Dysfunctional activity of classical DNA end-joining renders acquired resistance to carboplatin in human ovarian cancer cells.

Ovarian cancer is the deadliest gynecological malignancy worldwide. Although chemotherapy is required as the most standard treatment strategy for ovarian cancer, the survival rates are very low, largely because of high incidence of recurrence due to resistance to conventional surgery and genotoxic chemotherapies. Carboplatin-resistant ovarian cancer cells were generated by continuous treatment over six months. Carboplatin-resistance induced morphological alterations and promoted the rates of proliferation and migration of SKOV3 compared to the parental cells. Interestingly, carboplatin-resistant SKOV3 showed the high levels of γH2AX foci formed at the basal level, and the levels of γH2AX foci remained even after the recovery time, suggesting that the DNA damage response and repair machinery were severely attenuated by carboplatin-resistance. Surprisingly, the expression levels of XRCC4, a critical factor in non-homologous end joining (NHEJ) DNA repair, were significantly decreased in carboplatin-resistant SKOV3 compared with those in non-resistant controls. Furthermore, restoration of NHEJ in carboplatin-resistant SKOV3 by suppression of ABCB1 and/or AR re-sensitized carboplatin-resistant cells to genotoxic stress and reduced their proliferation ability. Our findings suggest that attenuation of the NHEJ DNA repair machinery mediated by resistance to genotoxic stress might be a critical cause of chemoresistance in patients with ovarian cancer.

Three-dimensional microengineered vascularised endometrium-on-a-chip.

STUDY QUESTION: Can we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model? SUMMARY ANSWER: Our representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner. WHAT IS KNOWN ALREADY: Organ-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems. STUDY DESIGN, SIZE, DURATION: The device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5-6 days (total ∼ 14 days) for the purpose of each experiment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Microfluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients. MAIN RESULTS AND THE ROLE OF CHANCE: We established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells. WIDER IMPLICATIONS OF THE FINDINGS: Our 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner. STUDY FUNDING/COMPETING INTEREST(S): This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.

CXCL12 enhances pregnancy outcome via improvement of endometrial receptivity in mice.

Successful pregnancy inevitably depends on the implantation of a competent embryo into a receptive endometrium. Although many substances have been suggested to improve the rate of embryo implantation targeting enhancement of endometrial receptivity, currently there rarely are effective evidence-based treatments to prevent or cure this condition. Here we strongly suggest minimally-invasive intra-uterine administration of embryo-secreted chemokine CXCL12 as an effective therapeutic intervention. Chemokine CXCL12 derived from pre- and peri-implanting embryos significantly enhances the rates of embryo attachment and promoted endothelial vessel formation and sprouting in vitro. Consistently, intra-uterine CXCL12 administration in C57BL/6 mice improved endometrial receptivity showing increased integrin ß3 and its ligand osteopontin, and induced endometrial angiogenesis displaying increased numbers of vessel formation near the lining of endometrial epithelial layer with higher CD31 and CD34 expression. Furthermore, intra-uterine CXCL12 application dramatically promoted the rates of embryo implantation with no morphologically retarded embryos. Thus, our present study provides a novel evidence that improved uterine endometrial receptivity and enhanced angiogenesis induced by embryo-derived chemokine CXCL12 may aid to develop a minimally-invasive therapeutic strategy for clinical treatment or supplement for the patients with repeated implantation failure with less risk.

TRIM28 functions as a negative regulator of aggresome formation.

Selective recognition and elimination of misfolded polypeptides are crucial for protein homeostasis. When the ubiquitin-proteasome system is impaired, misfolded polypeptides tend to form small cytosolic aggregates and are transported to the aggresome and eventually eliminated by the autophagy pathway. Despite the importance of this process, the regulation of aggresome formation remains poorly understood. Here, we identify TRIM28/TIF1ß/KAP1 (tripartite motif containing 28) as a negative regulator of aggresome formation. Direct interaction between TRIM28 and CTIF (cap binding complex dependent translation initiation factor) leads to inefficient aggresomal targeting of misfolded polypeptides. We also find that either treatment of cells with poly I:C or infection of the cells by influenza A viruses triggers the phosphorylation of TRIM28 at S473 in a way that depends on double-stranded RNA-activated protein kinase. The phosphorylation promotes association of TRIM28 with CTIF, inhibits aggresome formation, and consequently suppresses viral proliferation. Collectively, our data provide compelling evidence that TRIM28 is a negative regulator of aggresome formation.Abbreviations: BAG3: BCL2-associated athanogene 3; CTIF: CBC-dependent translation initiation factor; CED: CTIF-EEF1A1-DCTN1; DCTN1: dynactin subunit 1; EEF1A1: eukaryotic translation elongation factor 1 alpha 1; EIF2AK2: eukaryotic translation initiation factor 2 alpha kinase 2; HDAC6: histone deacetylase 6; IAV: influenza A virus; IP: immunoprecipitation; PLA: proximity ligation assay; polypeptidyl-puro: polypeptidyl-puromycin; qRT-PCR: quantitative reverse-transcription PCR; siRNA: small interfering RNA.

Non-invasive Intrauterine Administration of Botulinum Toxin A Enhances Endometrial Angiogenesis and Improves the Rates of Embryo Implantation.

Endometrial angiogenesis plays crucial roles in determining the endometrial receptivity. Defects in endometrial receptivity often cause repeated implantation failure, which is one of the major unmet needs for infertility and contributes a major barrier to the assisted reproductive technology. Despite the numerous extensive research work, there are currently no effective evidence-based treatments to prevent or cure this condition. As a non-invasive treatment strategy, botulinum toxin A (BoTA) was administered into one side of mouse uterine horns, and saline was infused into the other side of horns for the control. Impact of BoTA was assessed in the endometrium at 3 or 8 days after infusion. We demonstrated that BoTA administration enhances the capacity of endothelial cell tube formation and sprouting. The intrauterine BoTA administration significantly induced endometrial angiogenesis displaying increased numbers of vessel formation and expression levels of related marker genes. Moreover, BoTA intrauterine application promoted the endometrial receptivity, and the rates of embryo implantation were improved with BoTA treatment with no morphologically retarded embryos. Intrauterine BoTA treatment has a beneficial effect on vascular reconstruction of functional endometrium prior to embryo implantation by increasing endometrial blood flow near the uterine cavity suggesting BoTA treatment as a potential therapeutic strategy for patients who are suffering from repeated implantation failure with the problems with endometrial receptivity.

Intramembrane proteolysis of an extracellular serine protease, epithin/PRSS14, enables its intracellular nuclear function.

BACKGROUND: Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. RESULTS: In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. CONCLUSIONS: These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CßII.

Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α-converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA-mediated knockdown, and overexpression of dominant-negative PKCßII variants indicated that PKCßII is a major player in JNK inhibition- and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCßII and tumor necrosis factor-α-converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCßII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.

Arabidopsis cargo receptor NBR1 mediates selective autophagy of defective proteins.

Aggrephagy, a type of selective autophagy that sequesters protein aggregates for degradation in the vacuole, is an important protein quality control mechanism, particularly during cell stress. In mammalian cells, aggrephagy and several other forms of selective autophagy are mediated by dedicated cargo receptors such as NEIGHBOR OF BRCA1 (NBR1). Although plant NBR1 homologs have been linked to selective autophagy during biotic stress, it remains unclear how they impact selective autophagy under non-stressed and abiotic stress conditions. Through microscopic and biochemical analysis of nbr1 mutants expressing autophagy markers and an aggregation-prone reporter, we tested the connection between NBR1 and aggrephagy in Arabidopsis. Although NBR1 is not essential for general autophagy, or for the selective clearance of peroxisomes, mitochondria, or the ER, we found that NBR1 is required for the heat-induced formation of autophagic vesicles. Moreover, cytoplasmic puncta containing aggregation-prone proteins, which were rarely observed in wild-type plants, were found to accumulate in nbr1 mutants under both control and heat stress conditions. Given that NBR1 co-localizes with these cytoplasmic puncta, we propose that Arabidopsis NBR1 is a plant aggrephagy receptor essential for maintaining proteostasis under both heat stress and non-stress conditions.