secondary outcome was major bleeding. After propensity score matching, we defined 90 and 168 hyperthyroidism-related AF patients with mean (SD) age of 59.9 ± 13.5 and 59.2 ± 14.6 in the warfarin-treated group and untreated group separately. The mean (SD) CHA2DS2-VASc scores for the two groups were 2.1 ± 1.6 and 1.8 ± 1.5, respectively. Patients with hyperthyroidism-related AF receiving warfarin had no significant risk of ischemic stroke/TIA (adjusted HR: 1.16, 95% confidence interval [CI]: 0.52-2.56, p = 0.717) compared to nonusers. There was a comparable risk of major bleeding between those receiving warfarin or not (adjusted HR: 0.91, 95% CI: 0.56-1.47, p = 0.702). The active-comparator design also demonstrated that warfarin use had no significant association with the risk of stroke/TIA versus aspirin use (adjusted HR: 2.43; 95% CI: 0.68-8.70). In conclusion, anticoagulation therapy did not have a statistically significant benefit on ischemic stroke/TIA nor risk of bleeding, among patients with new-onset hyperthyroidism-related AF under a low CHA2DS2-VASc score, by comparing those without use.

RNF144A deficiency promotes PD-L1 protein stabilization and carcinogen-induced bladder tumorigenesis.

RNF144A is a DNA damage-induced E3 ubiquitin ligase that targets proteins involved in genome instability for degradation, e.g., DNA-PKcs and BMI1. RNF144A is frequently mutated or epigenetically silenced in cancer, providing the rationale to evaluate RNF144A loss of function in tumorigenesis. Here we report that RNF144A-deficient mice are more prone to the development of bladder tumors upon carcinogen exposure. In addition to DNA-PKcs and BMI1, we identify the immune checkpoint protein PD-L1 as a novel degradation target of RNF144A, since these proteins are expressed at higher levels in Rnf144a KO tumors. RNF144A interacts with PD-L1 in the plasma membrane and intracellular vesicles and promotes poly-ubiquitination and degradation of PD-L1. Therefore, Rnf144a KO stabilizes PD-L1 and leads to a reduction of tumor-infiltrating CD8+ T cell populations in the BBN-induced bladder tumors. The bladder tumors developed in WT and Rnf144a KO mice primarily express CK5 and CK14, markers of basal cancer subtype, as expected in BBN-induced bladder tumors. Intriguingly, the Rnf144a KO tumors also express GATA3, a marker for the luminal subtype, suggesting that RNF144A loss of function promotes features of cellular differentiation. Such differentiation features in Rnf144a KO tumors likely result from a decrease of EGFR expression, consistent with the reported role of RNF144A in maintaining EGFR expression. In summary, for the first time our study demonstrates the in vivo tumor suppressor activity of RNF144A upon carcinogenic insult. Loss of RNF144A promotes the expression of DNA-PKcs, BMI1 and PD-L1, likely contributing to the carcinogen-induced bladder tumorigenesis.

MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis.

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity.

Cell Cycle-Dependent Switch of TopBP1 Functions by Cdk2 and Akt.

Cdk2-dependent TopBP1-treslin interaction is critical for DNA replication initiation. However, it remains unclear how this association is terminated after replication initiation is finished. Here, we demonstrate that phosphorylation of TopBP1 by Akt coincides with cyclin A activation during S and G2 phases and switches the TopBP1-interacting partner from treslin to E2F1, which results in the termination of replication initiation. Premature activation of Akt in G1 phase causes an early switch and inhibits DNA replication. TopBP1 is often overexpressed in cancer and can bypass control by Cdk2 to interact with treslin, leading to enhanced DNA replication. Consistent with this notion, reducing the levels of TopBP1 in cancer cells restores sensitivity to a Cdk2 inhibitor. Together, our study links Cdk2 and Akt pathways to the control of DNA replication through the regulation of TopBP1-treslin interaction. These data also suggest an important role for TopBP1 in driving abnormal DNA replication in cancer.

Regulation of the LPA2 receptor signaling through the carboxyl-terminal tail-mediated protein-protein interactions.

While it is well known that lysophosphatidic acid (LPA) mediates diverse physiological and pathophysiological responses through the activation of G protein-coupled LPA receptors, the specificity and molecular mechanisms by which different LPA receptors mediate these biological responses remain largely unknown. Recent identification of several PDZ proteins and zinc finger proteins that interact with the carboxyl-terminal tail of the LPA2 receptor provides a considerable progress towards the understanding of the mechanisms how the LPA2 receptor specifically mediates LPA signaling pathways. These findings have led to the proposal that there are at least two distinct protein interaction motifs present in the carboxyl-terminus of the LPA2 receptor. Together, these data provide a new concept that the efficiency and specificity of the LPA2 receptor-mediated signal transduction can be achieved through the cross-regulation between the classical G protein-activated signaling cascades and the interacting partner-mediated signaling pathways.

c-Src-mediated phosphorylation of TRIP6 regulates its function in lysophosphatidic acid-induced cell migration.

TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in cell motility. Previously we have shown that TRIP6 binds to the LPA2 receptor and associates with several components of focal complexes in an agonist-dependent manner and, thus, enhances lysophosphatidic acid (LPA)-induced cell migration. Here we further report that the function of TRIP6 in LPA signaling is regulated by c-Src-mediated phosphorylation of TRIP6 at the Tyr-55 residue. LPA stimulation induces tyrosine phosphorylation of endogenous TRIP6 in NIH 3T3 cells and c-Src-expressing fibroblasts, which is virtually eliminated in Src-null fibroblasts. Strikingly, both phosphotyrosine-55 and proline-58 residues of TRIP6 are required for Crk binding in vitro and in cells. Mutation of Tyr-55 to Phe does not alter the ability of TRIP6 to localize at focal adhesions or associate with actin. However, it abolishes the association of TRIP6 with Crk and p130cas in cells and significantly reduces the function of TRIP6 to promote LPA-induced ERK activation. Ultimately, these signaling events control TRIP6 function in promoting LPA-induced morphological changes and cell migration.

ATM-mediated stabilization of hMutL DNA mismatch repair proteins augments p53 activation during DNA damage.

Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. Here we show that DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response.

Regulation of E2F1 by BRCT domain-containing protein TopBP1.

The E2F transcription factor integrates cellular signals and coordinates cell cycle progression. Our prior studies demonstrated selective induction and stabilization of E2F1 through ATM-dependent phosphorylation in response to DNA damage. Here we report that DNA topoisomerase IIbeta binding protein 1 (TopBP1) regulates E2F1 during DNA damage. TopBP1 contains eight BRCT (BRCA1 carboxyl-terminal) motifs and upon DNA damage is recruited to stalled replication forks, where it participates in a DNA damage checkpoint. Here we demonstrated an interaction between TopBP1 and E2F1. The interaction depended on the amino terminus of E2F1 and the sixth BRCT domain of TopBP1. It was specific to E2F1 and was not observed in E2F2, E2F3, or E2F4. This interaction was induced by DNA damage and phosphorylation of E2F1 by ATM. Through this interaction, TopBP1 repressed multiple activities of E2F1, including transcriptional activity, induction of S-phase entry, and apoptosis. Furthermore, TopBP1 relocalized E2F1 from diffuse nuclear distribution to discrete punctate nuclear foci, where E2F1 colocalized with TopBP1 and BRCA1. Thus, the specific interaction between TopBP1 and E2F1 during DNA damage inhibits the known E2F1 activities but recruits E2F1 to a BRCA1-containing repair complex, suggesting a direct role of E2F1 in DNA damage checkpoint/repair at stalled replication forks.

TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation.

The elevated lysophosphatidic acid signaling has been causally linked to cancer-associated inflammation and tumorigenesis through upregulation of nuclear factor-κB signaling. However, how this signaling event is regulated has not yet been fully understood. Here we demonstrate that TRIP6, an LPA2 receptor-interacting adaptor protein, functions as a positive regulator of nuclear factor-κB and JNK signaling through direct binding to and activation of the E3 ligase TRAF6. Upon lysophosphatidic acid stimulation, TRIP6 recruits TRAF6 to the LPA2 receptor and promotes lysophosphatidic acid-induced JNK and nuclear factor-κB activation in a TRAF6-dependent manner. TRIP6 antagonizes the recruitment of deubiquitinases A20 and CYLD to TRAF6, thus sustaining the E3 ligase activity of TRAF6 and augmenting lysophosphatidic acid-activated nuclear factor-κB signaling. In contrast, depletion of TRIP6 by TRIP6-specific shRNA or Cas9/sgRNA greatly enhances the association of TRAF6 with A20 and CYLD, and attenuates lysophosphatidic acid-induced muclear factor-κB and JNK/p38 activation in ovarian cancer cells. On the other hand, TRAF6 also regulates TRIP6 by facilitating its binding to nuclear factor-κB p65 and phosphorylation by c-Src. Together, TRIP6 cooperates with TRAF6 to regulate the LPA2 receptor signaling, which may ultimately contribute to chronic inflammation, apoptotic resistance and cell invasion.