Distinct molecular profiles drive multifaceted characteristics of colorectal cancer metastatic seeds.

Metastasis of primary tumors remains a challenge for early diagnosis and prevention. The cellular properties and molecular drivers of metastatically competent clones within primary tumors remain unclear. Here, we generated 10-16 single cell-derived lines from each of three colorectal cancer (CRC) tumors to identify and characterize metastatic seeds. We found that intrinsic factors conferred clones with distinct metastatic potential and cellular communication capabilities, determining organ-specific metastasis. Poorly differentiated or highly metastatic clones, rather than drug-resistant clones, exhibited poor clinical prognostic impact. Personalized genetic alterations, instead of mutation burden, determined the occurrence of metastatic potential during clonal evolution. Additionally, we developed a gene signature for capturing metastatic potential of primary CRC tumors and demonstrated a strategy for identifying metastatic drivers using isogenic clones with distinct metastatic potential in primary tumors. This study provides insight into the origin and mechanisms of metastasis and will help develop potential anti-metastatic therapeutic targets for CRC patients.

Structural variants involved in high-altitude adaptation detected using single-molecule long-read sequencing.

Structural variants (SVs), accounting for a larger fraction of the genome than SNPs/InDels, are an important pool of genetic variation, enabling environmental adaptations. Here, we perform long-read sequencing data of 320 Tibetan and Han samples and show that SVs are highly involved in high-altitude adaptation. We expand the landscape of global SVs, apply robust models of selection and population differentiation combining SVs, SNPs and InDels, and use epigenomic analyses to predict enhancers, target genes and biological functions. We reveal diverse Tibetan-specific SVs affecting the regulatory circuitry of biological functions, including the hypoxia response, energy metabolism and pulmonary function. We find a Tibetan-specific deletion disrupts a super-enhancer and downregulates EPAS1 using enhancer reporter, cellular knock-out and DNA pull-down assays. Our study expands the global SV landscape, reveals the role of gene-regulatory circuitry rewiring in human adaptation, and illustrates the diverse functional roles of SVs in human biology.

Mussel-Inspired Caries Management Strategy: Constructing a Tribioactive Tooth Surface with Remineralization, Antibiofilm, and Anti-inflammation Activity.

Dental caries is a common chronic oral disease in humans resulting from tooth demineralization caused by acid production of bacterial plaque, which leads to the destruction of enamel and dentin and oral inflammation. However, it is still a challenge that the function of natural active ingredients in currently available oral care products is not comprehensive, especially the lack of remineralization. Here, inspired by the strong biological adhesion ability of mussels and ancient oral disease plant therapy, a multifunctional strategy is proposed to construct a bioactive tooth surface to treat dental caries. It has been demonstrated that the Turkish gall extract (TGE) can inhibit adhesion of cariogenic bacteria Streptococcus mutans and Actinomyces viscosus and destroy biofilms on the tooth surface. Meanwhile, TGE can reduce the expression of inflammatory factors. Notably, the TGE coating can induce the growth of hydroxyapatite (HAP) crystals in vivo and in vitro, recovering the enamel mechanical properties under normal oral conditions. MD simulations interpreted the adsorption mechanism by which the hydroxyl groups in TGE bind to phosphate group (PO43-) on the tooth surface, attracting calcium ions (Ca2+) as nucleation sites for remineralization. This work underlines the importance of TGE coating in remineralization, antibiofilm, and anti-inflammation activity as a promising strategy for dental caries.

Overlooked Spherical Nanoparticles Exist in Plant Extracts: From Mechanism to Therapeutic Applications.

To date, plant medicine research has focused mainly on the chemical compositions of plant extracts and their medicinal effects. However, the therapeutic or toxic effects of nanoparticles in plant extracts remain unclear. In this study, large numbers of spherical nanoparticles were discovered in some plant extracts. Nanoparticles in Turkish galls extracts were used as an example to examine their pH responsiveness, free radical scavenging, and antibacterial capabilities. By utilizing the underlying formation mechanism of these nanoparticles, a general platform to produce spherical nanoparticles via direct self-assembly of Turkish gall extracts and various functional proteins was developed. The results showed that the nanoparticles retained both the antibacterial ability and intracellular carrier ability of the original protein or catechol. This work introduces a new member of the plant-derived edible nanoparticle (PDEN) family, establishes a simple and versatile platform for mass production nanoparticles, and provides new insight into the formation mechanism of nanoparticles during plant extraction.

Biosensor-based active ingredient recognition system for screening TNF-α inhibitors from lotus leaves.

Erhuangquzhi granules (EQG) have been clinically proven to be effective in nonalcoholic steatohepatitis (NASH) treatment. However, the active components and molecular mechanisms remain unknown. This study aimed to screen active components targeting tumor necrosis factor α (TNF-α) in EQG for the treatment of NASH by a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). The amine-coupling method was used to immobilize recombinant TNF-α protein on an SPR chip, the specificity of the TNF-α-immobilized chip was validated, and nine medicinal herbs in EQG were prescreened. Nuciferine (NF), lirinidine (ID), and O-nornuciferine (NNF) from lotus leaves were found and identified as TNF-α ligands by UPLC‒MS/MS, and the affinity constants of NF, ID, and NNF to TNF-α were determined by SPR experiments (Kd = 61.19, 31.02, and 20.71 µM, respectively). NF, ID, and NNF inhibited TNF-α-induced apoptosis in L929 cells, the levels of secreted IL-6 and IL-1ß were reduced, and the phosphorylation of IKKß and IκB was inhibited in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In conclusion, a class of new active small-molecule TNF-α inhibitors was discovered, which also provides a valuable reference for the material basis and mechanism of EQG action in NASH treatment.

Integrative analysis of transcriptome and metabolome reveals probiotic effects on cecal metabolism in broilers.

BACKGROUND: Probiotics play an important role in the host and have attracted widespread attention as an alternative to antibiotics. Arbor Acres broilers were used in the present experiment and fed different doses of compound probiotics at 1, 5, and 10 g kg-1 . The effects of compound probiotics on broiler growth performance and cecal transcriptome and metabolome were investigated. RESULTS: We discovered 425 differentially expressed genes (DEGs; upregulated: 256; downregulated: 169) in the cecal transcriptome study. These DEGs were assigned to fat metabolic pathways, such as the peroxisome proliferator-activated receptor (PPAR) signaling pathway, according to KEGG analysis. Probiotics downregulated LPL and upregulated PPARα expression in the cecum. In metabolome analysis of the cecum of cecum, we screened 86 differential metabolites and performed KEGG enrichment analysis of these metabolites. The KEGG analysis showed that these differentially expressed metabolites were annotated to nucleotide metabolism-related pathways, such as purine metabolism. In the cecum, probiotics upregulated the content of guanine, AMP, 3'-AMP, adenylosuccinate, deoxyguanosine, and ADP-ribose, whereas they downregulated the content of 5-hydroxyisourate. Comprehensive transcriptome and metabolome analysis revealed that glycolysis, gluconeogenesis, and glycerophospholipid metabolism pathways were jointly enriched in cecum of broilers fed a probiotic-containing diet. CONCLUSION: This study provides valuable information for studying the regulation and gene metabolism network of probiotics on cecal metabolism in broilers. © 2022 Society of Chemical Industry.

Characterization of the high-affinity anti-CTLA-4 monoclonal antibody JS007 for immune checkpoint therapy of cancer.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical inhibitory checkpoint molecule, and monoclonal antibodies (mAbs) targeting CTLA-4 that restore anti-tumor T cell immunity have achieved clinical success. Here, we report a humanized IgG1 mAb, namely JS007, with high binding affinity to CTLA-4. JS007 shows superior binding affinity and T-cell activating efficiency over ipilimumab. Moreover, it demonstrates substantial in vivo tumor suppression efficacy at low doses. The crystal structure of JS007/CTLA-4 complex (PDB: 8HIT) shows JS007 adopts a heavy-chain-dominant binding mode, and mainly contacts the BC loop, DE loop and FG loop of CTLA-4. Notably, two Tyr residues (VH-Y100 and VL-Y32) from the complementarity-determining region loops insert into the two cavities formed by the residues from the loops of CTLA-4, which may contribute to the stabilization of the binding. Comparative analysis with other anti-CTLA-4 mAbs indicates that the double "wedge-into-hole" binding mode is unique for JS007 and may be responsible for the high-affinity binding to CTLA-4. These findings have provided an important molecular understanding of the high-affinity CTLA-4 blockade mAbs and shed light on future development of agents targeting CTLA-4.

Transcriptome and metabolome analyses reveal the regulatory effects of compound probiotics on cecal metabolism in heat-stressed broilers.

The effect of compound probiotics on the caecum of broilers under heat stress was assessed in this study. A total of 400 twenty-eight-day-old AA male broilers were randomly divided into 4 treatment groups, where each group had 5 replicates of 20 broilers. The 4 treatment groups were a heat stress control group (broilers receiving a normal diet) and groups HP I, HP II, and HP Ⅲ, consisting of broilers receiving 1, 5, and 10 g of compound probiotics added to each kilogram of feed, respectively. Compound probiotics (L. casei, L. acidophilus, and B. lactis at a ratio of 1:1:2) were used to formulate a compound probiotic powder, with 1 × 1010 CFU/g of effective viable bacteria. Heat stress treatment was performed at 32 ± 1°C from 9:00 to 17:00 every day from 28 d to 42 d. In d 28 to 42, compared with the HC group, the ADG of broilers in the HP II and III groups was significantly increased (P < 0.05); the ADFI difference between groups was not significant (P > 0.05); the FCR of HP II and III broilers was significantly decreased (P < 0.05); and the FCR of the HP I group increased, but the difference was not significant (P > 0.05). Transcriptome results demonstrate that 665 differential genes were screened (DEGs; upregulated: 366, downregulated: 299). The DEGs were enriched in the B cell receptor signaling pathway, the intestinal immune network for IgA synthesis, the Fc epsilon RI signaling pathway, and other signaling pathways, according to KEGG enrichment analysis. Metabolome analysis identified 92 differential metabolites (DAMs; upregulated: 48, downregulated: 44). KEGG enrichment analysis indicated significant enrichment of Pantothenate and CoA biosynthesis and beta-Alanine metabolism. The combined transcriptome and metabolome analysis revealed that the DAMs and DEGs were mostly involved in beta-alanine metabolism, arginine biosynthesis, amino sugar and nucleotide sugar, and alanine, aspartate, and glutamate metabolism. The results of this study suggest that the addition of compound probiotics has a positive effect on intestinal metabolites, improving the growth performance and contributing to the overall health of broilers under heat stress.

Exploring the effect and mechanism of Aloin A against cancer cachexia-induced muscle atrophy via network pharmacology, molecular docking, molecular dynamics and experimental validation.

80% of advanced cancer patients suffer from cachexia, but there are no FDA-approved drugs. Therefore, it is imperative to discover potential drugs. OBJECTIVE: This study aims at exploring the effect and targets of Aloin A against cancer cachexia (CC)-induced muscle atrophy. METHODS: Network pharmacology, molecular docking, molecular dynamics (MD) and animal model of CC-induced muscle atrophy with a series of behavior tests, muscle quality, HE staining and RT-PCR were performed to investigate the anticachectic effects and targets of Aloin A and its molecular mechanism. RESULTS: Based on network pharmacology, 51 potential targets of Aloin A on CC-induced muscle atrophy were found, and then 10 hub genes were predicted by the PPI network. Next, KEGG and GO enrichment analysis showed that the anticachectic effect of Aloin A is associated with PI3K-AKT, MAPK, TNF, TLR, etc., pathways, and biological processes like inflammation, apoptosis and cell proliferation. Molecular docking and MD results showed good binding ability between the Aloin A and key targets. Moreover, experiments in vivo demonstrated that Aloin A effectively rescued muscle function and wasting by improving muscle quality, mean CSA, and distribution of muscle fibers by regulating HSP90AA1/AKT signaling in tumor-bearing mice. CONCLUSION: This study offers new insights for researchers to understand the effect and mechanism of Aloin A against CC using network pharmacology, molecular docking, MD and experimental validation, and Aloin A retards CC-induced muscle wasting through multiple targets and pathways, including HSP90AA1/AKT signaling, which provides evidence for Aloin A as a potential therapy for cancer cachexia in clinic.

Comparative transcriptome analysis of longissimus dorsi tissues with different intramuscular fat contents from Guangling donkeys.

BACKGROUND: Donkey meat has low fat and high protein contents and is rich in various unsaturated fatty acids and trace elements that are beneficial to human digestion and absorption. IMF (intramuscular fat), also known as marbling, is an important indicator of the lean meat to fat ratio, which directly affects the tenderness and juiciness of the meat. At present, the underlying molecular variations affecting IMF content among donkey breeds are unclear. The Guangling donkey is an indigenous species in China. This study explored candidate regulatory genes that affect IMF content in Guangling donkeys. The IMF content of the longissimus dorsi muscle in 30 Guangling donkeys was measured. Six donkeys of similar age were selected according to age factors and divided into two groups, the high (H) and low (L) fat groups, according to their IMF content. RESULTS: RNA-seq technology was used to compare the muscle transcriptome between the two groups. More than 75.0% of alternative splicing (AS) events were of the skipped exon (SE) type. A total of 887 novel genes were identified; only 386 novel genes were aligned to the annotation information of various databases. Transcriptomics analysis revealed 167 differentially expressed genes (DEGs), of which 64 were upregulated and 103 were downregulated between the H and L groups. Gene ontology analysis showed that the DEGs were enriched in multiple biological processes and pathways that are related to adipocyte differentiation, lipid synthesis, and neutral lipid metabolism. KEGG pathway analysis suggested that arachidonic acid metabolism, the HIF-1 signalling pathway, fructose and mannose metabolism, glycerophospholipid metabolism, and the AMPK signalling pathway were involved in lipid deposition. In addition, a gene-gene interaction network was constructed that revealed that the DEGs, including SCD, LEPR, CIDEA, DLK1, DGAT2, ITGAL, HMOX1, WNT10B, and DGKA, had significant roles in adipocyte differentiation and adipogenesis. The selected DEGs were further validated by qRT-PCR. CONCLUSION: This study improves the in-depth understanding of gene regulation and protein expression regarding IMF deposition and lays a basis for subsequent molecular breeding studies in Guangling donkeys.

Molecular Basis for the Recognition of HIV Nef138-8 Epitope by a Pair of Human Public T Cell Receptors.

Cross-recognized public TCRs against HIV epitopes have been proposed to be important for the control of AIDS disease progression and HIV variants. The overlapping Nef138-8 and Nef138-10 peptides from the HIV Nef protein are HLA-A24-restricted immunodominant T cell epitopes, and an HIV mutant strain with a Y139F substitution in Nef protein can result in immune escape and is widespread in Japan. Here, we identified a pair of public TCRs specific to the HLA-A24-restricted Nef-138-8 epitope using PBMCs from White and Japanese patients, respectively, namely TD08 and H25-11. The gene use of the variable domain for TD08 and H25-11 is TRAV8-3, TRAJ10 for the α-chain and TRBV7-9, TRBD1*01, TRBJ2-5 for the ß-chain. Both TCRs can recognize wild-type and Y2F-mutated Nef138-8 epitopes. We further determined three complex structures, including TD08/HLA-A24-Nef138-8, H25-11/HLA-A24-Nef138-8, and TD08/HLA-A24-Nef138-8 (2F). Then, we revealed the molecular basis of the public TCR binding to the peptide HLA, which mostly relies on the interaction between the TCR and HLA and can tolerate the mutation in the Nef138-8 peptide. These findings promote the molecular understanding of T cell immunity against HIV epitopes and provide an important basis for the engineering of TCRs to develop T cell-based immunotherapy against HIV infection.

Colorectal Cancer Patient-Derived 2D and 3D Models Efficiently Recapitulate Inter- and Intratumoral Heterogeneity.

Pre-existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two-dimensional (2D) and three-dimensional (3D) model for patient-derived cancer cells (PDCCs) and air-liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long-term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5-Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single-cell-derived clones() are efficiently established and pre-existing drug-resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre-existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre-existing drug-resistant or highly tumorigenic clones.

All-in-One Theranostic Platform Based on Hollow Microcapsules for Intragastric-Targeting Antiulcer Drug Delivery, CT Imaging, and Synergistically Healing Gastric Ulcer.

Bismuth-containing therapies are suggested as first-line and rescue alternatives for gastric ulcer (GU) treatment and Helicobacter pylori eradication. The current treatment strategy is called quadruple therapy and includes proton pump inhibitors, bismuth, and two broad-band antibiotics. This fact may affect medication compliance, leading to a resistance rate of more than 25% to clarithromycin or metronidazole. To counter this, from the perspective of natural products, an intragastric-targeting all-in-one theranostic platform is established: a drug carrier microcapsule composed of multiple synergistic antiulcer drugs, including bismuth, gallotannin, and antibiotics is obtained (BiG@MCs), and the therapeutic effects of BiG@MCs in rodent models are further evaluated. The results show that the BiG@MCs are spherical with homogeneous particle size (3 ± 0.5 µm) and can be response-released to the acidic environment of the stomach (pH 2.0-3.0), preventing the premature release of the BiG@MCs in physiological conditions. It is worth noting that the bismuth component can be easily identified by computed tomography and other detection instruments, which provide the possibility for drug tracing. In summary, these results indicate that BiG@MCs provide a versatile intragastric-targeting drug delivery platform for GU therapeutics.

N-glycosylation of PD-1 promotes binding of camrelizumab.

PD-1 is a highly glycosylated inhibitory receptor expressed mainly on T cells. Targeting of PD-1 with monoclonal antibodies (MAbs) to block the interaction with its ligand PD-L1 has been successful for the treatment of multiple tumors. However, polymorphisms at N-glycosylation sites of PD-1 exist in the human population that might affect antibody binding, and dysregulated glycosylation has been observed in the tumor microenvironment. Here, we demonstrate varied N-glycan composition in PD-1, and show that the binding affinity of camrelizumab, a recently approved PD-1-specific MAb, to non-glycosylated PD-1 proteins from E. coli is substantially decreased compared with glycosylated PD-1. The structure of the camrelizumab/PD-1 complex reveals that camrelizumab mainly utilizes its heavy chain to bind to PD-1, while the light chain sterically inhibits the binding of PD-L1 to PD-1. Glycosylation of asparagine 58 (N58) promotes the interaction with camrelizumab, while the efficiency of camrelizumab to inhibit the binding of PD-L1 is substantially reduced for glycosylation-deficient PD-1. These results increase our understanding of how glycosylation affects the activity of PD-1-specific MAbs during immune checkpoint therapy.

Intestinal stem cells and intestinal organoids.

The intestinal epithelium is one of the most rapidly renewing tissues, which is fueled by stem cells at the base of the crypts. Strategies of genetic lineage tracing and organoids, which capture major features of original tissues, are powerful avenues for exploring the biology of intestinal stem cells in vivo and in vitro, respectively. The combination of intestinal organoid-culturing system and genetic modification approaches provides an attractive platform to uncover the mechanism of colorectal cancer and genetic disorders in the human minigut. Here, we will provide a comprehensive overview of studies on intestinal epithelium and intestinal stem cells. We will also review the applications of organoids and genetic markers in intestinal research studies. Furthermore, we will discuss the advantages and drawbacks of organoids as disease models compared with mice models and cell lines.

Curcumin sensitizes lymphoma cells to DNA damage agents through regulating Rad51-dependent homologous recombination.

Curcumin is a natural compound isolated from the rhizome of Curcuma longa. It possesses anti-tumor activity through arresting cell cycles and promoting cell apoptosis. However, the effect of curcumin on DNA damage is not well defined. In this study, we investigated the effect of curcumin on inducing DNA damage and on sensitizing lymphoma cells to anti-tumoral DNA damage drugs. Western blot showed curcumin induced γ-H2AX foci in CH12F3 lymphoma cells, which suggests curcumin induces DNA breaks. In addition, curcumin decreased the expression of Rad51, which suggests curcumin induces DNA damage through regulating Rad51-dependant homologous recombination. Rad51-dependant homologous recombination is a vital DNA repair pathway for cancer cells to resist anti-tumoral DNA damage drugs, therefore, we studied the effect of curcumin on the sensitizing lymphoma cells to various chemotherapeutic drugs. We found low level of curcumin (5µM) sensitized lymphoma cells to anti-tumoral DNA damage agents including cisplatin, methyl methanesulfonate, hydroxyurea and camptothecin. We also found curcumin sensitized CH12F3 lymphoma cells to DNA-PK and PARP inhibitors. Flow cytometry analysis showed curcumin promoted apoptosis and western blot analysis confirmed curcumin activated caspase3-dependent apoptosis. Taken together, these results demonstrate that curcumin induces DNA damage through regulating Rad51-dependant homologous recombination and triggers caspase3-dependent apoptosis, more importantly, curcumin sensitizes lymphoma cells to various DNA damage drugs. Consequently, curcumin would be a potent agent for sensitizing lymphoma cells to anti-tumoral chemotherapeutic agents.

Triptolide induces DNA breaks, activates caspase-3-dependent apoptosis and sensitizes B-cell lymphoma to poly(ADP-ribose) polymerase 1 and phosphoinositide 3-kinase inhibitors.

Triptolide is the primary compound isolated from Tripterygium wilfordii, which has been reported to inhibit nucleotide excision repair as well as exhibit anti-inflammatory and antitumor activities. However, the action of triptolide in DNA breaks remains unknown. The present study investigated the effects of triptolide in the induction of DNA breaks and apoptosis in a murine B-cell lymphoma cell line, CH12F3. An MTT assay revealed that X-ray repair cross-complementing protein 1 (XRCC1)-/- CH12F3 cells were more sensitive to 6 nM triptolide compared with the wild-type CH12F3 cells, which suggests that low levels of triptolide induce DNA breaks in a manner that is dependent on the XRCC1-mediated repair pathway. Flow cytometric analysis identified that 50 nM triptolide increased the phospho-histone H2AX level, demonstrating that triptolide induces double-strand breaks. Western blot analysis revealed that triptolide up-regulated Rad51 and nuclear proliferating cell nuclear antigen. Annexin V/propidium iodide staining identified that triptolide promoted apoptosis and western blot analysis confirmed that triptolide activated caspase-3-dependent apoptosis. The results of the present study also demonstrated that 5 nM triptolide sensitized CH12F3 lymphoma cells to poly(ADP-ribose) polymerase 1 and phosphoinositide 3-kinase inhibitors, suggesting that triptolide may be a potent antitumor drug for sensitizing lymphoma cells to chemotherapeutic agents.

Inhibition of Rad51 sensitizes breast cancer cells with wild-type PTEN to olaparib.

PTEN is a tumor suppressor gene well characterized as a phosphatase. However, more evidences demonstrate PTEN functions in DNA repair independent of its phosphatase activity, which affects the efficacy of DNA damage anti-tumoral drugs in treating cancer cells with PTEN variations. Using BT549 breast cancer cells, we studied the roles of PTEN in DNA repair and in sensitization of breast cancer cells to olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor. Comet assay showed PTEN promoted DNA repair. PTEN-deficient BT549 cells are sensitive to olaparib, which shows the synthetic lethality between PTEN and PARP1. We expressed PTEN in BT549 cells and found PTEN-proficient BT549 cells resist to olaparib. Western blot showed that PTEN up-regulated Rad51 expression, suggesting PTEN promotes DNA repair through Rad51-dependnent homologous recombination. We used 5µM olaparib or 5µM RI-1, a Rad51 inhibitor, to treat PTEN-proficient BT549 cells respectively. The immunofluorescent analysis showed the combination of olaparib and RI-1 induced more than 4-fold of γH2AX foci than either of them. MTT assay showed 5µM RI-1 did not change the survival of PTEN-proficient BT549 cells, however, this dose of RI-1 sensitized PTEN-proficient BT549 cells to olaparib. Consequently, these results demonstrate that inhibition of Rad51 can sensitize BT549 cells with wild type PTEN to olaparib, which would contribute to using PARP inhibitors in individual treatment of breast cancer patients with PTEN variations.

Nuclear PTEN interferes with binding of Ku70 at double-strand breaks through post-translational poly(ADP-ribosyl)ation.

PTEN is a tumor suppressor gene characterized as a phosphatase that antagonizes the phosphatidylinositol 3-kinase signaling pathway in the cytoplasm. Nuclear PTEN plays roles in chromosomal stability, in which the double-strand breaks (DSB) repair mediated by homologous recombination (HR) and non-homologous end joining (NHEJ) is critical. Herein, the role of nuclear PTEN in DSB repair and the underlying molecular mechanism was investigated in this study. Using human breast cancer BT549 and MDA-MB-231 cell lines, we reveal a specific feature of PTEN that controls poly(ADP-ribosyl)ation of Ku70 and interferes with binding of Ku70 at DSB. Plasmid-based end joining and reporter assays showed that nuclear PTEN restrained NHEJ efficacy. Electrophoretic mobility shift assays showed that nuclear PTEN impaired Ku70 complex binding to DSB by 3-fold. Co-immunoprecipitation assay showed PTEN regulated poly(ADP-ribosyl)ation of Ku70 instead of directly interacting with Ku70, while PTEN promoted the poly(ADP-ribosyl)ation of PARP1 and induced the degradation of PARP1 in PTEN-WT cells exposed to DSB agents. Of note, the role of PTEN in DSB repair mostly depends on its nuclear localization rather than its phosphatase activity. As a result, the absence of nuclear PTEN rather than phosphatase-negative PTEN confers cell hypersensitivity to anti-tumor DNA damage drugs. This finding contributes to understanding the effect of PTEN in repair of DSB and using defined anti-tumor DSB drugs to treat tumor cells with aberrant PTEN.