The role of E3 ubiquitin ligase HECTD3 in cancer and beyond.

Ubiquitin modification plays significant roles in protein fate determination, signaling transduction, and cellular processes. Over the past 2 decades, the number of studies on ubiquitination has demonstrated explosive growth. E3 ubiquitin ligases are the key enzymes that determine the substrate specificity and are involved in cancer. Several recent studies shed light on the functions and mechanisms of HECTD3 E3 ubiquitin ligase. This review describes the progress in the recent studies of HECTD3 in cancer and other diseases. We propose that HECTD3 is a potential biomarker and a therapeutic target, and discuss the future directions for HECTD3 investigations.

USP3 promotes breast cancer cell proliferation by deubiquitinating KLF5.

The Krüppel-like factor 5 (KLF5) transcription factor is highly expressed in basal type breast cancer and promotes breast cancer cell proliferation, survival, migration, and tumorigenesis. KLF5 protein stability is regulated by ubiquitination. In this study, ubiquitin-specific protease 3 (USP3) was identified as a new KLF5 deubiquitinase by genome-wide siRNA library screening. We demonstrated that USP3 interacts with KLF5 and stabilizes KLF5 via deubiquitination. USP3 knockdown inhibits breast cancer cell proliferation in vitro and tumorigenesis in vivo, which can be partially rescued by ectopic expression of KLF5. Furthermore, we observed a positive correlation between USP3 and KLF5 protein expression levels in human breast cancer samples. These findings suggest that USP3 is a new KLF5 deubiquitinase and that USP3 may represent a potential therapeutic target for breast cancer.

Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency.

Ultrasound signal wavelet analysis to quantify the microstructures of normal and frozen tissues in vitro.

Cryosurgery has a number of advantages that make it particularly appealing in the treatment of liver cancer. However, a major problem for the wide clinical adoption of hepatic cryosurgery is the lack of a cost effective high resolution imaging way which is capable of both performing precise monitoring of the freezing process in situ and evaluating the postoperative effects after surgery. The mean scatterer spacing has been found to be an important parameter for describing the ultrasonic scattering and characterization of biological tissues. However, its potential values in the evaluation of cryosurgical effects of tissues reserved unclear so far. Here, we investigated the wavelet analysis to estimate the mean scatterer spacing parameter in normal and freeze-thawed tissues on porcine livers in vitro. The experimental results carried out at 10 MHz using weakly focused pulse-echo signal element transducer indicated that the mean scatterer spacing in normal liver tissues is 1.12 ± 0.13 mm whereas it is 1.67 ± 0.25 mm in several pre-frozen and then thawed tissues. These results disclosed the good correlation between the wavelet data and microstructures of the normal or thawed tissues, and hence demonstrated that the wavelet analysis holds promise to be used as an effective method for the characterization of thawed tissues scatterer spacing. The present method offers a potential pragmatic strategy for monitoring the transition zone between frozen and unfrozen tissues during the surgical therapy, and evaluating postoperative effects.

Open achilles tendon defects: a 12-year study on the injury mechanisms, reconstructions, and late complications.

BACKGROUND: Open injuries of the Achilles tendon, which can be complicated by skin and bone injuries, continue to be a great challenge for surgeons. This study aims to report our experience with treatment of open Achilles tendon defects, focusing on the injury mechanisms, soft tissue coverage and late complications. METHODS: A retrospective review was performed on 31 patients with open Achilles tendon defects between 1999 and 2011. The analyzed factors were injury mechanisms, surgeries, and long follow-up complications. The defect lengths of the Achilles tendons in the study ranged from 1 to 11 cm and the soft tissue defects ranged from 3 × 3 to 12 × 10 cm. Nine types of flaps were used for the coverage of concomitant skin defects. RESULTS: Motorcycle spoke injuries were the most common cause of injury. There was no complete flap loss or rerupture of the reconstructed Achilles tendon. At the latest follow-up, all limbs were preserved and all the patients had regained full walking abilities. The algorithm of one-stage reconstruction was established, according to the defect length of the Achilles tendon and the defect size of skin. Late complications included maximum dorsiflexion loss and failure of heel raising ability on the single reconstructed foot. CONCLUSION: Open Achilles tendon defects are characteristic of concurrent skin and bone injuries and the reconstruction protocols of the different tissues should not be separated.

HECTD3 inhibits NLRP3 inflammasome assembly and activation by blocking NLRP3-NEK7 interaction.

The NLRP3 inflammasome plays an important role in protecting the host from infection and aseptic inflammation, and its regulatory mechanism is not completely understood. Dysregulation of NLRP3 can cause diverse inflammatory diseases. HECTD3 is a E3 ubiquitin ligase of the HECT family that has been reported to participate in autoimmune and infectious diseases. However, the relationship between HECTD3 and the NLRP3 inflammasome has not been well studied. Herein, we show that HECTD3 blocks the interaction between NEK7 and NLRP3 to inhibit NLRP3 inflammasome assembly and activation. In BMDMs, Hectd3 deficiency promotes the assembly and activation of NLRP3 inflammasome and the secretion of IL-1ß, while the overexpression of HECTD3 inhibits these processes. Unexpectedly, HECTD3 functions in an E3 activity independent manner. Mechanically, the DOC domain of HECTD3 interacts with NACHT/LRR domain of NLRP3, which blocks NLRP3-NEK7 interaction and NLRP3 oligomerization. Furthermore, HECTD3 inhibits monosodium urate crystals (MSU)-induced gouty arthritis, a NLRP3-related disease. Thus, we reveal a novel regulatory mechanism of NLRP3 by HECTD3 and suggest HECTD3 could be a potential therapeutic target for NLRP3-dependent pathologies.

KLF5 inhibition potentiates anti-PD1 efficacy by enhancing CD8+ T-cell-dependent antitumor immunity.

Background: Immune checkpoint blockers (ICBs) are revolutionized therapeutic strategies for cancer, but most patients with solid neoplasms remain resistant to ICBs, partly because of the difficulty in reversing the highly immunosuppressive tumor microenvironment (TME). Exploring the strategies for tumor immunotherapy is highly dependent on the discovery of molecular mechanisms of tumor immune escape and potential therapeutic target. Krüppel-like Factor 5 (KLF5) is a cell-intrinsic oncogene to promote tumorigenesis. However, the cell-extrinsic effects of KLF5 on suppressing the immune response to cancer remain unclear. Methods: We analyzed the immunosuppressive role of KLF5 in mice models transplanted with KLF5-deleted/overexpressing tumor cells. We performed RNA sequencing, immunohistochemistry, western blotting, real time-PCR, ELISA, luciferase assay, chromatin immunoprecipitation (ChIP), and flow cytometry to demonstrate the effects of KLF5 on CD8+ T cell infiltration and related molecular mechanism. Single-cell RNA sequencing and spatial transcriptomics analysis were applied to further decipher the association between KLF5 expression and infiltrating immune cells. The efficacy of KLF5/COX2 inhibitors combined with anti-programmed cell death protein 1 (anti-PD1) therapy were explored in pre-clinical models. Finally, a gene-expression signature depending on KLF5/COX2 axis and associated immune markers was created to predict patient survival. Results: KLF5 inactivation decelerated basal-like breast tumor growth in a CD8+ T-cell-dependent manner. Transcriptomic profiling revealed that KLF5 loss in tumors increases the number and activated function of T lymphocytes. Mechanistically, KLF5 binds to the promoter of the COX2 gene and promotes COX2 transcription; subsequently, KLF5 deficiency decreases prostaglandin E2 (PGE2) release from tumor cells by reducing COX2 expression. Inhibition of the KLF5/COX2 axis increases the number and functionality of intratumoral antitumor T cells to synergize the antitumorigenic effects of anti-PD1 therapy. Analysis of patient datasets at single-cell and spatial resolution shows that low expression of KLF5 is associated with an immune-supportive TME. Finally, we generate a KLF5/COX2-associated immune score (KC-IS) to predict patient survival. Conclusions: Our results identified a novel mechanism responsible for KLF5-mediated immunosuppression in TME, and targeting the KLF5/COX2/PGE2 axis is a critical immunotherapy sensitizer.

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.

Histone Deacetylase Inhibitors (HDACi) Promote KLF5 Ubiquitination and Degradation in Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) accounts for approximately 15% of all breast cancer cases, and patients with BLBC have a low survival rate. Our previous study demonstrated that the KLF5 transcription factor promotes BLBC cell proliferation and tumor growth. In this study, we demonstrated that the histone deacetylase inhibitors (HDACi), suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), increased KLF5 acetylation at lysine 369 (K369), downregulated KLF5 protein expression levels, and decreased cell viability in BLBC cell lines. HDACi target KLF5 for proteasomal degradation by promoting KLF5 protein ubiquitination. K369 acetylation of KLF5 decreases the binding between KLF5 and its deubiquitinase, BAP1. These findings revealed a novel mechanism by which HDACi suppress BLBC, and a novel crosstalk between KLF5 protein acetylation and ubiquitination.

Targeting HECTD3-IKKα axis inhibits inflammation-related metastasis.

Metastasis is the leading cause of cancer-related death. The interactions between circulating tumor cells and endothelial adhesion molecules in distant organs is a key step during extravasation in hematogenous metastasis. Surgery is a common intervention for most primary solid tumors. However, surgical trauma-related systemic inflammation facilitates distant tumor metastasis by increasing the spread and adhesion of tumor cells to vascular endothelial cells (ECs). Currently, there are no effective interventions to prevent distant metastasis. Here, we show that HECTD3 deficiency in ECs significantly reduces tumor metastasis in multiple mouse models. HECTD3 depletion downregulates expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, in mouse primary ECs and HUVECs stimulated by inflammatory factors and inhibits adhesion of tumor cells to ECs both in vitro and in vivo. We demonstrate that HECTD3 promotes stabilization, nuclear localization and kinase activity of IKKα by ubiquitinating IKKα with K27- and K63-linked polyubiquitin chains at K296, increasing phosphorylation of histone H3 to promote NF-κB target gene transcription. Knockout of HECTD3 in endothelium significantly inhibits tumor cells lung colonization, while conditional knockin promotes that. IKKα kinase inhibitors prevented LPS-induced pulmonary metastasis. These findings reveal the promotional role of the HECTD3-IKKα axis in tumor hematogenous metastasis and provide a potential strategy for tumor metastasis prevention.

A Systematic Review and Meta-Analysis of Combined Antibiotic Spacer with Ilizarov Methods in the Treatment of Infected Nonunion of Tibia.

BACKGROUND: The objective of this systematic review was to evaluate current studies available reporting the antibiotic spacer combined with Ilizarov methods in the treatment of infected nonunion of tibia and to perform meta-analysis of bone results and infection recurrence to assess the efficacy of an antibiotic spacer combined with Ilizarov methods. METHODS: The MEDLINE, Embase, Cochrane Library, CNKI, and CBM (Chinese Biological Medicine) databases were searched for articles published between January 2000 and July 2020. Assessment of study quality was performed using a modified version of the Newcastle-Ottawa scale. Effect size and 95% confidence intervals were calculated for the main outcome. Heterogeneity was assessed. Fixed-effect modeling and Stata version 15.1 were used to analyze the data. Sensitivity analyses were conducted with the evidence of heterogeneity. RESULTS: 11 studies involving 210 patients with infected nonunion of tibia were finally included in our meta-analysis. Bone results and infection recurrence were analyzed based on the single-arm meta-analysis. The average of external fixation index (EFI) was 46.88 days/cm in all studies included. The excellent rate in bone results and the rate of infection recurrence was 65% (95% CI: [0.22, 0.97], I 2 = 0.0%, P = 0.932) and 6.99% (95% CI: [0.052, 0.325], I 2 = 0.0%, P = 1.000) in patients with infected nonunion of tibia treated with an antibiotic spacer combined with Ilizarov methods. CONCLUSIONS: Our meta-analysis revealed that the patients with infected nonunion of tibia treated with an antibiotic spacer combined with Ilizarov methods had a high rate of excellent bone results and a low rate of infection recurrence. Therefore, combining the antibiotic spacer with Ilizarov methods may be an applicable choice for repairing and reconstructing infected nonunion of tibia.

In adults, early mobilization may be beneficial for distal radius fractures treated with open reduction and internal fixation: a systematic review and meta-analysis.

OBJECTIVES: It remains debatable if early mobilization (EM) yields a better clinical outcome than the late mobilization (LM) in adults with an acute and displaced distal radial fracture (DRF) of open reduction internal fixation (ORIF). Therefore, we aimed to perform a systematic review and meta-analysis of randomized controlled trials (RCTs), comparing clinical results with the safety of EM with LM following ORIF. METHODS: Databases such as Medline, Cochrane Central Register, and Embase were searched from Jan 1, 2000, to July 31, 2021, and RCTs comparing EM with LM for DRF with ORIF were included in the analysis. The primary outcome of study included disabilities of the Arm, Shoulder, and Hand (DASH) score at different follow-up times. Wherever the secondary outcomes included patient-rated wrist evaluation (PRWE), grip strength (GS), visual analog scale (VAS), wrist range of motion (WROM), and associated complications, the two independent reviewers did data extraction for the analysis. Effect sizes of outcome for each group were pooled using random-effects models; thereafter, the results were represented in the forest plots. RESULTS: Nine RCTs with 293 EM and 303 LM participants were identified and included in the study. Our analysis showed that the DASH score of the EM group was significantly better than LM group at the six weeks postoperatively (- 10.15; 95% CI - 15.74 to - 4.57, P < 0.01). Besides, the EM group also had better outcomes in PRWE, GS and WROM at 6 weeks. However, EM showed potential higher rate for implant loosening and/or fracture re-displacement complication (3.00; 95% CI 1.02-8.83, P = 0.05). CONCLUSION: Functionally, at earlier stages, EM for patients with DRF of ORIF may have a beneficial effect than LM. The mean differences in the DASH score at 6 weeks surpassed the minimal clinically important difference; however, the potentially higher risk of implant loosening and/or fracture re-displacement cannot be ignored. Due to the lack of definitive evidence, multicenter and large sample RCTs are required for determining the optimal rehabilitation protocol for DRF with ORIF. PROSPERO registration number: CRD42021240214 2021/2/28.

Targeting ubiquitin conjugating enzyme UbcH5b by a triterpenoid PC3-15 from Schisandra plants sensitizes triple-negative breast cancer cells to lapatinib.

Increasing evidence suggested that a number of ubiquitin enzymes, including ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, E3 ubiquitin ligases and deubiquitination enzymes contribute to therapeutic resistance in triple-negative breast cancer (TNBC) cells. Inhibition of these enzymes with small molecule inhibitors may restore therapeutic sensitivity. Here, we demonstrated ubiquitin conjugating enzyme UbcH5b strongly supports HECTD3 auto-ubiquitination in vitro. Based on this, we developed a Fluorescence Resonance Energy Transfer (FRET) assay and identified three Schisandraceae triterpenoids, including PC3-15, to block HECTD3/UbcH5b auto-ubiquitination. Furthermore, we revealed that PC3-15 directly binds to UbcH5b and also inhibits UbcH5b-mediated p62 ubiquitination. We found that the UbcH5b-p62 axis confers TNBC cells resistance to lapatinib by promoting autophagy. Consistently, PC3-15 inhibits lapatinib-induced autophagy and increases lapatinib sensitivity in TNBC in vitro and in mouse xenografts. These findings suggest that the UbcH5b-p62 axis provides potential therapeutic targets and that Schisandraceae triterpenoids may be used for TNBC treatment in combination with lapatinib.

[The change in Toll-like receptor 4 gene expression in skeleton of endotoxemia mice].

OBJECTIVE: To explore the changes in gene expression of bone in endotoxemia in mice. METHODS: Lipopolysaccharide (LPS) was injected intraperitoneally to reproduce an endotoxemia model in mice. Forty-eight mice were randomly divided into eight groups: normal group, 4, 6, 8, 12, 24, 48 and 72 hours after LPS injection groups, with 6 mice in each group. To evaluate endotoxemia whole blood was collected for leukocytic count, eye sockets blood was obtained for liver and renal functions tests. mRNA expression level of Toll-like receptor 4 (TLR4) in bone was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay. The pathological changes of bone and tissue slides were prepared with hematoxylin and eosin (HE) staining for observing under microscope. RESULTS: Compared with normal group, leukocyte count of 4-hour LPS-treated group was significantly decreased (P<0.01). However, after 4 hours, leukocyte count became higher gradually and remained at high level at 72 hours compared with that of normal group (P<0.05). Compared with normal group, alanine aminotransferase (ALT) level reached to a high level in LPS-treated groups at 4 hours and 6 hours (both P<0.05), and then decreased gradually showing a tendency to return to normal level after 8 hours (P>0.05). Blood urea nitrogen (BUN) was increased at 6 hours (P<0.05), reaching to the highest level at 8 hours (P<0.05) and tended to become normal level after 12 hours (P>0.05). Six hours after LPS treatment, the expression of TLR4 mRNA was enhanced (P<0.01) and reached the peak at 24 hours (P<0.01), and it remained at a high level at 72 hours (P<0.05). However, there were no significant pathological changes in bone structure after LPS treatment. CONCLUSION: Expression level of TLR4 mRNA in bone is significantly increased in endotoxemia mice.

A Pro253Arg mutation in fibroblast growth factor receptor 2 (Fgfr2) causes skeleton malformation mimicking human Apert syndrome by affecting both chondrogenesis and osteogenesis.

Apert syndrome is one of the most severe craniosynostosis that is mainly caused by either a Ser252Trp(S252W) or Pro253Arg(P253R) mutation in fibroblast growth factor receptor 2 (FGFR2). As an autosomal dominant disorder, Apert syndrome is mainly characterized by skull malformation resulting from premature fusion of craniofacial sutures, as well as syndactyly, etc. A P253R mutation of FGFR2 results in nearly one-thirds of the cases of Apert syndrome. The pathogenesis of Apert syndrome resulting from P253R mutation of FGFR2 is still not fully understood. Here we reported a knock-in mouse model carrying P253R mutation in Fgfr2. The mutant mice exhibit smaller body size and brachycephaly. Analysis of the mutant skulls and long bones revealed premature fusion of coronal suture, shortened cranial base and growth plates of long bones. In vitro organ culture studies further revealed that, compared with wild-type littermates, the mutant mice have prematurely fused coronal sutures and retarded long bone growth. Treatment of the cultured calvaria and femur with PD98059, an Erk1/2 inhibitor, resulted in partially alleviated coronal suture fusion and growth retardation of femur respectively. Our data indicated that the P253R mutation in Fgfr2 directly affect intramembranous and endochondral ossification, which resulted in the premature closure of coronal sutures and growth retardation of long bones and cranial base. And the Erk1/2 signaling pathway partially mediated the effects of P253R mutation of Fgfr2 on cranial sutures and long bones.

[Generation of bone morphogenetic protein 4 conditional RNA interference mice].

To explore bone morphogenetic protein 4 (BMP4) function in the developing bone, a BMP4 conditional RNA interference (CRNAi) vector was constructed based on the pBSK/U6 vector with a LoxPneo cassette. The transgene fragment targeting bmp4 was obtained by Kpn and Afl double digestion and was purified before being microinjected into fertilized eggs from FVB/NJ mice. BMP4CRNAi transgenic mice were genotyped by PCR. And the PCR positive mice were crossed with Col2a1-Cre transgenic mice, whose Cre recombinase was specifically expressed in osteo-chondro-progenitor cells. Bmp4 mRNA expression in primary chondrocytes were examined by semi-quantitive RT-PCR to determine RNA interference efficiency. Results showed that BMP4(CRNAi) mice and BMP4 (Col2a1-CRNAi) mice were produced successfully, and bmp4 knockdown efficiency in primary chondrocytes of BMP4 Col2a1-CRNAi mice was 81%. This transgenic mouse line provides excellent model for studying the role of BMP4 in chondrocyte development, and BMP4CRNAi mouse may be a good model for studying the role of BMP4 in different cells, tissues and organs through crossing with different Cre transgenic mice.

Hypoxia induces miR-153 through the IRE1α-XBP1 pathway to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis.

It is well documented that hypoxia activates the hypoxia-inducible factor 1-alpha (HIF1α)/vascular endothelial growth factor A (VEGFA) axis to promote angiogenesis in breast cancer. However, it is unclear how this axis is negatively regulated. In this study, we demonstrated that miR-153 directly inhibits expression of HIF1α by binding to the 3'UTR of HIF1A mRNA, as well as suppresses tube formation of primary human umbilical vein endothelial cells (HUVECs) and breast cancer angiogenesis by decreasing the secretion of VEGFA. Importantly, expression of miR-153 was induced by hypoxia-stimulated ER stress, which activates IRE1α and its downstream transcription factor X-box binding protein 1 (XBP1). X-box binding protein 1 directly binds to the promoter of the miR-153 host gene PTPRN and activates transcription. These results indicate that hypoxia induces miR-153 to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis and miR-153 could be used for breast cancer anti-angiogenesis therapy.

HECTD3 mediates TRAF3 polyubiquitination and type I interferon induction during bacterial infection.

Lysine-63-linked (K63-linked) polyubiquitination of TRAF3 coordinates the engagement of pattern-recognition receptors with recruited adaptor proteins and downstream activator TBK1 in pathways that induce type I IFN. Whether autoubiquitination or other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient in the E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria Francisella novicida, Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impaired during bacterial infection both in vivo and in vitro. HECTD3 regulated type I IFN production by mediating K63-linked polyubiquitination of TRAF3 at residue K138. The catalytic domain of HECTD3 regulated TRAF3 K63 polyubiquitination, which enabled TRAF3-TBK1 complex formation. Our study offers insights into mechanisms of TRAF3 modulation and provides potential therapeutic targets against infections by intracellular bacteria and inflammatory diseases.

Metformin suppresses triple-negative breast cancer stem cells by targeting KLF5 for degradation.

Out of the breast cancer subtypes, triple-negative breast cancer (TNBC) has the poorest prognosis without effective targeted therapies. Metformin, a first-line drug for type 2 diabetes mellitus, was demonstrated to target breast cancer stem cells selectively. However, the efficiency and the mechanism of action of metformin in TNBC are unclear. In this study, we demonstrated that metformin decreased the percentage of TNBC stem cells partially through the downregulation of the expression of the stem cell transcription factor Krüppel-like factor 5 (KLF5) and its downstream target genes, such as Nanog and FGF-BP1, in TNBC cell lines. Metformin induced glycogen synthase kinase-3ß (GSK3ß)-mediated KLF5 protein phosphorylation and degradation through the inhibition of protein kinase A (PKA) activity in TNBC cells. Consistently, PKA activators increased the expression levels of KLF5. We observed a positive correlation between p-CREB, p-GSK3ß, KLF5 and FGF-BP1 protein levels in human TNBC samples. These findings suggest that metformin suppresses TNBC stem cells partially through the PKA-GSK3ß-KLF5 signaling pathway.