Migratory Capacity Research Articles

Modulation of SRC by SNTB1 activates the Hippo-YAP pathway during colon adenocarcinoma metastasis.

Colon adenocarcinoma (COAD) ranks as the third most prevalent and lethal cancer in 2020, with metastasis being the primary cause of cancer-related mortality. A comprehensive understanding of the mechanism underlying distant metastasis is imperative for enhancing the prognosis and quality of life of patients with COAD. This study employed gene set enrichment analysis (GSEA) on RNA-sequencing data from 408 patients with COAD in The Cancer Genome Atlas (TCGA) database. GSEA analysis was applied to find the significant hallmark gene set, and genes in the significant hallmark gene set was performed by univariate Cox regression to select the key gene. Then, multivariate Cox regression model was constructed. And various databases were utilized to validate and find the significant oncoprotein and hallmark gene set of the key gene. In addition, the expression of key regulators in para-carcinoma tissue, colon cancer and distant metastases samples were detected by real-time PCR, Immunohistochemistry and Western blot. Additionally, the biological functions were examined by in vitro and in vivo experiments. The scRNA-seq and CellphoneDB were performed to explore cell characters in COAD and the potential mechanism of metastasis. The regulatory network analysis revealed SRC/YAP1 as the most significant oncoprotein and signature gene set associated with SNTB1. Moreover, significant SNTB1 overexpression in COAD metastatic tissues was observed compared to para-carcinoma and primary COAD tissues. Co-immunoprecipitation assays demonstrated the formation of a complex between SNTB1 and SRC proteins. Furthermore, the overexpression of SNTB1 enhanced the proliferation, migration and invasion capacities of COAD cell lines. Caudal vein injection of COAD cells overexpressing SNTB1 in nude mice resulted in increased tumour growth and metastasis to the lung, liver and bone. Finally, single cell RNA-seq revealed alterations in the cellular subtypes of COAD, and CellphoneDB indicated that the interaction between cancer cells exhibiting high SNTB1 expression and enterocytes promoted EMT through cellular communications involving TGF-β, accelerating metastasis in COAD. This study postulates that SNTB1 interacts with SRC to activate the Hippo-YAP pathway, thereby promoting COAD metastasis. Furthermore, cellular communication with enterocytes promotes EMT, facilitating metastasis. These findings propose novel therapeutic targets for preventing or treating metastatic COAD.

CTHRC1 Attenuates Tendinopathy via Enhancing EGFR/MAPK Signaling Pathway.

Tendinopathy poses a formidable challenge due to the inherent limitations of tendon regenerative capabilities post-injury. At present, effective curative approaches for tendinopathy are still lacking. Collagen triple helix repeat-containing 1 (CTHRC1) is an extracellular matrix protein with significant roles in both physiological and pathological processes. The present study aims to investigate the function and underlying mechanism of CTHRC1 in tendinopathy. In this study, CTHRC1 is identified as a potential effector in promoting tendon regeneration through multi-proteomic analysis of Achilles tendon tissues in mice. In vitro, CTHRC1 enhances the proliferation, migration, and tenogenic differentiation of tendon stem/progenitor cell (TSPC). In vivo, CTHRC1 deletion impairs tendon healing, while its overexpression reverses the detrimental effects caused by CTHRC1 deficiency. Mechanistically, proteomics on TSPC stimulated with recombinant CTHRC1 reveal that CTHRC1 activates the mitogen-activated protein kinase (MAPK) signaling pathway via binding to epidermal growth factor receptor (EGFR), which in turn promotes the proliferative, migrative, and tenogenic capacities of TSPC to attenuate Achilles tendinopathy. Conversely, inhibiting EGFR reverses the tendon-healing effect of CRHRC1. The study demonstrates that CTHRC1 can promote the proliferative, migrative, and tenogenic capacities of TSPC, ultimately facilitating tendon healing through activating the EGFR/MAPK signaling pathway. CTHRC1 holds promise as a potential intervention for tendinopathy.

Optimizing cell migration assays: Critical roles of fluorescent labeling and chemoattractant gradients.

Cell migration assays, also known as chemotaxis assays, are widely used to measure the migratory capacities of cancer cells, leukocytes, macrophages, and other motile cell types. In these assays, fluorescently labeled cells are seeded onto cell culture inserts with microporous membranes that block light transmission from 490 to 700nm. The migrated cells are then observed and quantified from the bottom of the microporous membrane using a fluorescence microscope. In this study, we conducted cell migration assays using macrophages as the motile cells. We discovered that the commonly employed fluorescent labeling method using calcein acetoxymethyl ester (calcein AM) can lead to the time-dependent attenuation of fluorescent signals in certain cell types during migration assays, potentially compromising assay stability. This study overcame this limitation by utilizing PKH26, which fluorescently labels cell surfaces through a mechanism distinct from that of calcein AM. With this modification, we observed a consistent increase in the number of migrating macrophages over time. We also demonstrated that the gradient of chemoattractants is key to the success of cell migration assays. Our improved protocol provided reliable and stable results for cell migration assays.

Abstract B012: HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy

Abstract The high rate of locoregional/distant recurrence in triple-negative breast cancer (TNBC) is a major driver of the decreased survival observed in breast cancer patients. Radiation therapy is a reliable treatment modality to limit recurrence for many cancers, but TNBC patients have a higher chance for relapse despite receiving increased doses of radiation compared to patients with other breast cancer subtypes. This study investigated the differential pathways in radioresistant TNBC tumors that impact recurrence. We developed two radioresistant cell line models (468-RR and 4T1-RR) by administering a radiation dose of 50Gy over the course of 8 weeks to the human TNBC cell line, MDA-MB-468, and the mouse TNBC cell line, 4T1, respectively. Migration and invasion were assessed using the scratch-wound assay and transwell assay, respectively. We assessed in vivo metastasis by injecting the cells in the mammary fat pad, tail vein, or left ventricle. RNA-sequencing of the cell lines was done to identify global transcriptomic changes and regulatory pathways induced by radiation therapy. The mRNA stability of integrin b3 was assessed by inhibiting transcription using actinomycin D. Moreover, we implemented the newly developed InduroRT-mediated circRNA-sequencing (IMRT-seq) to identify the circular RNAs processed by HNRNPL that are present in radioresistant cells. We observed enhanced lung and bone metastasis, based on bioluminescent imaging, in both the spontaneous and vascular metastasis models for the radioresistant cells compared to the parental cells. Genset enrichment analysis identified mRNA stability and metabolism as upregulated pathways in radioresistant cells with HNRNPL, a ribonucleoprotein involved in mRNA stability, as one of the top genes. By knocking down HNRNPL, we observed decreased migration capacity of the radioresistant cells. Given the role of HNRNPL in integrin and extracellular matrix genes, which are key components associated with metastasis in cancer cells, we screened for these genes in our RNA-seq dataset and identified integrin b3 as a possible target. More specifically, knocking down HNRNPL decreased the mRNA stability of integrin b3 as assessed by actinomycin d treatment. By expressing integrin b3 in the HNRNPL-depleted cells, we were able to rescue the migration capacity of these cells. We believe a subset of HNRNPL-mediated circular RNAs are responsible for integrin b3 mRNA stability by sequestering miRNAs that target integrin b3. With IMRT-seq we are exploring the potential circRNA-miRNA interactions present in radioresistant TNBC responsible for its metastatic phenotype. The radioresistant cells derived from TNBC cell lines have an enhanced metastatic potential than the treatment-naive cell lines and our data suggest that RNA metabolism is pivotal to this phenotype. The upregulation of HNRNPL upon radiation by Nrf2 increases integrin b3 expression by stabilizing its transcripts and preventing its degradation. This novel mechanism of radioresistant metastasis provides a therapeutic approach to minimize TNBC recurrence. Citation Format: Ayush Kumar, Kensei Kishimoto, Christi Wisniewski, Shivam Goel, Arthur Mercurio. HNRNPL-mediated mRNA stability underlies metastasis triggered by resistance to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B012.

Abstract A054: Oncogenic functions of alternatively spliced MDM2-ALT2 isoform in retroperitoneal liposarcoma

Abstract Retroperitoneal liposarcoma (RPLPS) is one of the most common histologic subtypes of soft tissue sarcoma (STS). Complete surgical resection remains the mainstay treatment while the high rate of locoregional recurrence constitutes the predominant cause of mortality. Well- differentiated (WDLPS) and dedifferentiated (DDLPS) liposarcoma are the most frequent subtypes of RPLPS and present amplified MDM2 gene as a hallmark. However, there are few reports evaluating the role of alternatively spliced MDM2 transcripts in RPLPS. In this study, we assessed MDM2-ALT2 expression levels in a cohort of RPLPS patients and evaluated biological functions of MDM2-ALT2 isoform in vitro in DDLPS cell lines. Using BaseScope™ and qPCR, we demonstrated that MDM2-Full Length (MDM2-FL) and MDM2-ALT2 expression levels were upregulated in RPLPS patient-derived tissue samples compared to normal adjacent to tumor tissue (NAT). DDLPS cells overexpressing MDM2-FL or MDM2-ALT2 had higher proliferation rates and increased migration and invasion capacities, as well as increased protein levels of p- AKT, mTOR, p70S6K, MMP2 and cJun. Simultaneous overexpression of MDM2-ALT2 and AKT silencing showed that AKT inhibition impaired p-p70S6K and MMP2 protein increased levels and led to significantly decreased proliferation and migration rates compared to cells overexpressing MDM2-ALT2 only. Taken together, our data suggest that MDM2-ALT2 may promote RPLPS progression. Citation Format: Fernanda Costas C. de Faria, Safiya Khurshid, Patricia Sarchet, Sayumi Tahara, Lucia Casadei, Valerie Grignol, Roma Karna, Sydney Rentsch, Nippin Sp, Joal D Beane, Luciano Mazzoccoli, Matias Montes, Giovanni Nigita, Joe T Sharick, Jennifer L Leigth, Federica Calore, Dawn Chandler, Raphael E Pollock. Oncogenic functions of alternatively spliced MDM2-ALT2 isoform in retroperitoneal liposarcoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A054.

A comprehensive genome-based analysis identifies the anti-cancerous role of the anoikis-related gene ADH1A in modulating the pathogenesis of breast cancer.

Breast cancer (BC), a widespread and lethal neoplasm, is irrespective of the subtype of BC. Metastasis remains a crucial determinant for unfavorable outcome. The identification of novel diagnostic markers is instrumental in optimizing the treatment regime for BC. The direct correlation between anoikis and the progression/outcome of BC is well established. Nevertheless, the contribution of anoikis-related genes (ARGs) in BC remains obscure at present. We implemented the METABRIC dataset to scrutinize and assess differentially expressed ARGs in BC versus healthy breast tissues. An unsupervised consensus clustering approach for ARGs was employed to classify patients into diverse subtypes. ESTIMATE algorithms were utilized to assess immune infiltrative patterns. Prognostic gene expression patterns were derived from LASSO regression and univariate COX regression analysis. Subsequently, these signatures underwent examination via use of the Kaplan-Meier survival curve. 6 pairs of fresh tissue specimens (tumor and adjacent non-tumor) were employed to assess the expression of 7 ARGs genes via qPCR. Notably, DCN and FOS were not expressed in BC tissue, which had been excluded in our subsequent experiments. Also, among remaining 5 ARGs, solely the expression of ADH1A demonstrated a statistically remarkable disparity between freshly collected cancer tissues and the adjacent ones. ADH1A-overexpressed and ADH1A-sh vectors were transfected into MCF-7 and MCF-7-AR cell lines, respectively. The expression status of FABP4, CALML5, ADH1A, C1orf106, CIDEC, β-catenin, N-cadherin, and Vimentin in the clinical samples were scrutinized using RT-qPCR and western blotting techniques. Migration and invasion through transwell chambers were employed to assess the migratory and invasive potential of the cells. Detailed evaluation of cell proliferation was conducted utilizing a Cell Counting Kit-8 (CCK-8) assay. The apoptotic index of the cells was determined by flow cytometry analysis. An innovative anoikis-associated signature consisting of seven genes, namely ADH1A, DCN, CIEDC, FABP4, FOS, CALML5, and C1orf106, was devised to stratify BC patients into high- and low-risk cohorts. This unique risk assessment model, formulated via the distinctive signature approach, has been validated as an independent prognostic indicator. Additional analysis demonstrated that distinct risk subtypes manifested variances in the tumor microenvironment and drug sensitivities. Suppression of ADH1A enhanced the migratory and invasive capacities and reduced these tumorigenesis-related protein levels, underscoring the prognostic role of ADH1A in the progression of BC. Through our meticulous study, we have elucidated the possible molecular markers and clinical implications of ARGs in BC. Our model, which incorporate seven ARGs, has proven to accurately forecast the survival outcomes of BC patients. Moreover, the thorough molecular study of ADH1A has augmented our comprehension of ARGs in BC and opened a novel avenue for guiding personalized and precise therapeutic interventions for BC patients.

Removal of Phosphate from Water by Iron/Calcium Oxide-Modified Biochar: Removal Mechanisms and Adsorption Modeling

Phosphorus (P) pollution is a leading cause of water eutrophication, and metal-modified biochar is an effective adsorbent with the ability to alter the migration capacity of phosphorus. This study uses bamboo as the raw material to prepare metal-modified biochar (ZFCO-BC) loaded with Fe and Ca under N2 conditions at 900 °C, and investigates its adsorption characteristics for phosphate. Batch experimental results show the adsorption capacity of the ZFCO-BC gradually increases (from 4.0 to 69.1 mg/g) as the initial phosphate concentration increases (from 2 to 900 mg/L), mainly through multilayer adsorption. Additionally, as the pH increases from 1 to 7, the adsorption capacity of the ZFCO-BC climbs to reach its maximum value of 48.4 mg/g with an initial phosphate concentration of 150 mg/L. At this pH, phosphate primarily exists as H2PO4− and HPO42−, which both readily react with Fe3+ and Ca2+ in the biochar. Furthermore, the addition of CO32−, HCO3−, NO3−, SO42−, F−, and Cl− each affect the removal rate of phosphate by less than 10%, indicating the ZFCO-BC has a highly efficient and selective phosphate adsorption capacity. A multi-column adsorption experiment designed to achieve long-term and efficient phosphorus removal treated 275.5 pore volumes (PVs) of water over 366 h. The cyclic adsorption–desorption experiment results show that 0.5 M NaOH can effectively leach phosphate from the ZFCO-BC. Observations at the molecular level from P K-edge XANES spectra confirm the removal of low-concentration phosphate is primarily dominated by electrostatic attraction, while the main removal mechanism for high-concentration phosphate is chemical precipitation. This study demonstrates that ZFCO-BC has broad application prospects for phosphate removal from wastewater and as a potential slow-release fertilizer in agriculture.

The role of macrophage migratory behavior in development, homeostasis and tumor invasion

Tumor-associated macrophages (TAMs) recapitulate the developmental and homeostatic behaviors of tissue resident macrophages (TRMs) to promote tumor growth, invasion and metastasis. TRMs arise in the embryo and colonize developing tissues, initially to guide tissue morphogenesis and then to form complex networks in adult tissues to constantly search for threats to homeostasis. The macrophage growth factor, colony-stimulating factor-1 (CSF-1), which is essential for TRM survival and differentiation, is also responsible for the development of the unique motility machinery of mature macrophages that underpins their ramified morphologies, migratory capacity and ability to degrade matrix. Two CSF-1-activated kinases, hematopoietic cell kinase and the p110δ catalytic isoform of phosphatidylinositol 3-kinase, regulate this machinery and selective inhibitors of these proteins completely block macrophage invasion. Considering tumors co-opt the invasive capacity of TAMs to promote their own invasion, these proteins are attractive targets for drug development to inhibit tumor progression to invasion and metastasis.

MiR-155 promotes m6A modification of SOX2 mRNA through targeted regulation of HIF-1α and delays wound healing in diabetic foot ulcer in vitro models.

Diabetic foot ulcers (DFU) are one of the most destructive complications of diabetes mellitus. The aim of this study was to link miR-155 and SOX2 with DFU to explore the regulation of wound healing by DFU and its potential mechanism. Human keratinocytes (HaCaT) were induced with advanced glycation end products (AGEs) to construct DFU models in vitro. AGE-induced HaCaTcells were subjected to CCK-8 assays, flow cytometry, and wound healing assays to evaluate cell proliferation, apoptosis, and migration capacity, respectively. RT-qPCR and Western blotting were used to determine gene and protein expression levels, respectively. N6-methyladenosine (M6A) levels in total RNA were assessed using an M6A methylation quantification kit. Our results suggested that the inhibition of miR-155 promoted wound healing in an in vitro DFU model, while the knockdown of HIF-1α reversed this process, and that HIF-1α was a target protein of miR-155. In addition, knockdown of HIF-1α promoted the m6A level of SOX2 mRNA, inhibited the expression of SOX2, and inhibited the activation of the EGFR/MEK/ERK signaling pathway, thus inhibiting the proliferation and migration of HaCaTcells and promoting the apoptosis of HaCaTcells, while overexpression of SOX2 reversed this effect. We also found that METTL3 knockdown had the opposite effect of HIF-1α knockdown. Inhibition of miR-155 promoted the expression of HIF-1α and attenuated the m6A modification of SOX2 mRNA, thereby promoting the expression of SOX2 and activating the downstream EGFR/MEK/ERK signaling pathway to promote wound healing in an in vitro DFU model.

Mechanisms of microRNA Regulation of the Epithelial–Mesenchymal Transition (EMT) in Lung Cancer

Lung cancer remains the cancer with the highest mortality worldwide, largely due to a limited understanding of the precise molecular mechanisms that drive its progression. microRNAs (miRNAs) have emerged as crucial regulators of lung cancer progression by influencing key cellular processes, notably the epithelial–mesenchymal transition (EMT). EMT is a complex and potentially reversible process where epithelial cells lose their polarity and adhesion, reorganize their cytoskeleton, and transition to a mesenchymal phenotype, enhancing their migratory and invasive capacities. While EMT plays an essential role in normal physiological contexts such as tissue development and wound healing, it is also a critical mechanism underlying the progression and metastasis of lung cancer. This review aims to summarize the latest research findings on the role of endogenous and exosome-derived microRNAs in regulating EMT in lung cancer, focusing on studies conducted over the past five years. It also provides an overview of EMT’s essential molecular mechanisms to better understand how miRNAs regulate EMT in lung cancer.

A nanoencapsulated oral formulation of fenretinide promotes local and metastatic breast cancer dormancy in HER2/neu transgenic mice

BackgroundPrevention and treatment of metastatic breast cancer (BC) is an unmet clinical need. The retinoic acid derivative fenretinide (FeR) was previously evaluated in Phase I-III clinical trials but, despite its excellent tolerability and antitumor activity in preclinical models, showed limited therapeutic efficacy due to poor bioavailability. We recently generated a new micellar formulation of FeR, Bionanofenretinide (Bio-nFeR) showing enhanced bioavailability, low toxicity, and strong antitumor efficacy on human lung cancer, colorectal cancer, and melanoma xenografts. In the present study, we tested the effect of Bio-nFeR on a preclinical model of metastatic BC.MethodsWe used BC cell lines for in vitro analyses of cell viability, cell cycle and migratory capacity. For in vivo studies, we used HER2/neu transgenic mice (neuT) as a model of spontaneously metastatic BC. Mice were treated orally with Bio-nFeR and at sacrifice primary and metastatic breast tumors were analyzed by histology and immunohistochemistry. Molecular pathways activated in primary tumors were analyzed by immunoblotting. Stem cell content was assessed by flow cytometry, immunoblotting and functional assays such as colony formation ex vivo and second transplantation assay in immunocompromised mice.ResultsBio-nFeR inhibited the proliferation and migration of neuT BC cells in vitro and showed significant efficacy against BC onset in neuT mice. Importantly, Bio-nFeR showed the highest effectiveness against metastatic progression, counteracting both metastasis initiation and expansion. The main mechanism of Bio-nFeR action consists of promoting tumor dormancy through a combined induction of antiproliferative signals and inhibition of the mTOR pathway.ConclusionThe high effectiveness of Bio-nFeR in the neuT model of mammary carcinogenesis, coupled with its low toxicity, indicates this formulation as a potential candidate for the treatment of metastatic BC and for the adjuvant therapy of BC patients at high risk of developing metastasis.

Prognostic value of EMT-related genes and immune cell infiltration in thyroid carcinoma

BackgroundThe Epithelial–Mesenchymal Transition (EMT) is a very important process involved in cancer invasion and metastasis. Additionally, the Cathepsin K (CTSK) gene is closely related to the degradation of the extracellular matrix, which is a critical component of the EMT. The purpose of this study was to determine the relationships between EMT-related genes and immune cell infiltration and their prognostic value in Thyroid carcinoma (THCA). The effect of the CTSK gene on the aggressive biological features of THCA was assessed.MethodsWithin the framework of the present study, the THCA cohort was analyzed in detail based on data obtained from The TCGA database in the context of the EMT. The TCGA-THCA cohort was then divided into two groups, namely, high- and low-risk groups, based on the calculated EMT scores. Finally, based on the findings from the Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm, LASSO regression analysis, and Kaplan−Meier plotter, we selected five genes (CTSK, C3ORF80, FBLN2, PRELP and SRPX2) associated with patient prognosis. Furthermore, this study examined the presence of various immune cells within the THCA samples using three distinct algorithms, namely ssGSEA, xCell, and MCPcounter. Additional studies have been conducted to establish the roles of CTSK in THCA cell proliferation and migration using various assays, such as CCK8, colony formation, EdU proliferation, Transwell migration and wound healing assays. Additionally, the involvement of CTSK in the regulation of various EMT-related markers was confirmed using Western blot analysis.ResultsBased on EMT scores, TCGA-THCA patients were further divided into two groups, and the study revealed that patients in the high-risk group had a worse prognosis than those in the low-risk group. Among the five genes linked to the prognostic value of EMT (CTSK, C3ORF80, FBLN2, PRELP, and SRPX2), CTSK exhibited notably elevated expression in the high-risk cohort. This group also exhibited pronounced immune cell infiltration, with a marked correlation observed between CTSK expression and the levels of macrophages, MDSCs, and various T-cell subtypes. Furthermore, in vitro studies demonstrated that reducing CTSK expression led to significant reductions in THCA cell viability; clonogenic, proliferative, motility and migratory capacities; and the expression of key EMT-related proteins, including N-cadherin, vimentin, slug, and snail.ConclusionOur results suggest that the expression of CTSK, a gene associated with the EMT, may be associated with THCA onset and progression and thus may serve as a promising prognostic biomarker.

NKX3-2 Induces Ovarian Cancer Cell Migration by HDAC6-Mediated Repositioning of Lysosomes and Inhibition of Autophagy.

Several soluble factors secreted by the stromal cells and cancer cells within the tumor microenvironment facilitate the progression and invasiveness of ovarian cancer. In ovarian cancer cells, lysophosphatidic acid (LPA) modulates the transcriptome profile and promotes cell invasiveness by the downregulation of autophagy. Here, we further elucidate this mechanism by focusing on the molecular and cellular events regulating autophagy. Transcriptomic and Western blotting analyses revealed NKX3-2, a transcriptional factor, to be among the genes hyperexpressed in LPA-stimulated ovarian cancer cells. Bioinformatic analyses revealed that in ovarian cancer patients, the expression of NKX3-2 positively correlates with genes involved in cell motility and migration, while it negatively correlates with macromolecular catabolic pathways. In various ovarian cancer cell lines, NKX3-2 silencing abrogated LPA-induced cell migration. Mechanistically, this effect is linked to the restoration of the HDAC6-mediated relocation of the lysosomes in the para-golgian area, and this results in an increase in autolysosome formation and the overall upregulation of autophagy. Silencing the expression of ATG7 or BECN1, two autophagy genes, rescued the migratory phenotype of the NKX3-2-silenced ovarian cancer cells. Taken together, these data reveal the mechanism by which the LPA-NKX3-2 axis promotes the invasiveness of ovarian cancer cells and supports the possibility of targeting NKX3-2 to reduce the migratory capacity of cancer cells in response to a permissive microenvironment.

Xenotropic and polytropic retrovirus receptor 1 (XPR1) promotes progression of papillary thyroid carcinoma via the BRAF-ERK1/2-P53 signaling pathway.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Xenotropic and polytropic retrovirus receptor 1 (XPR1), identified as a cellular receptor, plays roles in many pathophysiological processes. However, the underlying function and molecular mechanisms of XPR1 in PTC remain unclear. Therefore, this study aimed to elucidate the role of XPR1 in the process of PTC and the potential mechanisms. RNA-sequencing was performed for gene differential expression analysis in PTC patients' tissues. Immunohistochemical assay, real-time PCR, and western blotting assay were used to determine the expression of XPR1, BRAF, and P53 in PTC tissues. The function of XPR1 on the progression of PTC was explored using in vitro and in vivo experiments. The molecular mechanism of XPR1 was investigated using gene silencing, ELISA, immunofluorescence, western blotting, and real-time PCR assays. We found that XPR1 was markedly upregulated in PTC tissues compared to adjacent noncancerous tissues, suggesting that high expression of XPR1 could be correlated with poor patient disease-free survival in PTC. In addition, the expression of BRAF and P53 in PTC tissues was substantially higher than in adjacent noncancerous tissues. Silencing of XPR1 reduced the proliferation, migration, and invasion capacities of TPC-1 cells in vitro and effectively inhibited the tumorigenecity of PTC in vivo. More importantly, silencing of XPR1 in TPC-1 cells significantly decreased the expression of XPR1, BRAF, and P53 both in vitro and in vivo. Interestingly, we demonstrated that XPR1 may positively activate the BRAF-ERK-P53 signaling pathway, further promoting PTC progression. The findings reveal a crucial role of XPR1 in PTC progression and prognosis via the BRAF-ERK1/2-P53 signaling pathway, providing potential therapeutic targets for treating PTC.

MTOR maintains endothelial cell integrity to limit lung vascular injury

The functional and structural integrity of the endothelium is essential for vascular homeostasis. Loss of barrier function in quiescent and migratory capacity in proliferative endothelium causes exuberant vascular permeability, a cardinal feature of many inflammatory diseases including acute lung injury (ALI). However, the signals governing these fundamental endothelial cell (EC) functions are poorly understood. Here, we identify Mechanistic Target of Rapamycin (MTOR) as an important link in preserving the barrier integrity and migratory/angiogenic responses in EC and preventing lung vascular injury and mortality in mice. Knockdown of MTOR in EC altered cell morphology, impaired proliferation and migration, and increased endocytosis of cell surface VE-Cadherin leading to disrupted barrier function. MTOR-depleted EC also exhibited reduced VE-Cadherin and VEGFR2 levels mediated in part by autophagy. Similarly, lungs from mice with EC-specific MTOR deficiency displayed spontaneous vascular leakage marked by decreased VE-Cadherin and VEGFR2 levels, indicating that MTOR deficiency in EC is sufficient to disrupt lung vascular integrity and may be a key pathogenic mechanism of ALI. Indeed, MTOR as well as VEGFR2 and VE-Cadherin levels were markedly reduced in injured mouse lungs or EC. Importantly, EC-targeted gene transfer of MTOR cDNA, either prophylactically or therapeutically, mitigated inflammatory lung injury, and improved lung function and survival in mouse models of ALI. These findings reveal an essential role of MTOR in maintaining EC function, identify loss of endothelial MTOR as a key mechanism of lung vascular injury, and show the therapeutic potential of EC-targeted MTOR expression in combating ALI and mortality in mice.

Lymphocyte Antigen 6 Family Member D (LY6D) Affects Stem Cell Phenotype and Progression of Pancreatic Adenocarcinoma.

The membrane-bound protein lymphocyte antigen 6 family member D (LY6D), a marker of early B cell lineage is reportedly expressed in several human malignancies and has been implicated in cancer stemness. However, its expression and role in cancer stemness remain largely unexplored in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to clarify the role of LY6D in PDAC. We conducted functional analysis of LY6D to evaluate its impact on the malignant features of PDAC cells in vitro. Using our in-house developed stem cell separation technique, which isolates cells with low proteasome activity and CD44 v9 cell surface marker for cancer stem cells, we performed sphere formation and chemosensitivity tests and tumor formation assay in mice, through knockdown of LY6D expression. Immuno-histopathological analysis was also conducted to reveal the clinical significance of LY6D in PDAC. In vitro functional assays demonstrated that LY6D was critically involved in promoting the cancer malignant phenotype, including increased invasive ability, drug resistance, migration capacity, and cancer stemness. Immunohistopathological analysis revealed that high LY6D expression levels were associated with high recurrence rates and poorer prognosis in PDAC. Our study showed that LY6D is a novel prognostic indicator and plays a key role in regulation of cancer stemness in PDAC.

Altingia chinensis petroleum ether extract suppresses NSCLC via induction of apoptosis, attenuation of EMT, and downregulation of PI3K/Akt pathway

BackgroundNon-small-cell lung cancer (NSCLC) is the primary type of lung cancer with the leading cause of fatalities from cancer, and the effective treatment is minimal. Altingia chinensis is a medicinal plant utilized as a traditional folk remedy to alleviate rheumatism, punch injury and paralysis. APE is the petroleum ether extract from A. Chinensis, whose antitumor effects are rarely studied. PurposeTo explore the antitumor effects of APE on NSCLC and its molecular mechanism. MethodsLLC and H1299 cells were used to explore the anticancer effect of APE on NSCLC in vitro. MTT assay and colony formation were employed to evaluate cell viability. Flow cytometry was used to evaluate apoptosis. Wound healing and transwell were employed to evaluate cell migration and invasive capacity. Meanwhile, an LLC tumor-bearing C57BL/6 mice model was established for assessing the anticancer effect of APE on NSCLC in vivo. H&E staining was used to assess histopathology. TUNEL assay was performed to assess apoptosis in tumor tissue. Network pharmacology, CESTA, and kinase assay were employed to analyze potential molecular mechanisms. Western blots were performed to detect proteins involved in apoptosis, EMT, and the PI3K/Akt pathway. ResultsThis is the first investigation to identify APE's antitumor potential in both NSCLC cells and tumor-bearing mice models. Significantly, APE dose-dependently decreased cell viability and caused morphological changes both in LLC and H1299 cells. Furthermore, APE (31.25, 62.5, and 125 μg/ml) induced apoptosis in NSCLC cells, as demonstrated by increased Annexin V-FITC/PI-stained cells, the cleaved-caspase 3 levels, and the Bax/Bcl-2 ratio. Additionally, APE suppressed cell migration and invasion by the increase of E-cadherin expression and the downregulation of vimentin, implying that APE inhibited cell metastasis via attenuation of EMT. Importantly, intragastric administration of 100 mg/kg APE significantly inhibited tumor growth without apparent side effects. TUNEL assay confirmed the apoptosis in tumor tissue. Western blots validated the alteration of EMT and apoptotic markers in tumor tissue, which matched the in vitro findings. Moreover, APE directly bound to PI3Kα and inhibited its activity, leading to inhibition of the PI3K-Akt pathway. ConclusionOverall, APE exhibits anti-tumor effects on NSCLC via induction of apoptosis, attenuation of EMT, and its mechanism involves the suppression of the PI3K/Akt pathway. Our study offers new insights for the identification of novel drug development for NSCLC.

Elevated expression levels of the protein kinase DYRK1B induce mesenchymal features in A549 lung cancer cells

BackgroundThe protein kinase DYRK1B is a negative regulator of cell proliferation but has been found to be overexpressed in diverse human solid cancers. While DYRK1B is recognized to promote cell survival and adaption to stressful conditions, the consequences of elevated DYRK1B levels in cancer cells are largely uncharted.MethodsTo elucidate the role of DYRK1B in cancer cells, we established a A549 lung adenocarcinoma cell model featuring conditional overexpression of DYRK1B. This system was used to characterize the impact of heightened DYRK1B levels on gene expression and to monitor phenotypic and functional changes.ResultsA549 cells with induced overexpression of wild type DYRK1B acquired a mesenchymal cell morphology with diminished cell-cell contacts and a reorganization of the pericellular actin cytoskeleton into stress fibers. This transition was not observed in cells overexpressing a catalytically impaired DYRK1B variant. The phenotypic changes were associated with increased expression of the transcription factors SNAIL and SLUG, which are core regulators of epithelial mesenchymal transition (EMT). Further profiling of DYRK1B-overexpressing cells revealed transcriptional changes that are characteristic for the mesenchymal conversion of epithelial cells, including the upregulation of genes that are related to cancer cell invasion and metastasis. Functionally, DYRK1B overexpression enhanced the migratory capacity of A549 cells in a wound healing assay.ConclusionsThe present data identify DYRK1B as a regulator of phenotypic plasticity in A549 cells. Increased expression of DYRK1B induces mesenchymal traits in A549 lung adenocarcinoma cells.

Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1

UVB radiation induces oxidative stress, DNA damage, and inflammation, leading to skin wrinkling, compromised barrier function, and an increased risk of carcinogenesis. Addressing or preventing photoaging may offer a promising therapeutic avenue for these conditions. Recent research indicated that mesenchymal stem cells (MSCs) exhibit significant therapeutic potential for various skin diseases. Given that extracellular vesicles (EV) can deliver diverse cargo to recipient cells and elicit similar therapeutic effects, we investigated the roles and underlying mechanisms of both adipose-derived MSC-derived EV (AMSC-EV) and umbilical cord-derived MSC-derived EV (HUMSC-EV) in photoaging. Our findings indicated that in vivo, treatment with AMSC-EV and HUMSC-EV resulted in improvements in wrinkles and skin hydration while also mitigating skin inflammation and thickness alterations in both the epidermis and dermis. Additionally, in vitro studies using human keratinocytes (HaCaTs), human dermal fibroblast cells (HDFs), and T-Skin models revealed that AMSC-EV and HUMSC-EV attenuated senescence, reduced levels of reactive oxygen species (ROS) and DNA damage, and alleviated inflammation induced by UVB. Furthermore, EV treatment enhanced cell viability and migration capacity in the epidermis and promoted extracellular matrix (ECM) remodeling in the dermis in photoaged cell models. Mechanistically, proteomics results showed that TIMP1 was highly expressed in both AMSC-EV and HUMSC-EV and could exert similar effects as MSC-EV. In addition, we found that EV and TIMP1 could inhibit Notch1 and downstream targets Hes1, P16, P21, and P53. Collectively, our data suggests that both AMSC-EV and HUMSC-EV attenuate skin photoaging through TIMP1/Notch1.

SAR1A Induces Cell Growth and Epithelial–Mesenchymal Transition Through the PI3K/AKT/mTOR Pathway in Head and Neck Squamous Cell Carcinoma: An In Vitro and In Vivo Study

Objectives: Head and neck squamous cell carcinoma (HNSCC) ranks sixth globally, with a 50% five-year survival rate. SAR1A exhibits high expression levels in various tumor types, yet its specific role in HNSCC remains to be clarified. Methods: In vitro assays, such as CCK8, EdU, colony formation, wound-healing, transwell, and Western blotting analyses, as well as in vivo assays, such as tumor xenografts and lung metastasis models, were conducted to evaluate the impacts of SAR1A on HNSCC proliferation, migration, and invasion. Transcriptome sequencing and KEGG enrichment pathway analysis revealed evident alterations in the PI3K/AKT/mTOR(PAM) pathways. LY294002 (a PI3K/AKT inhibitor) was used to investigate the role of the PAM pathway in proliferation, migration, and invasion in HNSCC. Results: Univariate and multivariate Cox regression were conducted to screen SAR1A as a gene prognostic biomarker in HNSCC, and it was validated in the Cancer Genome Atlas (TCGA) database. Functional assays demonstrated that the depletion of SAR1A leads to suppressed proliferation, migration, and invasion of HNSCC cells. This is accompanied by a decrease in the expression of epithelial–mesenchymal transition (EMT)-related markers in HNSCC cell lines. In addition, the diminished capacities of proliferation, migration, and invasion observed in SAR1A knockdown cells were reversed upon the overexpression of SAR1A. Furthermore, RNA-seq and KEGG enrichment analysis demonstrated a significant alteration in the PAM pathway following SAR1A knockdown. LY294002 effectively mitigated the increased proliferation, migration, and invasion induced by SAR1A overexpression. Conclusions: SAR1A facilitates HNSCC proliferation and EMT via the PI3K/AKT/mTOR pathway.