Hypoxic tumor-derived exosomal miR-4488 induces macrophage M2 polarization to promote liver metastasis of pancreatic neuroendocrine neoplasm through RTN3/FABP5 mediated fatty acid oxidation.

Tumor-associated macrophages (TAMs) represent a predominant cellular component within the tumor microenvironment (TME) of pancreatic neuroendocrine neoplasms (pNENs). There is a growing body of evidence highlighting the critical role of exosomes in facilitating communication between tumor cells and TAMs, thereby contributing to the establishment of the premetastatic niche. Nonetheless, the specific mechanisms through which exosomes derived from tumor cells influence macrophage polarization under hypoxic conditions in pNENs, and the manner in which these interactions support cancer metastasis, remain largely unexplored. Recognizing the capacity of exosomes to transfer miRNAs that can modify cellular behaviors, our research identified a significant overexpression of miR-4488 in exosomes derived from hypoxic pNEN cells. Furthermore, we observed that macrophages that absorbed circulating exosomal miR-4488 underwent M2-like polarization. Our investigations revealed that miR-4488 promotes M2-like polarization by directly targeting and suppressing RTN3 in macrophages. This suppression of RTN3 enhances fatty acid oxidation and activates the PI3K/AKT/mTOR signaling pathway through the interaction and downregulation of FABP5. Additionally, M2 polarized macrophages contribute to the formation of the premetastatic niche and advance pNENs metastasis by releasing MMP2, thereby establishing a positive feedback loop involving miR-4488, RTN3, FABP5, and MMP2 in pNEN cells. Together, these findings shed light on the role of exosomal miRNAs from hypoxic pNEN cells in mediating interactions between pNEN cells and intrahepatic macrophages, suggesting that miR-4488 holds potential as a valuable biomarker and therapeutic target for pNENs.

Hbxip is essential for murine embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1.

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

Antitumor effects of anlotinib in thyroid cancer.

There is no effective treatment for patients with poorly differentiated papillary thyroid cancer or anaplastic thyroid cancer (ATC). Anlotinib, a multi-kinase inhibitor, has already shown antitumor effects in various types of carcinoma in a phase I clinical trial. In this study, we aimed to better understand the effect and efficacy of anlotinib against thyroid carcinoma cells in vitro and in vivo. We found that anlotinib inhibits the cell viability of papillary thyroid cancer and ATC cell lines, likely due to abnormal spindle assembly, G2/M arrest, and activation of TP53 upon anlotinib treatment. Moreover, anlotinib suppresses the migration of thyroid cancer cells in vitro and the growth of xenograft thyroid tumors in mice. Our data demonstrate that anlotinib has significant anticancer activity in thyroid cancer, and potentially offers an effective therapeutic strategy for patients of advanced thyroid cancer type.

Genome-Wide RNAi Screen Identify Melanoma-Associated Antigen Mageb3 Involved in X Chromosome Inactivation.

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.

Pold3 is required for genomic stability and telomere integrity in embryonic stem cells and meiosis.

Embryonic stem cells (ESCs) and meiosis are featured by relatively higher frequent homologous recombination associated with DNA double strand breaks (DSB) repair. Here, we show that Pold3 plays important roles in DSB repair, telomere maintenance and genomic stability of both ESCs and spermatocytes in mice. By attempting to generate Pold3 deficient mice using CRISPR/Cas9 or transcription activator-like effector nucleases, we show that complete loss of Pold3 (Pold3-/-) resulted in early embryonic lethality at E6.5. Rapid DNA damage response and massive apoptosis occurred in both outgrowths of Pold3-null (Pold3-/-) blastocysts and Pold3 inducible knockout (iKO) ESCs. While Pold3-/- ESCs were not achievable, Pold3 iKO led to increased DNA damage response, telomere loss and chromosome breaks accompanied by extended S phase. Meanwhile, loss of Pold3 resulted in replicative stress, micronucleation and aneuploidy. Also, DNA repair was impaired in Pold3+/- or Pold3 knockdown ESCs. Moreover, Pold3 mediates DNA replication and repair by regulating 53BP1, RIF1, ATR and ATM pathways. Furthermore, spermatocytes of Pold3 haploinsufficient (Pold3+/-) mice with increasing age displayed impaired DSB repair, telomere shortening and loss, and chromosome breaks, like Pold3 iKO ESCs. These data suggest that Pold3 maintains telomere integrity and genomic stability of both ESCs and meiosis by suppressing replicative stress.

RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells.

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.

Rho differentially regulates the Hippo pathway by modulating the interaction between Amot and Nf2 in the blastocyst.

The Hippo pathway modulates the transcriptional activity of Yap to regulate the differentiation of the inner cell mass (ICM) and the trophectoderm (TE) in blastocysts. Yet how Hippo signaling is differentially regulated in ICM and TE cells is poorly understood. Through an inhibitor/activator screen, we have identified Rho as a negative regulator of Hippo in TE cells, and PKA as a positive regulator of Hippo in ICM cells. We further elucidated a novel mechanism by which Rho suppresses Hippo, distinct from the prevailing view that Rho inhibits Hippo signaling through modulating cytoskeleton remodeling and/or cell polarity. Active Rho prevents the phosphorylation of Amot Ser176, thus stabilizing the interaction between Amot and F-actin, and restricting the binding between Amot and Nf2. Moreover, Rho attenuates the interaction between Amot and Nf2 by binding to the coiled-coil domain of Amot. By blocking the association of Nf2 and Amot, Rho suppresses Hippo in TE cells.

Rtfc (4931414P19Rik) Regulates in vitro Thyroid Differentiation and in vivo Thyroid Function.

Thyroid is a one of the most important endocrine organs. Understanding the molecular mechanism underlying thyroid development and function, as well as thyroid diseases, is beneficial for the clinical treatment of thyroid diseases and tumors. Through genetic linkage analysis and exome sequencing, we previously identified an uncharacterized gene C14orf93 (RTFC, mouse homolog: 4931414P19Rik) as a novel susceptibility gene for familial non-medullary thyroid carcinoma, and demonstrated its function in promoting thyroid tumor. However, the role of RTFC in thyroid development and function remains unexplored. In this study, we found that knockout of Rtfc compromises the in vitro thyroid differentiation of mouse embryonic stem cells. In contrast, Rtfc-/- mice are viable and fertile, and the size and the morphology of thyroid are not affected by Rtfc knockout. However, female Rtfc-/- mice, but not male Rtfc-/- mice, display mild hypothyroidism. In summary, our data suggest the roles of Rtfc in in vitro thyroid differentiation of embryonic stem cells, and in vivo thyroid function.

C14orf93 (RTFC) is identified as a novel susceptibility gene for familial nonmedullary thyroid cancer.

The genetic causes for familial nonmedullary thyroid cancer (FNMTC) remain largely unknown. Through genetic linkage analysis and exome sequencing, C14orf93 (RTFC), PYGL, and BMP4 were identified as susceptibility gene candidates in a FNMTC family. By examining the expression and the oncogenic functions of these candidate genes, PYGL and BMP4 were excluded. We further characterized the functions of the uncharacterized gene RTFC in thyroid cancer. RTFC promotes thyroid cancer cell survival under starving conditions, and thyroid cancer cell migration. The R115Q, V205M and G209D RTFC mutants enhance the colony forming capacity of thyroid cancer cells, and are able to transform normal thyroid cells. In summary, our data suggest the roles of RTFC in thyroid carcinogenesis.

Roles of Klf5 Acetylation in the Self-Renewal and the Differentiation of Mouse Embryonic Stem Cells.

Transcription factor Krüppel-like factor 5 (Klf5) plays important roles in the formation of the inner cell mass (ICM) and the trophectoderm during embryogenesis, as well as the self-renewal and the differentiation of mouse embryonic stem cells (ESCs). Acetylation of KLF5 has been shown to reverse the transcriptional activity of KLF5 in human epidermal cells and prostate cancer cells. Whether Klf5 acetylation contributes to the lineage specification in the blastocyst and pluripotency maintenance in ESCs remains unexplored. Here, we showed the ubiquitous expression of acetylated Klf5 in the ICM and the trophectoderm, ruling out the possibility that differential acetylation status of Klf5 leads to the lineage specification in the blastocyst. We found that K358Q mutation, mimicking acetylation, enhances the transcriptional activity of Klf5 for pluripotency genes in ESCs, and that K358Q Klf5 is more potent in pluripotency maintenance and in somatic cell reprogramming, compared to K358R Klf5. In ESCs, Klf5 acetylation, stimulated by TGF-ß signaling, is involved in enhancing Sox2 expression. Moreover, upon ESC differentiation, acetylation of Klf5 facilitates the suppression of many differentiation genes, except for that K358Q Klf5 activates Cdx2, promoting trophectodermal differentiation. In summary, our results revealed the regulatory functions of Klf5 acetylation in ESC self-renewal and differentiation.

Systematic identification of factors for provirus silencing in embryonic stem cells.

Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.

Lysine residues 639 and 673 of mouse Ncoa3 are ubiquitination sites for the regulation of its stability.

Ncoa3 is a transcriptional coactivator involved in a wide range of biological processes. Regulation of Ncoa3 protein stability is important to control its activity precisely. Here, we found that deleting amino acid residues 614-740 of Ncoa3 enhances the protein expression level. Replacing two lysine residues, K639 and K673, within this region by arginine, increases the stability of the luciferase fusion protein as well as Ncoa3 protein. When these two lysine residues are mutated to arginine, the overall ubiquitination level of Ncoa3 decreases, indicating that lysine 639 and 673 are its ubiquitination sites. Taken together, we identified two ubiquitination sites at lysine 639 and 673 of Ncoa3. Ubiquitination of these two lysine residues leads to proteasomal degradation of Ncoa3.

Hepatitis C virus NS5A hijacks ARFGAP1 to maintain a phosphatidylinositol 4-phosphate-enriched microenvironment.

UNLABELLED: Phosphatidylinositol 4-phosphate (PI4P) is well known to be upregulated during hepatitis C virus (HCV) replication. The role of PI4 kinases in HCV has been extensively investigated. Whether the PI4P phosphatase Sac1 is altered by HCV remains unclear. Here, we identified ARFGAP1 to be a novel host factor for HCV replication. We further show that Sac1 interacts with ARFGAP1 and inhibits HCV replication. The elevation of PI4P induced by HCV NS5A is abrogated when the coatomer protein I (COPI) pathway is inhibited. We also found an interaction between NS5A and ARFGAP1. Furthermore, we identified a conserved cluster of positively charged amino acids in NS5A critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. Our data demonstrate that ARFGAP1 is a host factor for HCV RNA replication. ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the site of HCV replication to maintain high levels of PI4P. Our findings provide an additional mechanism by which HCV enhances formation of a PI4P-rich environment. IMPORTANCE: PI4P is enriched in the replication area of HCV; however, whether PI4P phosphatase Sac1 is subverted by HCV is not established. The detailed mechanism of how COPI contributes to viral replication remains unknown, though COPI components were hijacked by HCV. We demonstrate that ARFGAP1 is hijacked by HCV NS5A to remove COPI cargo Sac1 from the HCV replication area to maintain high-level PI4P generated by NS5A. Furthermore, we identify a conserved cluster of positively charged amino acids in NS5A, which are critical for interaction between NS5A and ARFGAP1, induction of PI4P, and HCV replication. This study will shed mechanistic insight on how other RNA viruses hijack COPI and Sac1.

The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF.

Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1α) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis.

Taiwanofungus camphoratus (Syn Antrodia camphorata) extract and amphotericin B exert adjuvant effects via mitochondrial apoptotic pathway.

The use of multiple drugs in cancer therapy increases the efficacy of the potential therapeutic effects. In this study, the authors investigated the adjuvant effects of an ethanol extract of solid-state cultivated Taiwanofungus camphoratus (TCEE) and amphotericin B (AmB) in the human cancer cell lines RPMI7951 and MG63. Taiwanofungus camphoratus is a well-known Chinese medicine in Taiwan, and AmB is a widely used antifungal agent. The authors demonstrated that TCEE pretreatment followed by AmB treatment effectively inhibited cell growth. The combination of sublethal doses of TCEE and AmB revealed a significant growth inhibitory effect in both cell lines. The combination of TCEE and AmB but not AmB alone induced phosphatidylserine externalization and loss of mitochondrial membrane potential. Cell cycle analyses revealed that combination of TCEE and AmB triggered G2/M arrest and significant apoptosis after 48 hours. These effects were greater than those achieved using TCEE or AmB alone. Furthermore, the authors demonstrated that the drugs increased the levels of p21(Cip1/Waf1) and pro-apoptotic protein Bax and reduced the level of anti-apoptotic protein Bcl-2. Taken together, the results showed that the combination treatment of TCEE and AmB displays strong adjuvant effects, which are indicated by the inhibition of cell proliferation in 2 human cancer cell lines, RPMI7951 and MG63. These findings suggest possible therapeutic applications and alternative medicines using this drug combination.

Role of nuclear receptor coactivator 3 (Ncoa3) in pluripotency maintenance.

Nuclear receptors, including Esrrb, Dax1, and Nr5a2, have been shown to be involved in pluripotency maintenance. Yet, the role of their coactivators in mouse embryonic stem cells remains unexplored. Here, we demonstrated that the nuclear receptor coactivator 3 (Ncoa3) is essential for pluripotency maintenance. Knockdown of Ncoa3 not only compromises the expression of pluripotency markers but also impairs in vitro and in vivo differentiation potential of mouse ESCs. Ncoa3 binds to the Nanog promoter and recruits the histone acetyltransferase CREB binding protein (CBP) and the histone arginine methyltransferase CARM1 to activate Nanog expression. Moreover, glycogen synthase kinase 3 GSK3 signaling down-regulates the Ncoa3 protein level to suppress Nanog expression. Thus, Ncoa3 not only contributes to self-renewal by activating Nanog but also facilitates ESC differentiation as a break point to disrupt the core transcriptional circuitry of pluripotency.

Pretreatment with an ethanolic extract of Taiwanofungus camphoratus (Antrodia camphorata) enhances the cytotoxic effects of amphotericin B.

Taiwanofungus camphoratus, a well-known Chinese medicine used in Taiwan, possesses several pharmacological functions, including anticancer effects. In the present study, we aimed to investigate a novel anticancer effect by pretreating cancer cells with an ethanolic extract of T. camphoratus (TCEE) followed by the administration of an antifungal agent amphotericin B (AmB). Both TCEE and AmB showed significant dose-dependent cytotoxicity in HT29 cells. Pretreatment with a nontoxic dose of TCEE enhanced the cytotoxicity of AmB. Furthermore, significant apoptotic cell death was found in cells treated with TCEE and AmB. A combination treatment with AmB plus TCEE resulted in a significant repression of tumor growth in HT29 xenografts. Collectively, our results indicated that combined treatment with AmB and TCEE effectively induced apoptosis and inhibited tumor growth. In the future, TCEE may serve as a potential complementary and alternative medicine to treat patients suffering from colorectal cancer.

Germline competency of parthenogenetic embryonic stem cells from immature oocytes of adult mouse ovary.

Parthenogenetic embryonic stem cells (pESCs) have been generated in several mammalian species from parthenogenetic embryos that would otherwise die around mid-gestation. However, previous reports suggest that pESCs derived from in vivo ovulated (IVO) mature oocytes show limited pluripotency, as evidenced by low chimera production, high tissue preference and especially deficiency in germline competence, a critical test for genetic integrity and pluripotency of ESCs. Here, we report efficient generation of germline-competent pESC lines (named as IVM pESCs) from parthenogenetic embryos developed from immature oocytes of adult mouse ovaries following in vitro maturation (IVM) and artificial activation. In contrast, pESCs derived from IVO oocytes show defective germline competence, consistent with previous reports. Further, IVM pESCs resemble more ESCs from fertilized embryos (fESCs) than do IVO pESCs on genome-wide DNA methylation and global protein profiles. In addition, IVM pESCs express higher levels of Blimp1, Lin28 and Stella, relative to fESCs, and in their embryoid bodies following differentiation. This may indicate differences in differentiation potentially to the germline. The mechanisms for acquisition of pluripotency and germline competency of IVM pESCs from immature oocytes remain to be determined.

Two "childhood" malignancies in an elderly individual: a case report and discussion.

Rhabdomyosarcoma (RMS) is the most common soft-tissue tumor in childhood, but is extremely rare in elderly. We present a rare case of cardiac RMS, which developed 1 year after the diagnosis and management of acute lymphoblastic leukemia in a 68-year-old female. The occurrence of such phenomena is intriguing, especially in an individual without prior history of malignancy at a younger age. Through the review of the existing literature, we attempt to approach the pathogenesis and clinical manifestations of this rare clinical entity.