Tumorsphere Formation Research Articles

CHAC1 blockade suppresses progression of lung adenocarcinoma by interfering with glucose metabolism via hijacking PKM2 nuclear translocation.

Patients with lung adenocarcinoma (LUAD) generally have poor prognosis. Abnormal cellular energy metabolism is a hallmark of LUAD. Glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) is a member of the γ-glutamylcyclotransferase family and an unfolded protein response pathway regulatory gene. Its biological function and molecular regulatory mechanism, especially regarding energy metabolism underlying LUAD, remain unclear. By utilizing tissue microarray and data from The Cancer Genome Atlas and Gene Expression Omnibus, we found that CHAC1 expression was markedly higher in LUAD tissues than in non-tumor tissues, and was positively correlated with poor prognosis. Phenotypically, CHAC1 overexpression enhanced the proliferation, migration, invasion, tumor sphere formation, and glycolysis ability of LUAD cells, resulting in tumor growth both in vitro and in vivo. Mechanistically, through a shotgun mass spectrometry-based proteomic approach and high-throughput RNA sequencing, we found that CHAC1 acted as a bridge connecting UBA2 and PKM2, enhancing the SUMOylation of PKM2. The SUMOylated PKM2 then transferred from the cytoplasm to the nucleus, activating the expression of glycolysis-related genes and enhancing the Warburg effect. Lastly, E2F Transcription Factor 1 potently activated CHAC1 transcription by directly binding to the CHAC1 promoter in LUAD cells. The results of this study implied that CHAC1 regulates energy metabolism and promotes glycolysis in LUAD progression.

6667 IGF1 Induces The Expression of CYR61 In Metastatic Prostate Cancer

Abstract Disclosure: G.L. Ortiz Hernández: None. M. Walker: None. L. Woods-Burnham: None. R.A. Kittles: None. C.A. Casiano: None. S.L. Neuhausen: None. The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Our goal was to assess the contribution of the interaction of CYR61-IGF1 to PCa cell proliferation. Their combined role may explain a proportion of aggressive PCa. This study was designed to characterize the role of IGF1-induced expression of CYR61 in metastatic PCa cellular models and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, migration, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. In addition, we found that IGF1 induces protein expression of CYR61 in a time-dependent manner, validated by both immunoblotting and confocal microscopy. PC3 cell lines are androgen receptor negative (AR-) and glucocorticoid receptor positive (GR+), which is characteristic of aggressive, advanced metastatic castration-resistant prostate cancer (mCRPC). Currently, we are conducting the same experiments in LNCaP cells, which are AR+/GR-, to determine if the CYR61-IGF1 interplay also occurs in PCa cells capable of AR signaling. In conclusion, we found that CYR61 expression is induced by IGF1 and its inhibition decreased the capacity of metastatic PCa cells to proliferate. Our results suggest that CYR61 inhibition may have the potential for treating metastatic PCa. Presentation: 6/1/2024

6667 IGF1 Induces the Expression of CYR61 in Metastatic Prostate Cancer

Abstract Disclosure: G.L. Ortiz Hernández: None. M. Walker: None. L. Woods-Burnham: None. R.A. Kittles: None. C.A. Casiano: None. S.L. Neuhausen: None. The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF1). High serum levels of IGF1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. The correlation between PCa aggressiveness and circulating levels of CYR61 and IGF1 is poorly studied. Our goal was to assess the contribution of the interaction of CYR61-IGF1 to PCa cell proliferation. Their combined role may explain a proportion of aggressive PCa. This study was designed to characterize the role of IGF1-induced expression of CYR61 in metastatic PCa cellular models and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, migration, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. In addition, we found that IGF1 induces protein expression of CYR61 in a time-dependent manner, validated by both immunoblotting and confocal microscopy. PC3 cell lines are androgen receptor negative (AR-) and glucocorticoid receptor positive (GR+), which is characteristic of aggressive, advanced metastatic castration-resistant prostate cancer (mCRPC). Currently, we are conducting the same experiments in LNCaP cells, which are AR+/GR-, to determine if the CYR61-IGF1 interplay also occurs in PCa cells capable of AR signaling. In conclusion, we found that CYR61 expression is induced by IGF1 and its inhibition decreased the capacity of metastatic PCa cells to proliferate. Our results suggest that CYR61 inhibition may have the potential for treating metastatic PCa. Presentation: 6/1/2024

Kinase library screening identifies IGF-1R as an oncogenic vulnerability in intrahepatic cholangiocarcinoma stem-like cells

BackgroundIntrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer of the peripheral bile ducts and is recognized by the abundance of cancer stem-like cells (CSCs) within the tumor mass. While CSC markers in iCCA are well-defined, the molecular vulnerabilities of this subpopulation remain elusive. MethodsThe 96-well, three dimensional (3D) tumorsphere culture was adapted from a well-established CSC model, validated for CSC markers through gene expression analysis. Kinase library screening was then conducted to reveal potential oncogenic vulnerable pathways. RNA interference was utilized to stably silence the candidate gene in three iCCA cell lines and its impact on iCCA cell proliferation and tumorsphere formation efficiency (TFE) was evaluated. ResultsKinase inhibitor library screening identified the top 50 kinase inhibitors crucial for tumorsphere viability, with 11 inhibitors targeting the IGF-1R/PI3K/AKT axis. Further dose-dependent analysis of the top ‘hit’ inhibitors confirmed IGF-1R as the candidate molecule. Upon stably silencing of IGF-1R, all three iCCA cell lines exhibited decreased AKT activation, impeded proliferation and reduced TFE, indicating a decline in CSC subpopulations. ConclusionsIGF-1R plays a critical role in maintaining iCCA-stem like cell populations. General significanceOur data highlight the potential utility of IGF-1R as a prognostic marker of iCCA and a therapeutic target for eliminating its CSC subpopulation.

8038 Targeting Breast Cancer Stem Cells With Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

8038 Targeting Breast Cancer Stem Cells with Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

SRSF6 modulates histone-chaperone HIRA splicing to orchestrate AR and E2F activity in prostate cancer.

Despite novel therapeutic strategies, advanced-stage prostate cancer (PCa) remains highly lethal, pointing out the urgent need for effective therapeutic strategies. While dysregulation of the splicing process is considered a cancer hallmark, the role of certain splicing factors remains unknown in PCa. This study focuses on characterizing the levels and role of SRSF6 in this disease. Comprehensive analyses of SRSF6 alterations (copy number/mRNA/protein) were conducted across eight well-characterized PCa cohorts and the Hi-MYC transgenic model. SRSF6 was up-regulated in PCa samples, correlating with adverse clinical parameters. Functional assays, both in vitro (cell proliferation, migration, colony, and tumorsphere formation) and in vivo (xenograft tumors), demonstrated the impact of SRSF6 modulation on critical cancer hallmarks. Mechanistically, SRSF6 regulates the splicing pattern of the histone-chaperone HIRA, consequently affecting the activity of H3.3 in PCa and breast cancer cell models and disrupting pivotal oncogenic pathways (AR and E2F) in PCa cells. These findings underscore SRSF6 as a promising therapeutic target for PCa/advanced-stage PCa.

Investigating SNHG3 as a potential therapeutic approach for HCC stem cells

IntroductionHepatocellular Carcinoma (HCC) is a common malignant tumor worldwide. Long Non-Coding RNA (lncRNA) has gained attention in tumor biology, and this study aims to investigate the role of lncRNA SNHG3 in HCC, specifically in the self-renewal and maintenance of liver cancer stem cells. MethodsThe expression of lncRNA SNHG3 was analyzed in HCC and adjacent normal tissue using the TCGA database. The expression levels of SNHG3 in HCC cell lines (Hep3B, HepG2, Huh7) were detected using qRT-PCR and Western blot techniques. Functional assays, including CCK-8, soft agar colony formation, and tumor sphere formation, were performed to evaluate the impact of SNHG3 on HCC stem cell functionality. MeRIP-qPCR was also used to investigate the regulatory role of SNHG3 in m6A modification of ITGA6 mRNA mediated by METTL3. ResultsThe study found that SNHG3 was significantly upregulated in HCC tissue and cell lines compared to normal liver tissue. SNHG3 expression correlated with the pathological stage, metastasis status, and tumor size of liver cancer. Inhibiting SNHG3 reduced proliferation, colony formation, and tumor sphere formation ability in HCC stem cells. SNHG3 also played a role in regulating the m6A modification and expression of ITGA6 through METTL3. ConclusionThis study emphasizes the upregulation of lncRNA SNHG3 and its role in HCC stem cell self-renewal. SNHG3 may regulate the m6A modification of ITGA6 mRNA through its interaction with METTL3, impacting the function of liver cancer stem cells. These findings support the potential of targeting SNHG3 as a therapeutic approach for HCC.

Abstract A077: The role of RhoV in basal-like pancreatic cancer

Abstract Background Pancreatic ductal adenocarcinoma is a devastating disease lacking targeted therapy. Basal-like pancreatic cancer is a tumor subtype with a worse clinical outcome than other subtypes. These cancers are characterized by the expression of basal keratins and mesenchymal markers. However, the genes that drive the basal-like phenotype are poorly understood. Ras homolog family member V (RhoV) is one of the Rho GTPases, and it has been demonstrated to drive the progression of lung and breast cancers. The current study aims to reveal the role of RhoV in basal-like pancreatic cancer. Design RhoV, p63, and CK5/6 were stained in sequential sections by immunohistochemistry. Expression of RhoV in pancreatic cancer and cell lines was reanalyzed based on the data from ProteinAtlas and Broad Insitute DepMap Portal. Basal cell line, BxPC3, and non-basal cell line, Panc1 were selected for functional evaluation based on their differentiated RhoV expression. RhoV gene was overexpressed and knocked down using the CRISPR/gRNA-based Lentiviral system, followed by sphere formation, colony formation, migration, and invasion assays. Results RhoV, p63, and CK5/6 were expressed in the same histologic location of basal pancreatic cancer. The gene expression level of RhoV was co-expressed with p63 (a classic basal marker) in four pancreatic cancer cell lines (broadinstitute.org). Knockout of RhoV by three runs of gRNAs in BxPC3 cells all significantly attenuated tumor sphere formation with less colony formation as compared to mock knockout. Overexpression of RhoV in non-basal-like cells, Panc 1, significantly increased the size of sphere formation, numbers of colony formation, and p38 activation compared to EV control. Cell migration and invasion assays showed the same RhoV-dependent migration and invasion in corresponding cells. Panc1 cells with RhoV knockout significantly generated smaller tumor xenografts in immunodeficient mice and RhoV overexpression led to larger tumor sizes. Conclusion Finding the driver of the basal-like phenotype helps to reveal the pathophysiology of the basal-like subtype of pancreatic cancer and facilitates the identification of new targets for therapeutic purposes. Our preliminary results indicated that RhoV, a small G protein from the Rho family of GTPases, played essential roles in tumor proliferation and progression, by activating the p38 signaling pathway. Further study will be focused on revealing Rhov's downstream pathways and exploring the role of RhoV in apoptosis/cell cycle, cell metabolism, autophagy, etc. in basel-like pancreatic cancer. Citation Format: Shang Wu, Jia Feng, Selina Gao, Ting Wang, Zebang Li, Yifei Liu, Chiung-Kuei Huang, Shaolei Lu. The role of RhoV in basal-like pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A077.

Integrated machine learning algorithms identify KIF15 as a potential prognostic biomarker and correlated with stemness in triple-negative breast cancer

Cancer stem cells (CSCs) have the potential to self-renew and induce cancer, which may contribute to a poor prognosis by enabling metastasis, recurrence, and therapy resistance. Hence, this study was performed to identify the association between CSC-related genes and triple-negative breast cancer (TNBC) development. Stemness gene sets were downloaded from StemChecker. Based on the online databases, a consensus clustering algorithm was conducted for unsupervised classification of TNBC samples. The variations between subtypes were assessed with regard to prognosis, tumor immune microenvironment (TIME), and chemotherapeutic sensitivity. The stemness-related gene signature was established and random survival forest analysis was employed to identify the core gene for validation experiments and tumor sphere formation assays. 499 patients with TNBC were classified into three subgroups and the Cluster 1 had a better OS than others. After that, WGCNA study was performed to identify genes important for Cluster 1 subtype. Out of all 8 modules, the subtype of Cluster 1 and the yellow module with 103 genes demonstrated the largest positive association. After that, a four-gene stemness-related signature was established. Based on the yellow module, the 39 potential pivotal genes were subjected to the random forest survival analysis to find out the gene that was relatively important for OS. KIF15 was confirmed as the targeted gene by LASSO and random survival forest analyses. In vitro experiments, the downregulation of KIF15 promoted the stemness of TNBC cells. The expression levels of stem cell markers Nanog, SOX2, and OCT4 were found to be elevated in TNBC cell lines after KIF15 inhibition. A stemness-associated risk model was constructed to forecast the clinical outcomes of TNBC patients. The downregulation of KIF15 expression in a subpopulation of TNBC stem cells may promote stemness and possibly TNBC progression.

The therapeutic effect of DX2 inhibition in nicotine-induced lung cancer progression

Alternative splicing products of AIMP2 and AIMP2-DX2 (DX2) have been reported to be associated with human lung cancer. In fact, DX2 expression is elevated in human lung cancers and DX2 transgenic mice also develop lung cancer, in particular, Small Cell Lung Cancer (SCLC). However, the mechanism by which DX2 is induced during cancer progression has not been clearly elucidated. Here, we show that DX2 is induced by nicotine, the main component of smoking-related chemicals, which can stabilize the human epidermal growth factor receptor 2 (HER2) protein and transcriptionally increase sonic hedgehog (Shh). Indeed, nicotine showed tumorigenicity via DX2 by promoting spheroid formation and in vivo lung and kidney cancer progression. Moreover, the elimination of DX2 using siRNA or an optimized inhibitor (SNU-14) blocked the induction of HER2, Shh and completely suppressed tumor sphere formation in response to nicotine. These results indicate that DX2 is critical for lung cancer progression, and a specific DX2 inhibitor would be useful for the treatment of human cancers, including SCLC and Non-Small Cell Lung Cancer (NSCLC).

The Protective Effects of an Aged Black Garlic Water Extract on the Prostate.

Chronic inflammation is a recognized risk factor for various cancers, including prostate cancer (PCa). We aim to explore the potential protective effects of aged black garlic extract (ABGE) against inflammation-induced prostate damage and its impact on prostate cancer cell lines. We used an ex vivo model of inflammation induced by Escherichia coli lipopolysaccharide (LPS) on C57BL/6 male mouse prostate specimens to investigate the anti-inflammatory properties of ABGE. The gene expression levels of pro-inflammatory biomarkers (COX-2, NF-κB, and TNF-α, IL-6) were measured. Additionally, we evaluated ABGE's therapeutic effects on the prostate cancer cell lines through in vitro functional assays, including colony formation, tumorsphere formation, migration assays, and phosphorylation arrays to assess the signaling pathways (MAPK, AKT, JAK/STAT, and TGF-β). ABGE demonstrated significant anti-inflammatory and antioxidant effects in preclinical models, partly attributed to its polyphenolic content, notably catechin and gallic acid. In the ex vivo model, ABGE reduced the gene expression levels of COX-2, NF-κB, TNF-α, and IL-6. The in vitro studies showed that ABGE inhibited cell proliferation, colony and tumorsphere formation, and cell migration in the prostate cancer cells, suggesting its potential as a therapeutic agent. ABGE exhibits promising anti-inflammatory and anti-cancer properties, supporting further investigation into ABGE as a potential agent for managing inflammation and prostate cancer.

The dual role of POSTN in maintaining glioblastoma stem cells and the immunosuppressive phenotype of microglia in glioblastoma

BackgroundGlioblastoma (GBM) is an immunosuppressive, universally lethal cancer driven by glioblastoma stem cells (GSCs). The interplay between GSCs and immunosuppressive microglia plays crucial roles in promoting the malignant growth of GBM; however, the molecular mechanisms underlying this crosstalk are unclear. This study aimed to investigate the role of POSTN in maintaining GSCs and the immunosuppressive phenotype of microglia.MethodsThe expression of POSTN in GBM was identified via immunohistochemistry, quantitative real-time PCR, and immunoblotting. Tumorsphere formation assay, Cell Counting Kit-8 assay and immunofluorescence were used to determine the key role of POSTN in GSC maintenance. ChIP-seq and ChIP-PCR were conducted to confirm the binding sequences of β-catenin in the promoter region of FOSL1. Transwell migration assays, developmental and functional analyses of CD4+ T cells, CFSE staining and analysis, enzyme-linked immunosorbent assays and apoptosis detection tests were used to determine the key role of POSTN in maintaining the immunosuppressive phenotype of microglia and thereby promoting the immunosuppressive tumor microenvironment. Furthermore, the effects of POSTN on GSC maintenance and the immunosuppressive phenotype of microglia were investigated in a patient-derived xenograft model and orthotopic glioma mouse model, respectively.ResultsOur findings revealed that POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/PI3K/AKT/β-catenin/FOSL1 pathway. In addition to its intrinsic effects on GSCs, POSTN can recruit microglia and upregulate CD70 expression in microglia through the αVβ3/PI3K/AKT/NFκB pathway, which in turn promotes Treg development and functionality and supports the formation of an immunosuppressive tumor microenvironment. In both in vitro models and orthotopic mouse models of GBM, POSTN depletion disrupted GSC maintenance, decreased the recruitment of immunosuppressive microglia and suppressed GBM growth.ConclusionOur findings reveal that POSTN plays critical roles in maintaining GSCs and the immunosuppressive phenotype of microglia and provide a new therapeutic target for treating GBM.

Circ_0008315 promotes tumorigenesis and cisplatin resistance and acts as a nanotherapeutic target in gastric cancer

IntroductionCisplatin-based chemotherapy is one of the fundamental therapeutic modalities for gastric cancer (GC). Chemoresistance to cisplatin is a great clinical challenge, and its underlying mechanisms remain poorly understood. Circular RNAs (circRNAs) are involved in the pathophysiology of multiple human malignancies.MethodsHigh-throughput sequencing was performed to determine the differentially expressed profile of circRNA in GC tissues and cisplatin-resistant GC cells. Quantitative real-time polymerase chain reaction and Fluorescence in situ hybridization was utilized to confirm the dysregulation of circ_0008315 in GC tissues. To evaluate the prognostic significance of circ_0008315 in GC, we used Kaplan-Meier plot. The self-renewal ability of drug-resistant GC cell was verified through tumor sphere formation assay. GC organoids were constructed to simulate the tumor microenvironment and verified the function of circ_0008315 in cisplatin resistance of gastric cancer. In vivo evaluation was conducted using patient-derived xenograft models. Dual-luciferase reporter gene, RNA immunoprecipitation and miRNA pull-down assays were employed to investigate the molecular mechanisms of circ_0008315 in GC.ResultsWe revealed that a novel circRNA hsa_circ_0008315 was upregulated in GC and cisplatin-resistant GC cells. Elevated circ_0008315 was also observed in cisplatin-resistant GC organoid model. High circ_0008315 expression predicted unfavorable survival outcome in GC patients. Downregulation of circ_0008315 expression inhibited proliferation, mobility, and epithelial-mesenchymal transition of GC cells in vitro and in vivo. Reducing circ_0008315 expression in cisplatin-resistant GC organoid model reversed cisplatin resistance. Mechanistically, circ_0008315 modulated the stem cell properties of GC through the miR-3666/CPEB4 signaling pathway, thereby promoting cisplatin resistance and GC malignant progression. Furthermore, we developed PLGA-PEG nanoparticles targeting circ_0008315, and the nanoparticles could effectively inhibit GC proliferation and cisplatin resistance.ConclusionCirc_0008315 exacerbates GC progression and cisplatin resistance, and can be used as a prognostic predictor. Circ_0008315 may function as a promising nanotherapeutic target for GC treatment.Graphical

Modulation of PD-L1 by Astragalus polysaccharide attenuates the induction of melanoma stem cell properties and overcomes immune evasion

BackgroundMelanoma is a highly aggressive form of skin cancer. The existence of cancer stem cells (CSCs) and tumor immune evasion are two major causes of melanoma progression, but no effective treatment has been found at present. Astragalus polysaccharide (APS) is a principal active component derived from Astragalus membranaceus, showing anti-tumor effects in various tumors including melanoma. However, the underlying mechanism is still unclear.MethodsThe regulation of APS on self-renewal ability and CSC markers expression in melanoma stem cells (MSCs) was measured by tumor sphere formation and tumorigenicity assays, RT-qPCR, and western blot. Flow cytometry was conducted to evaluate the activation of immune system by APS in melanoma mice. Further, the mechanism was explored based on PD-L1 overexpression and knock-down B16 cells.ResultsAPS attenuated the tumor sphere formation of MSCs in vitro as well as the tumorigenicity in vivo. It also decreased the expression of CD133, BMI1 and CD47. Based on the PD-L1 overexpression and knock-down B16 cells, it was confirmed that APS inhibited the induction of MSCs by down-regulating PD-L1 expression. Meanwhile, APS increased the infiltration of CD4+ and CD8+T cells in tumor tissues because of its inhibitory effect on PD-L1.ConclusionsAPS inhibited MSC induction and overcame tumor immune evasion through reducing PD-L1 expression. This study provided compelling evidence that APS could be a prospective therapeutic agent for treating melanoma.

Integrin αV Inhibition by GMI, a Ganoderma Microsporum Immunomodulatory Protein, Abolish Stemness and Migration in EGFR-Mutated Lung Cancer Cells Resistant to Osimertinib.

Integrins, the receptors of the extracellular matrix, are critical in the proliferation and metastasis of cancer cells. GMI, a Ganoderma microsporum immunomodulatory protein, possesses anticancer and antivirus abilities. The object of this study is to investigate the role of GMI in the integrins signaling pathway in lung cancer cells that harbor the EGFR L858R/T790M double mutation and osimertinib-resistance. Liquid chromatography-mass spectrometry and western blot assay were used to investigate the effect of GMI on inhibiting the protein expressions of integrins in H1975 cells. The migration ability and xenograft tumor growth of H1975 were suppressed by GMI. To elucidate the role of the integrin family in lung cancer resistant to osimertinib (AZD-9291, Tagrisso), H1975 cells were used to establish the osimertinib-resistant cells, named H1975/TR cells. The expressions of Integrin αV and stemness markers were much higher in H1975/TR cells than in H1975 cells. GMI suppressed cell viability, tumor spheroid growth, and the expressions of integrin αV and β1 in H1975/TR cells. Furthermore, GMI suppressed the expressions of stemness markers and formation of tumor spheres via blocking integrin αV signaling cascade. This is the first study to reveal the novel function of GMI in constraining cancer stem cells and migration by abolishing the integrin αV-related signaling pathway in EGFR-mutated and osimertinib-resistant lung cancer cells.

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

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

AB046. Dual role of POSTN in maintaining glioblastoma stem cells and immune-suppressive microglia in glioblastoma.

Glioblastoma (GBM) is an immunosuppressive, universally lethal cancer driven by GBM stem cells (GSCs). The interplay between GSCs and the immunosuppressive microglia plays crucial roles in promoting malignant growth of GBM, however, the molecular mechanisms underlying this crosstalk are incompletely understood. We performed RNA sequencing to explore the mechanism by which periostin (POSTN) regulates GSCs and microglia. The biological function of POSTN in GBM development was confirmed in vitro and in vivo. Specifically, tumorsphere formation assay, proliferation analysis, migration assays, enzyme-linked immunosorbent assay, immunoblotting, and intracranial mouse model were performed. We identified POSTN secreted from GSCs promotes GSC self-renewal and tumor growth via activation of the αVβ3/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/β-catenin/FOS like antigen 1 (FOSL1) pathway. In addition to its GSC intrinsic effects, POSTN is able to recruit microglia and upregulate cluster of differentiation 70 (CD70) expression through PI3K/AKT/nuclear factor-kappa B (NFκB) pathway in microglial cells, which in turn promotes the Treg development and functionality, and generates an immunosuppressive tumor microenvironment. Inhibition POSTN disrupts the GSC maintenance, inhibits recruitment of immunosuppressive microglial, reduces Treg development and function, and suppresses GBM growth, suggesting that targeting POSTN may effectively improve GBM treatment. In conclusion, our study defined POSTN as a key regulator in mediating the molecular crosstalk between GSCs and immune-suppressive Microglia in the tumor microenvironment in GBM. POSTN activates the PI3K/AKT/β-catenin/FOSL1 pathway in an autocrine manner to promote GSC self-renewal and tumor growth. At the same time, POSTN recruits microglia in a paracrine manner and upregulates the expression of CD70 in microglia through the PI3K/AKT/NFκB pathway, thereby promoting the development and function of Treg and generating an immunosuppressive tumor microenvironment. Our findings indicate that targeting the POSTN gene may be a promising approach to ablating GSCs, breaking the immunosuppressive environment and overcoming treatment resistance in GBM.

Progesterone Receptor Signaling Promotes Cancer Associated Fibroblast Mediated Tumorigenicity in ER+ Breast Cancer.

Breast cancer progression involves intricate interactions between cancer cells and the tumor microenvironment (TME). This study elucidates the critical role of progesterone receptor (PR) signaling in mediating the protumorigenic effects of cancer-associated fibroblasts (CAFs) on estrogen receptor-positive (ER+) luminal breast cancer cells. We demonstrate that CAFs produce physiologically relevant levels of estrogen and progesterone, which significantly contribute to breast cancer tumorigenicity. Specifically, CAF conditioned media (CM) promoted PR-dependent anchorage-independent growth, tumorsphere formation/stem cell expansion, and CD44 upregulation. CAF cells formed co-clusters more frequently with PR+ breast cancer cells relative to PR-null models. While both PR isoforms mediated these actions, PR-A was a dominant driver of tumorsphere formation/stemness, while PR-B induced robust CD44 expression and CAF/tumor cell co-cluster formation. CD44 knockdown impaired CAF/tumor cell co-clustering. Fibroblast growth factor 2 (FGF2), also secreted by CAFs, phosphorylated PR (Ser294) in a MAPK-dependent manner and activated PR to enhance CD44 expression and breast cancer tumorigenicity. The FGF receptor (FGFR) inhibitor PD173074 diminished CAF- and FGF2-dependent PR activation, tumorsphere formation, and co-clustering. In summary, this study reveals a novel mechanism through which stromal CAFs orchestrate elevated PR signaling in ER+ luminal breast cancer via secretion of both progesterone and FGF2, a potent activator of ERK1/2. Understanding tumor cell/TME interactions provides insights into potential therapeutic strategies aimed at disrupting PR- and/or FGF2/FGFR-dependent signaling pathways to prevent early metastasis in patients with ER+ breast cancer.

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

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