Domain-containing Protein Family Research Articles

SMYD family in cancer: epigenetic regulation and molecular mechanisms of cancer proliferation, metastasis, and drug resistance.

Epigenetic modifiers (miRNAs, histone methyltransferases (HMTs)/demethylases, and DNA methyltransferases/demethylases) are associated with cancer proliferation, metastasis, angiogenesis, and drug resistance. Among these modifiers, HMTs are frequently overexpressed in various cancers, and recent studies have increasingly identified these proteins as potential therapeutic targets. In this review, we discuss members of the SET and MYND domain-containing protein (SMYD) family that are topics of extensive research on the histone methylation and nonhistone methylation of cancer-related genes. Various members of the SMYD family play significant roles in cancer proliferation, metastasis, and drug resistance by regulating cancer-specific histone methylation and nonhistone methylation. Thus, the development of specific inhibitors that target SMYD family members may lead to the development of cancer treatments, and combination therapy with various anticancer therapeutic agents may increase treatment efficacy.

Comparative Proteome-Wide Abundance Profiling of Yeast Strains Deleted for Cdc48 Adaptors

The yeast ATPase Cdc48 (known as p97/VCP in human cells) plays an important role in the Ubiquitin Proteasome System. VCP is essential for cancer cell proliferation, and its dysregulation has been implicated in several neurodegenerative diseases. Cdc48 functions by extracting ubiquitylated proteins from membranes, protein complexes and chromatin by often facilitating their proteasomal degradation. Specific adaptors or cofactors, primarily belonging to the UBX domain-containing protein family (which has seven members in Saccharomyces cerevisiae) recruit Cdc48 to ubiquitylated proteins. Here, we employed sample multiplexing-based quantitative mass spectrometry to profile global protein abundance in p97 adaptor deletion strains, specifically comparing seven single deletion strains of UBX domain-containing proteins and the Cuz1 deletion strain, which belongs to the zinc finger AN1-type domain protein family. We observed that each strain showed unique sets of differentially abundant proteins compared to the wild type. Our analysis also revealed a role for Ubx3 in maintaining wild type levels of mitochondrial proteins. Overall, we identified ~1400 differentially abundant proteins in the absence of a specific Cdc48 adaptor. This unique dataset offers a valuable resource for studying the functions of these adaptors, aiming to achieve a better understanding of the cellular processes regulated by Cdc48 itself and to deepen our understanding of the Ubiquitin Proteasome System.

Autophagy Regulator Rufy 4 Promotes Osteoclastic Bone Resorption by Orchestrating Cytoskeletal Organization via Its RUN Domain

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4’s potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.

Exploring Protein S-Palmitoylation: Mechanisms, Detection, and Strategies for Inhibitor Discovery.

S-palmitoylation is a reversible and dynamic process that involves the addition of long-chain fatty acids to proteins. This protein modification regulates various aspects of protein function, including subcellular localization, stability, conformation, and biomolecular interactions. The zinc finger DHHC (ZDHHC) domain-containing protein family is the main group of enzymes responsible for catalyzing protein S-palmitoylation, and 23 members have been identified in mammalian cells. Many proteins that undergo S-palmitoylation have been linked to disease pathogenesis and progression, suggesting that the development of effective inhibitors is a promising therapeutic strategy. Reducing the protein S-palmitoylation level can target either the PATs directly or their substrates. However, there are rare clinically effective S-palmitoylation inhibitors. This review aims to provide an overview of the S-palmitoylation field, including the catalytic mechanism of ZDHHC, S-palmitoylation detection methods, and the functional impact of protein S-palmitoylation. Additionally, this review focuses on current strategies for expanding the chemical toolbox to develop novel and effective inhibitors that can reduce the level of S-palmitoylation of the target protein.

Endogenous RBM4 prevents Ang II-induced cardiomyocyte hypertrophy via downregulating the expression of PTBP1.

Aberrant gene expression in cardiomyocyte has been revealed to be the fundamental essence of pathological cardiac hypertrophy. However, the detailed mechanisms are not fully understood. The underlying regulators of gene expression involved in cardiac hypertrophy remain to be further identified. Here, we report that the RNA-binding protein RNA-binding motif protein 4 (RBM4) functions as an endogenic protector that is able to fight against cardiomyocyte hypertrophy in vitro. Under pro-hypertrophic stimulation of angiotensin II (Ang II), the protein level of RBM4 in cardiomyocyte and myocardium is elevated. Knockdown of RBM4 can further aggravate cardiomyocyte hypertrophy, while over-expression of RBM4 represses cardiomyocyte hypertrophy. Mechanistically, RBM4 is localized in the nucleus and down-regulates the expression of polypyrimidine tract-binding protein 1 (PTBP1), which has been shown to aggravate cardiomyocyte hypertrophy. In addition, we suggest that the up-regulation of RBM4 in cardiomyocyte hypertrophy is caused by N6-methyladenosine (m6A). Ang II induces m6A methylation of RBM4 mRNA, which further enhances the YTH domain-containing family protein 1 (YTHDF1)-mediated translation of RBM4. Thus, our results reveal a novel pathway consisting of m6A, RBM4 and PTBP1, which is involved in cardiomyocyte hypertrophy.

The YTH domain-containing protein family: Emerging players in immunomodulation and tumour immunotherapy targets.

The modification of N6-methyladenosine (m6A) plays a pivotal role in tumor by altering both innate and adaptive immune systems through various pathways, including the regulation of messenger RNA. The YTH domain protein family, acting as "readers" of m6A modifications, affects RNA splicing, stability, and immunogenicity, thereby playing essential roles in immune regulation and antitumor immunity. Despite their significance, the impact of the YTH domain protein family on tumor initiation and progression, as well as their involvement in tumor immune regulation and therapy, remains underexplored and lacks comprehensive review. This review introduces the molecular characteristics of the YTH domain protein family and their physiological and pathological roles in biological behavior, emphasizing their mechanisms in regulating immune responses and antitumor immunity. Additionally, the review discusses the roles of the YTH domain protein family in immune-related diseases and tumor resistance, highlighting that abnormal expression or dysfunction of YTH proteins is closely linked to tumor resistance. This review provides an in-depth understanding of the YTH domain protein family in immune regulation and antitumor immunity, suggesting new strategies and directions for immunotherapy of related diseases. These insights not only deepen our comprehension of m6A modifications and YTH protein functions but also pave the way for future research and clinical applications.

A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency.

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+ progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.

Knockdown of YAP1 Reduces YTHDF3 to Stabilize SMAD7 and thus Inhibit Bladder Cancer Stem Cell Stemness.

The previous study has proved the oncogenic role of Yes-associated protein 1 (YAP1) in bladder cancer (BLCA), thus this study focused on its impact on bladder cancer stem cells (BCSCs) and underlying mechanism. BCSCs were obtained by treating human BLCA cells UMUC3 with cisplatin and identified by measuring CD133+ in UMUC3/BCSCs via flow cytometry. YAP1 interaction proteins and mothers against decapentaplegic homolog 7 (SMAD7) N6-methyladenosine (m6A) site were analyzed by bioinformatics. BCSCs were transfected. SMAD7 m6A level, YTH domain-containing family proteins 3 (YTHDF3)-SMAD7 interaction, YAP1/YTHDF3 expression in BCSCs were assessed by methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) or quantitative reverse transcription PCR (qRT-PCR), respectively. BCSC proliferation was detected by 5-Bromo-2-deoxyuridine (BrdU) staining. UMUC3/BCSC migration/invasion and tumour sphere formation were determined by Transwell or tumour sphere formation assays. YAP1/YTHDF3/SMAD7/transforming growth factor (TGF)-β1/stemness marker expressions in UMUC3/BCSCs were measured by Western blot assay. BCSCs showed higher CD133+ ratio, expressions of stemness marker/YAP1/YTHDF3/TGF-β1, lower SMAD7 expression and greater invasion/migration/tumour sphere formation capabilities than UMUC3 cells. YAP1 knockdown decreased SMAD7 m6A level and impaired YTHDF3-SMAD7 interaction in BCSCs. YAP1 silencing inhibited cell growth/invasiveness/migration/tumour sphere formation and stemness-associated protein/YTHDF3/TGF-β1 expressions while upregulating SMAD7 expression in BCSCs, which was offset by YTHDF3 overexpression. The silencing of YAP1 in BCSCs impedes the YTHDF3-mediated degradation of m6A-modified SMAD7, culminating in diminished cell stemness.

Tandem LIM domain-containing proteins, LIMK1 and LMO1, directly bind to force-bearing keratin intermediate filaments

The cytoskeleton of the cell is constantly exposed to physical forces that regulate cellular functions. Selected members of the LIM (Lin-11, Isl-1, and Mec-3) domain-containing protein family accumulate along force-bearing actin fibers, with evidence supporting that the LIM domain is solely responsible for this force-induced interaction. However, LIM domain's force-induced interactions are not limited to actin. LIMK1 and LMO1, both containing only two tandem LIM domains, are recruited to force-bearing keratin fibers in epithelial cells. This unique recruitment is mediated by their LIM domains and regulated by the sequences outside the LIM domains. Based on invitro reconstitution of this interaction, LIMK1 and LMO1 directly interact with stretched keratin 8/18 fibers. These results show that LIM domain's mechano-sensing abilities extend to the keratin cytoskeleton, highlighting the diverse role of LIM proteins in force-regulated signaling.

A first-in-human, phase 1 study evaluating oral TACC3 inhibitor, AO-252, in advanced solid tumors.

TPS3176 Background: TACC3 is the most oncogenic member of the transforming acidic coiled-coil domain-containing protein (TACC) family, which have important roles in microtubule and centrosome-related processes. TACC3 is overexpressed in multiple cancers with centrosome amplification caused by genomic aberrations such as TP53 mutations, leading to chromosome instability and worse prognosis. AO-252 is a computationally designed, small molecule inhibitor of TACC3 protein-protein interactions. Binding assays and kinome screening suggested the specificity of AO-252 to TACC3. AO-252 targets the interactions of TACC3 with Clathrin/KIFC1, BRDA1 and MBD2/HAT complexes regulating mitosis, DNA damage response and epigenetic functions and was confirmed using co-immunoprecipitation (co-IP) assays. Gene expression analysis in multiple cell lines further confirmed the various TACC3 pathways affected by AO-252. In vitro, AO-252 showed low nanomolar potency in >200 cell line panel covering multiple different cancer indications and spared the normal cells. In vivo, AO-252 demonstrated strong tumor growth inhibition or regression in multiple xenograft models including triple-negative breast (TNBC), high-grade serous ovarian (HGSOC) and endometrial cancer with TP53 mutation/loss with a good therapeutic index. Methods: This first-in-human, multicenter, open-label phase I dose escalation and expansion study evaluates the safety, tolerability, maximum tolerated dose, and recommended phase 2 dose (RP2D) of AO-252 in solid tumors (NCT06136884). Patients with unresectable or metastatic TNBC, platinum-resistant HGSOC, primary peritoneal, fallopian-tube and serous endometrial cancers with documented TP53 mutation/loss, who have failed on at least 1 prior line of systemic therapy in advanced/metastatic setting (maximum 4 for TNBC and endometrial carcinoma) are eligible for enrollment. Part 1 follows an accelerated titration dose-escalation for cohort 1 and 2 and a traditional 3+3 study design from cohort 3. A maximum of 54 patients will be treated in the dose escalation, patients receiving AO-252 orally once or twice daily in a 28-day cycle. Part 2 consists of dose-expansion of a maximum of 30 patients into 2 RP2D levels of AO-252. Primary objectives are to evaluate the safety and tolerability as noted by dose-limiting toxicities, adverse events, serious adverse events, laboratory test results, electrocardiograms, vital signs, physical exams, and ECOG performance status. Secondary objectives are to determine the antitumor activity assessed by objective response rate per RECIST1.1, duration of response and disease control rate and to determine the pharmacokinetics of single and repeated doses of AO-252. Potential biomarkers relating to treatment outcome will be evaluated as exploratory endpoints. Enrollment in Cohort 1 is completed and enrollment to Cohort 2 will begin in February 2024. Clinical trial information: NCT06136884 .

YTHDF2 regulates MSS51 expression contributing to mitochondria dysfunction of granulosa cells in polycystic ovarian syndrome patients

Research questionGranulosa cells (GCs) dysfunction plays a crucial role in the pathogenesis of polycystic ovary syndrome (PCOS). It is reported that YTH domain-containing family protein 2 (YTHDF2) is upregulated in mural GCs of PCOS patients. What effect does the differential expression of YTHDF2 have in PCOS patients? DesignMural GCs and cumulus GCs from 15 patients with PCOS and 15 ovulatory controls and 4 cases of pathological sections in each group were collected. Real-time PCR, Western Blot, immunohistochemistry, and immunofluorescence experiments were conducted to detect gene and protein expression. RNA immunoprecipitation assay was performed to evaluate the binding relationship between YTHDF2 and MSS51. Mitochondrial morphology, cellular ATP and ROS levels and glycolysis-related gene expression were detected after YTHDF2 overexpression or MSS51 inhibition. ResultsIn the present study, we found that YTHDF2 was upregulated in GCs of PCOS patients while MSS51 was downregulated. YTHDF2 protein can bind to MSS51 mRNA and affect MSS51 expression. The reduction of MSS51 expression or the increase in YTHDF2 expression can lead to mitochondrial damage, reduced ATP levels, increased ROS levels and reduced expression of LDHA, PFKP and PKM. ConclusionsYTHDF2 may regulate the expression of MSS51, affecting the structure and function of mitochondria in GCs and interfering with cellular glycolysis, which may disturb the normal biological processes of GCs and follicle development in PCOS patients.

The tumor-intrinsic role of the m6A reader YTHDF2 in regulating immune evasion.

Tumors evade attacks from the immune system through various mechanisms. Here, we identify a component of tumor immune evasion mediated by YTH domain-containing family protein 2 (YTHDF2), a reader protein that usually destabilizes m6A-modified mRNA. Loss of tumoral YTHDF2 inhibits tumor growth and prolongs survival in immunocompetent tumor models. Mechanistically, tumoral YTHDF2 deficiency promotes the recruitment of macrophages via CX3CL1 and enhances mitochondrial respiration of CD8+ T cells by impairing tumor glycolysis metabolism. Tumoral YTHDF2 deficiency promotes inflammatory macrophage polarization and antigen presentation in the presence of IFN-γ. In addition, IFN-γ induces autophagic degradation of tumoral YTHDF2, thereby sensitizing tumor cells to CD8+ T cell-mediated cytotoxicity. Last, we identified a small molecule compound that preferentially induces YTHDF2 degradation, which shows a potent antitumor effect alone but a better effect when combined with anti-PD-L1 or anti-PD-1 antibodies. Collectively, YTHDF2 appears to be a tumor-intrinsic regulator that orchestrates immune evasion, representing a promising target for enhancing cancer immunotherapy.

Genome-Wide Identification and Expression Analysis of the YTH Domain-Containing RNA-Binding Protein Family in Cinnamomum camphora.

N6-methyladenosine (m6A) is one of the most abundant chemical modifications on mRNA in eukaryotes. RNA-binding proteins containing the YT521-B (YTH) domain play crucial roles in post-transcriptional regulation of plant growth, development, and stress response by reading the m6A mark. However, the YTH domain-containing RNA-binding protein family has not been studied in a valuable and medicinal tree such as Cinnamomum camphora (C. camphora) yet. In this study, we identified 10 YTH genes in C. camphora, located on eight out of 12 chromosomes. Phylogenetic analysis revealed that these genes can be classified into two major classes, YTHDF (CcDF) and YTHDC (CcDC). Closely related CcYTHs within the same class exhibited a similar distribution of conserved motifs and domain organization, suggesting functional similarities among these closely related CcYTHs. All CcYTH proteins possessed a highly conserved YTH domain, with CcDC1A containing an additional CCCH domain. The liquid-liquid phase separation (LLPS) predictions indicate that CcDC1A, CcDF1A, CcDF1C, CcDF3C, CcDF4C, and CcDF5C may undergo phase transitions. Quantitative expression analysis revealed that tissue-specific expression was observed fo CcYTHs. Notably, there were two genes, CcDF1A and CcDF5C; both exhibited significantly higher expression levels in various tissues than other genes, indicating that the m6A-YTH regulatory network in C. camphora might be quite distinct from that in most plants such as Arabidopsis thaliana (A. thaliana) with only one abundant YTH protein. According to the analysis of the up-stream cis-regulatory elements of these YTH genes, these genes could be closely related to stress, hormones, and development. The following stress response experiments further verified that their expression levels indeed changed under both PEG and NaCl treatments. These findings not only provide a foundation for future functional analysis of CcYTHs in C. camphora, but also provide insights into the functions of epigenetic mark m6A in forest trees.

Soot nanoparticles promote ferroptosis in dopaminergic neurons via alteration of m6A RNA methylation in Parkinson’s disease

Soot nanoparticles (SNPs) are black carbon prevalent in atmospheric environment with significant impacts on public health, leading to neurodegenerative diseases including development of Parkinson’s disease (PD). This study investigated the effects of SNPs exposure on PD symptoms, employing both in vivo and in vitro PD models. In the in vivo experiments, animal behavior assessments showed that SNPs exposure exacerbated motor and cognitive impairments in PD mice. Molecular biology techniques further unveiled that SNPs aggravated degeneration of dopaminergic neurons. In vitro experiments revealed that SNPs exposure intensified ferroptosis of PD cells by increasing reactive oxygen species and iron ion levels, while reducing glutathione levels and mitochondrial membrane potential. Sequencing tests indicated elevated N6-methyladenosine (m6A) alteration of the ferroptosis-related protein, acyl-CoA synthetase long chain family member 4 (ACSL4). This study demonstrates that SNPs may exacerbate the onset and progression of PD by recruiting YTH domain-containing family protein 1 (YTHDF1) protein, enhancing m6A methylation in the ACSL4 5′UTR, amplifying ACSL4 protein expression, and accelerating the ferroptosis process in dopaminergic neurons. These molecular mechanisms underlying SNPs exacerbation of PD development may provide crucial insights for formulating environmental safety regulations and potential therapeutic strategies addressing PD in populations residing in regions with varied air quality.

Tomato (Solanum lycopersicum L.) YTH Domain-Containing RNA-Binding Protein (YTP) Family Members Participate in Low-Temperature Treatment and Waterlogging Stress Responses

YT521-B homology (YTH) domain-containing RNA-binding proteins (YTPs) are important N6-methyladenosine (m6A) readers that have crucial roles in determining the destiny of m6A-modified RNAs, which are the most widespread RNA modifications in eukaryotes. Tomatoes (Solanum lycopersicum L.) hold significant importance in both dietary consumption patterns and scientific inquiries. While the YTP gene family has been characterized in tomatoes, their specific reactions to the low temperature and waterlogging stresses remain to be elucidated. In our study, nine tomato SlYTPs could be divided into five subclasses, YTHDFa-c and YTHDCa-b. After gene cloning and measuring their expression levels under stress conditions, it was revealed that SlYTP8 exhibited increased sensitivity to low-temperature treatment, while the expression levels of SlYTP9 were notably upregulated in leaf tissues subjected to waterlogging conditions. As members of the YTHDFc subfamily, SlYTP8 and SlYTP9 are both localized in the cytoplasm. Nevertheless, overexpression (OE) of SlYTP8 increased the sensitivity of tomato plants to low-temperature treatment, which was manifested by a higher accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and a weaker reactive oxygen species scavenging ability compared to wild-type (WT) tomatoes. However, in comparison to WT plants, the leaves of SlYTP9 OE tomatoes showed higher chlorophyll content and a stronger reactive oxygen species scavenging ability after 3 days of waterlogging treatment, thereby increasing the resistance of tomatoes to waterlogging stress. Moreover, in order to investigate the possible molecular mechanisms underlying their responses to the low temperature and waterlogging stresses, the transcription factors and interacting protein networks associated with SlYTP8/9 promoters and proteins were also predicted, respectively. These results could fill the gap in the understanding of tomato YTPs in response to the low temperature and waterlogging stresses, while also providing a theoretical and experimental basis for subsequent studies on their molecular mechanisms.

Salsolinol as an RNA m6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy.

JOURNAL/nrgr/04.03/01300535-202503000-00032/figure1/v/2024-06-17T092413Z/r/image-tiff Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, Sal) is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an environmental toxin that causes Parkinson's disease. However, the mechanism by which Sal mediates dopaminergic neuronal death remains unclear. In this study, we found that Sal significantly enhanced the global level of N6-methyladenosine (m6A) RNA methylation in PC12 cells, mainly by inducing the downregulation of the expression of m6A demethylases fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway. The m6A reader YTH domain-containing family protein 2 (YTHDF2) promoted the degradation of m6A-containing Yes-associated protein 1 (YAP1) mRNA, which is a downstream key effector in the Hippo signaling pathway. Additionally, downregulation of YAP1 promoted autophagy, indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity. These findings reveal the role of Sal on m6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy. Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.

Abstract PO4-18-03: Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis

Abstract Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) has been the most challenging subtype of BC, which consists of 20% of BC with an apparent correlation with poor prognosis. Despite that pyrotinib, a new HER2 inhibitor, has led to dramatic improvements in prognosis outcome, the efficacy of pyrotinib as monotherapy remain largely restricted due to its acquired resistance. Therefore, we aim at identifying a potent antitumor drug incorporated with pyrotinib for amplifying therapeutic efficacy for treating HER2-positive BC. Here, we reported a novel incorporation of pyrotinib in combination with chrysin, and explored its antitumor efficacy and the underlying mechanisms on HER2+ breast cancer. We determined that pyrotinib combined with chrysin yielded a potent synergistic effect to induce apoptosis and inhibit BT-474 and SK-BR-3 tumor cells, and suppressed in vivo tumor growth in tumor-bearing mice models. This may be mechanistically attributed to the induction of enhanced endoplasmic reticulum stress to increase the autophagy level. Furthermore, it was demonstrated that the combined treatment with pyrotinib and chrysin induced ubiquitination and G6PD degradation by regulating zinc finger and BTB/POZ domain-containing family protein 16 (ZBTB16) in tumorigenesis of BC. Besides, we identified that miR-16-5p is a potential upstream regulatory target of ZBTB16. Blocking miR-16-5p overexpression could inhibit HER2-positive tumorigenesis and significantly potentiate the efficacy of pyrotinib in combination with chrysin. Together, these findings demonstrate the utility of combined treatment with pyrotinib and chrysin as a potential option in the target treatment of HER2-positive BC through an unrecognized miR-16-5p/ZBTB16/G6PD axis. The miR-16-5p/ZBTB16/G6PD axis plays a crucial role in the pyrotinib plus chrysin-enabled anti HER2-positive BC Fig.1 a Cell viability of SK-BR-3 cells received various treatments. b Detection of cell cycle arrest of SK-BR-3 cells after various treatments. c Schematic diagram of the established protocol of the animal models. Photograph of resected tumor tissues, tumor volume (d), and tumor weight (e) of mice in different treatment groups during the whole testing period. f H&E, Ki67, and Tunel immunohistochemical staining of tumor sections of mice in various treatment groups. g Autophagy flux of SK-BR-3 cells labeled with mRFP-GFP-LC3 in the different treatment groups. h Expression level of ER stress markers in SK-BR-3 cells after various treatments via RT-qPCR and western blot. *P <0.05, compared with control group (DMSO), #P <0.05, compared with chrysin group, & P<0.05, compared with pyrotinib group. i Western blot analysis of the expression of G6PD in different treatment groups. j Fluorescence images of SK-BR-3 cells subjected to G6PD overexpression for the detection of autophagy level. k Ubibrowser database predicting the E3 ubiquitin ligases that may be involved in the regulation of G6PD ubiquitination. l Western blot analysis of G6PD protein half-life in SK-BR-3 cells with ZBTB16 silence. Cells were co-incubated with cycloheximide (CHX, 50μg/ml) for the indicated time. m Determination of the ubiquitination of G6PD in cells pretreated with 10 μM MG-132 for 3 h. Cells were transfected with ubiquitin after different treatments. The ubiquitinated G6PD was subjected to immunoprecipitation before western blot with ubiquitin antibody. n Dual-luciferase report verifying the targeting of ZBTB16 and miR-16-5p. o Transmission electron microscopy images of autophagosomes of tumors in various treatment groups (× 20000). p Immunohistochemical staining images of tumor slices for the determination of G6PD. q Schematic diagram for the underlying mechanism of pyrotinib combined chrysin against HER2-positive BC. Citation Format: Ting Luo, Xiaorong Zhong, Ping He, Dan Zheng, Yan Cheng, Kunrui Zhu. Combined Treatment with Pyrotinib and Chrysin Synergistically Improve the Autophagy Level in HER2-Positive Breast Cancer by Regulating the miR-16-5p/ZBTB16/G6PD Axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-18-03.

Identification of fibronectin type III domain containing 3B as a potential prognostic and therapeutic target for pancreatic cancer: a preliminary analysis

BackgroundFibronectin type III domain containing 3B (FNDC3B), a member of the fibronectin type III domain-containing protein family, has been indicated in various malignancies. However, the precise role of FNDC3B in the progression of pancreatic cancer (PC) still remains to be elucidated.MethodsIn this study, we integrated data from the National Center for Biotechnology Information, the Cancer Genome Atlas, Genotype-Tissue Expression database, and Gene Expression Omnibus datasets to analyze FNDC3B expression and its association with various clinicopathological parameters. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, along with Gene Set Enrichment Analysis (GSEA), single sample Gene Set Enrichment Analysis (ssGSEA) and estimate analysis were recruited to delve into the biological function and immune infiltration based on FNDC3B expression. Additionally, the prognostic estimation was conducted using Cox analysis and Kaplan–Meier analysis. Subsequently, a nomogram was constructed according to the result of Cox analysis to enhance the prognostic ability of FNDC3B. Finally, the preliminary biological function of FNDC3B in PC cells was explored.ResultsThe study demonstrated a significantly higher expression of FNDC3B in tumor tissues compared to normal pancreatic tissues, and this expression was significantly associated with various clinicopathological parameters. GSEA revealed the involvement of FNDC3B in biological processes and signaling pathways related to integrin signaling pathway and cell adhesion. Additionally, ssGSEA analysis indicated a positive correlation between FNDC3B expression and infiltration of Th2 cells and neutrophils, while showing a negative correlation with plasmacytoid dendritic cells and Th17 cells infiltration. Kaplan–Meier analysis further supported that high FNDC3B expression in PC patients was linked to shorter overall survival, disease-specific survival, and progression-free interval. However, although univariate analysis demonstrated a significant correlation between FNDC3B expression and prognosis in PC patients, this association did not hold true in multivariate analysis. Finally, our findings highlight the crucial role of FNDC3B expression in regulating proliferation, migration, and invasion abilities of PC cells.ConclusionDespite limitations, the findings of this study underscored the potential of FNDC3B as a prognostic biomarker and its pivotal role in driving the progression of PC, particularly in orchestrating immune responses.

The conserved GOLD domain in the Plasmodiophora brassicae effector Pb257 is required for triggering cell death and root swelling

Plasmodiophora brassicae is one of the biggest threats to cruciferous plants and one of the most economically significant diseases worldwide. However, our current understanding of its pathogenic mechanisms remains limited. In this study, we have identified an effector, Pb257, which plays a crucial role in the virulence of P. brassicae. The expression pattern analysis revealed continuous induction of Pb257 during both primary and secondary infections. Ectopic expression of Pb257 strongly triggered cell death in Nicotiana benthamiana and several other plants, which was dependent on SOBIR1 and the salicylic acid pathway but not BAK1, a central molecular regulator, to mediate classical defense pathways. Overexpression of Pb257 increased susceptibility of Brassica rapa to P. brassicae. Silencing of the Pb257-encoding gene led to reduce root swelling. Further investigation showed that the conserved GOLD domain within Pb257 was essential for inducing root enlargement, which is similar to the mild symptoms of clubroot, indicating that it is an important effector for promoting root cell enlargement. GOLD domain-containing family proteins were widely present in the kingdoms of protozoa, fungi, and animalia, and Pb257 was clustered into a clade of protozoa, suggesting the encoding gene as a potential marker gene for classification of P. brassicae. The GOLD domain-containing proteins are known regulators involved in human cell proliferation and immune system disruption, however, no previous reports have described a pathogen-derived GOLD domain protein with elicitor activity. Our findings demonstrate that Pb257 functions as a crucial effector contributing to the virulence of P. brassicae and could be used as a potential molecular target for controlling clubroot disease.

Bridge-like lipid transfer protein family member 2 suppresses ciliogenesis.

Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice (Mus musculus), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating β-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44, a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis in RPE-1 cells grown in serum-containing medium, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative LTP acting between tubular endosomes and ER that regulates primary cilium biogenesis.