CDH1 Expression Research Articles

CDH2 and CDH13 as potential prognostic and therapeutic targets for adrenocortical carcinoma

ABSTRACT Cadherin 2 (CDH2, N-cadherin) and cadherin 13 (CDH13, T-cadherin, H-cadherin) affect the progress and prognoses of many cancers. However, their roles in adrenocortical carcinoma (ACC), a rare endocrine cancer, remain unclear. To decipher the roles of these proteins in ACC and to identify their regulatory targets, we analyzed their expression levels, gene regulatory networks, prognostic value, and targets in ACC, using various bioinformatic analyses. CDH2 was strongly downregulated and CDH13 was strongly upregulated in patients with ACC; the expression levels of these genes affected the prognosis. In 75 patients, the expression of CDH2 and CDH13 was altered by 8% and 5%, respectively. CDH2 and CDH13, as well as their neighboring genes, were predicted to form a complex network of interactions, mainly through coexpression and physical and genetic interactions. CDH2 and its altered neighboring genes (ANGs) mainly affect tumor-related gene expression, cell cycle, and energy metabolism. The regulation of tumor-related integrin function, gene transcription, metabolism, and amide and phospholipid metabolism are the main functions of CDH13 and its ANGs. MiRNA and kinase targets of CDH2 and CDH13 in ACC were identified. CDH13 expression in patients with ACC was positively associated with immune cell infiltration. Anti-PD1/CTLA-4/PD-L1 immunotherapy significantly downregulated the expression of CDH13 in patients with ACC. Foretinib and elesclomol were predicted to exert strong inhibitory effects on SW13 cells by inhibiting the expression of CDH2 and CDH13. These data indicate that CDH2 and CDH13 are promising targets for precise treatment of ACC and may serve as new biomarkers for ACC prognosis.

The orchestration of coding and non-coding genes at the CDH13 locus in coronary artery disease

Abstract Background The CDH13 locus was associated with coronary artery disease (CAD) by genome-wide association studies (GWAS). However, the causal gene(s) and underlying disease mechanisms at this locus remain undetermined. Purpose We intended to pinpoint and functionally validate the causal coding and non-coding genes at the CDH13 locus and delineate the underlying mechanisms for CAD. Methods and results We conducted the transcriptome-wide association analysis (TWAS) using nine types of CAD-relevant tissues from Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) and Genotyped Tissue Expression (GTEx) projects and identified CDH13 (T-cadherin) and four long non-coding RNA (lncRNA) genes as candidate causal genes at The CDH13 locus. One of the antisense(AS) lncRNAs, CDH13-AS2, was predicted to bind on the 3’ untranslated region (UTR) of CDH13 using LncRRIsearch and RNAup servers. The binding interaction was validated by dCas13-medicated RNA immunoprecipitation. Weighted correlation network analysis (WGCNA) using transcriptomic data from CAD-relevant tissues of ~600 human subjects suggested a positive expression correlation between CDH13 and CDH13-AS2 in artery tissues. CRISPR/Cas9-based knockout (KO) of CDH13 or CDH13-AS2 in human umbilical vein endothelial cells (HUVECs) indicated synergistically atherogenic phenotypes, including reduced proliferation and migration, and increased apoptosis and monocyte adhesion. The data matched with the downregulation of CDH13 in artery samples of atherosclerosis patients. The cellular phenotypes were also in line with our in vivo data of Cdh13 and Apoe double-KO (Cdh13-/-/Apoe-/-) mice, which showed increased aortic lesion areas when fed on a high-fat diet compared to Apoe-/- mice. Furthermore, prediction using TargetScan, miRWalk, and scanMiRApp databases identified the potential binding of several EC-expression microRNAs (mir) on 3’UTR of CDH13. Several disease-associated variants were mapped to the predicted binding sites of a few microRNAs, including mir-let7, mir-30, and mir-125. The dual luciferase-based RNA interference (RNAi) assay confirmed the binding of mir-let7 on 3’UTR of CDH13. Transcriptional activation of CDH13-AS2 by the enzymatically inactivated Cas9 (dCas9) system diminished the binding of mir-let7 and increased CDH13 expression. Conclusions At the CDH13 locus, CDH13 and CDH13-AS2 are two causal genes, whose downregulation likely contributes to CAD. CDH13-AS2 bound and stabilized CDH13 partially by preventing mir-let7 mediated degradation of CDH13 mRNA, suggesting therapeutic potentials.

9378 Knockdown Of The Cdh2 Gene In Zebrafish Determines Its Role In Terminal Hormone Differentiation

Abstract Disclosure: B.V. Fernandes: None. C. MacRae: None. L.R. Carvalho: None. M. Junqueira: None. Introduction: Congenital hypopituitarism (CH) is characterized by the deficiency of one or more pituitary hormones. Whole exome sequencing (WES) has allowed the identification of novel/rare variants in genetic diseases. Among CH patients, 85% lack a molecular diagnosis. WES approach in a patients born to consanguineous parents identified the homozygous variant (c.865G>A, p. Val289Ile) in the CDH2 gene that produce N-Cadherin protein responsible for cell-cell adhesion. This variant was predicted as likely pathogenic allelic variant by ACMG criteria was found in a patient with GH, TSH, ACTH, LF/FSH deficiencies associated with an ectopic neurohypophysis and pituitary hypoplasia. CDH2 analysis in a cohort of 219 patients with CH, followed in a single Brazilian center, identified 3 unrelated families with heterozygous variants and incomplete penetrance where 2 were novel, in addition to the p. Val289Ile. The deleterious effect of the homozygous variant on cell adhesion properties was confirmed in permanent transfection assays in L1 fibroblast cell lines. To determine the role of the cdh2 gene in pituitary development and hormone production and secretion, we selected zebrafish as an animal model due to its 75% genetic homology with humans. Knockout of cdh2 using Crispr/cas9 genomic edition resulted in 85% lethality of animals at 48 hours post-fertilization (hpf), precluding analysis of its role during pituitary development. Objective: To produce a cdh2 knockdown in zebrafish for phenotypic and molecular analysis. Materials and Methods: The pET28B-RfxCas13d-His plasmid was transformed, linearized, and followed by in vitro transcription to produce Cas13 mRNA. Three guide RNAs targeting the cdh2 gene were designed to guide Cas13 to the target. The Cas13/RNA guide complex was injected into single-cell embryos, and phenotypic analyses were performed under a stereomicroscope until 96 hpf. To evaluate cdh2, prop1, pit1 and gh gene expression by RT-PCR, embryos were collected at 24, 48, 72, and 96 hpf. Results: Knockdown animals exhibited microcephaly, cardiac edema, and microphthalmia during embryonic development. CDH2 expression was decreased by 75% in the first 24 hours of life and began to recover by 48 hours, gradually increasing until 96 hpf when it equaled to WT. gh expression was statistically decreased at 48h compared to WT, with an increased gh expression similar to cdh2 increasing reaching similar WT levels by 96 hpf. There was no statistically significant difference in prop1 and pit1 expression compared to WT in the analyzed periods. Conclusion: The cdh2 knockdown in zebrafish using Cas13 enzyme was effective with cdh2 decreased expression by 75% presenting a phenotype similar to the knockout model. Decreased gh expression at 48h suggests an important role of cdh2 in hormonal terminal differentiation, which should be corroborated by analysis of other hormone expressions. Presentation: 6/2/2024

Metformin represses the carcinogenesis potentially induced by 50 Hz magnetic fields in aged mouse fibroblasts via inhibition of NF-kB.

Aging is a risk factor for various human disorders, including cancer. Current literature advocates that the primary principles of aging depend on the endogenous stress-induced DNA damage caused by reactive oxygen species 50 Hz low-frequency magnetic field was suggested to induce DNA damage and chromosomal instability. NF-kB, activated by DNA damage, is upregulated in age-related cancers and inhibition of NF-kB results in aging-related delayed pathologies. Metformin (Met), an NF-kB inhibitor, significantly reduces both NF-kB activation and expression in aging and cancer. This invitro study, therefore, was set out to assess the effects of 5mT MF in 50 Hz frequency and Met treatment on the viability and proliferation of aged mouse NIH/3T3 fibroblasts and expression of RELA/p65, matrix metalloproteinases MMP2 and MMP9, and E-cadherin (CDH1) genes. The trypan blue exclusion assay was used to determine cell viability and the BrdU incorporation assay to determine cell proliferation. The MMP-2/9 protein analysis was carried out by immunocytochemistry, NF-kB activity by ELISA and the expressions of targeted genes by qRT-PCR methods. Four doses of Met (500 uM, 1 mM, 2 mM and 10 mM) suppressed both the proliferation and viability of fibroblasts exposed to the MF in a dose-dependent pattern, and the peak inhibition was recorded at the 10 mM dose. Met reduced the expression of NF-kB, and MMP2/9, elevated CDH1 expression and suppressed NF-kB activity. These findings suggest that Met treatment suppresses the carcinogenic potential of 50 Hz MFs in aged mouse fibroblasts, possibly through modulation of NF-kB activation and epithelial-mesenchymal transition modulation.

Advancing 3D Spheroid Research through 3D Scaffolds Made by Two-Photon Polymerization.

Three-dimensional cancer cell cultures have been a valuable research model for developing new drug targets in the preclinical stage. However, there are still limitations to these in vitro models. Scaffold-based systems offer a promising approach to overcoming these challenges in cancer research. In this study, we show that two-photon polymerization (TPP)-assisted printing of scaffolds enhances 3D tumor cell culture formation without additional modifications. TPP is a perfect fit for this task, as it is an advanced 3D-printing technique combining a μm-level resolution with complete freedom in the design of the final structure. Additionally, it can use a wide array of materials, including biocompatible ones. We exploit these capabilities to fabricate scaffolds from two different biocompatible materials-PEGDA and OrmoClear. Cubic spheroid scaffolds with a more complex architecture were produced and tested. The biological evaluation showed that the human ovarian cancer cell lines SKOV3 and A2780 formed 3D cultures on printed scaffolds without a preference for the material. The gene expression evaluation showed that the A2780 cell line exhibited substantial changes in CDH1, CDH2, TWIST, COL1A1, and SMAD3 gene expression, while the SKOV3 cell line had slight changes in said gene expression. Our findings show how the scaffold architecture design impacts tumor cell culture 3D spheroid formation, especially for the A2780 cancer cell line.

GATA3 and markers of epithelial-mesenchymal transition predict long-term benefit from tamoxifen in ER-positive breast cancer

GATA binding protein 3 (GATA3) is essential for normal development of the mammary gland and associated with ER-positive breast cancer. Loss of GATA3 has been associated with epithelial-mesenchymal transition (EMT) in experimental studies. We investigated tumoral GATA3 in a cohort of postmenopausal patients with lymph-node negative breast cancer, randomized to adjuvant tamoxifen or control. Nuclear GATA3 expression was assessed with immunohistochemistry and GATA3 gene expression with Agilent microarrays. High GATA3 nuclear expression was associated with a lower rate of distant recurrence in ER-positive breast cancer (HR = 0.60, 95% CI 0.39–0.93). Low gene expression of GATA3 was associated with limited long-term benefit from adjuvant tamoxifen (interaction: p = 0.033). GATA3 gene expression was associated with the epithelial markers CDH1 (E-cadherin) and FOXA1, whereas negatively associated with several mesenchymal markers. Low expression of CDH1 was associated with marginal tamoxifen benefit (HR = 0.80 (0.43–1.49)), whereas patients with higher expression showed a significant benefit (HR = 0.33 (0.20–0.55), interaction: p = 0.029). In ER-positive breast cancer, diminished expression of GATA3 is associated with markers of EMT and poor long-term benefit from tamoxifen.

Fibrotic liver extracellular matrix induces cancerous phenotype in biomimetic micro-tissues of hepatocellular carcinoma model

BackgroundDespite considerable advancements in identifying factors contributing to the development of hepatocellular carcinoma (HCC), the pathogenesis of HCC remains unclear. In many cases, HCC is a consequence of prolonged liver fibrosis, resulting in the formation of an intricate premalignant microenvironment. The accumulation of extracellular matrix (ECM) is a hallmark of premalignant microenvironment. Given the critical role of different matrix components in regulating cell phenotype and function, this study aimed to elucidate the interplay between the fibrotic matrix and malignant features in HCC. MethodsLiver tissues from both control (normal) and carbon tetrachloride (CCl4)-induced fibrotic rats were decellularized using sodium dodecyl sulfate (SDS) and Triton X-100. The resulting hydrogel from decellularized ECM was processed into micro-particles via the water-in-oil emulsion method. Micro-particles were subsequently incorporated into three-dimensional liver biomimetic micro-tissues (MTs) comprising Huh-7 cells, human umbilical vein endothelial cells (HUVECs), and LX-2 cells. The MTs were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at day 11, immunofluorescence staining, immunoblotting, and spheroid migration assay at day 14 after co-culture. ResultsFibrotic matrix from CCl4-treated rat livers significantly enhanced the growth rate of the MTs and their expression of CCND1 as compared to the normal one. Fibrotic matrix, also induced the expression of epithelial-to-mesenchymal transition (EMT)-associated genes such as TWIST1, ACTA2, MMP9, CDH2, and VIMENTIN in the MTs as compared to the normal matrix. Conversely, the expression of CDH1 and hepatic maturation genes HNF4A, ALB, CYP3A4 was decreased in the MTs when the fibrotic matrix was used. Furthermore, the fibrotic matrix increased the migration of the MTs and their secretion of alpha-fetoprotein. ConclusionsOur findings suggest a regulatory role for the fibrotic matrix in promoting cancerous phenotype, which could potentially accelerate the progression of malignancy in the liver.

Small RNA activation of CDH13 expression overcome BCR-ABL1-independent imatinib-resistance and their signaling pathway studies in chronic myeloid leukemia

BCR-ABL1-independent resistance to imatinib has no effective treatment due to its complexity and diversity. We previously reported that the CDH13 oncogene was expressed at low levels in BCR-ABL1-independent resistant CML cell lines. However, its effects on CML resistant cells and mechanisms remain unknown. This study investigated the effects of saRNA-based CDH13 activation on BCR-ABL1-independent imatinib resistance in CML and its underlying mechanism, and proposes a unique treatment method to overcome imatinib resistance. Specifically, this study demonstrated that using the DSIR (Designer of Small Interfering RNA) website tool, saRNAs targeting the CDH13 promoter region were generated and validated using qPCR and western blotting. Among the predicted sequences, C2 and C3 efficiently elevated CDH13 mRNA and protein expression, as well as inhibited the relative vitality of cells and the ability to form clones. After promoting CDH13 expression in K562-IMR cells, it inhabited the NF-κB signaling pathway and induced apoptosis in imatinib-resistant CML cells. LNP-saRNA (C3) was also observed to limit the growth of K562-IMR cells in vivo. From the above, the activation of CDH13 expression by saRNA promotes cell apoptosis by inhibiting the NF-κB signaling pathway to overcome to BCR-ABL1-independent resistance to imatinib in patients with CML.

Exploring the therapeutic potential of Leuconostoc mesenteroides lysates in wound healing and immune modulation on keratinocyte cells.

The skin, being the body's largest organ, primarily functions as a formidable defense mechanism against potential microbial infections. The skin's microbiota, consisting of a complex assembly of microorganisms, exerts a pivotal influence on skin homeostasis by modulating keratinocytes and their cytokine secretion, thereby playing an integral role in promoting optimal cutaneous health. Leuconostoc mesenteroides finds extensive application in the production of fermented foods and bacteriocins. Empirical studies validate the effectiveness of L. mesenteroides treatments in enhancing immune function and demonstrating notable antioxidant characteristics. This study investigates the potential of L. mesenteroides in improving skin health and wound healing. It also aims to comprehend their impact on wound healing markers, cytokine production, and cell cycle regulation compared to ferulic acid, known for its wound healing effects. Our findings indicate that L. mesenteroides lysate possesses antibacterial properties against Staphylococcus aureus and Pseudomonas aeruginosa, along with the ability to mitigate their toxic effects in a pathogen-simulating model employing HaCaT keratinocyte cells. Additionally, the lysate demonstrated noteworthy wound closure after a 24-hour treatment, along with a significant reduction in interleukin-6 levels and oxidative stress index. Modulation of the cell cycle is evident by decreasing G0/G1 phases and increasing S and G2/M phases and enhanced expression of wound healing marker genes and proteins CDH1. In conclusion, L. mesenteroides lysate exhibits immune-modulating and antibacterial properties, offering potential alternatives to conventional treatments for various skin conditions. These findings contribute to the exploration of innovative approaches to enhancing human life through skin health and wound healing.

Adiponectin suppresses stiffness-dependent, profibrotic activation of lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible respiratory disease with limited therapeutic options. A hallmark of IPF is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We performed an unbiased, next-generation sequencing (NGS) screen to identify signaling pathways involved in stiffness-dependent lung fibroblast activation. Adipocytokine signaling was downregulated in primary lung fibroblasts (PFs) cultured on stiff matrices. Re-activating adipocytokine signaling with adiponectin suppressed stiffness-dependent activation of human PFs. Adiponectin signaling depended on CDH13 expression and p38 mitogen-activated protein kinase gamma (p38MAPKγ) activation. CDH13 expression and p38MAPKγ activation were strongly reduced in lungs from IPF donors. Our data suggest that adiponectin-signaling via CDH13 and p38MAPKγ activation suppresses profibrotic activation of fibroblasts in the lung. Targeting of the adiponectin signaling cascade may provide therapeutic benefits in IPF.NEW & NOTEWORTHY A hallmark of idiopathic pulmonary fibrosis (IPF) is excessive fibroblast activation and extracellular matrix (ECM) deposition. The resulting increase in tissue stiffness amplifies fibroblast activation and drives disease progression. Dampening stiffness-dependent activation of fibroblasts could slow disease progression. We found that activation of the adipocytokine signaling pathway halts and reverses stiffness-induced, profibrotic fibroblast activation. Specific targeting of this signaling cascade may therefore provide therapeutic benefits in IPF.

Glioma-associated oncogene (GLI)-specific decoy oligodeoxynucleotide induces apoptosis and attenuates proliferation, colony formation, and migration in liver cancer cells

Aim: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated death. The Sonic Hedgehog (SHH) signaling pathway participates in the initiation, progression, migration, and recurrence of HCC cancer stem cells. Furthermore, SHH regulates various cellular behaviors such as proliferation, differentiation, survival, self-renewal, epithelial-mesenchymal transition (EMT), and SHH autoregulation. Glioma-associated oncogene (GLI) family zinc finger are key transcription factors in the development of many organs and are deregulated in cancer. In this study, Huh-7 cells were treated with GLI-specific decoy oligodeoxynucleotide (ODN) to evaluate its anticancer impact. Methods: The transfection efficiency of GLI-specific decoy ODN was measured using fluorescent microscopy. Then, the effects of GLI-specific decoy ODN on apoptosis, viability, proliferation rate, colony formation, and migration capacities of Huh-7 cells were assessed. Furthermore, the expression of genes associated with the alteration of SHH was assessed. Results: Treatment of Huh-7 cells with GLI-specific decoy ODN decreased cell viability (56.36% ± 3%). Expression of certain genes such as c-MYC , SNAI2 , ZEB1 , and PROM1 decreased dramatically, while the expression of CDH1 increased significantly. Furthermore, the treated cells’ proliferation, colony formation, and migration capacity decreased considerably. This treatment induced apoptosis in the Huh-7 cells. Conclusion: Inhibition of the SHH signaling pathway using GLI-specific decoy ODN led to a decline in the growth rate of HCC cells, decreased migration, and attenuated EMT progression.

Identifying vital nodes for yeast network by dynamic network entropy

BackgroundThe progress of the cell cycle of yeast involves the regulatory relationships between genes and the interactions proteins. However, it is still obscure which type of protein plays a decisive role in regulation and how to identify the vital nodes in the regulatory network. To elucidate the sensitive node or gene in the progression of yeast, here, we select 8 crucial regulatory factors from the yeast cell cycle to decipher a specific network and propose a simple mixed K2 algorithm to identify effectively the sensitive nodes and genes in the evolution of yeast.ResultsConsidering the multivariate of cell cycle data, we first utilize the K2 algorithm limited to the stationary interval for the time series segmentation to measure the scores for refining the specific network. After that, we employ the network entropy to effectively screen the obtained specific network, and simulate the gene expression data by a normal distribution approximation and the screened specific network by the partial least squares method. We can conclude that the robustness of the specific network screened by network entropy is better than that of the specific network with the determined relationship by comparing the obtained specific network with the determined relationship. Finally, we can determine that the node CDH1 has the highest score in the specific network through a sensitivity score calculated by network entropy implying the gene CDH1 is the most sensitive regulatory factor.ConclusionsIt is clearly of great potential value to reconstruct and visualize gene regulatory networks according to gene databases for life activities. Here, we present an available algorithm to achieve the network reconstruction by measuring the network entropy and identifying the vital nodes in the specific nodes. The results indicate that inhibiting or enhancing the expression of CDH1 can maximize the inhibition or enhancement of the yeast cell cycle. Although our algorithm is simple, it is also the first step in deciphering the profound mystery of gene regulation.

Architectural organization and molecular profiling of 3D cancer heterospheroids and their application in drug testing.

3D cancer cell cultures have enabled new opportunities for replacing compound testing in experimental animals. However, most solid tumors are composed of multiple cell types, including fibroblasts. In this study we developed multicellular tumor heterospheroids composed of cancer and fibroblasts cell lines. We developed heterospheroids by combining HT-29, MCF-7, PANC-1 or SW480 with 1BR.3.G fibroblasts, which we have previously reported support spheroid formation. We also tested fibroblast cell lines, MRC-5, GM00498 and HIF, but 1BR.3.G was found to best form heterospheroids with morphological similarity to in vivo tumor tissue. The architectural organization of heterospheroids was based on histological examination using immunohistochemistry. We found that HT-29 and MCF-7 cells developed spheroids with the cancer cells surrounding the fibroblasts, whereas PANC-1 cells interspersed with the fibroblasts and SW480 cells were surrounded by fibroblasts. The fibroblasts also expressed collagen-1 and FAP-α, and whole transcriptomic analysis (WTA) showed abundant ECM- and EMT-related expression in heterospheroids, thus reflecting a representative tumor-like microenvironment. The WTA showed that PANC-1 heterospheroids possess a strong EMT profile with abundant Vimentin and CDH2 expression. Drug testing was evaluated by measuring cytotoxicity of 5FU and cisplatin using cell viability and apoptosis assays. We found no major impact on the cytotoxicity when fibroblasts were added to the spheroids. We conclude that the cancer cell lines together with fibroblasts shape the architectural organization of heterospheroids to form tumor-like morphology, and we propose that the various 3D tumor structures can be used for drug testing directed against the cancer cells as well as the fibroblasts.

Differential Impact of Valproic Acid on SLC5A8, SLC12A2, SLC12A5, CDH1, and CDH2 Expression in Adult Glioblastoma Cells.

Valproic acid (VPA) has anticancer, anti-inflammatory, and epigenetic effects. The study aimed to determine the expression of carcinogenesis-related SLC5A8, SLC12A2, SLC12A5, CDH1, and CDH2 in adult glioblastoma U87 MG and T98G cells and the effects of 0.5 mM, 0.75 mM, and 1.5 mM doses of VPA. RNA gene expression was determined by RT-PCR. GAPDH was used as a control. U87 and T98G control cells do not express SLC5A8 or CDH1. SLC12A5 was expressed in U87 control but not in T98G control cells. The SLC12A2 expression in the U87 control was significantly lower than in the T98G control. T98G control cells showed significantly higher CDH2 expression than U87 control cells. VPA treatment did not affect SLC12A2 expression in U87 cells, whereas treatment dose-dependently increased SLC12A2 expression in T98G cells. Treatment with 1.5 mM VPA induced SLC5A8 expression in U87 cells, while treatment of T98G cells with VPA did not affect SLC5A8 expression. Treatment of U87 cells with VPA significantly increased SLC12A5 expression. VPA increases CDH1 expression depending on the VPA dose. CDH2 expression was significantly increased only in the U87 1.5 mM VPA group. Tested VPA doses significantly increased CDH2 expression in T98G cells. When approaching treatment tactics, assessing the cell's sensitivity to the agent is essential.

YTHDF1 promotes the osteolytic bone metastasis of breast cancer via inducing EZH2 and CDH11 translation

Bone metastasis is common in breast cancer and more effective therapies are required, however, its molecular mechanism is poorly understood. Additionally, the role of the m6A reader YTHDF1 in bone metastasis of breast cancer has not been reported. Here, we reveal that the increased expression of YTHDF1 is clinically correlated with breast cancer bone metastases. YTHDF1 promotes migration, invasion, and osteoblast adhesion and induces osteoclast differentiation of cancer cells in vitro and vivo. Mechanically, RNA-seq, MeRIP-seq and RIP-seq analysis, and molecular biology experiments demonstrate that YTHDF1 translationally enhances EZH2 and CDH11 expression by reading m6A-enriched sites of their transcripts. Moreover, adeno-associated virus (AAV) was used to deliver shYTHDF1 (shYTHDF1-AAV) in intratibial injection models, eliciting a significant suppressive effect on breast cancer bone metastatic formation and osteolytic destruction. Overall, we uncovered that YTHDF1 promotes osteolytic bone metastases of breast cancer by inducing EZH2 and CDH11 translation.

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

Impact of HER2 low status on genomic signatures in triple negative breast cancer (TNBC).

1092 Background: HER2 status has become more important with new treatment regimens subcategorizing HER2 to into low (1+or 2+ FISH-) or absent HER2 expression. Through comprehensive genomic profiling (CGP), we aim to identify distinctive molecular features associated with various immunohistochemistry (IHC) categories of HER2 low tumors. Methods: Retrospective review of pathology specimens submitted to Foundation Medicine were done and 514 TNBC (ER-/PR-/HER20,1+,2+FISH-) and 191 3+/HER2 amplified clinically advanced BC underwent hybrid capture-based CGP to evaluate genomic alterations (GA) in 324 genes, MSI status and tumor mutational burden (TMB, mutations/Mb). PD-L1 expression was determined by IHC (Dako 22C3 TPS). Chi-square test and Mann Whitney U test were used in the statistical comparisons of the 2 groups. Statistical significance was defined as p < 0.05. Results: 53.5% were HER2 IHC 0+, 31.5% were HER2 IHC 1+ and 15.0% were IHC 2+/FISH negative. The HER2 3+/amplified BC were 59% ER+ and 41% ER-. Notable differences in GA frequencies included a significant increase in PIK3CA GA in the HER2 2+ vs the HER 0+ and 1+ groups (p=.0003). A lower frequency in BRCA1 GA in the HER2 2+ (5.2%) vs the HER2 0+ (9.8%) was significant when compared to the 1.0% in the HER2 3+/amplified cases (p<.0001). BRCA2 GA frequencies did not show statistically significant differences among the TNBC HER2 IHC score groups. TP53 and RB1 had the highest frequency in HER2 0+ samples, with decreasing prevalence in the HER2 1+, 2+ and 3+/amplified cases (p<.0001 for both). There was also a trend towards higher frequency of CDH1 GA as the HER2 IHC score increased from 0 to 2+ among TNBC. There were no significant differences in immunotherapy biomarkers (MSI, TMB and PD-L1) based on HER2 status among the TNBC groups. TMB >= 10 mutations/Mb was significantly higher in the HER2 3+/amplified group (p=.004). Conclusions: We found that TNBC HER2 2+ positive FISH show key molecular differences from their HER2 low (1+) or absent (0+) counterparts. The most substantial difference was an increase in GA in PIK3CA. While this is targetable in the hormone positive setting, its potential role as a target in the hormone negative setting is uncertain. Also noted but did not reach significance was a decrease in GA within BRCA1/2 and a positive linear relationship with CDH1 and HER2 IHC expression. These GA differences may indicate potential treatment options for different HER2 subgroups and warrant further investigation. Limitations of our study include the retrospective nature and the lack of clinical data. [Table: see text]

RNA expression-based risk stratification of stage III cutaneous melanoma.

e21551 Background: Prognostication of locally advanced cutaneous melanoma (CM) relies on accurate assessment of primary tumor invasion depth and extent of lymph node involvement. However, precision is often limited by detection limits of positron emission computed tomography and sentinel lymph node biopsy, and the estimated disease-specific survival (DSS) can vary widely. In this study, we explored tumor gene expression-based risk stratification strategies and found that CDH1 (E-cadherin) and BRAF transcript level profiles can predict DSS in stage III CM. Methods: The Cancer Genome Atlas Skin Cutaneous Melanoma (TCGA-SKCM) project’s normalized RNA-Seq transcriptome profile (n = 288) was analyzed with MATLAB software. TCGA-SKCM adopted AJCC 7th edition staging. "High” or “low” level of each gene was determined by the median FPKM of the cohort used as cutoff. P-values were obtained from Cox proportional hazards regression model. Results: In stage III (n = 96), 3-year DSS for high- and low-CDH1 groups were 45% and 75%, respectively (p = 1.5x10-5). High-BRAF group was weakly associated with superior 3-year DSS compared to low-BRAF group (75% vs. 55%; p = 0.06), although high-BRAF group was composed of more BRAF hotspot mutants compared to low-BRAF group (56% vs. 30%). The 3-year DSS for high-CDH1/low-BRAF and low-CDH1/high-BRAF groups were 28% and 84%, respectively (p = 4.4x10-5). Composition of stages [IIIA, IIIB, IIIC, IIINOS] in high-CDH1/low-BRAF and low-CDH1/high-BRAF groups were [5%, 18%, 45%, 32%] and [23%, 13%, 37%, 27%], respectively. For high-CDH1/high-BRAF group, 3-year DSS was 54% with stage composition [6%, 33%, 39%, 22%]. DSS was not associated with CDH1/BRAF levels in stage I and II. Stage IV was not analyzed due to the small sample size. Conclusions: We demonstrate CDH1/BRAF transcript level-based prognostication of CM cases with regional metastasis (metastatic node, in-transit or satellite metastasis) defined as stage III. Our findings are contrary to the notion that CDH1 is a tumor/invasion suppressor gene, although we note the emerging evidence that CDH1 expression boosts establishment of cancer at a secondary site once they escape from the primary site1). The association between high BRAF level and superior survival may also seem counterintuitive given the well-accepted oncogenicity of activating BRAF mutations in variety of malignancies. However, we speculate that wild-type and oncogenic BRAF, which RNA-Seq cannot easily differentiate, may play independent roles in tumorigenesis similar to what has been previously reported in wild-type and oncogenic RAS2).

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

Ribosomal Dysregulation in Metastatic Laryngeal Squamous Cell Carcinoma: Proteomic Insights and CX-5461's Therapeutic Promise.

One of the main barriers to the successful treatment of laryngeal squamous cell carcinoma (LSCC) is postoperative progression, primarily due to tumor cell metastasis. To systematically investigate the molecular characteristics and potential mechanisms underlying the metastasis in laryngeal cancer, we carried out a TMT-based proteomic analysis of both cancerous and adjacent non-cancerous tissues from 10 LSCC patients with lymph node metastasis (LNM) and 10 without. A total of 5545 proteins were quantified across all samples. We identified 57 proteins that were downregulated in LSCC with LNM, which were enriched in cell adhesion pathways, and 69 upregulated proteins predominantly enriched in protein production pathways. Importantly, our data revealed a strong correlation between increased ribosomal activity and the presence of LNM, as 18 ribosomal subunit proteins were found to be upregulated, with RPS10 and RPL24 being the most significantly overexpressed. The potential of ribosomal proteins, including RPS10 and RPL24, as biomarkers for LSCC with LNM was confirmed in external validation samples (six with LNM and six without LNM) using Western blotting and immunohistochemistry. Furthermore, we have confirmed that the RNA polymerase I inhibitor CX-5461, which impedes ribosome biogenesis in LSCC, also decreases the expression of RPS10, RPL24, and RPS26. In vitro experiments have revealed that CX-5461 moderately reduces cell viability, while it significantly inhibits the invasion and migration of LSCC cells. It can enhance the expression of the epithelial marker CDH1 and suppress the expression of the mesenchymal markers CDH2, VIM, and FN at a dose that does not affect cell viability. Our study broadens the scope of the proteomic data on laryngeal cancer and suggests that ribosome targeting could be a supplementary therapeutic strategy for metastatic LSCC.