Xenograft Experiments Research Articles

Radiotherapy-resistant prostate cancer cells escape immune checkpoint blockade through the senescence-related ataxia telangiectasia and Rad3-related protein.

The majority of patients with prostate cancer (PCa) exhibit intrinsic resistance to immune checkpoint blockade (ICB) following radiotherapy (RT). This resistance is generally attributed to the limited antigen presentation of heterogeneous cells within tumors. Here, we aimed to isolate and characterize these diverse subgroups of tumor post-RT to understand the molecular mechanisms of their resistance to ICB. Single-cell RNA-sequencing (scRNA-seq) was used to profile senescent cancer cell clusters induced by RT in LNCaP cells. The expression and phosphorylation levels of ataxia telangiectasia and Rad3-related protein (ATR) were assessed by immunohistochemistry in clinical samples from patients with or without RT. Co-immunoprecipitation, mutagenesis, and Western blotting were used to measure the interactions between proteins. Xenograft experiments were performed to assess the tumor immune response in the mice. We identified a subset of PCa cells that exhibited resistance to RT, characterized by a reduced antigen presentation capability, which enhanced their ability to evade immune detection and resist cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockade. scRNA-seq revealed that the senescent state was a transient phase of PCa cells post-RT, particularly in CTLA-4 blockade treatment-resistant cells. This state was marked by increased cytosolic ATR level. Cytosolic ATR phosphorylated CD86 in its cytosolic domain and enhanced the interaction between CD86 and its E3 ligase MARCH1 through electrostatic attraction. Depletion or inhibition of Atr increased the sensitivity to immune attack and improved responses to anti-Ctla-4 antibody treatment in a mouse model. Our findings indicate that the activation of cytosolic ATR, which is associated with cellular senescence, impedes the effectiveness of combined RT and ICB treatments. This discovery may provide valuable insights for improving the efficacy of combined RT and ICB therapies in PCa.

Vitamin A and D3 combinations reduce breast cancer tumor load in a postmenopausal MCF-7 xenograft mouse model in a dose- and time- dependent manner

Introduction: Earlier, we documented that a combination of vitamins A and D3 synergistically inhibited the growth of MCF-7, T48:A18 and SKBR3 breast cancer cells with the best activity seen in the ER+ cell line MCF-7. Transcriptomic analysis of treated MCF-7 cells also showed that the combination significantly upregulated the apoptosis and unfolded protein response canonical pathways, and reduced estrogen signaling. Objective: This study aimed to explore the impact of increasing vitamin A and D3 dose combinations over time in a postmenopausal model of breast cancer using ovariectomized athymic female mice bearing MCF-7 xenografts and further analyze mechanisms of action in MCF-7 cells using RNA-seq analysis. Methods: MCF-7 breast cancer cells were grown in culture for the xenograft experiments. Athymic female mice were injected with MCF-7 cells (1 x 10^6 in 100 µl of 50% Matrigel mixed with sterile PBS) via subcutaneous injection. Once the tumors reached an average volume of 100 mm³, the mice were randomly divided into four groups and treated with different vitamin A and D3 combinations. Tumor sizes and mouse body weights were monitored on a biweekly basis. After the treatment period, the mice were euthanized, and the tumors were surgically removed and measured. RNA-seq data from the treated MCF-7 cells were then further evaluated using IPA. Results: As compared with controls, treatment with vitamin A (25,000 IU) and vitamin D (10,000 IU) led to a significant reduction in tumor volume >70%, (p < 0.05-0.01) in OVX athymic mice with MCF-7 xenografts as determined by a two-tailed Student T test. Over the treatment period, the tumor volume in mice treated with vitamin A (10,000 IU) and vitamin D (5,000 IU) or vitamin A (25,000 IU) and vitamin D (5,000 IU) also trended downward and was statistically significant using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (p<0.05 and p<0.0001, respectively) but was not significant using a two-tailed Student T test. In cultured MCF-7 cells, Ingenuity Pathway Analysis of mRNA-seq data showed that the vitamin A and D combination significantly altered the expression of 101 genes out of 864 in the molecular mechanisms in cancer canonical pathway, downregulating gene expression in the integrin/P13K/Akt/mTOR pathway. Conclusions: The findings showed that the combination of vitamins A and D3 effectively reduced tumor burden in a postmenopausal MCF-7 xenograft mouse model, with effects that were both dose-dependent and time-dependent. The combination also significantly altered the expression of genes in the molecular mechanisms of cancer canonical pathway in cultured MCF-7 cells. These preclinical data support the use of vitamins A and D3 in the management of estrogen-dependent breast cancers, with the caveat that higher doses and longer treatment periods may be needed to observe anti-tumor effects. Keywords: Apoptosis, autophagy, breast cancer, cell cycle, integrin, postmenopausal, P13K, tumor load, xenograft

Regulation of AUF1 alternative splicing by hnRNPA1 and SRSF2 modulate the sensitivity of ovarian cancer cells to cisplatin.

Clarification of cisplatin resistance may provide new targets for therapy in cisplatin resistant ovarian cancer. The current study aims to explore involvement of isoforms of AU-rich element RNA-binding protein 1 (AUF1) in cisplatin resistance in ovarian cancer. The cancer stem cell-like features were analyzed using colony formation assay, tumor sphere formation assay and nude mouse xenograft experiments. AUF1 isoforms expression was analyzed using immunoblotting, qRT-PCR, and immunohistochemistry. RIP and Biotin pulldown was used to analyze the interaction of SRSF2 and hnRNPA1 with AUF1 transcript. Transcriptome regulated by AUF1 isoforms was analyzed by RNA-seq. The current study demonstrated differential expression of AUF1 isoforms in cisplatin sensitive and resistant ovarian cancer tissues and cells. P37 isoform promoted proliferation, while p45 isoform enhanced responsiveness of ovarian cancer cells to cisplatin. the clonal formation capacity of the cells, and the restoration of p45 expression reduced the capacity with cisplatin treatment. The competitive binding of phosphorylated hnRNPA1 and O-GlcNAc-modified SRSF2 on AUF1 exon 2 and exon 7 regulated the alternative splicing of AUF1. The competitive binding of phosphorylated hnRNPA1 and O-GlcNAc modified SRSF2 on exon 2 and exon 7 regulated the alternative splicing of AUF1 and subsequent isoform expression. P37 isoform played a "cancer promoter" role, p42 and p45, especially p45 played a "cancer suppressor" role in ovarian cancer. This study provides a new target for exploring the drug resistance mechanism of ovarian cancer.

Shikonin Inhibits Oral Cancer Progression Through Suppression of Metastasis and Angiogenesis and Induction of Autophagy and Apoptosis

Background: Shikonin, a compound extracted from Lithospermum erythrorhizon, has demonstrated therapeutic effects on cancer; however, its effects on oral cancer remain unclear. Objectives: This study aimed to explore the therapeutic value of shikonin for the treatment of oral cancer. Methods: MTT, colony formation, and Transwell assays were employed to evaluate the inhibitory effects of shikonin on the proliferation and migration abilities of human oral cancer cell lines (SCC-4 and SAS). Apoptosis and cell viability were assessed using the TdT-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. To investigate the potential anticancer mechanisms of shikonin, RNA sequencing, quantitative PCR, and western blotting were performed to analyze changes in the expression levels of oral cancer cell-related genes and proteins (matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), VEGF-A, VEGF-C, Beclin-1, autophagy-related genes (ATG5), and light chain-3 (LC-3)). Additionally, animal xenograft experiments were conducted to examine the in vivo antitumor effects of shikonin. Results: The findings revealed that the external application of shikonin specifically targeted oral cancer cells without affecting normal cells and led to a dose-dependent inhibition of their growth. Even at non-lethal doses, shikonin effectively suppressed the production of metalloproteinases, thereby inhibiting cancer cell migration and wound healing. Furthermore, shikonin treatment reduced levels of tumor progression factors, such as vascular endothelial growth factor (VEGF)-A and VEGF-C, which are released during the early stages of cancer cell angiogenesis and lymphangiogenesis. Meanwhile, higher doses of shikonin induced cell autophagy and activated proteins such as ATG-5, LC-3B, and Beclin-1. At lethal doses, shikonin further decreased mitochondrial membrane potential, released calcium ions, and triggered apoptotic pathways. However, the administration of a calcium ion chelator (BAPTA-AM) inhibited shikonin-induced apoptosis. Conclusions: These results demonstrate that shikonin induces autophagy and activates apoptotic pathways in oral cancer cells. Shikonin treatment significantly inhibited oral cancer growth and induced apoptosis in a rat model. In conclusion, shikonin effectively inhibited oral cancer cell growth, metastasis, and the expression of tumor progression-related proteins. Given the ease of drug delivery to the affected area in oral cancer, shikonin holds substantial potential for future applications that may improve patient recovery and enhance cure rates.

PDE4D drives rewiring of the MAPK pathway in BRAF-mutated melanoma resistant to MAPK inhibitors

BackgroundPhosphodiesterase type 4D (PDE4D) breaks down cyclic AMP (cAMP) reducing the signaling of this intracellular second messenger which plays a major role in melanocyte pathophysiology. In advanced melanoma, expression of PDE4D is increased, plays a role in tumor invasion and is negatively associated with survival. In the current work, we investigated the role of PDE4D in the resistance of BRAF-mutated melanoma to mitogen-activated protein kinase (MAPK) pathway-targeted therapy.MethodsEstablished human melanoma cell line sensitive and resistant to BRAF and MEK inhibitors and tumor tissues from melanoma patients were used in this study. Immunoblotting was used to analyze protein expression and quantitative reverse transcription-PCR was used to analyze mRNA expression. DNA methylation analysis was evaluated via bisulfite treatment followed by quantitative PCR. Cell viability was measured by clonogenic assays or spheroid cultures. Cell xenograft experiments in immunodeficient mice were used to validate the results in vivo.ResultsAnalysis of baseline tumors from patients with BRAFV600E-mutated melanoma treated with MAPK inhibitors showed that higher PDE4D expression in situ predicted worse survival in patients. Furthermore, acquired resistance to BRAF and MEK inhibitors was associated with overexpression of PDE4D in situ and ex vivo. The overexpression of the PDE4D5 isoform in melanoma cells resistant to targeted therapies was explained by demethylation or deletion of a CpG island located upstream of the PDE4D5 promoter. We further showed that PDE4D overexpression allowed RAF1 activation, promoting a switch from BRAF to RAF1 isoform in BRAF-mutated melanoma, favoring resistance to BRAF and MEK inhibitors. As a result, pharmacological inhibition of PDE4 activity impeded the proliferation of resistant cells ex vivo and in vivo. The anti-tumorigenic activity of PDE4 inhibitor was achieved via inhibition of the Hippo pathway which plays an important role in resistance to targeted therapies.ConclusionsIn summary, our research showed that PDE4D drives rewiring of the MAPK pathway in BRAF-mutated melanoma resistant to MAPK inhibitors and suggests that PDE4 inhibition is a novel therapeutic option for treatment of BRAF-mutated melanoma patients.

Identification of the Oncogenic Role of the Circ_0001326/miR-577/VDAC1 Cascade in Prostate Cancer.

Prostate cancer (PCa) is one of the leading causes of cancer death among men worldwide. Circular RNAs (circRNAs) have been implicated in the pathogenesis of PCa. However, the precise action of circ_0001326 in PCa malignant progression is still unknown. The levels of circ_0001326, miR-577 and voltage dependent anion channel 1 (VDAC1) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation, colony formation, apoptosis, migration and invasion were evaluated by the Cell Counting Kit-8 (CCK-8), EdU staining, colony formation, flow cytometry, wound-healing and transwell assays, respectively. Targeted relationships among circ_0001326, miR-577 and VDAC1 were confirmed by dual-luciferase reporter assays. Xenograft experiments were performed to detect the role of circ_0001326 in tumor growth. Our data revealed that circ_0001326 was overexpressed in PCa tissues and cells. Circ_0001326 depletion repressed PCa cell proliferation, migration, and invasion and enhanced apoptosis in vitro, as well as hampered tumor growth in vivo. Mechanistically, circ_0001326 directly targeted miR-577, and VDAC1 was directly targeted and suppressed by miR-577. Moreover, the effects of circ_0001326 knockdown on PCa cell functional behaviors were mediated by miR-577. VDAC1 silencing phenocopied miR-577 overexpression in regulating PCa cell functional behaviors in vitro. Furthermore, circ_0001326 regulated VDAC1 expression through sponging miR-577. Our findings showed that circ_0001326 regulated PCa cell functional behaviors at least partly through targeting the miR-577/VDAC1 axis.

SSX2IP promotes cell proliferation and migration in breast cancer by regulating FANCI.

Synovial sarcoma X breakpoint 2 interacting protein (SSX2IP) is expressed in various normal tissues and participates in the progression of human cancers. Nevertheless, the specific functions and underlying molecular mechanisms of SSX2IP in cancer, particularly in breast cancer, remain poorly understood. In this study, we aimed to explore the functional role of SSX2IP in breast cancer. Immunohistochemical staining, quantitative real-time PCR, and western blotting blot analysis were used to assess genes expression levels. By manipulating SSX2IP expression levels and conducting functional assays including Celigo cell counting assay or CCKCCK-8-8 assay, flow cytometry, wound healing assay, and Transwell assay, we explored the impact of SSX2IP on the malignant phenotype of breast cancer cells. Additionally, the in vivo tumor-suppressive ability of SSX2IP was investigated by tumor xenograft experiment. Our results revealed an upregulation of SSX2IP in the breast cancer. Functional assays demonstrated that SSX2IP knockdown inhibited cell proliferation and migration, induced apoptosis in vitro, as well as suppressed the tumor growth in vivo. Conversely, SSX2IP overexpression contributed to the malignant phenotype of breast cancer cells. Co-expression analysis showed that FA Complementation Group I (FANCI) was co-expressed with SSX2IP. Additionally, SSX2IP positively regulated FANCI expression and its interaction was verified by Co-IP.Co-IP. Furthermore, FANCI overexpression partially reversed the effects of SSX2IP knockdown on cell proliferation and metastasis. In summary, our findings revealed that SSX2IP contributes to the progression of breast cancer by regulating FANCI, hinting at its potential as a novel biomarker and therapeutic target for the treatment of breast cancer.

Activation of Hedgehog pathway by circEEF2/miR-625-5p/TRPM2 axis promotes prostate cancer cell proliferation through mitochondrial stress.

The purpose of this study was to identify the role played by circEEF2 (has-circ-0048559) in prostate cancer (PCa) development and to determine the potential mechanism involved. circEEF2, miR-625-5p, and the transient receptor potential M2 channel protein (TRPM2) were determined using RT-qPCR in PCa. Cell proliferation was determined by CCK-8 assay and colony formation assay, whereas migration and invasion were assessed by Transwell assay, and apoptosis was evaluated by flow cytometry after annexin V-FITC and propidium iodide staining. The interactions between circEEF2 and miRNAs were investigated through the Circular RNA Interactome database, and the downstream targets of miR-625-5p were forecasted using TargetScan. The interaction was confirmed using both the dual luciferase reporter gene assay and RNA pull-down assay. TRPM2, Hedgehog signaling pathway proteins (GLI1 and GLI2), ubiquinone oxidase subunit B8, and cytochrome C oxidase subunit IV (COX4) were analyzed by protein blotting. JC-1 fluorescence detection was applied for mitochondrial membrane potential changes, fluorescent probe assay for intracellular ROS levels, and immunofluorescence staining for γ-H2AX expression. The role of circEEF2 in PCa tumor growth was tested by xenograft experiments. CircEEF2 expression was upregulated in PCa (p<0.05). Cells of PCa were inhibited in proliferation, migration, invasion, and enhanced in apoptosis by depleting circEEF2 (p<0.05). circEEF2 directly targeted adsorbed miR-625-5p. TRPM2 bound to miR-625-5p. Upregulating TRPM2 likewise reversed the therapeutic effect of depleting circEEF2 on cancer development in PCa cells. circEEF2 activated the Hedgehog pathway through the miR-625-5p/TRPM2 axis, promotes mitochondrial stress, and promotes PCa development in vivo. circEEF2 upregulates mitochondrial stress to promote PCa by activating the Hedgehog pathway through the miR-625-5p/TRPM2 axis.

Spatial and Single Cell Analyses Reveal Heterogeneity of DNAM-1 Receptor-Ligand Interactions that Instructs Intratumoral γδT-Cell Activity.

The dynamic interplay between tumor cells and γδT cells within the tumor microenvironment (TME) significantly influences disease progression and immunotherapy outcome. Here, we delved into the modulation of γδT-cell activation by tumor cell ligands CD112 and CD155, which interact with the activating receptor DNAM-1 on γδT cells. Spatial and single cell RNA sequencing (scRNA-seq), as well as spatial metabolome analysis, from neuroblastoma (NB) revealed that the expression levels and localization of CD112 and CD155 varied across and within tumors, correlating with differentiation status, metabolic pathways, and ultimately disease prognosis and patient survival. Both in vivo tumor xenograft experiments and in vitro co-culture experiments demonstrated that a high CD112/CD155 expression ratio in tumors enhanced γδT-cell-mediated cytotoxicity, while a low-ratio fostered tumor resistance. Mechanistically, CD112 sustained DNAM-1-mediated γδT-cell activation, whereas CD155 downregulated DNAM-1 expression via TRIM21-mediated ubiquitin proteasomal degradation. By interacting with tumor cells differentially expressing CD112 and CD155, intratumoral γδT cells exhibited varying degrees of activation and DNAM-1 expression, representing three major functional subsets. This study underscores the complexity of tumor-immune crosstalk, offering insights into how tumor heterogeneity shapes the immune landscape.

The methyltransferase KIAA1429 potentiates cervical cancer tumorigenesis via modulating LARP1 mRNA m6A modification and stability.

Cervical cancer (CC) is one of the most common gynecological malignancies in the world and poses a great threat to public health. There is inadequate knowledge of the molecular mechanisms underlying CC. This study aimed to explore the prognostic value of KIAA1429 (VIRMA, vir-Like m6A methyltransferase associated) in patients with CC and analyze its molecular mechanisms. The level of KIAA1429 in tumor specimens was tested using RT-qPCR and western blotting. Cellular biological processes were assessed using CCK-8 and Transwell assays. Xenograft experiments were used to verify the function of KIAA1429 in CC in vivo. The results manifested that KIAA1429 expression was enhanced in CC. Downregulation of KIAA1429 hindered the viability, migration, and invasion of CC cells. Moreover, LARP1 (La-related protein 1) was uncovered to be positively modulated by KIAA1429. Further, the anti-tumor impacts of KIAA1429 depletion on the phenotype of CC cells were counteracted by LARP1 amplification. Additionally, KIAA1429 deficiency suppressed the stability of LARP1 through methylating LARP1. Collectively, KIAA1429 can boost the tumorigenesis of CC via modifying LARP1 through m6A methylation to promote its stability. This work highlights the promoting effects of KIAA1429 on CC development and presents new targets for its treatment.

ZNF281 Facilitates the Invasion of Cervical Cancer Cell Both In Vivo and In Vitro †.

Background: Cervical cancer is the fourth most common cancer among women worldwide. The zinc finger transcription factor 281 (ZNF281)/ZBP-99 protein specifically binds to GC-rich DNA sequences and regulates gene expression, and it has been shown to promote tumor progression. In this study, we aim to investigate the function and molecular mechanism of ZNF281 in uterine cervical carcinoma. Methods: We conducted immunohistochemistry and Western blot assays to determine the expression of ZNF281 in eight human cervical cancer tissues. And, xenograft experiments involving the injection of HeLa cells into nude mice was used to determine the function of ZNF281 on proliferation. Transwell assays were used to detect the migration and invasion of HeLa cells after indicated that ZNF281 overexpression. Results: Our results indicated that ZNF281 protein levels were higher in cervical cancer tissues compared to normal cervical tissues. Additionally, ZNF281 was expressed in human cervical carcinoma cell lines, including HeLa, SiHa, C-33 A, CaSki, and HT-3, and is localized in both the cell nucleus and cytoplasm. ZNF281 overexpression did not influence HeLa cell proliferation or tumor size in situ. Moreover, nude mice injected with ZNF281-overexpressing cell lines developed more tumor lesions in the lungs compared to those injected with control cell lines. Conclusions: These findings suggest that ZNF281 is associated with tumor metastasis without affecting cell proliferation, both in vivo and in vitro.

IL-21/IL-21R signaling renders acute myeloid leukemia stem cells more susceptible to cytarabine treatment and CAR T cell therapy

Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4+ Tcell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling, resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML, serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR Tcell treatment on LSCs invitro. Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore, promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML.

Repurposed genipin targeting UCP2 exhibits antitumor activity through inducing ferroptosis in glioblastoma.

Uncoupling protein-2 (UCP2) controls the antioxidant response and redox homeostasis in cancer and is considered a potent molecular target for cancer treatment. However, the specific mechanism of UCP2 inhibition and its role in glioblastoma (GBM) have not yet been elucidated. Here, we attempt to identify a UCP2 inhibitor and study the underlying molecular mechanism in GBM. Bioinformatics analysis and immunohistochemistry are used to validate the high expression of UCP2 in GBM and its prognostic significance. Drug intervention and tumor xenograft experiments are conducted to determine the inhibitory effect of genipin, a UCP2 inhibitor, on UCP2. The mitochondrial membrane potential and key ferroptosis genes are examined to determine the occurrence of ferroptosis. High expression of UCP2 in GBM is associated with poor prognosis, and inhibiting UCP2 can alleviate the malignant behavior of GBM tumors. Genipin can downregulate the expression of GPX4 and upregulate the expression of ACSL4 by inhibiting UCP2, leading to ferroptosis and alleviating the malignant behavior of tumors. In summary, UCP2 is a potential therapeutic target for GBM. Genipin, which targets UCP2, effectively inhibits GBM development by inducing ferroptosis in vivo and in vitro. These findings indicate that genipin treatment based on UCP2 targeting has potential therapeutic applications with a clinical perspective for the treatment of GBM patients.

Enhancing cisplatin efficacy in hepatocellular carcinoma with selenocystine: The suppression of DNA repair and inhibition of proliferation in hepatoma cells

Cisplatin (cDDP) is a crucial chemotherapy drug for treating various cancers, including hepatocellular carcinoma (HCC). However, its effectiveness is often hindered by side effects and drug resistance. Selenocystine (SeC) demonstrates potential as an anticancer agent, particularly by inhibiting DNA repair mechanisms. This study explored the synergistic potential of SeC combined with cDDP for treating HCC. Our results show that SeC pretreatment followed by cDDP significantly suppresses HCC cell proliferation more effectively than either treatment alone, with minimal toxicity to normal liver cells. The combination induces significant DNA damage by inhibiting homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Xenograft experiments confirmed that the combined therapy strongly inhibits tumor growth. SeC boost the effectiveness of cDDP by amplifying DNA damage and inhibiting DNA repair, presenting a promising approach to enhancing liver cancer treatment.

ALKBH4 functions as a hypoxia-responsive tumor suppressor and inhibits metastasis and tumorigenesis.

The human AlkB homolog (ALKBH) dioxygenase superfamily plays a crucial role in gene regulation and is implicated in cancer progression. Under hypoxic conditions, hypoxia-inducible factors (HIFs) dynamically regulate methylation by controlling various dioxygenases, thereby modulating gene expression. However, the role of hypoxia-responsive AlkB dioxygenase remains unclear. The molecular events were examined using real-time PCR and Western blot analysis. Tumor cell aggressiveness was evaluated through migration, invasion, MTT, trypan blue exclusion, and colony formation assays. In vivo metastatic models and xenograft experiments were conducted to evaluate tumor progression. Here, we examined the expression of the ALKBH superfamily under hypoxic conditions and found that ALKBH4 expression was negatively regulated by hypoxia. Knockdown of ALKBH4 enhanced the epithelial-mesenchymal transition (EMT), cell migration, invasion, and growth in vitro. The silencing of ALKBH4 enhanced metastatic ability and tumor growth in vivo. Conversely, overexpression of ALLKBH4 reversed these observations. Furthermore, overexpression of ALKBH4 significantly reversed hypoxia/HIF-1α-induced EMT, cell migration, invasion, tumor metastasis, and tumorigenicity. Notably, high expression of ALKBH4 was associated with better outcomes in head and neck cancer and breast cancer patients. Enrichment analysis also revealed that ALKBH4 was negatively enriched in hypoxia-related pathways. Clinically, a negative correlation between ALKBH4 and HIF-1α protein expression has been observed in tissues from both head and neck cancers and breast cancers. These findings collectively suggest that ALKBH4 acts as a tumor suppressor and holds therapeutic potential for hypoxic tumors.

Ubiquitin-specific protease 10 determines colorectal cancer outcome by modulating epidermal growth factor signaling via inositol polyphosphate-4-phosphatase type IIB

Although there have been advances in understanding colorectal cancer (CRC) pathogenesis, significant gaps still exist, highlighting the need for deeper insights. Dysregulated protein homeostasis, including perturbations in the epidermal growth factor receptor (EGFR) pathway, remains a focal point in CRC pathogenesis. Within this context, the roles of ubiquitin ligases and deubiquitinases have attracted attention, but exploration of their precise contributions is still in its early stages. To address this gap, we investigated the involvement of the deubiquitinase USP10 in CRC. Our in vitro and in vivo study reveals a new paradigm in CRC biology and unravels a novel mechanistic axis, demonstrating for the first time the involvement of inositol polyphosphate 4-phosphatase type II B (INPP4B) in USP10-mediated CRC modulation. Specifically, our study demonstrates that the loss of USP10 results in reduced sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and osimertinib. This is accompanied by a decrease in the activation of the AKT1/PKB pathway upon EGF stimulation, which is mediated by INPP4B. Importantly, in vivo xenograft experiments validate these findings and highlight the crucial role of USP10, particularly in conjunction with INPP4B, in driving CRC progression. The findings enhance our understanding of CRC pathobiology and reveal a new regulatory axis involving USP10 and INPP4B in CRC progression. This unique insight identifies USP10 and INPP4B as potential therapeutic targets in CRC.

Cold plasma irradiation inhibits skin cancer via ferroptosis

Cold atmospheric plasma (CAP) has been extensively utilized in medical treatment, particularly in cancer therapy. However, the underlying mechanism of CAP in skin cancer treatment remains elusive. In this study, we established a skin cancer model using CAP treatmentin vitro. Also, we established the Xenograft experiment modelin vivo. The results demonstrated that treatment with CAP induced ferroptosis, resulting in a significant reduction in the viability, migration, and invasive capacities of A431 squamous cell carcinoma, a type of skin cancer. Mechanistically, the significant production of reactive oxygen species (ROS) by CAP induces DNA damage, which then activates Ataxia-telangiectasia mutated (ATM) and p53 through acetylation, while simultaneously suppressing the expression of Solute Carrier Family 7 Member 11 (SLC7A11). Consequently, this cascade led to the down-regulation of intracellular Glutathione peroxidase 4 (GPX4), ultimately resulting in ferroptosis. CAP exhibits a favorable impact on skin cancer treatment, suggesting its potential medical application in skin cancer therapy.

PD1CD28 chimeric molecule enhances EGFRvⅢ specific CAR-T cells in xenograft experiments in mouse models.

Over the years, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, this efficacy has not been replicated in the context of glioblastoma (GBM). In this study, a PD1CD28 chimeric molecule was introduced into EGFRvⅢ-directed CAR-T cells, generating EGFRvⅢ-P2A-PD1CD28 CAR-T cells. Notably, this modification significantly increased IL-2 secretion and enhanced antigen-dependent activation of CAR-T cells, especially when programmed cell death ligand 1 (PD-L1) was present in vitro. In addition, the in vivo xenograft experiments revealed that the PD1CD28 chimeric molecule played a pivotal role in reducing recurrence rates, effectively controlling recurrent tumor volume, and ultimately prolonging the survival of mice. Collectively, these findings suggest that EGFRvⅢ-directed CAR-T cells co-expressing the PD1CD28 chimeric molecule have the potential to significantly enhance the treatment efficacy against GBM.

The promotion of cell proliferation and invasion in cutaneous squamous cell carcinomas after ARNT downregulation is associated with CXCL3

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor associated with adaptive responses to cellular stress. Its role in cutaneous squamous cell carcinoma (cSCC) remains poorly understood. The aim of this study was to investigate the role of ARNT in cSCC. Immunohistochemistry revealed downregulation of ARNT in cSCC, precancerous lesions (actinic keratosis), and cells. Knockdown of ARNT in A431 and SCL-1 cells significantly enhanced cell growth and metastasis. Microarray analysis and Ingenuity Pathway Analysis confirmed that loss of ARNT in A431 cells was highly correlated with cell growth and movement and upregulated CXCL3 expression. Cellular and xenograft experiments further confirmed that ARNT regulates cSCC proliferation and invasiveness in a CXCL3-dependent manner. ARNT may regulate CXCL3 expression through ROS-STAT3 pathway. In conclusion, this study demonstrates that ARNT plays a critical role in the development of cSCC and significantly affects the proliferation and metastatic ability of cSCC cells. It has the potential to serve as an ideal treatment target for cSCC.

The Y44 residue point mutation in CXCL14 reveals the significance of CXCL14 protein stability in anti-tumor activity

Abstract Objectives CXCL14, a member of the CXC chemokine family, plays a significant role in tumor development, progression, and metastasis, making it a potential target for cancer diagnosis and treatment. This study aims to investigate the impact of a point mutation in the (41VSRYR45) motif of CXCL14 on its anti-tumor activity. Methods Phylogenetic analysis, tertiary structure prediction, homology modeling, and cell culture experiments were employed to assess the effects of CXCL14 mutations on protein stability and anti-tumor activity. Phylogenetic analysis identified conserved regions critical for function, while computational tools predicted structural changes due to mutations. Homology modeling provided structural insights, and cell culture experiments involved transfecting HeLa cells with wild-type or mutant CXCL14 plasmids, assessing stability and anti-tumor effects. Additionally, in vivo, xenograft experiments using nude mice were conducted to evaluate the anti-tumor efficacy of CXCL14 variants. Results The conserved Y44 site within the (41VSRYR45) motif was identified as a key ubiquitination site. Mutations Y44V and Y44F enhanced CXCL14 stability, reducing HeLa cell proliferation and migration while increasing apoptosis. In vivo, tumor xenograft experiments confirmed the stronger inhibitory effect of these CXCL14 variants on tumor growth compared to the wild-type protein. Conclusion The Y44 site in CXCL14 is crucial for its stability and anti-tumor activity. Mutations Y44V and Y44F enhance CXCL14 stability and anti-tumor effects, suggesting that targeting this site could be a promising therapeutic strategy for cervical cancer.