Tumorsphere Formation Research Articles

BTB and CNC homology 1 (Bach1) induces lung cancer stem cell phenotypes by stimulating CD44 expression

BackgroundGrowing evidence suggests that cancer stem cells (CSCs) are responsible for cancer initiation in tumors. Bach1 has been identified to contribute to several tumor progression, including lung cancer. The role of Bach1 in CSCs remains poorly known. Therefore, the function of Bach1 on lung CSCs was focused currently.MethodsThe expression of Bach1, CD133, CD44, Sox2, Nanog and Oct4 mRNA was assessed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). Protein expression of Bach1, CD133, CD44, Sox2, Nanog, Oct4, p53, BCL2, BAX, p-p38, p-AKT1, c-Fos and c-Jun protein was analyzed by western blotting. 5-ethynyl-29-deoxyuridine (EdU), colony formation, Flow cytometry analysis and transwell invasion assay were carried out to analyze lung cancer cell proliferation, apoptosis and invasion respectively. Tumor sphere formation assay was utilized to evaluate spheroid capacity. Flow cytometry analysis was carried out to isolate CD133 or CD44 positive lung cancer cells. The relationship between Bach1 and CD44 was verified using ChIP-qPCR and dual-luciferase reporter assay. Xenograft tumor tissues were collected for hematoxylin and eosin (HE) staining and IHC analysis to evaluate histology and Ki-67.ResultsThe ratio of CD44 + CSCs from A549 and SPC-A1 cells were significantly enriched. Tumor growth of CD44 + CSCs was obviously suppressed in vivo compared to CD44− CSCs. Bach1 expression was obviously increased in CD44 + CSCs. Then, via using the in vitro experiment, it was observed that CSCs proliferation and invasion were greatly reduced by the down-regulation of Bach1 while cell apoptosis was triggered by knockdown of Bach1. Loss of Bach1 was able to repress tumor-sphere formation and tumor-initiating CSC markers. A repression of CSCs growth and metastasis of shRNA-Bach1 was confirmed using xenograft models and caudal vein injection. The direct interaction between Bach1 and CD44 was confirmed by ChIP-qPCR and dual-luciferase reporter assay. Furthermore, mitogen-activated protein kinases (MAPK) signaling pathway was selected and we proved the effects of Bach1 on lung CSCs were associated with the activation of the MAPK pathway. As manifested, loss of Bach1 was able to repress p-p38, p-AKT1, c-Fos, c-Jun protein levels in lung CSCs. Inhibition of MAPK signaling remarkably restrained lung CSCs growth and CSCs properties induced by Bach1 overexpression.ConclusionIn summary, we imply that Bach1 demonstrates great potential for the treatment of lung cancer metastasis and recurrence via activating CD44 and MPAK signaling.

Activating transcription factor 5 (ATF5) promotes tumorigenic capability and activates the Wnt/b-catenin pathway in bladder cancer

BackgroundIn bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear.MethodsQuantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses.ResultsATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/β-catenin pathway.ConclusionsThis study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

GLO 1 and PKCλ Regulate ALDH1-positive Breast Cancer Stem Cell Survival.

We examined the inhibitory effects of both glyoxalase 1 (GLO 1) and protein kinase C (PKC)λ in aldehyde dehydrogenase 1 (ALDH1)-positive breast cancer stem cells (CSCs). Breast cancer genomics datasets (TCGA, n=593; METABRIC, n=1904) were downloaded and statistically analyzed. The effects of GLO 1 and PKCλ on trypan blue staining and tumor-sphere formation by ALDH1high cells derived from triple negative breast cancer (TNBC) and basal-like breast cancer were examined. GLO 1high, PKCλhigh, and ALDH1A3high tumors were enriched in stage I/II/III/IV samples, associated with the HER2 and TNBC subtypes according to receptor status, and associated with the HER2-enriched and basal-like subtypes according to PAM50. Inhibition of either GLO 1 (TLSC702) or PKCλ (ANF) suppressed tumor-sphere formation and enhanced death in ALDH1high cells. TLSC702 also effectively inhibited tumor-sphere formation and induced death in PKCλ knockout ALDH1high cells. GLO 1 and PKCλ are important for the survival of ALDH1-positive breast CSCs, and may represent potential therapeutic targets for the treatment of ALDH1-positive breast CSCs.

In Vitro and Clinical Compassionate Use Experiences with the Drug-Repurposing Approach CUSP9v3 in Glioblastoma.

Background: Glioblastoma represents the most common primary brain tumor in adults. Despite technological advances, patients with this disease typically die within 1–2 years after diagnosis. In the search for novel therapeutics, drug repurposing has emerged as an alternative to traditional drug development pipelines, potentially facilitating and expediting the transition from drug discovery to clinical application. In a drug repurposing effort, the original CUSP9 and its derivatives CUSP9* and CUSP9v3 were developed as combinations of nine non-oncological drugs combined with metronomic low-dose temozolomide. Methods: In this work, we performed pre-clinical testing of CUSP9v3 in different established, primary cultured and stem-like glioblastoma models. In addition, eight patients with heavily pre-treated recurrent glioblastoma received the CUSP9v3 regime on a compassionate use basis in a last-ditch effort. Results: CUSP9v3 had profound antiproliferative and pro-apoptotic effects across all tested glioblastoma models. Moreover, the cells’ migratory capacity and ability to form tumor spheres was drastically reduced. In vitro, additional treatment with temozolomide did not significantly enhance the antineoplastic activity of CUSP9v3. CUSP9v3 was well-tolerated with the most frequent grade 3 or 4 adverse events being increased hepatic enzyme levels. Conclusions: CUSP9v3 displays a strong anti-proliferative and anti-migratory activity in vitro and seems to be safe to apply to patients. These data have prompted further investigation of CUSP9v3 in a phase Ib/IIa clinical trial (NCT02770378).

Aptamer-mediated doxorubicin delivery reduces HCC burden in 3D organoids model

Epithelial cell adhesion molecule (EpCAM) is a surface marker which is frequently overexpressed in hepatocellular carcinoma (HCC) but minimally expressed on mature hepatocytes. We developed a specific aptamer against EpCAM (EpCAM-apt) and tested its potential as a drug delivery agent for HCC. The targeting ability of EpCAM-apt was confirmed in vitro and in vivo after which the complex was conjugated with doxorubicin (Dox) to form EpCAM-apt-Dox. The targeting efficacy of the drug-loaded complex against liver cancer stem-like cells (LCSCs) and therapeutic effects in HCC were evaluated. EpCAM-expressing (EpCAM+) HCC cells showed characteristics of stem like cells including greater proliferative capacity and tumour sphere formation. EpCAM-apt-Dox selectively delivered Dox to EpCAM+ HCC cells with high drug retention and accumulation versus control. EpCAM-apt-Dox reduced the self-renewal capacity and stem-like cell frequency in vitro. Elimination of cancer stem-like cells (CSCs) with EpCAM-apt-Dox significantly inhibited the growth of HCC cells and patient-derived HCC organoids but exerted minimal cytotoxicity to normal liver organoids. Moreover, EpCAM-apt-Dox suppressed the growth of xenograft tumours derived from HCC organoids in vivo and prolonged mouse survival without inducing adverse effects to major organs. Thus, aptamer-based drug delivery to the stem-like cell population is a promising strategy for HCC treatment.

TMPRSS4 promotes cancer stem\u2013like properties in prostate cancer cells through upregulation of SOX2 by SLUG and TWIST1

BackgroundTransmembrane serine protease 4 (TMPRSS4) is a cell surface–anchored serine protease. Elevated expression of TMPRSS4 correlates with poor prognosis in colorectal cancer, gastric cancer, prostate cancer, non–small cell lung cancer, and other cancers. Previously, we demonstrated that TMPRSS4 promotes invasion and proliferation of prostate cancer cells. Here, we investigated whether TMPRSS4 confers cancer stem–like properties to prostate cancer cells and characterized the underlying mechanisms.MethodsAcquisition of cancer stem–like properties by TMPRSS4 was examined by monitoring anchorage-independent growth, tumorsphere formation, aldehyde dehydrogenase (ALDH) activation, and resistance to anoikis and drugs in vitro and in an early metastasis model in vivo. The underlying molecular mechanisms were evaluated, focusing on stemness-related factors regulated by epithelial–mesenchymal transition (EMT)-inducing transcription factors. Clinical expression and significance of TMPRSS4 and stemness-associated factors were explored by analyzing datasets from The Cancer Genome Atlas (TCGA).ResultsTMPRSS4 promoted anchorage-independent growth, ALDH activation, tumorsphere formation, and therapeutic resistance of prostate cancer cells. In addition, TMPRSS4 promoted resistance to anoikis, thereby increasing survival of circulating tumor cells and promoting early metastasis. These features were accompanied by upregulation of stemness-related factors such as SOX2, BMI1, and CD133. SLUG and TWIST1, master EMT-inducing transcription factors, made essential contributions to TMPRSS4-mediated cancer stem cell (CSC) features through upregulation of SOX2. SLUG stabilized SOX2 via preventing proteasomal degradation through its interaction with SOX2, while TWIST1 upregulated transcription of SOX2 by interacting with the proximal E-box element in the SOX2 promoter. Clinical data showed that TMPRSS4 expression correlated with the levels of SOX2, PROM1, SNAI2, and TWIST1. Expression of SOX2 was positively correlated with that of TWIST1, but not with other EMT-inducing transcription factors, in various cancer cell lines.ConclusionsTogether, these findings suggest that TMPRSS4 promotes CSC features in prostate cancer through upregulation of the SLUG- and TWIST1-induced stem cell factor SOX2 beyond EMT. Thus, TMPRSS4/SLUG–TWIST1/SOX2 axis may represent a novel mechanism involved in the control of tumor progression.

Zinc-finger protein 750 mitigates malignant biological behavior of oral CSC-like cells enriched from parental CAL-27 cells.

Oral squamous cell carcinoma (OSCC) is the most commonly occurring oral malignancy. Cancer stem cells (CSCs) are known to be responsible for cancer recurrence and metastasis. Zinc-finger protein 750 (ZNF750) has been reported to inhibit OSCC cell proliferation and invasion. The present study aimed to elucidate the role of ZNF750 in the inhibition of the renewal ability of CSCs derived from the OSCC cell line, CAL-27. The effects of ZNF750 on CSC-like properties were examined using aldehyde dehydrogenase (ALDH), tumor sphere formation and colony formation assays. Reverse transcription-quantitative PCR and western blotting were performed to detect the expression levels of octamer-binding transcription factor 4, sex-determining region Y-box 2, the enhancer of zeste homolog 2 (Ezh2), embryonic ectoderm development (EED) and SUZ12 polycomb repressive complex 2 subunit (SUZ12), and for the identification of genes associated with metastasis. ZNF750 effectively attenuated CSC-like cell self-renewal abilities; ZNF750 decreased the ALDH-positive cell population, tumor sphere and colony formation abilities, cell viability and stemness factors. Furthermore, the expression levels of Ezh2, EED and SUZ12 were decreased by ZNF750. ZNF750 inhibited MMP1, 3, 9 and 13 expression levels, and decreased the cell invasion and migratory abilities. Moreover, the expression of tissue inhibitors of matrix metalloproteinases-1 was increased by ZNF750. However, opposite effects were observed following the knockdown of the ZNF750 gene. Overall, the present study demonstrated that ZNF750 has the potential to inhibit the renewal of CSC-like cells enriched from parental CAL-27 cells.

Kinome-Wide siRNA Screening Identifies DYRK1B as a Potential Therapeutic Target for Triple-Negative Breast Cancer Cells.

Simple SummaryTherapeutic target is limited for patients with triple-negative breast cancer (TNBC). Through kinome-wide siRNA (709 genes) screening, DYRK1B was identified as a potential gene essential for cell proliferation and mobility of TNBC cells, particularly in DYRK1B highly expressed TNBC cells. TNBC patients with high expression of DYRK1B had poor overall survival and disease-free survival. CCDC97 and ZNF581 were positively correlated with DYRK1B expression and might be involved in DYRK1B-mediated tumor malignancy in TNBC patients, providing DYRK1B as a potential theranostic target for TNBC. Aims: The selective molecules for targeted therapy of triple-negative breast cancer (TNBC) are limited. Several kinases play pivotal roles in cancer development and malignancy. The study aims to determine if any kinases confer to malignancy of TNBC cells, which could serve as a theranostic target for TNBC. Methods: Kinome siRNA library was used to screen selective genes required for the proliferation of TNBC cells. The involvement of DYRK1B in cancer malignancy was evaluated with migration, invasion assays, and spheroid culture. The expression of DYRK1B was confirmed with quantitative PCR and immunoblotting. The clinical correlation of DYRK1B in TNBC patients was examined with tissue microarray and The Cancer Genome Atlas (TCGA) database. Results: Our results showed that silencing DYRK1B significantly suppressed cell viability in DYRK1B-high expressed TNBC cells, likely by arresting the cell cycle at the G1 phase. Nevertheless, silencing DYRK1B had marginal effects on DYRK1B-low expressed TNBC cells. Similarly, the knockdown of DYRK1B decreased tumorsphere formation and increased cell death of the tumorsphere. Moreover, inactivation of DYRK1B by either specific inhibitor or ectopic expressing catalytic mutant of DYRK1B inhibited cell viability and metastatic characteristics, including migration and invasion. In addition, DYRK1B protein expression was elevated in tumor tissues compared to that in adjacent normal tissues of TNBC patients. Further, DYRK1B gene expression was highly correlated with CCDC97 or ZNF581 genes in TNBC cells and patients. High co-expression of DYRK1B with CCDC97 or ZNF581 was significantly associated with unfavorable overall survival and disease-free survival of TNBC patients. Conclusions: our results suggest DYRK1B might be essential for promoting tumor progression and could be a theranostic target for TNBC. Silencing or inactivation of DYRK1B might be a potential targeted therapy for TNBC.

Abstract PO-045: Targeting HNF1A-dependent cell proliferation and stemness in PDAC using BET inhibitors

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis, with an overall survival rate of only 10%. Standard of care involves chemotherapy which improves survival by only a few weeks. Several barriers to therapeutic efficacy impede increasing survival outcome, including intrinsic resistance, owing to presence of therapy-resistant pancreatic cancer stem-like cells (PCSCs). Therefore, developing therapeutics to target molecular networks that maintain PCSC populations is critical. Microarray analysis from our lab previously established that pancreas lineage transcription factor HNF1A and its targets are enriched in CD44+CD24+EPCAM+ human PCSCs and that genetic depletion of HNF1A ablates this population and importantly, diminished tumor growth in primary PDAC xenografts. However, there are no therapeutics to directly target HNF1A. In this project, we propose to indirectly ablate HNF1A and its network by targeting epigenetic readers, bromodomain and extra-terminal domain protein family (BET) that maintain expression of transcription factor networks. We hypothesize that BET inhibitors decrease stemness and tumor growth in a HNF1A-dependent manner. Studies in our lab established that BET inhibitors decrease colony-formation, tumorsphere formation and cell-cycle progression in PDAC cells, that is rescued by overexpressing HNF1A. These results indicate that BET inhibitors decrease stemness and proliferation in pancreatic cancer cells in a HNF1A-dependent manner. These results suggest that BET inhibitors target multiple HNF1A-dependent tumor-promoting programs, such as stemness and tumor growth which could impact utilization of these drugs in clinical settings. The ultimate goal of our project is to provide rationale for the use of BET inhibitors in clinical setting in pancreatic cancer. Citation Format: Bharani Muppavarapu, Ethan Abel, Melanie Mayberry. Targeting HNF1A-dependent cell proliferation and stemness in PDAC using BET inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-045.

Cellular retinol binding protein-1 inhibits cancer stemness via upregulating WIF1 to suppress Wnt/\u03b2-catenin pathway in hepatocellular carcinoma

BackgroundCRBP-1, a cytosolic chaperone of vitamin A, is identified in a serious number of cancers; however, its biological role in hepatocellular carcinoma (HCC) needs to be further explored. The aim of our present study is to explore the roles and mechanisms of CRBP-1 in regulating liver cancer by using in vitro and in vivo biology approaches.MethodsThe expression level of CRBP-1 was detected using immunohistochemistry in HCC and matching adjacent non-tumorous liver tissues. Following established stable CRBP-1 overexpressed HCC cell lines, the cell growth and tumorigenicity were investigated both in vitro and in vivo. Intracellular retinoic acid was quantified by ELISA. The relationship between CRBP-1 and WIF1 was validated by using dual luciferase and ChIP analyses.ResultsThe low expression of CRBP-1 was observed in HCC tissues compared to the normal liver tissues, while high CRBP-1 expression correlated with clinicopathological characteristics and increased overall survival in HCC patients. Overexpression of CRBP-1 significantly inhibited cell growth and tumorigenicity both in vitro and in vivo. Moreover, overexpression of CRBP-1 suppressed tumorsphere formation and cancer stemness related genes expression in HCC. Mechanically, CRBP-1 inhibited Wnt/β-catenin signaling pathway to suppress cancer cell stemness of HCC. Furthermore, our results revealed that CRBP-1 could increase the intracellular levels of retinoic acid, which induced the activation of RARs/RXRs leading to the transcriptional expression of WIF1, a secreted antagonist of the Wnt/β-catenin signaling pathway, by physically interacting with the region on WIF1 promoter.ConclusionOur findings reveal that CRBP-1 is a crucial player in the initiation and progression of HCC, which provide a novel independent prognostic biomarker and therapeutic target for the diagnosis and treatment of HCC.

CSIG-05. DISSOCIATION OF THE INTRAMOLECULARLY CLEAVED N- AND C-TERMINAL FRAGMENTS OF ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 (ADGRD1) INCREASES ITS CANONICAL SIGNALING IN GLIOBLASTOMA

Abstract We previously demonstrated that GPR133 (ADGRD1), an adhesion GPCR that signals via cytosolic cAMP increase, is de novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells. Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic xenograft initiation in vivo. GPR133’s requirement for GBM growth and its absence in non-malignant brain suggest its therapeutic potential, yet its mechanisms of action and activation remained unclear. Here, we demonstrate in patient-derived GBM cultures and HEK293T cells that GPR133 gets intramolecularly cleaved into N-terminal and C-terminal fragments (NTF and CTF) right after synthesis in the endoplasmic reticulum. The resulting NTF and CTF remain non-covalently bound to each other, until the mature receptor reaches the plasma membrane, where we observe dissociation of the extracellular NTF from the transmembrane-spanning CTF. While cleavage is not required for correct subcellular trafficking, the cleaved wild-type GPR133 generates significantly higher cytosolic cAMP levels than an uncleavable point mutant GPR133 (H543R), suggesting that cleavage and dissociation are involved in receptor activation. To test this hypothesis in a more controllable proxy system, we generated a fusion of the CTF of GPR133 and the N-terminus of human protease-activated receptor 1 (hPAR1). Indeed, acute thrombin-induced cleavage and shedding of the hPAR1 NTF increases intracellular cAMP levels generated by the GPR133 CTF. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. To test whether extracellular binding proteins could influence NTF shedding and/or GPR133 signaling activation, we conducted ligand discovery screens and indeed found a new GPR133 binding protein in GBM cells, which is capable of influencing receptor signaling. Together, these findings provide critical insights into GPR133’s mechanism of activation, that will guide future approaches of therapeutic targeting of GPR133 in GBM.

Death-Associated Protein Kinase 1 Inhibits Progression of Thyroid Cancer by Regulating Stem Cell Markers.

The activation of metastatic reprogramming is vital for cancer metastasis, but little is known about its mechanism. This study investigated the potential role of death-associated protein kinase 1 (DAPK1) in thyroid cancer progression. We generated knockdown (KD) DAPK1 using siRNA or shRNA in 8505C and KTC-1 cell lines, which we transiently or stably overexpressed in MDA-T32 and BCPAP cell lines. DAPK1 KD in 8505C and KTC-1 cells significantly increased cell proliferation and colony formation compared with controls. We observed significant inhibition of cancer cell invasion in cells overexpressing DAPK1, but the opposite effect in KD cells. Tumorsphere formation significantly increased after inhibition of DAPK1 expression in 8505C cells and was significantly suppressed in DAPK1-overexpressing MDA-T32 and BCPAP cells. DAPK1 overexpression inhibited mRNA and protein levels of stem markers (OCT4, Sox2, KLF4, and Nanog). Furthermore, the expression of these markers increased after KD of DAPK1 in 8505C cells. Mechanistic studies suggest that DAPK1 may modulate the expression of stem cell markers through the inhibition of β-catenin pathways. These findings were consistent with the public data and our thyroid tissue analysis, which showed higher DAPK1 expression was associated with advanced-stage papillary thyroid cancer with a higher stemness index and lower disease-free survival.

Propranolol inhibits stemness of hemangioma through Jagged1.

BackgroundPropranolol is used clinically to treat infantile hemangioma (IH), although the exact mechanism that underlies its effectiveness is not fully understood. The Jagged1/Notch signaling pathway is downstream of the β2-adrenergic receptor (β2-AR). Propranolol is a non-selective β2-AR blocker that was shown to inhibit demethylation adrenaline-induced Jagged1 expression. A previous study has shown that propranolol dose-dependently inhibits the growth of IH. However, the effects of propranolol on stemness of IH are not known and are thus addressed in the current study.MethodsWe analyzed the expression of Jagged1 and Notch3 in IH specimens, using genetic tools to alter Notch signaling. The transduced IH cells were treated with different doses of propranolol, and the effects on IH cell proliferation, migration, and potential for tumor sphere formation were investigated. The effects of altered Notch signaling on tumor formation in vivo were also assessed.ResultsNotch3 and Jagged1 were significantly upregulated in IH. Augmented Notch signaling in IH cells increased cell proliferation, migration, the potential for tumor sphere formation and in vivo tumor formation. On the other hand, reduced Notch signaling in IH cells decreased cell proliferation, migration, the potential for tumor sphere formation and in vivo tumor formation.ConclusionsJagged1/Notch signaling regulated the stemness of IH, and propranolol inhibited it through suppression of Notch signaling.

Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells.

To evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands. UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation. A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity. Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres.

MiR-21-5p inhibition attenuates Warburg effect and stemness maintenance in osteosarcoma cells via inactivation of Wnt/β-catenin signaling.

MicroRNA (miR)-21 has been found to be overexpressed in osteosarcoma (OS). The aim of the present study was to investigate the effect of miR-21-5p on the Warburg effect and stemness maintenance in OS cells and its potential molecular mechanism. Herein, miR-21-5p was overexpressed or inhibited in MG-63 cells via transfection with mimics or inhibitors. The effect of miR-21-5p on cell viability, apoptosis, Warburg effect and stemness maintenance were explored in OS cells. The results demonstrated that miR-21-5p inhibition suppressed MG-63 cell viability and enhanced their apoptosis. Additionally, miR-21-5p inhibition attenuated the stemness maintenance of MG-63 cells, as demonstrated by the reduced proportion of CD133-positive MG-63 cells, the decrease in tumorsphere formation capacity, and the downregulation of Sox2, Oct4, and Nanog proteins. Moreover, miR-21-5p inhibition suppressed the Warburg effect in MG-63 cells, as indicated by the decrease in glucose uptake, lactic acid production, and ATP level and the downregulation of proteins involved in the Warburg effect (GLUT1, LDHA, HK2, and PKM2). Furthermore, the results suggested that the effect of miR-21-5p suppression on stemness and the Warburg effect may be associated with the decreased activity of the Wnt/β-catenin pathway in OS cells. Our findings suggest a novel potential biomarker for OS therapy.

GOLPH3/CKAP4 promotes metastasis and tumorigenicity by enhancing the secretion of exosomal WNT3A in non-small-cell lung cancer

Cancer metastasis is the main cause of mortality associated with non-small-cell lung cancer (NSCLC), accounting for up to 70% of deaths among patients. The mechanisms underlying distal metastasis remain largely unknown. Golgi phosphoprotein 3 (GOLPH3) correlates negatively with overall survival in multiple tumors. In this study, we evaluated the function of GOLPH3 in NSCLC distal metastasis. GOLPH3 was expressed at high levels in samples from patients with NSCLC and was positively associated with clinicopathologic characteristics including clinical stage (P < 0.001), T (P = 0.001), N (P = 0.007), and M (P = 0.001) classification. Functionally, Transwell and wound-healing assays suggested that GOLPH3 overexpression enhances NSCLC cell migration and invasion abilities. Tumor-sphere formation and flow cytometry assays demonstrated that GOLPH3 overexpression enhances a stem cell-like phenotype of NSCLC cells. Metastasis models established by tail vein and intracardiac injection confirmed the pro-metastatic function of GOLPH3 in vivo. A subcutaneous tumor formation model confirmed that GOLPH3 overexpression increased the tumorigenicity of NSCLC cells. Mechanistically, gene set enrichment analysis revealed a positive association of GOLPH3 mRNA expression with WNT-activated gene signatures. Luciferase-reporter and nuclear extract assays showed that GOLPH3 overexpression enhances metastasis and tumorigenicity through activation of the WNT/β-catenin pathway. Immunoprecipitation-mass spectrometry and gene ontology analysis demonstrated that GOLPH3 interacts with cytoskeleton-associated protein 4 (CKAP4) in exosome-mediated distal metastasis. We found that GOLPH3 decreased the amount of plasma membrane-localized CKAP4 and increased the amount of exosome-localized CKAP4 to promote the formation of CKAP4-containing exosomes. Furthermore, we demonstrated that CKAP4 binds exosomal WNT3A to enhance its secretion. Therefore, the GOLPH3/CKAP4 axis plays a crucial role in promoting exosomal-WNT3A secretion to enhance and maintain the stem-like phenotype and metastasis in NSCLC, thus indicating the therapeutic potential of GOLPH3 in patients with NSCLC metastasis.

Gamma-mangostin isolated from garcinia mangostana suppresses colon carcinogenesis and stemness by downregulating the GSK3β/β-catenin/CDK6 cancer stem pathway

BackgroundDespite advances in chemotherapies and targeted drugs, colorectal cancer (CRC) remains challenging to treat due to drug resistance. Emerging evidence indicates that cancer-associated fibroblasts (CAFs) facilitate the generation of cancer stem-like cells (CSCs) and drug resistance. Glycogen synthase kinase-3 (GSK) associated signaling pathways have been implicated in the generation of CSCs and represent a target for therapeutics development. HypothesisGamma-mangostin (gMG) isolated from Garcinia mangostana was evaluated for its ability to downregulate GSK3β-associated signaling in CRC cells and overcome CAF-induced 5-fluorouracil resistance and CSC generation. MethodsBioinformatics analysis, in silico molecular docking, in vitro assays, including cell viability tests, colony- and tumor sphere-formation assays, transwell migration assays, ELISA, SDS-PAGE, Western blotting, miR expression, qPCR, and flow cytometry, as well as in vivo mouse xenograft models were used to evaluate the antitumor effects of gMG. ResultsBioinformatics analyses indicated that GSK3β/CDK6/β-catenin mRNA signature was significantly higher in colon cancer patients. Additional algorithms predicted a higher miR-26b level was associated with significantly higher survival in CRC patients and GSK3β and CDK6 as targets of miR-26b-5p. To validate these findings in vitro, we showed that CAF-cocultured CRC cells expressed an increased expression of GSK3β, β-catenin, CDK6, and NF-κB. Therapeutically, we demonstrated that gMG treatment suppressed GSK3β-associated signaling pathways while concomitantly increased the miR-26b-5p level. Using a xenograft mouse model of CAFs cocultured HCT116 tumorspheres, we showed that gMG treatment reduced tumor growth and overcame CAF-induced 5-fluorouracil resistance. ConclusionsPharmacological intervention with gMG suppressed CRC carcinogenesis and stemness via downregulating GSK3/β-catenin/CDK6 and upregulating the miR-26b-5p tumor suppressor. Thus, gMG represents a potential new CRC therapeutic agent and warrants further investigation.

MiR-144-3p inhibits gastric cancer progression and stemness via directly targeting GLI2 involved in hedgehog pathway

BackgroundGastric cancer (GC) is the fifth most commonly diagnosed cancer worldwide. Due to the dismal prognosis, identifying novel therapeutic targets in GC is urgently needed. Evidences have shown that miRNAs played critical roles in the regulation of tumor initiation and progression. GLI family zinc finger 2 (GLI2) has been reported to be up-regulated and facilitate cancer progression in multiple malignancies. In this study, we focused on identifying GLI2-targeted miRNAs and clarifying the underlying mechanism in GC.MethodsPaired fresh gastric cancer tissues were collected from gastrectomy patients. GLI2 and miRNAs expression were detected in gastric cancer tissues and cell lines. Bioinformatics analysis was used to predict GLI2-targeted miRNAs and dual-luciferase reporter assay was applied for target verification. CCK-8, clone formation, transwell and flow cytometry were carried out to determine the proliferation, migration, invasion and cell cycle of gastric cancer cells. Tumorsphere formation assay and flow cytometry were performed to detail the stemness of gastric cancer stem cells (GCSCs). Xenograft models in nude mice were established to investigate the role of the miR-144-3p in vivo.ResultsGLI2 was frequently upregulated in GC and indicated a poor survival. Meanwhile, miR-144-3p was downregulated and negatively correlated with GLI2 in GC. GLI2 was a direct target gene of miR-144-3p. MiR-144-3p overexpression inhibited proliferation, migration and invasion of gastric cancer cells. Enhanced miR-144-3p expression inhibited tumorsphere formation and CD44 expression of GCSCs. Restoration of GLI2 expression partly reversed the suppressive effect of miR-144-3p. Xenograft assay showed that miR-144-3p could inhibit the tumorigenesis of GC in vivo.ConclusionsMiR-144-3p was downregulated and served as an essential tumor suppressor in GC. Mechanistically, miR-144-3p inhibited gastric cancer progression and stemness by, at least in part, regulating GLI2 expression.

The LEDGF/p75 Integrase Binding Domain Interactome Contributes to the Survival, Clonogenicity, and Tumorsphere Formation of Docetaxel-Resistant Prostate Cancer Cells.

Patients with prostate cancer (PCa) receiving docetaxel chemotherapy invariably develop chemoresistance. The transcription co-activator lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 and PSIP1, is upregulated in several human cancers, including PCa and promotes resistance to docetaxel and other drugs. The C-terminal region of LEDGF/p75 contains an integrase binding domain (IBD) that tethers nuclear proteins, including the HIV-1 integrase and transcription factors, to active chromatin to promote viral integration and transcription of cellular survival genes. Here, we investigated the contribution of the LEDGF/p75 IBD interactome to PCa chemoresistance. Quantitative immunoblotting revealed that LEDGF/p75 and its IBD-interacting partners are endogenously upregulated in docetaxel-resistant PCa cell lines compared to docetaxel-sensitive parental cells. Using specific human autoantibodies, we co-immunoprecipitated LEDGF/p75 with its endogenous IBD-interacting partners JPO2, menin, MLL, IWS1, ASK1, and PogZ, as well as transcription factors c-MYC and HRP2, in docetaxel-resistant cells, and confirmed their nuclear co-localization by confocal microscopy. Depletion of LEDGF/p75 and selected interacting partners robustly decreased the survival, clonogenicity, and tumorsphere formation capacity of docetaxel-resistant cells. These results implicate the LEDGF/p75 IBD interactome in PCa chemoresistance and could lead to novel therapeutic strategies targeting this protein complex for the treatment of docetaxel-resistant tumors.

L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

The l-type amino acid transporter 1 (LAT1) is a membranous transporter that transports neutral amino acids for cells and is dysregulated in various types of cancer. Here, we first observed increased LAT1 expression in pemetrexed-resistant non-small cell lung cancer (NSCLC) cells with high cancer stem cell (CSC) activity, and its mRNA expression level was associated with shorter overall survival in the lung adenocarcinoma dataset of the Cancer Genome Atlas database. The inhibition of LAT1 by a small molecule inhibitor, JPH203, or by RNA interference led to a significant reduction in tumorsphere formation and the downregulation of several cancer stemness genes in NSCLC cells through decreased AKT serine/threonine kinase (AKT)/mammalian target of rapamycin (mTOR) activation. The treatment of the cell-permeable leucine derivative promoted AKT/mTOR phosphorylation and reversed the inhibitory effect of JPH203 in the reduction of CSC activity in pemetrexed-resistant lung cancer cells. Furthermore, we observed that LAT1 silencing caused the downregulation of programmed cell death 1 ligand 1 (PD-L1) on lung cancer cells. The PD-L1+/LAT1+ subpopulation of NSCLC cells displayed great CSC activity with increased expression of several cancer stemness genes. These data suggest that LAT1 inhibitors can serve as anti-CSC agents and could be used in combination with immune checkpoint inhibitors in lung cancer therapy.