Sphere Formation Research Articles

Restriction of YWHAB-mediated YAP cytoplasmic retention as a novel mechanism underlying stemness maintenance and chemo-resistance in peritoneal metastasis of ovarian cancer: A comparative proteomic study.

122 Background: Ovarian cancer (OC) peritoneal metastasis (OCPM) is a significant cause of high mortality of OC. To investigate the mechanisms underlying OCPM stemness maintenance and resistance, we characterized proteomic alterations in residual OCPM tissues after neoadjuvant chemotherapy (NACT), and verified restriction of YWHAB-mediated YAP cytoplasmic retention as a novel important mechanism. Methods: Tumor specimens from HGSOC patients underwent proteomic analysis using TMT and REACTOME for pathway and GO analysis. The OVCAR3 cell line, derived from malignant ascites, and formalin-fixed samples were used for Immunohistochemistry. Ovarian cancer stem-like cells, including cisplatin-resistant cells, were cultured, and ALHD activity was measured by qPCR and ALDEFLUOR Kit. Western blot and Co-IP were used for protein analysis. Sphere formation and limiting dilution assays were performed in vitro and in vivo using BALB/c nude mice, and FACS quantified OCSCs. YWHAB-knockdown cells were created via plasmids, lentivirus, and transfection. Statistical tests included paired and unpaired t-tests, one-way ANOVA, and ELDA for limiting dilution. Data are shown as mean ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001 indicating significance. Results: TMT-based proteomics identified 324 differentially expressed proteins in post-NACT OCPM tissues, with 179 upregulated and 145 downregulated. Bioinformatics revealed novel targets in key pathways, including TUBB, VCP in Hedgehog, YWHAB in Hippo, TLA1/2, SPTA1 in MAPK, and FASN in NOTCH. YWHAB downregulation was confirmed in pPR OCPM tissues and cells, serving as a marker to differentiate pNR from pCR/pPR OCPM (AUC = 0.7673). YWHAB inhibition in pCR OCPM cells enhanced stemness, as seen in sphere formation, OCSC percentages, CD133 expression, ALDH activity, and tumorigenicity. It also induced cisplatin resistance. YWHAB inhibition reduced cytoplasmic YAP retention and increased nuclear YAP, enhancing transcriptional activity. YAP5SA expression negated the effects of YWHAB depletion on stemness and resistance in pCR OCPM cells. Conclusions: In summary, this study explored the potential mechanisms underlying OCPM stemness maintenance and resistance by employing proteomic analysis, and revealed a novel YWHAB-mediated mechanism. This finding indicates that YAP would an important target for eradicating YWHAB-restricted OCSCs in OCPM.

PSYCHOLOGICAL AND PEDAGOGICAL CONDITIONS FOR THE FORMATION OF THE EMOTIONAL-VOLITIONAL SPHERE IN JUNIOR SCHOOLCHILDREN DEPRIVED OF PARENTAL PROTECTION

The scientific article examines psychological assistance, especially in the psychological aspect, in the logical and pedagogical aspect of the formation of the emotional-volitional sphere in younger schoolchildren. The article argues that the formation of the emotional-volitional sphere of a primary school student thanks to supervision is a complex system of relationships when, on the one hand, the child needs emotional support and comfort. On the other hand, the requirement to follow rules and norms limits the choice of behavior.

Hypermethylated RASAL1’s promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

BackgroundPatients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.MethodsThe expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.ResultsThe expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.ConclusionTaken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

ATG10-dependent autophagy is required for DDX10 to regulate cell proliferation, apoptosis and stemness in colorectal cancer

Colorectal cancer (CRC) remains a highly prevalent gastrointestinal neoplasm, presenting significant prevalence and lethality rate. DEAD/H box RNA helicase 10 (DDX10) has been proposed as a potential oncogene in CRC, the specific action mechanism by which DDX10 modulates the aggressive biological cellular events in CRC remains implicitly elucidated, however. During this study, DDX10 expression was detected via RT-qPCR and Western blotting. Cell proliferation was estimated via EDU staining. TUNEL staining and Western blotting appraised cell apoptosis. Cell stemness was evaluated by sphere formation assay, RT-qPCR, Western blotting as well as immunofluorescence staining. Relevant assay kit examined aldehyde dehydrogenase (ALDH) activity. Western blotting and immunofluorescence staining also detected autophagy. DDX10 was hyper-expressed in CRC cells. Down-regulation of DDX10 hampered cell proliferation, aggravated the apoptosis while eliminated the ability to form spheroid cells in CRC. In addition, DDX10 deletion improved ATG10 expression and therefore activated autophagy in CRC cells. Consequently, ATG10 depletion or treatment with autophagy inhibitor 3-Methyladenine (3-MA) partially compensated the influences of DDX10 silencing on the proliferation, apoptosis and stemness of CRC cells. Accordingly, DDX10 deficiency may aggravate autophagy mediated by ATG10 to impede cell proliferation, stemness and facilitate cell apoptosis, hence blocking the progression of CRC.

Extracellular vesicle-based formulation of doxorubicin: drug loading optimization, characterization, and cytotoxicity evaluation in tumor spheroids

Doxorubicin (DOX) is a chemotherapeutic with considerable efficacy, but its application is limited due to cardiotoxicity. Nanoparticles can improve DOX efficacy and prevent its adverse effects. Herein, DOX-loaded extracellular vesicles (DOX-EVs) were prepared using different loading methods including incubation, electroporation, and sonication in different hydration buffers to permeabilize nanostructures or desalinize DOX for improved entrapment. Different protein:drug (µg:µg) ratios of 1:10, 1:5, and 1:2, and incubation parameters were also investigated. The optimal formulation was characterized by western blotting, electron microscopy, Zetasizer, infrared spectroscopy, and release study. The cellular uptake and efficacy were investigated in MCF-7 spheroids via MTS assay, spheroid formation assay (SFA), confocal microscopy, and flow cytometry. The percentage of entrapment efficiency (EE) of formulations was improved from 1.0 ± 0.1 to 22.0 ± 1.4 using a protein:drug ratio of 1:2 and sonication in Tween 80 (0.1%w/v) containing buffer. Characterization studies verified the vesicles’ identity, spherical morphology, and controlled drug release properties. Cellular studies revealed the accumulation and cytotoxicity of DOX-EVs in the spheroids, and SFA and confocal microscopy confirmed the efficacy and cellular localization. Flow cytometry results revealed a comparable and amplified efficacy for DOX-EV formulations with different cell origins. Overall, the EV formulation of DOX can be applied as a promising alternative with potential advantages.

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1

BackgroundCancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity.MethodsCSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry.ResultsWe observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity.ConclusionsThis study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

ОСОБЕННОСТИ И ПРИНЦИПЫ РАЗВИТИЯ СЕЛЬСКИХ АГЛОМЕРАЦИЙ

Agglomeration is a form of territorial space and is one of the tools for the sustainable development of rural territories, and their stable development provides the necessary conditions for the for-mation of the social sphere, infrastructure and the creation of additional jobs. One of the factors for the effective development of rural territories is rationally organized local government, one of the activities of which is the creation of rural agglomerations. The rural agglomeration is charac-terized by a high proportion of the population engaged in agriculture. Farmers, entrepreneurs, and specialists of various fields work here, who are engaged in the production and processing of manufactured products. The territory houses warehouses, processing and packaging plants. The rural agglomeration supplies agricultural products, raw materials and food to the trading net-works of cities, thereby playing an important role in the development of the country and increasing the economic well-being of rural territories.

Low level of tumor necrosis factor α in tumor microenvironment maintains self-renewal of glioma stem cells by Vasorin-mediated glycolysis.

Self-renewal of glioma stem cells (GSCs) is responsible for glioblastoma (GBM) therapy-resistant and recurrence. Tumor necrosis factor α (TNFα) and TNF signaling pathway display an antitumor activity in preclinical models and in tumor patients. However, TNFα exhibits no significance for glioma clinical prognosis based on Glioma Genome Atlas database. This study aimed to explore whether TNFα of tumor microenvironment maintains self-renewal of GSCs and promotes worse prognosis in glioma patient. Spatial transcriptomics, immunoblotting, sphere formation assay, extreme limiting dilution, and gene expression analysis were used to determine the role of TNFα on GSC's self-renewal. Mass spectrometry, RNA-sequencing detection, bioinformatic analyses, qRT-RNA, immunofluorescence, immunohistochemistry, single cell RNA sequencing, in vitro and in vivo models were used to uncover the mechanism of TNFα-induced GSC self-renewal. Low level of TNFα displays a promoting effect on GSC self-renewal and worse glioma prognosis. Mechanistically, Vasorin (VASN) mediated TNFα-induced self-renewal by potentiating glycolysis. Lactate produced by glycolysis inhibits the TNFα secretion of tumor-associated macrophages (TAMs) and maintains TNFα in a low level. TNFα-induced GSC self-renewal mediated by VASN provides a possible explanation for the failures of endogenous TNFα effect on GBM. Combination of targeting VASN and TNFα anti-tumor effect may be an effective approach for treating GBM.

COLEC10 inhibits the stemness of hepatocellular carcinoma by suppressing the activity of β-catenin signaling.

Liver cancer stem cells (CSCs) contribute to tumor initiation, progression, and recurrence in hepatocellular carcinoma (HCC). The Wnt/β-catenin pathway plays a crucial role in liver cancer stemness, progression, metastasis, and drug resistance, but no clinically approved drugs have targeted this pathway efficiently so far. We aimed to elucidate the role of COLEC10 in HCC stemness. The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases were employed to search for the association between COLEC10 expression and HCC stemness. Colony formation, sphere formation, side population, and limiting dilution tumor initiation assays were used to identify the regulatory role of COLEC10 overexpression in the stemness of HCC cell lines. Wnt/β-catenin reporter assay and immunoprecipitation were performed to explore the underlying mechanism. COLEC10 level was negatively correlated with HCC stemness. Elevated COLEC10 led to decreased expressions of EpCAM and AFP (alpha-fetoprotein), two common markers of liver CSCs. Overexpression of COLEC10 inhibited HCC cells from forming colonies and spheres, and reduced the side population numbers in vitro, as well as the tumorigenic capacity in vivo. Mechanically, we demonstrated that overexpression of COLEC10 suppressed the activity of Wnt/β-catenin signaling by upregulating Wnt inhibitory factor WIF1 and reducing the level of cytoplasmic β-catenin. COLEC10 overexpression promoted the interaction of β-catenin with the component of destruction complex CK1α. In addition, KLHL22 (Kelch Like Family Member 22), a reported E3 ligase adaptor predicted to interact with CK1α, could facilitate COLEC10 monoubiquitination and degradation. COLEC10 inhibits HCC stemness by downregulating the Wnt/β-catenin pathway, which is a promising target for liver CSC therapy.

Cancer-associated fibroblast-derived WNT5A promotes cell proliferation, metastasis, stemness and glycolysis in gastric cancer via regulating HK2

BackgroundGastric cancer (GC) is one of the most common cancers worldwide. Tumor microenvironment plays an important role in tumor progression. This study aims to explore the role of cancer-associated fibroblasts (CAFs) in GC and the underlying mechanism.MethodsCell viability, proliferation, invasion and migration were assessed by MTT, EdU, transwell and wound healing assays, respectively. Sphere formation assay was used to evaluate cell stemness. Glucose consumption, lactate production and ATP consumption were measured to assess glycolysis. In addition, The RNA and protein expression were detected by qRT-PCR and western blot. The interaction between wingless Type MMTV Integration Site Family, Member 5 A (WNT5A) and hexokinase 2 (HK2) was verified by Co-immunoprecipitation. The xenograft model was established to explore the function of CAFs on GC tumor growth in vivo.ResultsCAFs promoted the proliferation, metastasis, stemness and glycolysis of GC cells. WNT5A was upregulated in CAFs, and CAFs enhanced WNT5A expression in GC cells. Knockdown of WNT5A in either GC cells or CAFs repressed the progression of GC cells. In addition, WNT5A promoted HK2 expression, and overexpression of HK2 reversed the effect of WNT5A knockdown in CAFs on GC cells. Besides, knockdown of WNT5A in CAFs inhibits tumor growth in vivo.ConclusionCAF-derived WNT5A facilitates the progression of GC via regulating HK2 expression.

MiR-21-5p knockdown inhibits epithelial to mesenchymal transition in A549 lung adenocarcinoma cells by upregulating RhoB.

MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.

Fibroblasts Promote Resistance to KRAS Silencing in Colorectal Cancer Cells.

Colorectal cancer (CRC) responses to KRAS-targeted inhibition have been limited due to low response rates, the mechanisms of which remain unknown. Herein, we explored the cancer-associated fibroblasts (CAFs) secretome as a mediator of resistance to KRAS silencing. CRC cell lines HCT15, HCT116, and SW480 were cultured either in recommended media or in conditioned media from a normal colon fibroblast cell line (CCD-18Co) activated with rhTGF-β1 to induce a CAF-like phenotype. The expression of membrane stem cell markers was analyzed by flow cytometry. Stem cell potential was evaluated by a sphere formation assay. RNAseq was performed in KRAS-silenced HCT116 colonospheres treated with either control media or conditioned media from CAFs. Our results demonstrated that KRAS-silencing up-regulated CD24 and down-regulated CD49f and CD104 in the three cell lines, leading to a reduction in sphere-forming efficiency. However, CAF-secreted factors restored stem cell marker expression and increased stemness. RNA sequencing showed that CAF-secreted factors up-regulated genes associated with pro-tumorigenic pathways in KRAS-silenced cells, including KRAS, TGFβ, NOTCH, WNT, MYC, cell cycle progression and exit from quiescence, epithelial-mesenchymal transition, and immune regulation. Overall, our results suggest that resistance to KRAS-targeted inhibition might derive not only from cell-intrinsic causes but also from external elements, such as fibroblast-secreted factors.

Protein phosphatase 6 promotes stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, demanding a more profound comprehension of its molecular foundations for the development of improved therapeutic strategies. This study aimed to elucidate the role of protein phosphatase 6 (PP6), a member of the type 2A protein phosphatase family, in CRC. Protein phosphatase 6 functions as a heterotrimer with a catalytic subunit (PP6c), regulatory subunits (PP6Rs; PP6R1, PP6R2, and PP6R3), and scaffold subunits (ANKRD28, ANKRD44, and ANKRD52). Elevated PP6c expression has been identified in CRC tissues compared to normal mucosa, aligning with its potential involvement in CRC pathogenesis. PP6c knockdown resulted in decreased colony-forming ability and invivo proliferation of various CRC cell lines. Transcriptome analysis revealed that PP6c knockdown resulted in altered expression of genes associated with cancer stemness. Notably, the PP6c-PP6R3 complex is a key player in regulating cancer stem cell (CSC) markers. Additionally, increased PP6c expression was observed in CSC-like cells induced by sphere formation, implicating the role of PP6c in CSC maintenance. This study highlights the role of PP6c in CRC and suggests that it is a potential therapeutic target disrupting a pathway critical for CRC progression and stem cell maintenance.

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Luteolin enhances drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in paclitaxel‑resistant esophageal squamous cell carcinoma.

Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stem‑like cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong anti‑tumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)‑resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit‑8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMT‑related proteins and stem cell markers after sphere formation. Parental cells and drug‑resistant cells were screened by high‑throughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTX‑resistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drug‑resistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTX‑resistant ESCC cells but did not show significant toxicity in vivo. Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTX‑resistant ESCC and could be a promising agent for the treatment of PTX‑resistant ESCC cancers.

E2F1 Facilitates the Proliferation and Stemness of Gastric Cancer Cells by Activating CDC25B Transcription and Modulating the MAPK Pathway.

Gastric cancer (GC) is a health problem that concerns people around the world. CDC25B is an essential cell cycle regulatory factor that is overexpressed in a variety of tumor cells. CDC25B plays a vital part in the progression and proliferation of malignant tumors. However, it is not yet clear that how CDC25B affects the stemness of GC cells. The study used bioinformatics to detect the expression of E2F1 and CDC25B in GC tissues and their correlation, as well as pathways enriched by CDC25B. We detected the expression of E2F1 and CDC25B in GC cell lines using quantitative reverse transcription polymerase chain reaction and tested the combination relationship between E2F1 and CDC25B using chromatin immunoprecipitation (ChIP) and dual-luciferase assays. We measured cell viability using CCK-8 assay, evaluated sphere-forming efficiency using sphere formation assay, and determined cell proliferation ability using colony formation assay. We also analyzed the expression of stemness markers and MAPK pathway-related proteins using western blot. In GC tissues and cells, CDC25B was upregulated. Silencing CDC25B could affect the MAPK pathway, thereby repressing the proliferation and stemness of GC cells. As predicted by bioinformatics, CDC25B had an upstream transcription factor, E2F1, which also had a high expression level in GC. Dual-luciferase and ChIP assays confirmed the combination relationship between the two. Rescue experiments uncovered that overexpression of CDC25B could reverse the impact induced by E2F1 knockdown on proliferation and stemness of cells. In conclusion, E2F1 could activate CDC25B transcription to regulate the MAPK pathway and enhance the proliferation and stemness of GC cells. We revealed a potential regulatory pathway of stemness of GC cells that was mediated by CDC25B, providing new ideas for improving and innovating GC treatment.

Jmjd2c maintains the ALDHbri+ cancer stemness with transcription factor SOX2 in lung squamous cell carcinoma

ABSTRACT Lung squamous cell carcinoma (LSCC) is a deadly cancer in the world. Histone demethylase Jmjd2c is a key epigenetic regulator in various tumors, while the molecular mechanism underlying Jmjd2c regulatory in LSCC is still unclear. We used the aldehyde dehydrogenasebright (ALDHbri+) subtype as a research model for cancer stem cells (CSCs) in LSCC and detected the sphere formation ability and the proportion of ALDHbri+ CSCs with Jmjd2c interference and caffeic acid (CA) treatment. Additionally, we carried out bioinformatic analysis on the expression file of Jmjd2c RNAi mice and performed western blotting, qRT-PCR, Co-IP and GST pull-down assays to confirm the bioinformatic findings. Moreover, we generated Jmjd2c-silenced and Jmjd2c-SOX2-silenced ALDHbri+ tumor-bearing BALB/c nude mice to detect the effects on tumor progression. The results showed that Jmjd2c downregulation inhibited the sphere formation and the proportion of ALDHbri+ CSCs. The SOX2 decreased expression significantly in Jmjd2c RNAi mice, and they were positively co-expressed according to the bioinformatic analysis. In addition, SOX2 expression decreased in Jmjd2c shRNA ALDHbri+ CSCs, Jmjd2c and SOX2 proteins interacted with each other. Furthermore, Jmjd2c interference revealed significant blocking effect, and Jmjd2c-SOX2 interference contributed even stronger inhibition on ALDHbri+ tumor progression. The Jmjd2c and SOX2 levels were closely related to the development and prognosis of LSCC patients. This study indicated that Jmjd2c played key roles on maintaining ALDHbri+ CSC activity in LSCC by interacting with transcription factor SOX2. Jmjd2c might be a novel molecule for therapeutic targets and biomarkers in the diagnosis and clinical treatment of lung cancer.

FERMT1 suppression induces anti-tumor effects and reduces stemness in glioma cancer cells

ObjectiveGlioma is a leading cause of mortality worldwide, its recurrence poses a major challenge in achieving effective treatment outcomes. Cancer stem cells (CSCs) have emerged as key contributors to tumor relapse and chemotherapy resistance, making them attractive targets for glioma cancer therapy. This study investigated the potential of FERMT1 as a prognostic biomarker and its role in regulating stemness through cell cycle in glioma.MethodsUsing data from TCGA-GBM, GSE4290, GSE50161 and GSE147352 for analysis of FERMT1 expression in glioma tissues. Then, the effects of FERMT1 knockdown on cell cycle, proliferation, sphere formation ability, invasion and migration were investigated. The influences of FERMT1 on expression of glycolysis-related proteins and levels of ATP, glucose, lactate and G6PDH were also explored. Furthermore, the effects of FERMT1 knockdown on cellular metabolism were evidenced.ResultsSignificant upregulation of FERMT1 in glioma tissues was observed. Silencing FERMT1 not only affected the cell cycle but also led to a notable reduction in proliferation, invasion and migration. The expression of glycolysis-associated proteins including GLUT1, GLUT3, GLUT4, and SCO2 were reduced by FERMT1 knockdown, resulted in increased ATP and glucose as well as decreased lactic acid and G6PDH levels. FERMT1 knockdown also inhibited cellular metabolism. Moreover, FERMT1 knockdown significantly reduced sphere diameter, along with inhibiting the expression of transcription factors associated with stemness in glioma cells.ConclusionThese findings demonstrated that FERMT1 could be an ideal target for the advancement of innovative strategies against glioma treatment via modulating cellular process involved in stemness regulation and metabolism.

Co‑expression of SLC20A1 and ALDH1A3 is associated with poor prognosis, and SLC20A1 is required for the survival of ALDH1‑positive pancreatic cancer stem cells.

Solute carrier family 20 member 1 (SLC20A1) is a sodium/inorganic phosphate symporter, which has been identified as a prognostic marker in several types of cancer, including pancreatic cancer. However, to the best of our knowledge, the association between SLC20A1 expression and cancer stem cell (CSC) markers, such as aldehyde dehydrogenase 1 (ALDH1), in pancreatic ductal adenocarcinoma (PDAC), and the role of SLC20A1 in PDAC CSCs remains unclear. In the present study, a genomic dataset of primary pancreatic cancer (The Cancer Genome Atlas, Pan-Cancer Atlas) was downloaded and analyzed. Kaplan-Meier analysis and multivariate Cox regression analysis were performed to evaluate the overall survival, disease-specific survival (DSS), disease-free interval (DFI) and progression-free interval (PFI). Subsequently, SLC20A1 small interfering RNA (siRNA) knockdown (KD) was induced in the PANC-1 and MIA-PaCa-2 PDAC cell lines, and in sorted high ALDH1 activity (ALDH1high) cells, after which, cell viability, in vitro tumor sphere formation, cell death and caspase-3 activity were examined. The results revealed that patients with high expression of SLC20A1 (SLC20A1 high) at tumor stage I had a poor prognosis compared with patients with low expression of SLC20A1 (SLC20A1 low) in terms of DSS, DFI and PFI. In addition, patients with high expression of SLC20A1 and ALDH1A3 (SLC20A1 high ALDH1A3 high) exhibited poorer clinical outcomes than patients with high expression of SLC20A1 and low expression of ALDH1A3 (SLC20A1 high ALDH1A3 low), low expression of SLC20A1 and high expression of ALDH1A3 (SLC20A1 low ALDH1A3 high) and SLC20A1 low ALDH1A3 low. SLC20A1 siRNA KD in ALDH1high cells isolated from PANC-1 and MIA-PaCa-2 cell lines resulted in suppression of in vitro tumorsphere formation, and enhancement of cell death and caspase-3 activity. These results suggested that SLC20A1 was involved in cell survival via the suppression of caspase-3-dependent apoptosis, and contributed to cancer progression and poor clinical outcomes in PDAC. In conclusion, SLC20A1 may be used as a prognostic marker and novel therapeutic target of ALDH1-positive pancreatic CSCs.

Evaluation of Boric Acid Treatment on microRNA-127-5p and Metastasis Genes Orchestration of Breast Cancer Stem Cells.

Coregulation of microRNAs (miRNAs) and cancer stem cells (CSCs) is very important in carcinogenesis. miR-127-5p is known to be downregulated in breast cancer. In this study, we aimed to investigate how boric acid (BA), known for its previously unstudied anti-cancer properties, would affect the expression of miR127-5p and genes responsible for breast cancer stem cells (BC-SCs) metastasis. BC-SCs were isolated from human breast cancer cells (MCF-7) by immunomagnetic cell separation and characterized with flow cytometry and sphere formation. The viability of BC-SCs and the determination of its IC50 value in response to boric acid (BA) were assessed via the MTT assay. Boric acid exhibited dose- and time-dependent inhibition of cell viability in cells. The IC50 doses of boric acid in MCF-7 cells and BC-SCs were 45.69mM and 41.27mM, respectively. The impact of BA on the expression of metastatic genes and miR127-5p was elucidated through RT-qPCR analysis. While the expression of the COL1A1 (p < 0.05) and VIM (p < 0.01) was downregulated, the expression of the miR-127-5p, ZEB1 (p < 0.01), CDH1 (p < 0.05), ITGB1 (p < 0.05), ITGA5 (p < 0.05), LAMA5 (p < 0.01), and SNAIL (p < 0.05), was up-regulated in dose-treated BC-SCs (p < 0.001) to the RT-qPCR results. Our findings suggest that boric acid could induce miR-127-5p expression. However, it cannot be said that it improves the metastasis properties of breast cancer stem cells.