Xenograft Tumor Formation Research Articles

Mechanism of RIP2 enhancing stemness of glioma cells induces temozolomide resistance.

We aimed to investigate the role of receptor-interacting protein 2 (RIP2) in regulation of stemness of glioma cells and chemotherapy resistance. Plasmid transfection was used to overexpress RIP2. Chemical inhibitors were used to inhibit RIP2 or NF-κB activity. Cancer stemness of glioma cells was investigated by sphere formation assays, clone formation assays, and xenograft tumor formation assays. The expression of RIP2, p-NF-κB, IκBα, CD133, or SOX-2 was detected by Western blotting and immunofluorescence. Apoptosis was detected by flow cytometry. Immunohistochemical staining was used to detect the expression of RIP2, CD133, and SOX-2 in xenograft tumor tissue. The effect of the RIP2/NF-κB pathway on temozolomide (TMZ) resistance was evaluated by xenograft tumor assay. Transfection with RIP2 plasmid enhanced the sphere formation capability of U251 cells, clone formation capability, and xenograft tumor formation capability. RIP2 could mediate TMZ resistance by upregulating the expression of CD133 and SOX-2 by activating the NF-κB pathway. Both RIP2 inhibitor GSK583 and the NF-κB inhibitor SC75741 could reverse the resistance of U251 cells to TMZ. RIP2 mediates TMZ resistance by regulating the maintenance of stemness in glioma cells through NF-κB. Interventions targeting the RIP2/NF-κB pathway may be a new strategy for TMZ-resistant gliomas.

Involvement of the oncogenic small nucleolar RNA SNORA24 in regulation of p53 stability in colorectal cancer.

Colorectal cancer (CRC) is a common malignant cancer worldwide. Although the molecular mechanism of CRC carcinogenesis has been studied extensively, the details remain unclear. Small nucleolar RNAs (snoRNAs) have recently been reported to have essential functions in carcinogenesis, although their roles in CRC pathogenesis are largely unknown. In this study, we found that the H/ACA snoRNA SNORA24 was upregulated in various cancers, including CRC. SNORA24 expression was significantly associated with age and history of colon polyps in CRC patient cohorts, with high expression associated with a decreased 5-year overall survival. Our results indicated that the oncogenic function of SNORA24 is mediated by promoting G1/S phase transformation, cell proliferation, colony formation, and growth of xenograft tumors. Furthermore, SNORA24 knockdown induced massive apoptosis. RNA-sequencing and gene ontology (GO) enrichment analyses were performed to explore its downstream targets. Finally, we confirmed that SNORA24 regulates p53 protein stability in a proteasomal degradation pathway. Our study clarifies the oncogenic role of SNORA24 in CRC and advance the current model of the role of the p53 pathway in this process.

The MBNL1/circNTRK2/PAX5 pathway regulates aerobic glycolysis in glioblastoma cells by encoding a novel protein NTRK2-243aa

Glioblastoma multiforme (GBM) is the most common tumor of the human central nervous system. Aerobic glycolysis has been strongly related to tumor development and malignant behavior. In this study, we found that MBNL1, circNTRK2, and NTRK2-243aa were markedly downregulated and inhibited glycolysis in GBM, whereas PAX5 was upregulated and promoted glycolysis. Functionally, MBNL1 promoted the expression of circNTRK2 by binding to NTRK2 pre-mRNA, as validated using RNA pull-down and nascent RNA immunoprecipitation assays. Mass spectrometry, western blotting, and immunofluorescence staining methods were used to detect the expression of NTRK2-243aa. NTRK2-243aa—encoded by circNTRK2—phosphorylated PAX5 at Y102, leading to the attenuation of the half-life of PAX5, as validated by in vitro kinase and MG132 rescue assays. Besides, PAX5 transcriptionally facilitated the expression of PKM2 and HK2 by binding to their promoter regions, as verified by luciferase reporter and chromatin immunoprecipitation assays. Finally, overexpression of MBNL1 and circNTRK2 combined with PAX5 knockdown effectively inhibited the formation of GBM xenograft tumors and significantly prolonged the survival of orthotopic nude mice. We have delineated that the MBNL1/circNTRK2/PAX5 pathway plays a crucial role in regulating GBM glycolysis and could provide potential targets and alternative strategies for the treatment of GBM.

Abstract 5711: Targeting FTO suppresses pancreatic carcinogenesis via cancer stem cell maintenance

Abstract Background: Pancreatic cancer (PC) remains a lethal disease worldwide and is estimated to become the 2nd leading cause of cancer-related deaths in the United States by 2030. Recently, existence of cancer stem cells (CSC) has been reported in several malignancies, including PC and have been considered as seeds for cancer initiating cells. However, the molecular mechanism governing the maintenance of CSC traits and their self-renewal remain unclear. Upregulated expression of fat mass and obesity-associated protein (FTO), which demethylates N6-methyladenosine (the most prevalent eukaryotic RNA modification), has been associated with cancer progression of various types. Nevertheless, our current understanding on the role of FTO in modulating biological processes relevant to PC is in infancy. Methods: In this study, we first analyzed and compared the FTO expression in various PC cells vs normal pancreatic ductal epithelial (HPDE) cells. We then depleted FTO in PC cells using lentiviral-mediated and pharmacological inhibitor (CS1) approaches and analyzed the effects on cell growth, proliferation, apoptosis, cell cycle, migration, invasion, epithelial-mesenchymal transition, CSC growth and self-renewal, and xenograft tumor formation. Results: We found that PC cells expressed markedly higher mRNA and protein levels of FTO compared to HPDE cells. FTO depleted PC cells showed impaired growth in both the solid and semi-solid medium, and also a G1 phase cell cycle arrest which correlated with the increment in the expression of CDK inhibitors: p21cip1 and p27kip1. FTO loss in the PC cells also led to an increase in early apoptotic cell population with a concomitant increase in the expression of caspase 3 and 9. Of note, FTO depleted PC cells displayed marked delay in the wound closure and reduced capabilities to invade through matrigel. This further corroborated with the enhanced expression levels of epithelial (E-cadherin) and decrease in the mesenchymal (N-cadherin, vimentin, and fibronectin) markers. Mechanistically, MET in FTO depleted cells correlated with impaired tumor sphere formation and reduced expression of markers of cancer stemness (CD44, NanoG, Sox2, ALDH1, and CD133). Replating of single cell derived from these spheroids revealed that FTO loss hampered the secondary spheres formation; thus, FTO is necessary for spheroid formation, their maintenance and self-renewal potential of CSC. Additionally, FTO loss in PC cells led to delayed as well as decreased tumor growth in nude mice. Tumor extracts from FTO-depleted xenografts displayed induction of apoptosis, decreased expression of proliferation marker Ki67, and a MET phenotype. Conclusion: Through a comprehensive mechanistic analysis we explicitly demonstrate the biological and functional significance of FTO in governing PC tumorigenesis and maintenance of CSC; thus, targeting FTO may represent an attractive therapeutic approach for PC. Citation Format: Rachana Garg, Laleh Melstorm, Jianjun Chen, Chuan He, Ajay Goel. Targeting FTO suppresses pancreatic carcinogenesis via cancer stem cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5711.

Abstract 5297: ONC201 suppresses cancer cell growth in a reconstructed tumor microenvironment that includes chemotherapy-induced senescent fibroblasts

Abstract Cellular senescence and its associated secretory phenotype (SASP) can promote cancer progression in the tumor microenvironment (TME). The TME includes tumor cells, stromal cells, immune cells, endothelial cells, and extracellular matrix. Senescent cancer-associated fibroblasts (CAF) may contribute to tumor growth and therapy resistance. Targeting senescent CAF by means of removal, modulation of the SASP, or through cellular reprogramming might provide therapeutic avenues for treating cancer. We investigated the impact of chemotherapy-induced fibroblast senescence in the TME on tumor growth and response to cancer therapy. Expression of cytokines in chemo-induced senescent fibroblasts and cancer cells was assessed by bulk and single cell cytokine profiling. As expected, there were alterations in SASP factors with increased pro-tumorigenic immune factors and decreased anti-tumor cytokines during IMR90 etoposide-induced fibroblast senescence. We co-cultured luciferase-labeled HT29 cancer cells with senescent IMR90 and found that the senescent fibroblasts promoted HT29 cell growth in culture and accelerated xenograft tumor formation in mice. We next inhibited cellular senescence and its SASP with the senolytic drug ABT263 or the senostatic/senomorphic drug lamivudine (3TC). Both ABT263 and 3TC significantly reduced bioluminescence of HT29-Luc cells co-cultured with senescent IMR90 compared to non-treated IMR90 cells. Therapy-induced senescence confers 5-Fluorouracil (5-FU) resistance in colorectal cancer. We found that 5-FU treatment significantly reduced colony formation of HT29 cells in the presence of proliferating or senescent IMR90 cells, with a lesser reduction in the presence of the senescent IMR90 cells. This suggests that a microenvironment that includes senescent cells promotes tumor cell resistance to 5-FU. We hypothesize that SASP factors might confer cancer cell resistance to 5-FU treatment. Cytokine profiling showed that TRAIL expression is reduced in senescent cells. Treatment with the TRAIL-inducer ONC201 reduced colony formation and cell viability of HT29 cells co-cultured with senescent IMR90 fibroblasts. Single-cell cytokine profiling showed subpopulations of cancer cells with increased polyfunctionality strength index (PSI, secretion of more than 2 dominant types of cytokines per cell in a population). Combined treatment with ABT263 and ONC201 synergically reduced viability HT29 cells co-cultured with senescent IMR90, and this correlated with reduced PSI. Our results indicate that TME targeting by increasing antitumor cytokines in conjunction with senolytic therapies can inhibit tumor growth. We are continuing to unravel the cytokine landscape of chemotherapy-induced cell senescence to gain insights into therapeutic strategies targeting chemotherapy-induced TME-senescence and drug resistance. Citation Format: Shengliang Zhang, Kelsey E. Huntington, Bianca Kun, Lanlan Zhou, Jill Kreiling, John M. Sedivy, Wafik S. El-Deiry. ONC201 suppresses cancer cell growth in a reconstructed tumor microenvironment that includes chemotherapy-induced senescent fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5297.

G-quadruplex stabilizer Tetra-Pt(bpy) disrupts telomere maintenance and impairs FAK-mediated migration of telomerase-positive cells

G-quadruplex regulates a wide spectrum of biological processes, including telomere maintenance, DNA replication and transcription. The development of small molecules to selectively target G-quadruplex and their application remain hotspots in cancer therapy. Here, we explored the biological effect of G-quadruplexes stabilizer Tetra-Pt(bpy) in telomerase-positive cancer cells. Telomere maintenance was evaluated by telomerase repeat amplification protocol, chromosome orientation fluorescence in situ hybridization and telomere restriction fragment assays. We found that Tetra-Pt(bpy) accelerates telomere shortening through dual inhibition of telomerase activity and telomere sister chromatin exchanges mediated by telomeric G-quadruplexes. Consequently, Tetra-Pt(bpy)-treated cancer cells became enriched with extremely short telomeres and produced a strong telomeric DNA damage response following long-term treatment, leading to cell proliferation inhibition and senescence. Experimental evidence from RNA seq and cell migration-related assays showed that Tetra-Pt(bpy) decreased cell-matrix adhesion and inhibited the migration of non-senescent tumor cells. Mechanistically, Tetra-Pt(bpy) induced the formation of G-quadruplexes in focal adhesion kinase (FAK)-encoding gene PTK2, resulting in FAK transcription inhibition. Tetra-Pt(bpy) reduced xenograft tumor formation and inhibited tumor cell growth and migration in mice. This study further elucidates the function of G-quadruplexes in the human genome and reveals the potential of Tetra-Pt(bpy) as a novel chemotherapeutic agent for targeting telomerase-positive cancer cells.

MODL-08. Cellular yield and xenograft creation from probe washing after stereotactic biopsy of deep-seated CNS tumors in children

Abstract Intracranial tumors located in otherwise difficult-to-resect regions of the brain are routinely biopsied to obtain tissue diagnosis and guide clinical decision making. However, tissue yield from these stereotactic biopsy samples is limited and leaves little remaining for research purposes or tumor banking. Given the importance of molecular diagnostics and rise of targeted therapies, research into the genetic and immune landscape of these lesions is of paramount importance, especially for inoperable and deep-seated tumors. We sought to obtain viable cellular material from otherwise discarded biopsy probes without visible tumor sample within the probe. Cells were obtained from clinically challenging pediatric tumors of the brainstem, thalamus, and pineal region by washing biopsy probes in FBS media immediately after completion of a stereotactic biopsy. The wash was then filtered and re-suspended in media before evenly distributing in a 24 well plate for culture purposes. A total of 15 samples were collected from probe washes, including 6 diffuse midline gliomas of the brain stem, 7 supratentorial diffuse midline gliomas, and 2 tumors in the pineal region. Viable tumor cells were obtained from 9/15 samples, ranging from 500 to approximately 525,000 cells per sample wash (219,222 ± 181,951). Orthotopic implantation into matching locations was completed with 7 tumor samples. Formation of a patient-derived orthotopic xenograft was confirmed in one thalamic high-grade glioma (165,000 cells). Three of the five diffuse midline gliomas of the brainstem showed early signs of tumor formation (yielding >2 million cells) and will be validated. In conclusion, viable tumor cells can be collected from biopsy probe washes after stereotactic biopsy, without visible tumor within the probe. Tumor cells can be used for patient-derived orthotopic xenograft tumor formation, which allows for new avenues in the development of animal models of difficult to resect brain tumors.

5‑Aza‑dC suppresses melanoma progression by inhibiting GAS5 hypermethylation.

The in-depth study of melanoma pathogenesis has revealed that epigenetic modifications, particularly DNA methylation, is a universal inherent feature of the development and progression of melanoma. In the present study, the analysis of the tumor suppressor gene growth arrest-specific transcript 5 (GAS5) demonstrated that its expression was downregulated in melanoma, and its expression level had a certain negative association with its methylation modification level. The promoter of GAS5 presented with detectable CpG islands, and methylation-specific polymerase chain reaction analysis demonstrated that GAS5 was actually modified by methylation in melanoma tissues and cells; however, no methylation modification of GAS5 was detected in normal tissues. Following the treatment of melanoma cells with 5-Aza-2′-deoxycytidine (5-Aza-dC), GAS5 methylation was significantly reversed. The analysis of melanoma cell proliferation revealed that 5-Aza-dC inhibited A375 and SK-MEL-110 cell proliferation in a time-dependent manner. Further analysis of apoptosis demonstrated that 5-Aza-dC significantly increased the apoptosis level of the two cell lines. Moreover, migration analysis of melanoma cells revealed that 5-Aza-dC significantly reduced cell migration. Furthermore, 5-Aza-dC significantly decreased the invasive ability of the two cell lines. However, when the expression of GAS5 was silenced, the effects of 5-Aza-dC on cell proliferation, apoptosis, invasion and migration were not significant. Furthermore, the subcutaneous injection of A375 cells in nude mice successfully resulted in xenograft tumor formation. However, following an intraperitoneal injection of 5-Aza-dC, the volume and weight of xenograft tumors and Ki-67 expression were significantly reduced, and caspase-3 activity and GAS5 expression were enhanced; following the silencing of GAS5, the antitumor effect of 5-Aza-dC was significantly blocked. On the whole, the present study demonstrates that 5-Aza-dC inhibits the growth of melanoma, and its function may be related to the methylation modification of GAS5.

Circular RNA circLRCH3 Inhibits Proliferation, Migration, and Invasion of Colorectal Cancer Cells Through miRNA-223/LPP Axis.

PurposeColorectal cancer (CRC) is one of the most common carcinomas worldwide with a high mortality rate. Numerous studies suggest that circular RNA (circRNA) plays a crucial role in the progression of various carcinomas, including CRC. The present work focused on exploring the role and underlying molecular mechanism of action of the circRNA circLRCH3 in CRC.MethodsReal-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to detect expression levels of circLRCH3, miR-233, and lipoma preferred partner (LPP). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the proliferation of CRC cells and the transwell assay was used to evaluate cell migration and invasion capacity. A flow cytometry assay was used to analyze the effect of circLRCH3 on the distribution of the cell cycle and apoptosis of CRC cells. The expression of LPP was analyzed using Western blotting or an RT-qPCR assay. The relationship between miR-223 and circLRCH3, and that between miR-223 and LPP, was predicted and examined using bioinformatics analysis and luciferase reporter gene experiments. A xenograft tumor formation assay was also performed.ResultsWe found that the expression level of circLRCH3 was downregulated in CRC cells and negatively correlated with miR-223. The overexpression of circLRCH3 or silencing of miR-223 inhibited the growth, invasion, and migration of CRC cells, but promoted their apoptosis. In contrast, overexpression of miR-223 and depletion of LPP severally abrogated the tumor suppressive roles of circLRCH3 and miR-223 knockdown in CRC cells in vitro. The xenograft experiments in nude mice also proved the antitumor effect of circLRCH3.ConclusionThese results suggested that the circLRCH3/miR-223/LPP axis likely plays a critical role in CRC.

PD07-09 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING

You have accessJournal of UrologyCME1 May 2022PD07-09 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING Srikanth Perike, Calvin VanOpstall, Mathias Morales, Sophia Lamperis, Steve Kregel, Ryan Brown, and Donald J. Vander Griend Srikanth PerikeSrikanth Perike More articles by this author , Calvin VanOpstallCalvin VanOpstall More articles by this author , Mathias MoralesMathias Morales More articles by this author , Sophia LamperisSophia Lamperis More articles by this author , Steve KregelSteve Kregel More articles by this author , Ryan BrownRyan Brown More articles by this author , and Donald J. Vander GriendDonald J. Vander Griend More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is one of the most frequently diagnosed malignancies in men, and its incidence and mortality continue to be a significant clinical problem. Recent evidence from our lab identified MEIS1, an important HOX protein cofactor, as a potential tumor suppressor. Patients bearing MEIS1-positive prostate tumors were significantly less likely to have biochemical recurrence and metastasis compared to men bearing MEIS-negative tumors. Androgen receptor (AR), the major oncogene in PCa, has been shown to interact with HOXB13 to promote prostate cancer progression, but the function of MEIS proteins to antagonize AR/HOXB13 interactions are unknown. We hypothesize that MEIS1 proteins interact with HOXB13 to suppress cancer initiation and progression, and loss of MEIS1 expression in a portion of prostate tumors enables oncogenic AR/HOXB13 interactions. METHODS: We determined the impact of MEIS1 expression and dependency of HOXB13 on AR signaling using cell lines ectopically expressing MEIS1 and/or CRISPR-mediated HOXB13 deletion in both androgen-sensitive LAPC4 and castration-resistant CWR22Rv1 cells. Western blots, qPCR, Proximity Ligation Assay and co-IPs were performed to evaluate the relationship between expression of MEIS1, HOXB13 and AR. Hormonally-intact and castrated male nude mice were used to test the in vivo capability of MEIS1-mediated tumor xenograft formation and rate of tumor growth in the presence and absence of AR ligand. RNA sequencing and informatics analyses was performed on tumor xenografts to determine the global impact of MEIS expression on AR gene targeting in PCa. RESULTS: We found that AR expression was significantly increased when MEIS1 was ectopically expressed compared to controls and HOXB13 knock-out lines. Re-expression of MEIS1 enhanced the binding between HOXB13-MEIS and AR-MEIS, and reduced AR-HOXB13 interaction. In castrated mice, re-expression of MEIS1 significantly decreased the tumor formation and tumor growth rate compared to hormonally intact nude mice. Conversely, xenografts of HOXB13-knockout tumors showed an increased rate of tumor growth and tumor formation compared to MEIS1-expressing cells and controls in both hormonally intact and castrated nude mice. Our preliminary RNA-Seq analysis demonstrate that when MEIS is present, AR pathway activation in response to androgen was associated with increased differentiation and growth suppression. CONCLUSIONS: Our collective data supports our hypothesis that increased MEIS1 expression reduces the AR/HOXB13 interaction and increases the in vivo sensitivity to host castration; this suggests that MEIS-positive cells have decreased oncogenic AR signaling. Future RNA-Seq studies combined with AR ChIP-Seq data will enable us to identify the MEIS-AR targeted genes that are involved in AR pathway modulation that inhibit prostate cancer cell proliferation and suppress tumor progression. These data provide us a strong rationale to support the potential utility of MEIS proteins as predictive clinical biomarkers of metastatic progression. Source of Funding: Funding: This work was supported by the Urology Care Foundation Research Scholar Award sponsored by Chesapeake Urology Associates, DOD grant PC130587, and DOD grant PC180414 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e102 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Srikanth Perike More articles by this author Calvin VanOpstall More articles by this author Mathias Morales More articles by this author Sophia Lamperis More articles by this author Steve Kregel More articles by this author Ryan Brown More articles by this author Donald J. Vander Griend More articles by this author Expand All Advertisement PDF DownloadLoading ...

Loss of DRO1/CCDC80 in the tumor microenvironment promotes carcinogenesis.

Tumors are composed of the tumor cells and the surrounding microenvironment. Both are closely interwoven and interact by a complex and multifaceted cross-talk which plays an integral part in tumor initiation, growth, and progression. Dro1/Ccdc80 has been shown to be a potent suppressor of colorectal cancer and ubiquitous inactivation of Dro1/Ccdc80 strongly promoted colorectal carcinogenesis in ApcMin/+ mice and in a chemically-induced colorectal cancer model.The aim of the present study was to investigate whether Dro1/Ccdc80’s tumor suppressive function is tumor-cell-autonomous. Expression of Dro1/Ccdc80 in cancer cells had no effect on both colon tumor development in ApcMin/+ mice and formation of xenograft tumors. In contrast, DRO1/CCDC80 loss in the microenvironment strongly increased tumor growth in xenograft models, inhibited cancer cell apoptosis, and promoted intestinal epithelial cell migration. Moreover, stromal Dro1/Ccdc80 inactivation facilitated formation of intestinal epithelial organoids. Expression analyses showed Dro1/Ccdc80 to be significantly down-regulated in murine gastric cancer associated fibroblasts, in ApcMin/+ colon tumor primary stromal cells and in microdissected stroma from human colorectal cancer compared to normal, non-tumor stroma. Our results demonstrate epithelial derived DRO1/CCDC80 to be dispensable for intestinal tissue homeostasis and identify Dro1/Ccdc80 as tumor suppressor in the tumor microenvironment.

A novel HIF1α-STIL-FOXM1 axis regulates tumor metastasis

BackgroundMetastasis is the major cause of morbidity and mortality in cancer that involves in multiple steps including epithelial–mesenchymal transition (EMT) process. Centrosome is an organelle that functions as the major microtubule organizing center (MTOC), and centrosome abnormalities are commonly correlated with tumor aggressiveness. However, the conclusive mechanisms indicating specific centrosomal proteins participated in tumor progression and metastasis remain largely unknown.MethodsThe expression levels of centriolar/centrosomal genes in various types of cancers were first examined by in silico analysis of the data derived from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and European Bioinformatics Institute (EBI) datasets. The expression of STIL (SCL/TAL1-interrupting locus) protein in clinical specimens was further assessed by Immunohistochemistry (IHC) analysis and the oncogenic roles of STIL in tumorigenesis were analyzed using in vitro and in vivo assays, including cell migration, invasion, xenograft tumor formation, and metastasis assays. The transcriptome differences between low- and high-STIL expression cells were analyzed by RNA-seq to uncover candidate genes involved in oncogenic pathways. The quantitative polymerase chain reaction (qPCR) and reporter assays were performed to confirm the results. The chromatin immunoprecipitation (ChIP)-qPCR assay was applied to demonstrate the binding of transcriptional factors to the promoter.ResultsThe expression of STIL shows the most significant increase in lung and various other types of cancers, and is highly associated with patients’ survival rate. Depletion of STIL inhibits tumor growth and metastasis. Interestingly, excess STIL activates the EMT pathway, and subsequently enhances cancer cell migration and invasion. Importantly, we reveal an unexpected role of STIL in tumor metastasis. A subset of STIL translocate into nucleus and associate with FOXM1 (Forkhead box protein M1) to promote tumor metastasis and stemness via FOXM1-mediated downstream target genes. Furthermore, we demonstrate that hypoxia-inducible factor 1α (HIF1α) directly binds to the STIL promoter and upregulates STIL expression under hypoxic condition.ConclusionsOur findings indicate that STIL promotes tumor metastasis through the HIF1α-STIL-FOXM1 axis, and highlight the importance of STIL as a promising therapeutic target for lung cancer treatment.

Rapid Isolation of Gastric Adenocarcinoma Cancer Stem Cells as a Target for Autologous Dendritic Cell-Based Immunotherapy.

Gastric cancer (GC) is a malignancy cause associated with a high death rate in the world. Cancer stem cells (CSCs) are a rare immortal subpopulation of cells within tumors with characteristics of the ability to self-renew, initiate tumor, and differentiate into defined progenies as well as and high resistance to conventional therapies. Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Therefore, rapid isolation of CSCs in order to therapeutic targets, especially immunotherapy is very important. Cancerous cell suspension isolated from patients with GC was cultured in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions to generate spheres. Expression of mRNA level stemness transcription factors (OCT4, SOX2, SALL4, and Cripto-1), CD44 variable isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) of spheroid-forming single cells compared with gastric normal tissue cells using real time PCR and molecules of CD44, CD54, and EpCAM as gastric CSC markers, and stemness factor Oct4 using flow cytometry, as well as tumorgenicity using subcutaneous injection of sphere-forming cells to nude mice were investigated. Few cancerous cells isolated from patients with GC were able to generate three-dimensional spheroid colonies in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions, and form xenograft tumors in immunodeficient nude mice after subcutaneous injection. Spheroid-forming single cells upregulated stemness transcription factors OCT4, SOX2, SALL4, and Cripto-1 that are associated with pluripotency and self-renewal and CD44 isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) compared with gastric normal tissue cells. Finally, molecules of CD44, CD54, and EpCAM as gastric CSC markers and stemness factor Oct4 were expressed in sphere-forming cells. We suggested that the sphere formation and tumorigenicity assays are two procedures, leading to the rapid isolation of cancer cells with certain stem-like properties in order to target CSCs using autologous dendritic cell therapy, especially in patients with advanced disease.

Targeting PI3Kα/δ and the ErbB Family of Protein-Tyrosine Kinases in Cisplatin-Resistant Head and Neck Squamous Cell Carcinomas

<h3>Purpose/Objective(s)</h3> The mortality rate for advanced head and neck squamous cell carcinoma (HNSCC) remains very high due to cancer recurrence and metastasis. Cisplatin is a commonly used anti-cancer drug for treatment of recurrent and metastatic HNSCC, but the majority of patients will eventually develop resistance. Therefore, there is an urgent, yet still unmet, need for improved therapies for cisplatin-resistant HNSCC. Increasing evidence has demonstrated that ErbB family kinase and PI3K/Akt are the most important regulators and targets in HNSCC. Our goal is to find new therapeutics to treat cisplatin resistant HNSCC without use of cisplatin. Here, we determined the efficacy of a combination of the PI3Kinase inhibitor, Copanlisib with the ErbB inhibitor, Afatinib, on suppressing cisplatin resistant HNSCC <i>in vitro</i> and <i>in vivo</i>. <h3>Materials/Methods</h3> Both cisplatin sensitive (Cal27 and FaDu) and resistant (Cal27CP and FaDu-CP) HNSCC cells were treated with copanlisib, afatinib, or a combination of copanlisib and afatinib. The effects on cell signaling pathways were tested by Western blot assay and RT-PCR. Cell proliferation was determined by MTT assay. Apoptosis was assayed by Annexin V. Migration and invasion was monitored via the xCELLigence real-time cell system tic cells. Xenograft tumor formation was observed in nude mice. <h3>Results</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals. <h3>Conclusion</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals.

Hsa_circ_0041268 promotes NSCLC progress by sponging miR-214-5p/ROCK1.

Circular RNAs hold significant regulatory functions during various tumors. However, the exact hsa_circ_0041268 roles in non‐small cell lung cancer (NSCLC) along with regulatory mechanism are unknown. In this study, RT‐qPCR was used to perceive hsa_circ_0041268 expressions in NSCLC cell lines. Our team constructed small interfering RNA for hsa_circ_0041268. NSCLC cell proliferation, migration, and tumorigenesis in nude mice were assayed to confirm hsa_circ_0041268 activities in NSCLC cells. We then used bioinformatics and luciferase reporter analyses to characterize the hsa_circ_0041268 downstream targets. The result shows that the expressions of hsa_circ_0041268 incremented in NSCLC cell lines and hsa_circ_0041268 downregulation decreased cell proliferation and migration. ROCK1 and miR‐214‐5p were hsa_circ_0041268 downstream targets. miR‐214‐5p downregulation or ROCK1 overexpression restored migration and proliferation abilities after hsa_circ_0041268 silencing. ROCK1 overexpression renovated migration and proliferation abilities after miR‐214‐5p overexpression. In vivo investigations confirmed that hsa_circ_0041268 downregulation inhibited tumor formation and metastasis in nude mice xenografts. Together, results demonstrated that hsa_circ_0041268 acted as tumor promoter through novel hsa_circ_0041268/miR‐214‐5p/ROCK1 axis, which highlighted its potential as NSCLC therapeutic agent.

Combination of Resminostat with Ruxolitinib Exerts Antitumor Effects in the Chick Embryo Chorioallantoic Membrane Model for Cutaneous T Cell Lymphoma.

Simple SummaryThe combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in vitro. The aim of the present study was to validate their antitumor effects in vivo using the chick embryo chorioallantoic membrane (CAM) model, which allows quick and efficient monitoring of tumor growth, migration, invasion, and metastatic potential. The drug combination exhibited a significant inhibition of primary tumor size, and inhibited intravasation and extravasation of tumor cells to the liver and lung. It also exerted an inhibitory effect in the migration and invasion of tumor cells and significantly reduced key signaling pathway activation. Our data demonstrate that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing, and that the combination of Resminostat and Ruxolitinib exerts significant antitumor effects in CTCL progression that need to be further evaluated in a clinical setting.The combination of Resminostat (HDACi) and Ruxolitinib (JAKi) exerted cytotoxic effects and inhibited proliferation of CTCL cell lines (MyLa, SeAx) in previously published work. A xenograft tumor formation was produced by implanting the MyLa or SeAx cells on top of the chick embryo chorioallantoic membrane (CAM). The CAM assay protocol was developed to monitor the metastatic properties of CTCL cells and the effects of Resminostat and/or Ruxolitinib in vivo. In the spontaneous CAM assays, Resminostat and Ruxolitinib treatment inhibited the cell proliferation (p < 0.001) of MyLa and SeAx, and induced cell apoptosis (p < 0.005, p < 0.001, respectively). Although monotherapies reduced the size of primary tumors in the metastasis CAM assay, the drug combination exhibited a significant inhibition of primary tumor size (p < 0.0001). Furthermore, the combined treatment inhibited the intravasation of MyLa (p < 0.005) and SeAx cells (p < 0.0001) in the organs, as well as their extravasation to the liver (p < 0.0001) and lung (p < 0.0001). The drug combination also exerted a stronger inhibitory effect in migration (p < 0.0001) rather in invasion (p < 0.005) of both MyLa and SeAx cells. It further reduced p-p38, p-ERK, p-AKT, and p-STAT in MyLa cells, while it decreased p-ERK and p-STAT in SeAx cells in CAM tumors. Our data demonstrated that the CAM assay could be employed as a preclinical in vivo model in CTCL for pharmacological testing. In agreement with previous in vitro data, the combination of Resminostat and Ruxolitinib was shown to exert antitumor effects in CTCL in vivo.

YAP ISGylation increases its stability and promotes its positive regulation on PPP by stimulating 6PGL transcription

Yes-associated protein (YAP) activation is crucial for tumor formation and development, and its stability is regulated by ubiquitination. ISGylation is a type of ubiquitination like post-translational modification, whereas whether YAP is ISGylated and how ISGylation influences YAP ubiquitination-related function remains uncovered. In addition, YAP can activate glucose metabolism by activating the hexosamine biosynthesis pathway (HBP) and glycolysis, and generate a large number of intermediates to promote tumor proliferation. However, whether YAP stimulates the pentose phosphate pathway (PPP), another tumor-promoting glucose metabolism pathway, and the relationship between this stimulation and ISGylation needs further investigation. Here, we found that YAP was ISGylated and this ISGylation inhibited YAP ubiquitination, proteasome degradation, interaction with-beta-transducin repeat containing E3 ubiquitin-protein ligase (βTrCP) to promote YAP stability. However, ISGylation-induced pro-YAP effects were abolished by YAP K497R (K, lysine; R, arginine) mutation, suggesting K497 could be the major YAP ISGylation site. In addition, YAP ISGylation promoted cell viability, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) tumor formation. YAP ISGylation also increased downstream genes transcription, including one of the key enzymes of PPP, 6-phosphogluconolactonase (6PGL). Mechanistically, YAP promoted 6PGL transcription by simultaneously recruiting SMAD family member 2 (SMAD2) and TEA domain transcription factor 4 (TEAD4) binding to the 6PGL promoter to activate PPP. In clinical lung adenocarcinoma (LUAD) specimens, we found that YAP ISGylation degree was positively associated with 6PGL mRNA level, especially in high glucose LUAD tissues compared to low glucose LUAD tissues. Collectively, this study suggested that YAP ISGylation is critical for maintaining its stability and further activation of PPP. Targeting ISGylated YAP might be a new choice for hyperglycemia cancer treatment.

Up-regulation of miR-30b suppresses glioblastoma by negatively regulating MEF2D through Wnt/β-catenin signaling pathway

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To study miR-30b’ significance on glioblastoma, and its underlying mechanism of action.&#x0D; Methods: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) while 3-(4,5)- dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT), Transwell, and xenograft tumor formation assays were carried out to study miR-30b’s effect on glioblastoma while luciferase reporter assay was employed to study the interaction between MEF2D and miR-30b. Glioblastoma cells treatment with miR-30 mimic or inhibitor were subjected to Western blot assay to study the effect of Wnt/β-catenin signaling on miR-30b/MEF2D axis-mediated cell progression.&#x0D; Results: MiR-30b was lowly expressed in glioblastoma tissues (p = 0.007), and this was associated with poor prognosis of patients (p = 0.022). The direct target of miR-30b was identified as MEF2D (p = 0.036). Increasing miR-30b blocked MEF2D expression in glioblastoma cells (p = 0.029). Moreover, MEF2D overturned miR-30b’ inhibitory effect on glioblastoma cell progression (p = 0.041; p = 0.006; p = 0.037). In vivo, restoration of miR-30b inhibited tumor growth (p = 0.01) and MEF2D. Interestingly, restoration of miR-30b inhibited epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin signaling pathways.&#x0D; Conclusion: These results indicate the critical role of miR-30b/MEF2D axis in glioblastoma via EMT and Wnt/β-catenin pathways. Thus, the miR-30b/MEF2D axis might be a beneficial therapeutic target for the management of glioblastoma patients.&#x0D; &#x0D; &#x0D; &#x0D;

Circular RNA circ_UBAP2 facilitates the progression of osteosarcoma by regulating microRNA miR-637/high-mobility group box (HMGB) 2 axis

ABSTRACT Circular RNA circ_UBAP2 has been reported to be closely associated with various tumors. The present work focused on exploring the roles of circ_UBAP2 and its molecular mechanism in osteosarcoma (OS). Circ_UBAP2, miR-637, and high-mobility group box (HMGB) 2 levels in OS cells and tissues were detected by quantitative real-time polymerase chain reaction. The relationship between miR-637 and circ_UBAP2, as well as between miR-637 and HMGB2, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), Transwell and flow cytometry assays, respectively. HMGB2 protein levels were measured using Western blotting. Xenograft tumor formation assay was also performed. Circ_UBAP2 showed high expression levels in OS tissues and cells, which was directly proportional to metastasis and clinical stage of OS. The overexpression of circ_UBAP2 enhanced the growth, invasion, and migration of OS cells, but suppressed their apoptosis. In contrast, circ_UBAP2 silencing had opposite effects. Furthermore, miR-637 served as a downstream target of circ_UBAP2, which played opposite roles to circ_UBAP2 in OS. More importantly, HMGB2 served as miR-637ʹs downstream target. The xenograft experiments in nude mice also proved that knockdown of circ_UBAP2 could increase miR-637 expression, but decrease HMGB2 expression, thus alleviating OS progression. Mechanistically, circ_UBAP2 exerts a cancer-promoting effect on OS by downregulating miR-637 and upregulating the expression of HMGB2. Circ_UBAP2 plays a promoting role in OS, and the circ_UBAP2/miR-637/HMGB2 axis is involved in OS progression.

KRT13 promotes stemness and drives metastasis in breast cancer through a plakoglobin/c-Myc signaling pathway

BackgroundKeratins (KRTs) are intermediate filament proteins that interact with multiple regulatory proteins to initiate signaling cascades. Keratin 13 (KRT13) plays an important role in breast cancer progression and metastasis. The objective of this study is to elucidate the mechanism by which KRT13 promotes breast cancer growth and metastasis.MethodsThe function and mechanisms of KRT13 in breast cancer progression and metastasis were assessed by overexpression and knockdown followed by examination of altered behaviors in breast cancer cells and in xenograft tumor formation in mouse mammary fat pad. Human breast cancer specimens were examined by immunohistochemistry and multiplexed quantum dot labeling analysis to correlate KRT13 expression to breast cancer progression and metastasis.ResultsKRT13-overexpressing MCF7 cells displayed increased proliferation, invasion, migration and in vivo tumor growth and metastasis to bone and lung. Conversely, KRT13 knockdown inhibited the aggressive behaviors of HCC1954 cells. At the molecular level, KRT13 directly interacted with plakoglobin (PG, γ-catenin) to form complexes with desmoplakin (DSP). This complex interfered with PG expression and nuclear translocation and abrogated PG-mediated suppression of c-Myc expression, while the KRT13/PG/c-Myc signaling pathway increased epithelial to mesenchymal transition and stem cell-like phenotype. KRT13 expression in 58 human breast cancer tissues was up-regulated especially at the invasive front and in metastatic specimens (12/18) (p < 0.05). KRT13 up-regulation in primary breast cancer was associated with decreased overall patient survival.ConclusionsThis study reveals that KRT13 promotes breast cancer cell growth and metastasis via a plakoglobin/c-Myc pathway. Our findings reveal a potential novel pathway for therapeutic targeting of breast cancer progression and metastasis.