Effects of super-enhancers in cancer metastasis: mechanisms and therapeutic targets.

Metastasis remains the principal cause of cancer-related lethality despite advancements in cancer treatment. Dysfunctional epigenetic alterations are crucial in the metastatic cascade. Among these, super-enhancers (SEs), emerging as new epigenetic regulators, consist of large clusters of regulatory elements that drive the high-level expression of genes essential for the oncogenic process, upon which cancer cells develop a profound dependency. These SE-driven oncogenes play an important role in regulating various facets of metastasis, including the promotion of tumor proliferation in primary and distal metastatic organs, facilitating cellular migration and invasion into the vasculature, triggering epithelial-mesenchymal transition, enhancing cancer stem cell-like properties, circumventing immune detection, and adapting to the heterogeneity of metastatic niches. This heavy reliance on SE-mediated transcription delineates a vulnerable target for therapeutic intervention in cancer cells. In this article, we review current insights into the characteristics, identification methodologies, formation, and activation mechanisms of SEs. We also elaborate the oncogenic roles and regulatory functions of SEs in the context of cancer metastasis. Ultimately, we discuss the potential of SEs as novel therapeutic targets and their implications in clinical oncology, offering insights into future directions for innovative cancer treatment strategies.

Patterns of Occult Metastasis to Level Va and Vb in Clinically Lateral Node-Positive Papillary Thyroid Carcinoma.

BACKGROUND: The optimal extent of therapeutic lateral neck dissection (ND) in papillary thyroid carcinoma (PTC) continues to be debated. We analyzed the frequency, patterns, and predictive factors of occult level Va and Vb metastasis in clinically lateral node-positive PTC patients. METHODS: We reviewed the data of PTC patients who underwent thyroidectomy and therapeutic lateral ND from level II to V between May 2008 and August 2020. In our study, 46 patients without clinically positive metastatic lymph nodes (LNs) at level V on the preoperative evaluation were included to analyze occult metastasis at level Va and Vb, respectively. Patient demographics, including age, sex, distribution of pathologic LNs, and characteristics of the primary tumors, were reviewed. In addition, clinicopathologic factors associated with occult level Va and Vb metastasis were analyzed. RESULTS: Of the 46 patients, 14 (30.4%) patients had occult metastases at level Vb. No occult metastases were found at level Va. Clinically positive level II metastasis (p = 0.015) and simultaneous level II, III, and IV metastases (p = 0.010) in the preoperative evaluation were significantly associated with occult level Vb metastasis. Patients without LN metastasis at level IV or with three or fewer metastatic LNs in the lateral neck never had occult LN metastases at level Vb. CONCLUSIONS: Occult metastasis at level Va is rare in PTC with lateral LN metastasis. Occult metastasis at level Vb may occur in PTC patients with multilevel involvement, including level II and/or four or more lateral LN metastases.

SHMT2 Induces Stemness and Progression of Head and Neck Cancer.

Various enzymes in the one-carbon metabolic pathway are closely related to the development of tumors, and they can all be potential targets for cancer therapy. Serine hydroxymethyltransferase2 (SHMT2), a key metabolic enzyme, is very important for the proliferation and growth of cancer cells. However, the function and mechanism of SHMT2 in head and neck cancer (HNC) are not clear. An analysis of The Cancer Genome Atlas (TCGA) data showed that the expression of SHMT2 was higher in tumor tissue than in normal tissue, and its expression was significantly associated with male sex, aggressive histological grade, lymph node metastasis, distant metastasis, advanced TNM stage, and lymphovascular invasion in HNC. SHMT2 knockdown in FADU and SNU1041 cell lines significantly inhibited cell proliferation, colony formation, migration, and invasion. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using TCGA data revealed that SHMT2 was closely related to cancer stem cell regulation and maintenance. Furthermore, we found that silencing SHMT2 inhibited the expression of stemness markers and tumor spheroid formation compared with a control group. On the contrary, stemness markers were significantly increased after SHMT2 overexpression in HEP-2 cells. Interestingly, we found that knocking down SHMT2 reduced the expression of genes related to the Notch and Wnt pathways. Finally, silencing SHMT2 significantly reduced tumor growth and decreased stemness markers in a xenograft model. Taken together, our study suggests that targeting SHMT2 may play an important role in inhibiting HNC progression.

Transcriptional Regulation of GDF15 by EGR1 Promotes Head and Neck Cancer Progression through a Positive Feedback Loop.

Growth and differentiation factor 15 (GDF15), a divergent member of the transforming growth factor-ß (TGF-ß) superfamily, has been reported to be overexpressed in different kinds of cancer types. However, the function and mechanism of GDF15 in head and neck cancer (HNC) remains unclear. The Cancer Genome Atlas (TCGA) data show that the expression of GDF15 is significantly associated with tumor AJCC stage, lymph vascular invasion and tumor grade in HNC. In this study, we confirmed that knockdown of GDF15 attenuated: cell proliferation, migration and invasion via regulation of EMT through a canonical pathway; SMAD2/3 and noncanonical pathways; PI3K/AKT and MEK/ERK in HNC cell lines. Furthermore, we found that early growth response 1 (EGR1) was a transcription factor of GDF15. Interestingly, we also demonstrated that GDF15 could regulate the expression of EGR1, which meant a positive feedback loop occurred between these two factors. Moreover, combined inhibition of both GDF15 and EGR1 in a HNC mouse xenograft model showed significantly decreased tumor volume compared to inhibition of EGR1 or GDF15 alone. Our study showed that the GDF15-EGR1 signaling axis may be a good target in HNC patients.

MYB transcription factor PdMYB118 directly interacts with bHLH transcription factor PdTT8 to regulate wound-induced anthocyanin biosynthesis in poplar.

BACKGROUND: R2R3-MYB transcription factors (TFs) play important roles in plant growth and development, and response to biotic and abiotic stresses. However, their regulatory mechanisms in wound-induced anthocyanin biosynthesis in woody plants are largely unknown. RESULTS: In this work, we report that expression of anthocyanin biosynthesis genes (ABGs) were activated by PdMYB118, a MYB TF encoding gene from Populus deltoids, and the activation of PdMYB118 was significantly enhanced by PdTT8, a bHLH protein, through its direct interaction with PdMYB118. PdMYB118 and some ABGs were evidently induced by wound induction and methyl jasmonate (MeJA) treatment. Overexpression of PdMYB118 promoted anthocyanin accumulation in transgenic poplar upon wound induction. Furthermore, a poplar JASMONATE ZIM-domain (JAZ) protein, PtrJAZ1, repressed the transcriptional function of PdMYB118/PdTT8 complex by binding to PdTT8, and wound stimulated the biosynthesis of jasmonic acid (JA) and the degradation of PtrJAZ1. CONCLUSIONS: Based on these observations, we proposed that PtrJAZ1 degradation triggered the expression of ABGs, leading to increased biosynthesis of anthocyanins in the wounded leaves of transgenic poplar. Therefore, our findings not only illustrate the crucial role of PdMYB118 in wound-induced anthocyanin biosynthesis in poplar, but also provide a molecular basis for the genetic engineering of colorful tree species.

PdMYB118, isolated from a red leaf mutant of Populus deltoids, is a new transcription factor regulating anthocyanin biosynthesis in poplar.

KEY MESSAGE: A new anthocyanin biosynthesis transcription factor PdMYB118, which could be used for the genetic engineering of colorful tree species, was indentified from a red leaf mutant of Populus deltoids. In higher plants, the biosynthesis of anthocyanins is regulated by several classes of transcription factors (TFs), including R2R3-MYB, bHLH and WD-repeat proteins. In this work, we isolated an MYB gene regulating anthocyanin biosynthesis from a red leaf mutant of Populus deltoids, which accumulated more anthocyanins in the leaves and showed higher expression levels of anthocyanin biosynthesis genes than did the wild type. Gene expression analyses of all TFs regulating anthocyanin biosynthesis demonstrated that only a MYB118 homologous gene, PdMYB118, was up-regulated in the mutant compared with the wide type. Subcellular localization analyses in poplar leaf mesophyll protoplasts showed that PdMYB118-YFP fusion protein was specifically located in nucleus. When transiently expressed in poplar leaf protoplasts, PdMYB118 specifically promoted the expression of anthocyanidin biosynthesis genes. Dual-luciferase assays revealed that PdMYB118 can directly activate the promoters of these genes. When overexpressed in Shanxin Yang (P. davidiana × P. bolleana), a hybrid clone commercially grown for landscaping in the northern part of China, transgenic plants overexpressing PdMYB118 produced more anthocyanins in the leaves and turned their color into redness when grown in both greenhouse and field. Consistently, transcripts of some important anthocyanidin biosynthesis genes were significantly increased in the leaves of transgenic plants. All these results indicate that PdMYB118 functions as an essential transcription factor regulating anthocyanin biosynthesis in poplar and could be used for the genetic engineering of colorful tree species.

Numerical Study for a Large-Volume Droplet on the Dual-Rough Surface: Apparent Contact Angle, Contact Angle Hysteresis, and Transition Barrier.

The profile, apparent contact angle (ACA), contact angle hysteresis (CAH), and wetting state transmission energy barrier (WSTEB) are important static and dynamic properties of a large-volume droplet on the hierarchical surface. Understanding them can provide us with important insights into functional surfaces and promote the application in corresponding areas. In this paper, we establish three theoretical models (models 1-3) and the corresponding numerical methods, which were obtained by the free energy minimization and the nonlinear optimization algorithm, to predict the profile, ACA, CAH, and WSTEB of a large-volume droplet on the horizontal regular dual-rough surface. In consideration of the gravity, the energy barrier on the contact circle, the dual heterogeneous structures and their roughness on the surface, the models are more universal and accurate than the previous models. It showed that the predictions of the models were in good agreement with the results from the experiment or literature. The models are promising to become novel design approaches of functional surfaces, which are frequently applied in microfluidic chips, water self-catchment system, and dropwise condensation heat transfer system.

Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees.

Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. In Arabidopsis, AtCYP85A2 works as a bifunctional cytochrome P450 monooxygenase to catalyse the conversion of castasterone to brassinolide, a final rate-limiting step in the BR-biosynthetic pathway. Here, we report the functional characterizations of PtCYP85A3, one of the three AtCYP85A2 homologous genes from Populus trichocarpa. PtCYP85A3 shares the highest similarity with AtCYP85A2 and can rescue the retarded-growth phenotype of the Arabidopsis cyp85a2-2 and tomato dx mutants. Constitutive expression of PtCYP85A3, driven by the cauliflower mosaic virus 35S promoter, increased the endogenous BR levels and significantly promoted the growth and biomass production in both transgenic tomato and poplar. Compared to the wild type, plant height, shoot fresh weight and fruit yield increased 50%, 56% and 43%, respectively, in transgenic tomato plants. Similarly, plant height and stem diameter increased 15% and 25%, respectively, in transgenic poplar plants. Further study revealed that overexpression of PtCYP85A3 enhanced xylem formation without affecting the composition of cellulose and lignin, as well as the cell wall thickness in transgenic poplar. Our finding suggests that PtCYP85A3 could be used as a potential candidate gene for engineering fast-growing trees with improved wood production.

Identification of EGF-NF-κB-FOXC1 signaling axis in basal-like breast cancer.

BACKGROUND: The pathogenesis of human basal-like breast cancer (BLBC) is not well understood and patients with BLBC have a poor prognosis. Expression of the epidermal growth factor receptor (EGFR) and nuclear factor-κB (NF-κB) is well-known to be upregulated in BLBC. The forkhead box C1 (FOXC1) transcription factor, an important prognostic biomarker specific for BLBC, has been shown to be induced by EGF and is critical for EGF effects in breast cancer cells. How FOXC1 is transcriptionally activated in BLBC is not clear. METHODS: Luciferase reporter assays were performed to show that NF-κB-p65 enhances FOXC1 promoter activity in BLBC cells (MDA-MB-468). Electrophoretic mobility shift assay, biotinylated oligonucleotide precipitation assay, and chromatin immunoprecipitation assay were used to show that NF-κB interacts and binds to the promoter region of FOXC1. RESULTS: In this study, we demonstrate that NF-κB is a pivotal mediator of the EGF/EGFR regulation of FOXC1 expression by binding to the FOXC1 promoter to activate FOXC1 transcription. Loss or inhibition of NF-κB diminished FOXC1 expression. CONCLUSION: Collectively, our findings reveal a novel EGFR-NF-κB-FOXC1 signaling axis that is critical for BLBC cell function, supporting the notion that intervention in the FOXC1 pathway may provide potential modalities for BLBC treatment.

Numerical Calculation Method of Apparent Contact Angles on Heterogeneous Double-Roughness Surfaces.

Double-roughness surfaces can be used to mimic lotus surfaces. The apparent contact angles (ACAs) of droplets on these surfaces were first calculated by Herminghaus. Then Patankar utilized the pillar model to improve the Herminghaus approach and put forward the formulas for ACAs calculation of the homogeneous double-roughness surfaces where the dual-scale structures and the bases were the same wettable materials. In this paper, we propose a numerical calculation method of ACAs on the heterogeneous double-roughness surfaces where the dual-scale structures and the bases are made of different wettable materials. This numerical calculation method has successfully enhanced the Herminghaus approach. It is promising to become a novel design approach of heterogeneous superhydrophobic surfaces, which are frequently applied in technical fields of self-cleaning, anti-icing, antifogging, and enhancing condensation heat transfer.

Tenovin-6-mediated inhibition of SIRT1/2 induces apoptosis in acute lymphoblastic leukemia (ALL) cells and eliminates ALL stem/progenitor cells.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a heterogeneous group of malignant disorders derived from B- or T-cell lymphoid progenitor cells. ALL often is refractory to or relapses after treatment; thus, novel targeted therapy for ALL is urgently needed. In the present study, we initially found that the level of SIRT1, a class III histone deacetylase, was higher in primary ALL cells from patients than in peripheral blood mononuclear cells from healthy individuals. But it is not clear whether inhibition of SIRT1 by its selective small molecule inhibitor Tenovin-6 is effective against ALL cells. METHODS: We tested the effect of Tenovin-6 on ALL cell lines (REH and NALM-6) and primary cells from 41 children with ALL and 2 adult patients with ALL. The effects of Tenovin-6 on cell viability were determined by MTS assay; colony-forming assays were determined by soft agar in ALL cell lines and methylcellulose medium in normal bone marrow cells and primary ALL blast cells; cell apoptosis and cell cycling were examined by flow cytometry; the signaling pathway was determined by Western blotting; ALL stem/progenitor cells were seperated by using MACS MicroBead kit. RESULTS: The results showed that Tenovin-6 treatment activated p53, potently inhibited the growth of pre-B ALL cells and primary ALL cells, and sensitized ALL cells to frontline chemotherapeutic agents etoposide and cytarabine. Tenovin-6 induced apoptosis in REH and NALM-6 cells and primary ALL cells and diminished expression of Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP) in such cells. Furthermore, inhibition of SIRT1 by Tenovin-6 inhibited the Wnt/ß-catenin signaling pathway and eliminated ALL stem/progenitor (CD133 + CD19-) cells. CONCLUSION: Our results indicate that Tenovin-6 may be a promising targeted therapy for ALL and clinical trials are warranted to investigate its efficacy in ALL patients.

Characterization of Arabidopsis Tubby-like proteins and redundant function of AtTLP3 and AtTLP9 in plant response to ABA and osmotic stress.

Tubby and Tubby-like proteins (TLPs) play essential roles in the development and function of mammal neuronal cells. In addition to the conserved carboxyl (C)-terminal Tubby domain, which is required for their plasma membrane (PM) tethering, plant TLPs also possess an amino (N)-terminal F-box domain to interact with specific Arabidopsis Skp1-like (ASK) proteins as functional SCF-type E3 ligases. Here, we report the molecular characterization of Arabidopsis TLPs (AtTLPs). ß-Glucuronidase staining showed overlapped but distinct expression patterns of AtTLPs in Arabidopsis. Yeast two-hybrid assays further revealed that AtTLP1, AtTLP3, AtTLP6, AtTLP7, AtTLP9, AtTLP10 and AtTLP11 all interacted with specific ASKs, but AtTLP2, AtTLP5 and AtTLP8 did not. Subcellular localization observations in both Arabidopsis protoplasts and tobacco pollen tubes indicated that all GFP-AtTLP fusion proteins, except GFP-AtTLP8 which lacks the conserved phosphatidylinositol 4,5-bisphosphate binding sites, were targeted to the PM. Detailed studies on AtTLP3 demonstrated that AtTLP3 is a PM-tethered PIP2 binding protein which functions redundantly with AtTLP9 in abscisic acid (ABA)- and osmotic stress-mediated seed germination. Our results suggest that AtTLPs possibly work in multiple physiological and developmental processes in Arabidopsis, and AtTLP3 is also involved in ABA signaling pathway like AtTLP9 during seed germination and early seedling growth.

Ponatinib efficiently kills imatinib-resistant chronic eosinophilic leukemia cells harboring gatekeeper mutant T674I FIP1L1-PDGFRα: roles of Mcl-1 and ß-catenin.

BACKGROUND: T674I FIP1L1-PDGFRα in a subset of chronic eosinophilic leukemia (CEL) is a gatekeeper mutation that is resistant to many tyrosine kinase inhibitors (TKIs) (e.g., imatinib, nilotinib and dasatinib), similar to T315I Bcr-Abl. Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed. Ponatinib (AP24534) is a novel orally bioavailable TKI against T315I Bcr-Abl, but it is not clear whether ponatinib is effective against T674I FIP1L1-PDGFRα. The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα. METHODS: Molecular docking analysis in silico was performed. The effects of ponatinib on PDGFRα signaling pathways, apoptosis and cell cycling were examined in EOL-1, BaF3 cells expressing either wild type (WT) or T674I FIP1L1-PDGFRα. The in vivo antitumor activity of ponatinib was evaluated with xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice models. RESULTS: Molecular docking analysis revealed that ponatinib could bind to the DFG (Asp-Phe-Gly)-out state of T674I PDGFRα. Ponatinib potently inhibited the phosphorylation of WT and T674I FIP1L1-PDGFRα and their downstream signaling molecules (e.g., Stat3, Stat5). Ponatinib strikingly inhibited the growth of both WT and T674I FIP1L1-PDGFRα-carrying CEL cells (IC50: 0.004-2.5 nM). It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of ß-catenin to decrease its stability and pro-survival functions. In vivo, ponatinib abrogated the growth of xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice. CONCLUSIONS: Ponatinib is a pan-FIP1L1-PDGFRα inhibitor, and clinical trials are warranted to investigate its efficacy in imatinib-resistant CEL.

FOXC1 is a critical mediator of EGFR function in human basal-like breast cancer.

BACKGROUND: Human basal-like breast cancer (BLBC) has a poor prognosis and is often identified by expression of the epidermal growth factor receptor (EGFR). BLBC remains a major clinical challenge because its pathogenesis is not well understood, thus hindering efforts to develop targeted therapies. Recent data implicate the forkhead box C1 (FOXC1) transcription factor as an important prognostic biomarker and functional regulator of BLBC, but its regulatory mechanism and impact on BLBC tumorigenesis remain unclear. METHODS: The association between FOXC1 and EGFR expression in human breast cancer was examined by immunohistochemistry in formalin-fixed tissues and analysis of the TCGA database. The regulation of FOXC1 by EGFR activation was investigated in MDA-MB-468 cells using immunoblotting, qRT-PCR, and luciferase activity assays. This EGFR effect on FOXC1 expression was confirmed using the MDA-MB-468 xenograft model. RESULTS: Both FOXC1 mRNA and protein levels significantly correlated with EGFR expression in human breast tumors. EGFR activation induced FOXC1 transcription through the ERK and Akt pathways in BLBC. EGFR inhibition in vivo reduced FOXC1 expression in xenograft tumors. We also found that FOXC1 knockdown impaired the effects of EGF on BLBC cell proliferation, migration, and invasion. CONCLUSIONS: Our findings uncover a novel EGFR-FOXC1 signaling axis critical for BLBC cell functions, supporting the notion that intervention in the FOXC1 pathway may provide potential modalities for BLBC treatment.

Low platelet count at diagnosis of anti-neutrophil cytoplasmic antibody-associated vasculitis is correlated with the severity of disease and renal prognosis.

Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is an autoimmune disease that involves inflammation of blood vessels. There is increasing evidence that platelets play a crucial role not only in hemostasis but also in inflammation and innate immunity. In this study, we explored the relationship between platelet count, clinical characteristics, and the prognosis of patients with AAV. We divided 187 patients into two groups based on their platelet count. Clinicopathological data and prognostic information were retrospectively gathered from medical records. Univariate and multivariate regression analyses were used to identify risk factors for prognosis, including end-stage renal disease (ESRD) and mortality. The cutoff point for platelet count was set at 264.5 × 109/L, as determined by the receiver operating characteristic (ROC) curve for predicting progression to ESRD in patients with AAV. We observed patients with low platelet count (platelets < 264.5 × 109/L) had lower leukocytes, hemoglobin, complement, acute reactants, and worse renal function (P for eGFR < 0.001). They were also more likely to progress to ESRD or death compared to the high platelet count group (platelets > 264.5 × 109/L) (P < 0.0001, P = 0.0338, respectively). Low platelet count was potentially an independent predictor of poor renal prognosis in the multivariate regression analysis [HR 1.670 (95% CI 1.019-2.515), P = 0.014]. Lower platelet count at diagnosis is associated with more severe clinical characteristics and impaired renal function. Therefore, platelet count may be an accessible prognostic indicator for renal outcomes in patients with AAV.

Protocol for isolation and analysis of the leukemia stem cells in BCR-ABL-driven chronic myelogenous leukemia mice.

Practical procedures for sorting and analysis of leukemia stem cells (LSCs) are to improve our understanding of chronic myelogenous leukemia (CML). Here, we present a detailed magnetic-bead-based sorting and flow-cytometry-based analysis protocol for LSCs in BCR-ABL-driven CML mice. We describe steps for sorting and functional analysis of BCR-ABL-expressing c-Kit+ cells (GFP+c-Kit+) from CML mice as well as antibody staining and gating strategies for characterization of leukemia stem/progenitor cells and myeloid leukemia cells. For complete details on the use and execution of this protocol, please refer to Liu et al. (2022).1.

Mitochondrial Ribosomal Protein L14 Promotes Cell Growth and Invasion by Modulating Reactive Oxygen Species in Thyroid Cancer.

OBJECTIVES: The mitochondrial ribosomal protein L14 (MRPL14) is encoded by a nuclear gene and participates in mitochondrial protein translation. In this study, we aimed to investigate the role of MRPL14 in thyroid cancer. METHODS: We investigated the association between MRPL14 expression and clinicopathological features using The Cancer Genome Atlas (TCGA) and Chungnam National University Hospital (CNUH) databases. Functional studies of MRPL14, including proliferation, migration, invasion, mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production, were performed in papillary thyroid cancer (PTC) cell lines (B-CPAP and KTC-1). RESULTS: Based on the TCGA dataset, PTC tissues lost mitochondrial integrity and showed dysregulated expression of overall mitoribosomal proteins (MRPs) compared with normal thyroid tissues. Of 78 MRPs, MRPL14 was highly expressed in thyroid cancer tissues. MRPL14 overexpression was significantly associated with advanced tumor stage, extrathyroidal extension, and lymph node metastasis. MRPL14 increased cell proliferation of thyroid cancer and promoted cell migration via epithelial-mesenchymal transition-related proteins. Moreover, MRPL14 knockdown reduced the expression of oxidative phosphorylation complex IV (MTCO1) and increased the accumulation of ROS. Cotreatment with a ROS scavenger restored cell proliferation and migration, which had been reduced by MRPL14 knockdown, implying that ROS functions as a key regulator of the oncogenic effects of MRPL14 in thyroid cancer cells. CONCLUSION: Our findings indicate that MRPL14 may promote cell growth, migration, and invasion by modulating ROS in thyroid cancer cells.