Unraveling the role of CD24 in Hepatocellular carcinoma: Involvement of inactivated Hippo signaling and SOX4-mediated regulation.

Hepatocellular carcinoma (HCC) is a prevalent type of liver cancer, and CD24 gene is reportedly involved in HCC progression. However, the precise regulatory mechanisms of CD24 in HCC remain unclear. In this study, we established a primary HCC mouse model and observed that CD24, induced by inactivation of the Hippo pathway, was highly expressed in HCC. Using a systematic molecular and genomic approach, we identified the Hippo-YAP1-SOX4 pathway as the mechanism through which YAP1 induces CD24 upregulation in HCC cells. CD24 knockdown significantly attenuated YAP1 activation-induced HCC. These findings shed light on the link between CD24 and HCC progression, particularly in the Hippo-inactivated subclass of HCC. Therefore, CD24 may serve as a potential target for specific treatment of this HCC subclass.

Immunogenic chemotherapy: great potential for improving response rates.

The activation of anti-tumor immunity is critical in treating cancers. Recent studies indicate that several chemotherapy agents can stimulate anti-tumor immunity by inducing immunogenic cell death and durably eradicate tumors. This suggests that immunogenic chemotherapy holds great potential for improving response rates. However, chemotherapy in practice has only had limited success in inducing long-term survival or cure of cancers when used either alone or in combination with immunotherapy. We think that this is because the importance of dose, schedule, and tumor model dependence of chemotherapy-activated anti-tumor immunity is under-appreciated. Here, we review immune modulation function of representative chemotherapy agents and propose a model of immunogenic chemotherapy-induced long-lasting responses that rely on synergetic interaction between killing tumor cells and inducing anti-tumor immunity. We comb through several chemotherapy treatment schedules, and identify the needs for chemotherapy dose and schedule optimization and combination therapy with immunotherapy when chemotherapy dosage or immune responsiveness is too low. We further review tumor cell intrinsic factors that affect the optimal chemotherapy dose and schedule. Lastly, we review the biomarkers indicating responsiveness to chemotherapy and/or immunotherapy treatments. A deep understanding of how chemotherapy activates anti-tumor immunity and how to monitor its responsiveness can lead to the development of more effective chemotherapy or chemo-immunotherapy, thereby improving the efficacy of cancer treatment.

Discovery of Dual TAF1-ATR Inhibitors and Ligand-Induced Structural Changes of the TAF1 Tandem Bromodomain.

Bromodomains regulate chromatin remodeling and gene transcription through recognition of acetylated lysines on histones and other proteins. Bromodomain-containing protein TAF1, a subunit of general transcription factor TFIID, initiates preinitiation complex formation and cellular transcription. TAF1 serves as a cofactor for certain oncogenic transcription factors and is implicated in regulating the p53 tumor suppressor. Therefore, TAF1 is a potential target to develop small molecule therapeutics for diseases arising from dysregulated transcription, such as cancer. Here, we report the ATR kinase inhibitor AZD6738 (Ceralasertib) and analogues thereof as bona fide inhibitors of TAF1. Crystallographic and small-angle X-ray scattering studies established that newly identified and previously reported inhibitors stabilize distinct structural states of the TAF1 tandem bromodomain through "open-closed" transitions and dimerization. Combined with functional studies on p53 signaling in cancer cell lines, the data provide new insights into the feasibility and challenges of TAF1 inhibitors as chemical probes and therapeutics.

Identification and characterization of novel compound heterozygous variants in FSHR causing primary ovarian insufficiency with resistant ovary syndrome.

Primary ovarian insufficiency (POI) is among the foremost causes of women infertility due to premature partial or total loss of ovarian function. Resistant ovary syndrome (ROS) is a subtype of POI manifested as normal ovarian reserve but insensitive to gonadotropin stimulation. Inactivating variants of follicle-stimulating hormone receptor (FSHR), a class A G-protein coupled receptor, have been associated with POI and are inherited via an autosomal recessive pattern. In this study, we investigated the genetic causes of a primary infertility patient manifested as POI with ROS, and elucidated the structural and functional impact of variants of uncertain significance. Next-generation sequencing (NGS) combined with Sanger sequencing revealed novel compound heterozygous FSHR variants: c.1384G>C/p.Ala462Pro and c.1862C>T/p.Ala621Val, inherited from her father and mother, respectively. The two altered amino acid sequences, localized in the third and seventh transmembrane helix of FSHR, were predicted as deleterious by in silico prediction. In vitro experiments revealed that the p.Ala462Pro variant resulted in barely detectable levels of intracellular signaling both in cAMP-dependent CRE-reporter activity and ERK activation and displayed a severely reduced plasma membrane receptor expression. In contrast, the p.Ala621Val variant resulted in partial loss of receptor activation without disruption of cell surface expression. In conclusion, two unreported inactivating FSHR variants potentially responsible for POI with ROS were first identified. This study expands the current phenotypic and genotypic spectrum of POI.

Rational Design of Right-Handed Heterogeneous Peptidomimetics as Inhibitors of Protein-Protein Interactions.

Peptidomimetics have gained great attention for their function as protein-protein interaction (PPI) inhibitors. Herein, we report the design and investigation of a series of right-handed helical heterogeneous 1:1 α/Sulfono-γ-AA peptides as unprecedented inhibitors for p53-MDM2 and p53-MDMX. The most potent helical heterogeneous 1:1 α/Sulfono-γ-AA peptides were shown to bind tightly to MDM2 and MDMX, with Kd of 19.3 and 66.8 nM, respectively. Circular dichroism spectra, 2D-NMR spectroscopy, and the computational simulations suggested that these helical sulfono-γ-AA peptides could mimic the critical side chains of p53 and disrupt p53/MDM2 PPI effectively. It was noted that these 1:1 α/Sulfono-γ-AA peptides were completely resistant to proteolytic degradation, boosting their potential for biomedical applications. Furthermore, effective cellular activity is achieved by the stapled 1:1 α/Sulfono-γ-AA peptides, evidenced by significantly enhanced p53 transcriptional activity and much more induced level of MDM2 and p21. The 1:1 α/Sulfono-γ-AA peptides could be an alternative strategy to antagonize a myriad of PPIs.

Islet-cell autoantigen 69 accelerates liver regeneration by downregulating Tgfbr1 and attenuating Tgfß signaling in mice.

Regeneration is a unique defense mechanism of liver tissue in response to functional cell loss induced by toxic chemicals or surgical resection. In this study, we found that Islet-cell autoantigen 69 (Ica69) accelerates liver regeneration in mice. Following 70% partial hepatectomy, both Ica69 mRNA and protein are significantly upregulated in mouse hepatocytes at the early stage of liver regeneration. Compared with the wild-type mice, Ica69-deficient mice have more severe liver injury, delayed liver regeneration, and high surgical accidental mortality following hepatectomy. Mechanistically, Ica69 interacts with Pick1 protein to regulate Tgfbr1 protein expression and Tgfß-induced Smad2 phosphorylation. Our findings suggest that Ica69 in liver tissue is a new potential target for promoting liver regeneration.

α-Helix-Mimicking Sulfono-γ-AApeptide Inhibitors for p53-MDM2/MDMX Protein-Protein Interactions.

The use of peptidomimetic scaffolds is a promising strategy for the inhibition of protein-protein interactions (PPIs). Herein, we demonstrate that sulfono-γ-AApeptides can be rationally designed to mimic the p53 α-helix and inhibit p53-MDM2 PPIs. The best inhibitor, with Kd and IC50 values of 26 nM and 0.891 µM toward MDM2, respectively, is among the most potent unnatural peptidomimetic inhibitors disrupting the p53-MDM2/MDMX interaction. Using fluorescence polarization assays, circular dichroism, nuclear magnetic resonance spectroscopy, and computational simulations, we demonstrate that sulfono-γ-AApeptides adopt helical structures resembling p53 and competitively inhibit the p53-MDM2 interaction by binding to the hydrophobic cleft of MDM2. Intriguingly, the stapled sulfono-γ-AApeptides showed promising cellular activity by enhancing p53 transcriptional activity and inducing expression of MDM2 and p21. Moreover, sulfono-γ-AApeptides exhibited remarkable resistance to proteolysis, augmenting their biological potential. Our results suggest that sulfono-γ-AApeptides are a new class of unnatural helical foldamers that disrupt PPIs.

Mutant p53 Sequestration of the MDM2 Acidic Domain Inhibits E3 Ligase Activity.

Missense p53 mutants often accumulate in tumors and drive progression through gain of function. MDM2 efficiently degrades wild-type p53 but fails to degrade mutant p53 in tumor cells. Previous studies revealed that mutant p53 inhibits MDM2 autoubiquitination, suggesting that the interaction inhibits MDM2 E3 activity. Recent work showed that MDM2 E3 activity is stimulated by intramolecular interaction between the RING and acidic domains. Here, we show that in the mutant p53-MDM2 complex, the mutant p53 core domain binds to the MDM2 acidic domain with significantly higher avidity than wild-type p53. The mutant p53-MDM2 complex is deficient in catalyzing ubiquitin release from the activated E2 conjugating enzyme. An MDM2 construct with extra copies of the acidic domain is resistant to inhibition by mutant p53 and efficiently promotes mutant p53 ubiquitination and degradation. The results suggest that mutant p53 interferes with the intramolecular autoactivation mechanism of MDM2, contributing to reduced ubiquitination and increased accumulation in tumor cells.

MDMX acidic domain inhibits p53 DNA binding in vivo and regulates tumorigenesis.

The MDM2 homolog MDMX oncoprotein is indispensable for inhibition of p53 during normal embryonic development and malignant transformation, yet how MDMX harnesses p53 functions is unclear. In addition to a canonical N-terminal p53-binding domain, recent work suggests the central acidic domain of MDMX regulates p53 interaction through intramolecular mimicry and engages in second-site interaction with the p53 core domain in vitro. To test the physiological relevance of these interactions, we generated an MDMX knockin mouse having substitutions in a conserved WW motif necessary for these functions (W201S/W202G). Notably, MDMXSG cells have normal p53 level but increased p53 DNA binding and target gene expression, and rapidly senesce. In vivo, MDMXSG inhibits early-phase disease in Eµ-Myc transgenic mice but accelerates the onset of lethal lymphoma and shortens overall survival. Therefore, MDMX is an important regulator of p53 DNA binding, which complements the role of MDM2 in regulating p53 level. Furthermore, the results suggest that the WW motif has dual functions that regulate p53 and inhibit Myc-driven lymphomas independent of p53.

Tumor cell senescence response produces aggressive variants.

Tumors often respond favorably to initial chemotherapy but eventually relapse with drug resistance and increased metastatic potential. Cellular senescence is a major therapeutic outcome of cancer chemotherapy, which leads to tumor stasis or regression through immune clearance of senescent cells. However, senescent tumor cells have been shown to resume proliferation at low frequency. We found that subjecting arrested senescent tumor cells to cytotoxic treatments stimulates the clonogenic proliferation of remaining survivors. The senescence revertants showed a reduced rate of proliferation but increased migration and invasion potential in vitro, and increased tumorigenic potential in vivo. Gene expression profiling showed that the senescence revertants are distinct from both parental and senescent cells. A subset of senescence-activated genes remains active in the revertants. These genes are implicated in regulating cell motility, invasion, and metastasis, which may collectively contribute to the aggressiveness of the revertants. The findings suggest that although therapy-induced senescence has short-term benefits, the response also causes reprogramming of gene expression and activates invasion-related genes that accelerate tumor progression.

Nucleolar repression facilitates initiation and maintenance of senescence.

Tumor cells with defective apoptosis pathways often respond to chemotherapy by entering irreversible cell cycle arrest with features of senescence. However, rare cells can bypass entry to senescence, or re-enter cell cycle from a senescent state. Deficiency in senescence induction and maintenance may contribute to treatment resistance and early relapse after therapy. Senescence involves epigenetic silencing of cell cycle genes and reduced rRNA transcription. We found that senescence-inducing treatments such as DNA damage and RNA polymerase I inhibition stimulate the binding between the nucleolar protein NML (nucleomethylin) and SirT1. The NML complex promotes rDNA heterochromatin formation and represses rRNA transcription. Depletion of NML reduced the levels of H3K9Me3 and H3K27Me3 heterochromatin markers on rDNA and E2F1 target promoters in senescent cells, increased rRNA transcription, and increased the frequency of cell cycle re-entry. Depletion of the nucleolar transcription repressor factor TIP5 also promoted escape from senescence. Furthermore, tumor tissue staining showed that breast tumors without detectable nucleolar NML expression had poor survival. The results suggest that efficient regulation of nucleolar rDNA transcription facilitates the maintenance of irreversible cell cycle arrest in senescent cells. Deficiency in nucleolar transcription repression may accelerate tumor relapse after chemotherapy.

hMOF acetylation of DBC1/CCAR2 prevents binding and inhibition of SirT1.

The NAD(+)-dependent deacetylase SirT1 regulates gene silencing and genomic stability in response to nutrient deprivation and DNA damage. An important regulator of SirT1 in mammalian cells is DBC1 (deleted in breast cancer 1; KIAA1967 or CCAR2), which binds to SirT1 and inhibits the deacetylation of substrates. Recent studies have revealed that ATM/ATR-mediated phosphorylation of DBC1 promotes binding to SirT1. Here we show that DBC1 is modified by acetylation on two N-terminal lysine residues (K112 and K215). The MYST family histone acetyltransferase hMOF (human MOF) is responsible for DBC1 acetylation. Acetylation of K112 and K215 inhibits DBC1-SirT1 binding and increases SirT1 deacetylase activity. SirT1 also promotes DBC1 deacetylation, suggesting the presence of a negative-feedback mechanism that stabilizes the SirT1-DBC1 complex and limits SirT1 activity. hMOF binding and acetylation of DBC1 are inhibited after DNA damage in an ATM-dependent fashion, contributing to increased SirT1-DBC1 binding after DNA damage. Furthermore, a DBC1 mutant that mimics the acetylated state fails to promote apoptosis after DNA damage. These results suggest that acetylation of DBC1 inhibits binding to SirT1 and serves as a mechanism that connects DNA damage signaling to SirT1 and cell fate determination.

Regulation of SirT1-nucleomethylin binding by rRNA coordinates ribosome biogenesis with nutrient availability.

Nucleomethylin (NML), a novel nucleolar protein, is important for mediating the assembly of the energy-dependent nucleolar silencing complex (eNoSC), which also contains SirT1 and SUV39H1. eNoSC represses rRNA transcription during nutrient deprivation, thus reducing energy expenditure and improving cell survival. We found that NML is an RNA binding protein that copurifies with 5S, 5.8S, and 28S rRNA. The SirT1 and RNA binding regions on NML showed partial overlap, and the NML-SirT1 interaction was competitively inhibited by rRNA. Nutrient deprivation triggered downregulation of rRNA transcription, reduced the level of NML-associated rRNA, and stimulated NML-SirT1 binding. Assembly of eNoSC facilitated repression of pre-rRNA transcription. These results suggest that nascent rRNA generates a positive-feedback signal by suppressing the assembly of eNoSC and protecting active ribosomal DNA units from heterochromatin formation. This RNA-mediated mechanism enables the eNoSC to amplify the effects of upstream nutrient-responsive regulators.

The NAD+ synthesis enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT1) regulates ribosomal RNA transcription.

The chromosomal region encoding the nuclear NAD(+) synthesis enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT1) is frequently deleted in human cancer. We describe evidence that NMNAT1 interacts with the nucleolar repressor protein nucleomethylin and is involved in regulating rRNA transcription. NMNAT1 binds to nucleomethylin and is recruited into a ternary complex containing the NAD(+)-dependent deacetylase SirT1. NMNAT1 expression stimulates the deacetylase function of SirT1. Knockdown of NMNAT1 enhances rRNA transcription and promotes cell death after nutrient deprivation. Furthermore, NMNAT1 expression is induced by DNA damage and plays a role in preventing cell death after damage. Heterozygous deletion of NMNAT1 in lung tumor cell lines correlates with low expression level and increased sensitivity to DNA damage. These results suggest that NMNAT1 deletion in tumors may contribute to transformation by increasing rRNA synthesis, but may also increase sensitivity to nutrient stress and DNA damage.

Reactive astrocytes contribute to increased epileptic susceptibility induced by subthreshold dose of pilocarpine.

Seizures may influence epileptogenesis, but it is not yet clearly established whether subthreshold stimulations that are not sufficient to induce visible behavioral seizures change epileptic susceptibility, and the possible underlying mechanisms have not been completely understood. We assessed the susceptibility to epilepsy after subthreshold dose of pilocarpine, as well as glial fibrillary acidic protein (GFAP) expression using immunohistochemistry. An increase in the susceptibility to pentylenetetrazole (PTZ)-induced seizures was observed in rats previously subjected to subthreshold dose of pilocarpine. The immunoreactivity of GFAP was also increased, indicating that astrocytes became reactive in some brain subfields. The increased epileptic susceptibility was significantly reduced by L-alpha-aminoadipic acid (L-AAA), an inhibitor of astrocytic function. Our results suggest that subthreshold stimulation may increase the susceptibility to subsequent development of epilepsy, and reactive astrocytes might be an important contributor to this process. Adequate inhibition of astrocytic function may be a potential preventive approach against epileptogenesis.

Synthesis and structure-activity relationship studies of cytotoxic cinnamic alcohol derivatives.

Three series of di- and trisubstituted derivatives of cinnamic alcohol and its conjugated dienol analogues were designed and synthesised. The derivatives were screened for cytotoxicity against nine tumour cell lines: KB, A549, Hela, CNE, PC-3, BEL-7404, HL-60, BGC823 and P388D1. Most of the cinnamic alcohol derivatives showed cytotoxic activity. The compound 7-(4',5'-dichlorobenzyloxy)-6,8-dihydroxycinnamic alcohol (55) exhibited significant cytotoxicity to seven human tumour cell lines on a micromolar range, especially with regard to the KB and P388D1 cell lines, showing IC(50) values of 0.4 and 0.5 µM, respectively. The structure-activity relationships of the derivatives are discussed.

Preparation of C-23 esterified silybin derivatives and evaluation of their lipid peroxidation inhibitory and DNA protective properties.

A diverse series of C-23 esterified silybin derivatives (1a-n) were designed and synthesized. The antioxidative properties of these compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical scavenging, ferrous ion chelation, and inhibition of rat liver homogenate lipid peroxidation. Their protective effects on the prevention of hydrogen peroxide induced DNA damage were also investigated. Most of the synthesized compounds exhibited more effective antioxidant activities than silybin. The esterified silybin analogues displayed satisfactory performance especially on iron chelation and antiperoxidative activity. Compound 1n in particular exhibited remarkable antiperoxidative effect with an IC(50) value of 0.2+/-0.1 microM, which was stronger than that of quercetin (IC(50)=1.8+/-0.6 microM). Compounds 1c, 1e, 1g, 1h and 1k displayed potent, dose-dependent protective properties against DNA cleavage. The results of the bioassays support the antioxidative and DNA protective effects of these synthesized silybin derivatives.

[Antioxidative and cytotoxic properties of diarylheptanoids isolated from Zingiber officinale].

OBJECTIVE: To investigate the antioxidant and cytotoxic properties of five diarylheptanoids (1-5) isolated from the rhizomes of Zingiber officinale. METHOD: Various models such as scavenging superoxide anions and 1,1-diphenyl-2- picrylhydrazyl (DPPH) radicals, inhibiting lipid peroxidation, as well as protecting of rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H2O2) were employed to assay the antioxidative effects of the diarylheptanoids. The cytotoxicities of compounds 1-5 were measured with MTT assays. RESULT: The test compounds (1-5) showed promising DPPH inhibitory activities, and compound 5 exhibited the strongest DPPH scavenging activity with an IC50 value of (22.6+/-2.4) micromol x L(-1). Compounds 1, 3 and 4 showed potential anti-peroxidative effects with inhibitory rates of (66.3+/-15.4)%, (68.7+/-15.8)% and (72.2+/-10.6)%, respectively, at 100 microg x mL(-1). It could be observed that compounds 1, 3 and 4 demonstrated significant neuroprotective activities in a dose-dependent manner. Moreover, compound 3 exhibited certain cytotoxicities against human chronic myelogenous leukemia cells (K562) and its adriamycin-resistant cells (K562/ADR) with IC50 values of (34.9+/-0.6), (50.6+/-23.5) micromol x L(-1), respectively. CONCLUSION: In vitro results demonstrated that five diarylheptanoids (1-5) isolated from the roots of Z. officinale were capable of scavenging radicals, inhibiting lipid peroxidation and protecting PC12 cells against the insult by H2O2. Additionally, compound 3 could inhibit the growth of K562 and K562/ADR cells.