Activation of ERß hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor ß (ERß) in TNBC, but the detailed molecular mechanisms downstream ERß activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERß agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERß were critical for the binding between U2AF1 and ERß. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERß-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERß activation in TNBC, which provides rationale for ERß activation-based single or combined therapy for patients with TNBC.

VPS34 Acetylation Controls Its Lipid Kinase Activity and the Initiation of Canonical and Non-canonical Autophagy.

The class III phosphoinositide 3-kinase VPS34 plays a key role in the regulation of vesicular trafficking and macroautophagy. So far, we know little about the molecular mechanism of VPS34 activation besides its interaction with regulatory proteins to form complexes. Here, we report that VPS34 is specifically acetylated by the acetyltransferase p300, and p300-mediated acetylation represses VPS34 activity. Acetylation at K771 directly diminishes the affinity of VPS34 for its substrate PI, while acetylation at K29 hinders the VPS34-Beclin 1 core complex formation. Inactivation of p300 induces VPS34 deacetylation, PI3P production, and autophagy, even in AMPK-/-, TSC2-/-, or ULK1-/- cells. In fasting mice, liver autophagy correlates well with p300 inactivation/VPS34 deacetylation, which facilitates the clearance of lipid droplets in hepatocytes. Thus, p300-dependent VPS34 acetylation/deacetylation is the physiological key to VPS34 activation, which controls the initiation of canonical autophagy and of non-canonical autophagy in which the upstream kinases of VPS34 can be bypassed.

Application Prospect of Induced Pluripotent Stem Cells in Organoids and Cell Therapy.

Since its inception, induced pluripotent stem cell (iPSC) technology has been hailed as a powerful tool for comprehending disease etiology and advancing drug screening across various domains. While earlier iPSC-based disease modeling and drug assessment primarily operated at the cellular level, recent years have witnessed a significant shift towards organoid-based investigations. Organoids derived from iPSCs offer distinct advantages, particularly in enabling the observation of disease progression and drug metabolism in an in vivo-like environment, surpassing the capabilities of iPSC-derived cells. Furthermore, iPSC-based cell therapy has emerged as a focal point of clinical interest. In this review, we provide an extensive overview of non-integrative reprogramming methods that have evolved since the inception of iPSC technology. We also deliver a comprehensive examination of iPSC-derived organoids, spanning the realms of the nervous system, cardiovascular system, and oncology, as well as systematically elucidate recent advancements in iPSC-related cell therapies.

AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation.

Eukaryotes initiate autophagy to cope with the lack of external nutrients, which requires the activation of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase Sirtuin 1 (Sirt1). However, the mechanisms underlying the starvation-induced Sirt1 activation for autophagy initiation remain unclear. Here, we demonstrate that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a conventional glycolytic enzyme, is a critical mediator of AMP-activated protein kinase (AMPK)-driven Sirt1 activation. Under glucose starvation, but not amino acid starvation, cytoplasmic GAPDH is phosphorylated on Ser122 by activated AMPK. This causes GAPDH to redistribute into the nucleus. Inside the nucleus, GAPDH interacts directly with Sirt1, displacing Sirt1's repressor and causing Sirt1 to become activated. Preventing this shift of GAPDH abolishes Sirt1 activation and autophagy, while enhancing it, through overexpression of nuclear-localized GAPDH, increases Sirt1 activation and autophagy. GAPDH is thus a pivotal and central regulator of autophagy under glucose deficiency, undergoing AMPK-dependent phosphorylation and nuclear translocation to activate Sirt1 deacetylase activity.

THE PRESENT AND FUTURE OF THE MASS SPECTROMETRY-BASED INVESTIGATION OF THE EXOSOME LANDSCAPE.

Exosomes are critical intercellular messengers released upon the fusion of multivesicular bodies with the cellular plasma membrane that deliver their cargo in the form of extracellular vesicles. Containing numerous nonrandomly packed functional proteins, lipids, and RNAs, exosomes are vital intercellular messengers that contribute to the physiologic processes of the healthy organism. During the post-genome era, exosome-oriented proteomics have garnered great interest. Since its establishment, mass spectrometry (MS) has been indispensable for the field of proteomics research and has advanced rapidly to interrogate biological samples at a higher resolution and sensitivity. Driven by new methodologies and more advanced instrumentation, MS-based approaches have revolutionized our understanding of protein biology. As the access to online proteomics database platforms has blossomed, experimental data processing occurs with more speed and accuracy. Here, we review recent advances in the technological progress of MS-based proteomics and several new detection strategies for MS-based proteomics research. We also summarize the use of integrated online databases for proteomics research in the era of big data. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Pharmacological activation of estrogen receptor beta augments innate immunity to suppress cancer metastasis.

Metastases constitute the greatest causes of deaths from cancer. However, no effective therapeutic options currently exist for cancer patients with metastasis. Estrogen receptor ß (ERß), as a member of the nuclear receptor superfamily, shows potent tumor-suppressive activities in many cancers. To investigate whether modulation of ERß could serve as a therapeutic strategy for cancer metastasis, we examined whether the selective ERß agonist LY500307 could suppress lung metastasis of triple-negative breast cancer (TNBC) and melanoma. Mechanistically, while we observed that LY500307 potently induced cell death of cancer cells metastasized to lung in vivo, it does not mediate apoptosis of cancer cells in vitro, indicating that the cell death-inducing effects of LY500307 might be mediated by the tumor microenvironment. Pathological examination combined with flow cytometry assays indicated that LY500307 treatment induced significant infiltration of neutrophils in the metastatic niche. Functional experiments demonstrated that LY500307-treated cancer cells show chemotactic effects for neutrophils and that in vivo neutrophil depletion by Ly6G antibody administration could reverse the effects of LY500307-mediated metastasis suppression. RNA sequencing analysis showed that LY500307 could induce up-regulation of IL-1ß in TNBC and melanoma cells, which further triggered antitumor neutrophil chemotaxis. However, the therapeutic effects of LY500307 treatment for suppression of lung metastasis was attenuated in IL1B-/- murine models, due to failure to induce antitumor neutrophil infiltration in the metastatic niche. Collectively, our study demonstrated that pharmacological activation of ERß could augment innate immunity to suppress cancer metastatic colonization to lung, thus providing alternative therapeutic options for cancer patients with metastasis.

[An association study between gene polymorphism of the key enzyme's folacin metabolism pathway and plasmatic homocysteine levels in fertile woman].

OBJECTIVE: The effect of the gene polymorphism for the key enzyme's folacin metabolism pathway on plasmatic homocysteine (Hcy) levels in fertile woman was observed. METHODS: The subjects were from Shaoxing City, Jiangsu province in 2012, the selection criteria for the women of childbearing age were between 20-45 years old, with an average age of 28.2 (95%CI:27.8-28.6) years old. Sample collection continued uninterrupted lasted seven days, a total of 535 samples were collected, venous blood with EDTA addition or sodium citrate to anticoagulant. After separation, the blood cells and blood plasma were cryopreserved. DNA was extracted using spin column method. All the samples were selected for the gene polymorphism testing of the key enzyme's on folate metabolism and monitoring of plasmatic Hcy level. RESULTS: Eight single nucleotide polymorphism (SNP) sites of methylenetetrahydrofolate reductase gene (MTHFR) , methionine synthase gene (MS) , synthetic methionine reductase gene (MSR) and cystathionine ß synthase gene (CBS) were detected. It was found the genotype AA of the SNP sites-rs1801131 would result higher plasmatic Hcy levels (8.99 µmol/L) than the genotypes CC (7.81 µmol/L) and CA(8.38 µmol/L) (P < 0.01) . Similarly, the genotype TT of the SNP sites-rs1801133 was significantly responded to the increasing of Hcy levels (11.10 µmol/L) than the genotype CC (8.15 µmol/L) and CT (8.45 µmol/L), (P < 0.01) . The two sites of genotype combination of AA-TT could also result in the significant increase of Hcy levels (11.02 µmol/L) than other combined genotypes (genotypes CC-CC, CA-CC, CA-CT, AA-CC, AA-CT), especially the genotype CC-CC. And the risk factor was 1.41 (95CI:1.20-1.66) times over the genotype CC-CC. CONCLUSION: The gene mutations of two SNP sites rs1801131 and rs1801133 in MTHFR would increase Hcy levels.

Targeting PKM2 signaling cascade with salvianic acid A normalizes tumor blood vessels to facilitate chemotherapeutic drug delivery.

Aberrant tumor blood vessels are prone to propel the malignant progression of tumors, and targeting abnormal metabolism of tumor endothelial cells emerges as a promising option to achieve vascular normalization and antagonize tumor progression. Herein, we demonstrated that salvianic acid A (SAA) played a pivotal role in contributing to vascular normalization in the tumor-bearing mice, thereby improving delivery and effectiveness of the chemotherapeutic agent. SAA was capable of inhibiting glycolysis and strengthening endothelial junctions in the human umbilical vein endothelial cells (HUVECs) exposed to hypoxia. Mechanistically, SAA was inclined to directly bind to the glycolytic enzyme PKM2, leading to a dramatic decrease in endothelial glycolysis. More importantly, SAA improved the endothelial integrity via activating the ß-Catenin/Claudin-5 signaling axis in a PKM2-dependent manner. Our findings suggest that SAA may serve as a potent agent for inducing tumor vascular normalization.

The genomic and immunogenomic landscape of mechanics pathway informs clinical prognosis and response to mechanotherapy.

Mechanics shape cell and tissue plasticity and maintain their homeostasis. In cancers, mechanical signals regulate cancer hallmarks via mechanotransduction pathways, such as proliferation, metastasis and metabolic reprogramming. However, comprehensive characterization of mechanotransduction pathway genes and their clinical relevance across different cancer types remains untouched. Herein, we systematically portrayed the alterations of mechanotransduction pathway genes across 31 cancer types using The Cancer Genome Atlas (TCGA) databases. All the cancer types could be categorized into 6 subtypes based upon the transcriptional pattern of mechanics pathway genes. Each subtype has its own unique molecular expression pattern, mutation landscapes, immune infiltrates, and patient clinical outcome. We further found that the responses of two subtypes of cancers, one with the optimal outcome and the other with the worst prognosis, to a classical mechanotherapeutic agent (Fasudil, RhoA/ROCK inhibitor) were totally different, indicating that our cancer stratification system based upon mechanotransduction pathway genes could inform clinical responses of patients to mechanotherapeutic agents. Collectively, our study provides a novel pan-cancer landscape of the mechanotransduction pathways and underscores its potential clinical significance in the prediction of clinical prognosis and therapeutic responses to mechanotherapy among cancer patients.

Suppressing MDSC Infiltration in Tumor Microenvironment Serves as an Option for Treating Ovarian Cancer Metastasis.

It is still a big puzzle how ovarian cancer cells and the tumor microenvironment (TME) attract lymphocytes infiltration for facilitating metastasis, a leading cause of death from gynecological malignancies. Using genome-wide LncRNA microarray assay, here we report that a LncRNA associated with ovarian cancer metastasis (LncOVM) is highly correlated with poor prognosis and survival. LncOVM interacts with and stabilizes PPIP5K2 by suppressing ubiquitinated degradation to promote complement C5 secretion from ovarian cancer cells. The TME-enriched complement C5 attracts myeloid-derived suppressor cells (MDSCs) infiltration in TME to facilitate metastasis. Knockdown of LncOVM or PPIP5K2 inhibits tumor progression in xenograft models. Application of C5aR antibody or inhibitor (CCX168) inhibits MDSC recruitment and restores the suppression of tumorigenesis and metastasis in vivo. Our study reveals that suppression of ovarian cancer metastasis can be achieved by targeting MDSC infiltration in TME through disrupting LncOVM-PPIP5K2-complement axis, providing an option for treating ovarian cancer patients.

Acetylation of STX17 (syntaxin 17) controls autophagosome maturation.

The fusion of autophagosomes and endosomes/lysosomes, also called autophagosome maturation, ensures the degradation of autophagic cargoes. It is an important regulatory step of the macroautophagy/autophagy process. STX17 is the key autophagosomal SNARE protein that mediates autophagosome maturation. Here, we report that the acetylation of STX17 regulates its SNARE activity and autophagic degradation. The histone acetyltransferase CREBBP/CBP and the deacetylase HDAC2 specifically regulate the acetylation of STX17. In response to cell starvation and MTORC1 inhibition, the inactivation of CREBBP leads to the deacetylation of STX17 at its SNARE domain. This deacetylation promotes the interaction between STX17 and SNAP29 and the formation of the STX17-SNAP29-VAMP8 SNARE complex with no effect on the recruitment of STX17 to autophagosomal membranes. Deacetylation of STX17 also enhances the interaction between STX17 and the tethering complex HOPS, thereby further promoting autophagosome-lysosome fusion. Our study suggests a mechanism by which acetylation regulates the late-stage of autophagy, and possibly other STX17-related intracellular membrane fusion events.Abbreviations: ACTB: actin beta; CREBBP/CBP: CREB binding protein; Ctrl: control; GFP: green fluorescent protein; GST: glutathione S-transferase; HDAC: histone deacetylase; HOPS: homotypic fusion and protein sorting complex; KO: knockout; LAMP1/2: lysosomal associated membrane protein 1/2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MS: mass spectrometry; MTORC1: mechanistic target of rapamycin kinase complex 1; NAM: nicotinamide; PtdIns3K: phosphatidylinositol 3-kinase; RFP: red fluorescent protein; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TSA: trichostatin A; TSC1/2: TSC complex subunit 1/2; VAMP8: vesicle associated membrane protein 8; WT: wild type.

The RNA binding protein SORBS2 suppresses metastatic colonization of ovarian cancer by stabilizing tumor-suppressive immunomodulatory transcripts.

BACKGROUND: Ovarian cancer constitutes one of the most lethal gynecologic malignancies for females. Currently, early detection strategies and therapeutic options for ovarian cancer are far from satisfactory, leading to high diagnosis rates at late stages and disease relapses. New avenues of therapy are needed that target key processes in ovarian cancer progression. While a variety of non-coding RNAs have been proven to regulate ovarian cancer metastatic progression, the functional roles of RNA-binding proteins (RBPs) in this process are less well defined. RESULTS: In this study, we identify that the RBP sorbin and SH3 domain containing 2 (SORBS2) is a potent suppressor of ovarian cancer metastatic colonization. Mechanistic studies show that SORBS2 binds the 3' untranslated regions (UTRs) of WFDC1 (WAP four-disulfide core domain 1) and IL-17D (Interleukin-17D), two secreted molecules that are shown to act as metastasis suppressors. Enhanced expression of either WFDC1 or IL-17D potently represses SORBS2 depletion-mediated cancer metastasis promotion. By enhancing the stability of these gene transcripts, SORBS2 suppresses ovarian cancer invasiveness and affects monocyte to myeloid-derived suppressor cell and M2-like macrophage polarization, eliciting a tumor-suppressive immune microenvironment. CONCLUSIONS: Our data illustrate a novel post-transcriptional network that links cancer progression and immunomodulation within the tumor microenvironment through SORBS2-mediated transcript stabilization.

Small GTPase Arl6 controls RH30 rhabdomyosarcoma cell growth through ciliogenesis and Hedgehog signaling.

BACKGROUND: Rhabdomyosarcoma (RMS) originates from skeletal muscle precursors that fail to differentiate. Hedgehog (Hh) signaling and primary cilia contribute to the pathobiology of RMS. RESULTS: Here we showed ADP ribosylation factor like GTPase 6 (ARL6) localizes at the base of primary cilium, controls ciliogenesis and Hh signaling. The transcription of Arl6 is dynamic during the differentiation of myoblasts, companying with the growth and elimination of primary cilia. Arl6 expression is significantly up regulated in cilia-dependent RMS cells and tissues. Knockdown of Arl6 inhibits proliferation and promotes apoptosis of RMS RH30 cells through defected ciliogenesis and reduced Hh activity. CONCLUSIONS: Taken together, the functions of Arl6 in ciliogenesis and Hh signaling suggest it as a potential RMS drug target.

Requirement of Smurf-mediated endocytosis of Patched1 in sonic hedgehog signal reception.

Cell surface reception of Sonic hedgehog (Shh) must ensure that the graded morphogenic signal is interpreted accordingly in neighboring cells to specify tissue patterns during development. Here, we report endocytic sorting signals for the receptor Patched1 (Ptch1), comprising two 'PPXY' motifs, that direct it to degradation in lysosomes. These signals are recognized by two HECT-domain ubiquitin E3 ligases, Smurf1 and Smurf2, which are induced by Shh and become enriched in Caveolin-1 lipid rafts in association with Ptch1. Smurf-mediated endocytic turnover of Ptch1 is essential for its clearance from the primary cilium and pathway activation. Removal of both Smurfs completely abolishes the ability of Shh to sustain the proliferation of postnatal granule cell precursors in the cerebellum. These findings reveal a novel step in the Shh pathway activation as part of the Ptch1 negative feedback loop that precisely controls the signaling output in response to Shh gradient signal.