Phenotypic Heterogeneity Analysis of APC-Mutant Colon Cancer by Proteomics and Phosphoproteomics Identifies RAI14 as a Key Prognostic Determinant in East Asians and Westerners.

Adenomatous polyposis coli (APC) is an important tumor suppressor and is mostly linked to the regulation of the Wnt/ß-catenin signaling pathway. APC mutation has been identified as an early event in more than 80% of sporadic colorectal cancers (CRCs). Moreover, prognostic differences are observed in CRC patients with APC mutations. Although previous genomics studies have investigated the roles of concomitant gene mutations in determining the phenotypic heterogeneity of APC-mutant tumors, valuable prognostic determinants for APC-mutant CRC patients are still lacking. Based on the proteome and phosphoproteome data, we classified APC-mutant colon cancer patients and revealed genomic, proteomic, and phosphoproteomic heterogeneity in APC-mutant tumors. More importantly, we identified RAI14 as a key prognostic determinant for APC-mutant but not APC-wildtype colon cancer patients. The heterogeneity and the significance of prognostic biomarkers in APC-mutant tumors were further validated in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) colon cancer cohort. In addition, we found that colon cancer patients with high expression of RAI14 were less responsive to chemotherapy. Knockdown of RAI14 in cell lines led to reduced cell migration and changes in epithelial-mesenchymal transition (EMT)-related markers. Mechanistically, knockdown of RAI14 remodeled the phosphoproteome associated with cell adhesion, which might affect EMT marker expression and promote F-actin degradation. Collectively, this work describes the phenotypic heterogeneity of APC-mutant tumors and identifies RAI14 as an important prognostic determinant for APC-mutant colon cancer patients. The prognostic utility of RAI14 in APC-mutant colon cancer will provide early warning and increase the chance of successful treatment.

Deciphering the Clinical Significance and Kinase Functions of GSK3α in Colon Cancer by Proteomics and Phosphoproteomics.

GSK3α and GSK3ß are two GSK3 isoforms with 84% overall identity and 98% identity in their catalytic domains. GSK3ß plays important roles in the pathogenesis of cancer, while GSK3α has long been considered a functionally redundant protein of GSK3ß. Few studies have specifically investigated the functions of GSK3α. In this study, unexpectedly, we found that the expression of GSK3α, but not GSK3ß, was significantly correlated with the overall survival of colon cancer patients in 4 independent cohorts. To decipher the roles of GSK3α in colon cancer, we profiled the phosphorylation substrates of GSK3α and uncovered 156 phosphosites from 130 proteins specifically regulated by GSK3α. A number of these GSK3α-mediated phosphosites have never been reported before or have been incorrectly identified as substrates of GSK3ß. Among them, the levels of HSF1S303p, CANXS583p, MCM2S41p, POGZS425p, SRRM2T983p, and PRPF4BS431p were significantly correlated with the overall survival of colon cancer patients. Further pull-down assays identified 23 proteins, such as THRAP3, BCLAF1, and STAU1, showing strong binding affinity to GSK3α. The interaction between THRAP3 and GSK3α was verified by biochemical experiments. Notably, among the 18 phosphosites of THRAP3, phosphorylation at S248, S253, and S682 is specifically mediated by GSK3α. Mutation of S248 to D (S248D), which mimics the effect of phosphorylation, obviously increased cancer cell migration and the binding affinity to proteins related to DNA damage repair. Collectively, this work not only discloses the specific function of GSK3α as a kinase but also suggests GSK3α as a promising therapeutic target for colon cancer.

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis.

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.

MAPK p38/Ulk1 pathway inhibits autophagy and induces IL-1ß expression in hepatic stellate cells.

In the past, hepatic stellate cells (HSCs) were considered to be noninflammatory cells and to contribute to liver fibrosis by producing extracellular matrix. Recently, it was found that HSCs can also secrete cytokines and chemokines and therefore participate in hepatic inflammation. Autophagy participates in many immune response processes in immune cells. It is unclear whether autophagy is involved in inflammatory cytokine induction in HSCs. MAPK p38, Ulk1 phosphorylation, and the Ulk1-Atg13 complex were analyzed in HSC-T6 cells after LPS treatment. The relationship between autophagy inhibition and inflammation was investigated in primary rat HSCs. We discovered that LPS inhibited autophagy through MAPK p38. The activation of MAPK p38 induced Ulk1 phosphorylation, which disrupted the Ulk1-Atg13 complex and therefore inhibited autophagy. Furthermore, in primary rat HSCs, we demonstrated that autophagy inhibition regulated IL-1ß induction, which depended on the MAPK p38/Ulk1 pathway. Our results reveal a continuous signaling pathway, MAPK p38-Ulk1 phosphorylation-Ulk1-Atg13 disruption, which inhibits autophagy and induces IL-1ß expression in HSCs.NEW & NOTEWORTHY LPS inhibits autophagy in a concentration- and dose-dependent manner in HSC-T6 cells. MAPK p38 induces phosphorylation of Ulk1, which disrupts the Ulk1-Atg13 complex and is therefore required for the inhibition of autophagy by LPS. LPS induces IL-1ß expression via the MAPK p38/Ulk1 pathway in HSCs.

Obesity interacts with hyperuricemia on the severity of non-alcoholic fatty liver disease.

BACKGROUND: A series of evidence revealed that body mass index was an important confounding factor in the research of uric acid and ischemic heart disease/hypertension. The objective of this study was to investigate whether obesity status can modify the association between serum uric acid and the severity of liver damage in NAFLD, and the possible interactive effect of hyperuricemia and obesity. METHODS: We conducted a cross-sectional study in a total of 557 ultrasound diagnosed-NAFLD. The hepatic steatosis and liver fibrosis were quantitatively evaluated by transient elastography. Hyperuricemia was defined as serum uric acid > 420 µmol/L in men, > 360 µmol/L in women and obesity was defined as body mass index ≥ 25 kg/m2. The adjusted OR values of hyperuricemia and obesity were analyzed by multivariate logistic regression analysis, and the additive model was used to investigate the possible interactive effect. RESULTS: Multivariate regression analysis showed that hyperuricemia was associated with serious hepatic steatosis (1.74[1.09-2.79]) and elevated ALT (2.17[1.38-3.41]), but not with advanced fibrosis (1.61[0.91-2.85]). The association was further investigated in different BMI group. Hyperuricemia was associated with higher odds of serious hepatic steatosis (2.02[1.14-3.57]) and elevated ALT (2.27[1.37-3.76]) only in obese NAFLD, not in non-obese subjects. Similarly, patients with hyperuricemia had higher odds of advanced fibrosis in obese subjects (2.17[1.13-4.18]), not in non-obese subjects (0.60[0.14-2.70]). Furthermore, there was an additive interaction between hyperuricemia and obesity on the odds of serious hepatic steatosis (AP: 0.39[0.01-0.77]) and advanced fibrosis. (AP: 0.60[0.26-0.95]). CONCLUSIONS: Hyperuricemia and obesity had a significantly synergistic effect on the hepatic steatosis and fibrosis. Thus, management of uric acid may need to be targeted in obese NAFLD.

Short-term and long-term safety and efficacy of tenofovir alafenamide, tenofovir disoproxil fumarate and entecavir treatment of acute-on-chronic liver failure associated with hepatitis B.

BACKGROUND & AIMS: There is limited evidence on the efficacy and safety of nucleos(t) ide analogues (NAs) in the treatment of HBV-ACLF. Our objective was to evaluate the outcomes among TAF, TDF and ETV, three first-line antivirals against chronic hepatitis B, in patients with HBV-ACLF. METHODS: Patients with HBV-related ACLF were recruited and received daily TAF (25 mg/d), TDF (300 mg/d) and ETV (0.5 mg/d). They were prospectively followed-up. The primary endpoint was overall survival at week 12 and week 48, the secondary endpoints were virological response and biochemical response. RESULTS: Forty gender and age matched eligible subjects were recruited and divided into three groups: TAF group, TDF group and ETV group. By week 48, 8 (80%) patients in TAF group, 6 (60%) patients in TDF group and 17 (85%) patients in ETV group survived without liver transplantation (P = 0.251). After 4 weeks of NAs treatment, all three groups showed paralleling reduction of HBV DNA levels. All three groups presented similar biochemical responses at week 4, patients treated with TAF showed a priority in total bilirubin reduction, albumin and cholesterol maintenance. Additionally, although there was no significant difference in changes of serum urea, serum creatinine, serum cystatin C and estimated GFR among the three groups by treatment week 4, TDF showed unfavorable renal safety even in short -term treatment. The treatment using NAs was well-tolerated and there was no serious drug-related adverse event reported. CONCLUSIONS: TAF, TDF and ETV are of similar efficacy and safety in short-term and long-term treatment of HBV-ACLF. TRIAL REGISTRATION: This study is ongoing and is registered with ClinicalTrials.gov , NCT03640728 (05/02/2019).

Roles of RNF126 and BCA2 E3 ubiquitin ligases in DNA damage repair signaling and targeted cancer therapy.

The dysfunction of E3 ubiquitin ligases is important in the pathogenesis of many human diseases, as they play important roles in multiple cellular processes. In this review, we evaluated the structures, functions and clinical significance of two RING-type E3 ubiquitin ligases from the same subfamily, ring-finger protein 126 (RNF126) and breast cancer associated gene 2 (BCA2). Interestingly, the expression of RNF126 and BCA2 are regulated by multiple signaling pathways, including EGFR, ERK, AKT, and NF-κB. RNF126 and BCA2 appear to be functional mediators for not only DNA damage repair but also cancer development. Due to their significant functions in cell proliferation and DNA damage repair, RNF126 and BCA2 may be two potential diagnostic biomarkers and therapeutic targets for cancers.

Rapamycin could increase the effects of melatonin against age-dependent bone loss.

Previous studies have demonstrated the beneficial effect of melatonin (MEL) on bone tissue and bone metabolism. Rapamycin (RAP) promotes osteoblast proliferation and inhibits osteoclast proliferation, and positively affects bone regeneration; however, reports about effects of RAP on bone loss for aged female rats with MEL administration are limited. This study investigated the impact of treatment with RAP on bone loss for aged female rats with MEL administration. Female Sprague-Dawley rats weighing approximately 520 g were randomly divided into 3 groups of 10: group CON, group MEL and group MEL + RAP and received saline, MEL, RAP plus MEL treatment until death at 12 weeks, respectively. The results of maintaining bone mass and bone strength with RAP plus MEL administration were evaluated by histology, microcomputerized tomography (Micro-CT), gene expression analysis and biomechanical testing. Results from this study indicated that MEL + RAP had stronger effects on the prevention and treatment of osteoporosis than MEL administration. Administration of MEL + RAP produced the strongest effects on bone parameters and strength for distal femurs and regulation of OPG/RANKL signalling pathway-related gene expression. These results seemed to indicate that RAP could increase the effects of MEL on age-dependent bone loss.

Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

Recurrent hyperkalemia in patients with chronic kidney disease and hepatitis C treated with direct antiviral agents.

BACKGROUND: Sofosbuvir is the keystone of direct antiviral agents for the chronic hepatitis C (CHC). The safety of sofosbuvir in patients with stage 4-5 chronic kidney disease (CKD) needs further observation in real world. CASE PRESENTATION: Thirty-three patients with stage 5 CKD and hepatitis C virus (HCV) infection from 2 hemodialysis centers accepted sofosbuvir based treatment as we reported previously. Serum potassium concentrations were tested every 4 weeks or on demand. Ten of 33 patients showed recurrence of hyperkalemia. We summarized the characteristics of hyperkalemia occurrence in these 10 patients. Overall, 24 episodes of hyperkalemia were observed in these 10 patients, 21 were under treatment and 3 were after treatment. Patients with or without hyperkalemia before sofosbuvir treatment didn't show significantly differences in the median frequencies of hyperkalemia episodes during the observation period (3.5 vs. 2, p = 0.264). CONCLUSIONS: Patients with stage 5 CKD and HCV infection treated with sofosbuvir based regimens, even halved sofosbuvir, should be taken caution and closely monitoring serum potassium and renal function is necessary.

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes.

Plasmodium vivax merozoites only invade reticulocytes, a minor though heterogeneous population of red blood cell precursors that can be graded by levels of transferrin receptor (CD71) expression. The development of a protocol that allows sorting reticulocytes into defined developmental stages and a robust ex vivo P vivax invasion assay has made it possible for the first time to investigate the fine-scale invasion preference of P vivax merozoites. Surprisingly, it was the immature reticulocytes (CD71(+)) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71(-)), principally found in the peripheral blood, were rarely invaded. Invasion assays based on the CD71(+) reticulocyte fraction revealed substantial postinvasion modification. Thus, 3 to 6 hours after invasion, the initially biomechanically rigid CD71(+) reticulocytes convert into a highly deformable CD71(-) infected red blood cell devoid of host reticular matter, a process that normally spans 24 hours for uninfected reticulocytes. Concurrent with these changes, clathrin pits disappear by 3 hours postinvasion, replaced by distinctive caveolae nanostructures. These 2 hitherto unsuspected features of P vivax invasion, a narrow preference for immature reticulocytes and a rapid remodeling of the host cell, provide important insights pertinent to the pathobiology of the P vivax infection.

A Basis for Rapid Clearance of Circulating Ring-Stage Malaria Parasites by the Spiroindolone KAE609.

Recent clinical trials revealed a surprisingly rapid clearance of red blood cells (RBCs) infected with malaria parasites by the spiroindolone KAE609. Here, we show that ring-stage parasite-infected RBCs exposed to KAE609 become spherical and rigid, probably through osmotic dysregulation consequent to the disruption of the parasite's sodium efflux pump (adenosine triphosphate 4). We also show that this peculiar drug effect is likely to cause accelerated splenic clearance of the rheologically impaired Plasmodium vivax- and Plasmodium falciparum-infected RBCs.

Glycophorin C (CD236R) mediates vivax malaria parasite rosetting to normocytes.

Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.

In vivo splenic clearance correlates with in vitro deformability of red blood cells from Plasmodium yoelii-infected mice.

Recent experimental and clinical studies suggest a crucial role of mechanical splenic filtration in the host's defense against malaria parasites. Subtle changes in red blood cell (RBC) deformability, caused by infection or drug treatment, could influence the pathophysiological outcome. However, in vitro deformability measurements have not been directly linked in vivo with the splenic clearance of RBCs. In this study, mice infected with malaria-inducing Plasmodium yoelii revealed that chloroquine treatment could lead to significant alterations to RBC deformability and increase clearance of both infected and uninfected RBCs in vivo. These results have clear implications for the mechanism of human malarial anemia, a severe pathological condition affecting malaria patients.

Punicalagin Ameliorates Diabetic Liver Injury by Inhibiting Pyroptosis and Promoting Autophagy via Modulation of the FoxO1/TXNIP Signaling Pathway.

Diabetic liver injury (DLI) is one of the complications of diabetes mellitus, which seriously jeopardizes human health. Punicalagin (PU), a polyphenolic compound mainly found in pomegranate peel, has been shown to ameliorate metabolic diseases such as DLI, and the mechanism needs to be further explored. In this study, a HFD/STZ-induced diabetic mouse model is established to investigate the effect and mechanism of PU on DLI. The results show that PU intervention significantly improves liver histology and serum biochemical abnormalities in diabetic mice, significantly inhibits the expression of pyroptosis-related proteins such as NLRP3, Caspase1, IL-1ß, and GSDMD in the liver of diabetic mice, and up-regulated the expression of autophagy-related proteins. Meanwhile, PU treatment significantly increases FoxO1 protein expression and inhibits TXNIP protein expression in the liver of diabetic mice. The above results are further verified in the HepG2 cell injury model induced by high glucose. AS1842856 is a FoxO1 specific inhibitor. The intervention of AS1842856 combined with PU reverses the regulatory effects of PU on pyroptosis and autophagy in HepG2 cells. In conclusion, this study demonstrates that PU may inhibit pyroptosis and upregulate autophagy by regulating FoxO1/TXNIP signaling, thereby alleviating DLI.

E3 ubiquitin ligase BCA2 promotes breast cancer stemness by up-regulation of SOX9 by LPS.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. Breast cancer stem cells (BCSCs) are believed to play a crucial role in the carcinogenesis, therapy resistance, and metastasis of TNBC. It is well known that inflammation promotes stemness. Several studies have identified breast cancer-associated gene 2 (BCA2) as a potential risk factor for breast cancer incidence and prognosis. However, whether and how BCA2 promotes BCSCs has not been elucidated. Here, we demonstrated that BCA2 specifically promotes lipopolysaccharide (LPS)-induced BCSCs through LPS induced SOX9 expression. BCA2 enhances the interaction between myeloid differentiation primary response protein 88 (MyD88) and Toll-like receptor 4 (TLR4) and inhibits the interaction of MyD88 with deubiquitinase OTUD4 in the LPS-mediated NF-κB signaling pathway. And SOX9, an NF-κB target gene, mediates BCA2's pro-stemness function in TNBC. Our findings provide new insights into the molecular mechanisms by which BCA2 promotes breast cancer and potential therapeutic targets for the treatment of breast cancer.

Viologen-based ionic conjugated mesoporous polymer as the electron conveyer for efficient polysulfide trapping and conversion.

The commercialization of lithium-sulfur (Li-S) batteries has been hindered by the shuttle effect and sluggish redox kinetics of lithium polysulfides (LiPSs). Herein, we reported a viologen-based ionic conjugated mesoporous polymer (TpV-Cl), which acts as the cathode host for modifying Li-S batteries. The viologen component serves as a reversible electron conveyer, leading to a comprehensive enhancement in the adsorption of polysulfides and improved conversion rate of polysulfides during the electrochemical process. As a result, the S@TpV-PS cathode exhibits outstanding cycling performance, achieving 300 cycles at 2.0 C (1 C = 1675 mA g-1) with low decay rate of 0.032% per cycle. Even at a high sulfur loading of 4.0 mg cm-2, S@TpV-PS shows excellent cycling stability with a Coulombic efficiency of up to 98%. These results highlight the significant potential of S@TpV-PS in developing high-performance Li-S batteries.

Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum.

Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long-term survival of the parasite.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.