High Levels Of CD44 Research Articles

Hedgehog signaling promotes sorafenib resistance in hepatocellular carcinoma patient-derived organoids

BackgroundThe mechanism underlying sorafenib resistance in hepatocellular carcinoma (HCC) remains unclear. Accumulating evidence suggests that tumor-initiating cells (TICs) are a pivotal driving force. Both CD44 and Hedgehog signaling play crucial roles in TIC properties in HCC. In this study, we explored the roles of CD44 and Hedgehog signaling in sorafenib resistance and evaluated the therapeutic effect of cotreatment with sorafenib and Hedgehog signaling inhibitors in HCC patient-derived organoid (PDO) models to improve treatment efficacy.MethodsWe collected HCC specimens to establish PDO models. Cell viability and malignant transformation properties were investigated after treatment with different TIC-related inhibitors alone or in combination with sorafenib to evaluate the therapeutic effect in PDOs and cell lines by in vitro and in vivo experiments. Expression levels of Hedgehog signaling proteins and CD44 were monitored to reveal potential relationships.ResultsWe demonstrated that our HCC PDO models strongly maintained the histological features of the corresponding tumors and responded to drug treatment. Furthermore, CD44-positive HCC PDOs were obviously resistant to sorafenib, and sorafenib increased CD44 levels. A drug screen showed that compared with Notch, Hippo and Wnt signaling inhibitors, a Hedgehog signaling inhibitor (GANT61) potently suppressed HCC PDO cell viability. In addition, there was a highly synergistic effect in vitro and in vivo on the suppression of cell viability and malignant properties when sorafenib and GANT61 were added to CD44-positive HCC PDOs and cell lines, respectively. Furthermore, the upregulation of CD44 and Hedgehog signaling induced by sorafenib was reversed by GANT61.ConclusionsGANT61 significantly suppressed Hedgehog signaling to reverse sorafenib resistance in CD44-positive HCC. The combination of sorafenib and Hedgehog signaling inhibitors might be effective in HCC patients with high CD44 levels as a personalized-medicine approach.

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Characterizing CD44 regulatory microRNAs as putative therapeutic agents in human melanoma.

The multistructural and multifunctional transmembrane glycoprotein CD44 is overexpressed in many tumors of distinct origin including malignant melanoma and contributes to a poor prognosis by affecting cell proliferation, cell migration, and also the sensitivity for apoptosis induction. Previous studies reported so far 15 CD44 regulatory microRNAs (miRs) in different cell systems.Using a novel method for miR affinity purification miR-143-3p was identified as most potent binder to the 3’ untranslated region (UTR) of CD44. Overexpression of miR-143-3p in melanoma cells inhibits CD44 translation, which is accompanied by a reduced proliferation, migration and enhanced daunorubicin induced apoptosis of melanoma cells in vitro.Analyses of discordant CD44 and miR-143-3p expression levels in human melanocytic nevi and dermal melanoma samples demonstrated medium to high CD44 levels with no association to tumor grading or staging. The CD44 expression correlated to PD-L1, but not to MART-1 expression in malignant melanoma. Interestingly, the CD44 expression was inversely correlated to the infiltration of pro-inflammatory immune effector cells.In conclusion, the tumor suppressive miR-143-3p was identified as the most potent CD44 inhibitory miR, which affects growth characteristics of melanoma cells suggesting the implementation of miR-143-3p as as a potential anti-CD44 therapy of malignant melanoma.

Synergistic Enhancement of Cellular Uptake With CD44-Expressing Malignant Pleural Mesothelioma by Combining Cationic Liposome and Hyaluronic Acid–Lipid Conjugate

Malignant pleural mesothelioma (MPM) is a highly aggressive form of cancer, with a median survival of less than 1 year. It is well known that the hyaluronan (HA) receptor CD44 is highly expressed by MPM cells and is reported to be correlated with a poor prognosis. We herein report on the development of a new type if drug delivery system against CD44 that involves the use of lipid nanoparticles (LNPs) equipped with a new type of HA derivative. In this study, we evaluated HA-lipid conjugation (HAL) via the end of the HA molecule through reductive amination, a process that allowed the carboxylate group to remain intact. As a result, the HAL-modified LNP appears to be a potent nanoparticle for dealing with MPM. Surprisingly, the use of a combination of a cationic lipid and HAL had a synergistic effect on cellular uptake in MPM and consequently permitted an anti-cancer drug such as cis-diamminedichloro-platinum(II) (CDDP). Intrapleural injection of CDDP-loaded HAL-LNP (1.5 mg/kg as CDDP) per week significantly suppressed the progression of this type of cancer in an MPM orthotopic model. These results suggest that HAL-modified LNP represents a potent delivery system for MPM cells that express high levels of CD44.

The effect of sodium valproate on differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in two-dimensional culture and fibrin scaffold conditions.

Differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes is a complex phenomenon, and attempts to find an effective inducing agent are still ongoing. We studied the effect of fibrin scaffold and sodium valproate (VPA, as a histone deacetylase inhibitor) on the differentiation of human adipose-derived stem cells (hADSCs) into cardiomyocyte-like cells. The cells were cultured in culture flask (2D) and in fibrin scaffold (3D), fabricated of human plasma fibrinogen, with and without VPA (1mM). QRT-PCR, Western blot, and immunochemistry assays were used to evaluate the expression of cardiac markers at gene and protein levels. High levels of CD44, CD90, CD73, and CD105 were expressed on the surface of hADSCs. Treated encapsulated hADSCs (3D) presented significantly higher mRNA expression of HAND1 (1.54-fold), HAND2 (1.59-fold), cTnI (1.76-fold), MLC2v (1.4-fold), Cx43 (1.38-fold), βMHC (1.34-fold), GATA4 (1.48-fold), and NKX2.5 (1.66-fold) in comparison to 2D conditions at four weeks after induction. The protein expressions of NKX2.5 (0.78 vs 0.65), cTnI (1.04 vs 0.81), and Cx43 (1.11 vs 1.08) were observed in the differentiated cells both in 3D and 2D groups, while control cells were absolutely negative for these proteins. The frequency of cTnI and Cx43-positive cells was significantly higher in 3D (24.2 ± 15 and 12 ± 3%) than 2D conditions (19.8 ± 3 and 10 ± 2%). Overall, the results showed that VPA can increase cardiomyogenesis in hADSCs and that fibrin scaffold enhances the inductive effect of VPA. Results of this study may improve cell-based protocols for implementation of more successful cardiac repair strategies.

Low CD8⁺ T Cell Infiltration and High PD-L1 Expression Are Associated with Level of CD44⁺/CD133⁺ Cancer Stem Cells and Predict an Unfavorable Prognosis in Pancreatic Cancer.

Cancer immunotherapy targeting immune checkpoints has exhibited promising clinical outcomes in many cancers, but it offers only limited benefits for pancreatic cancer (PC). Cancer stem cells (CSCs), a minor subpopulation of cancer cells, play important roles in tumor initiation, progression, and drug resistance. Accumulating evidence suggests that CSCs employ immunosuppressive effects to evade immune system recognition. However, the clinical implications of the associations among CD8+ T cells infiltration, programmed death receptor ligand-1 (PD-L1) expression, and CSCs existence are poorly understood in PC. Immunostaining and quantitative analysis were performed to assess CD8+ T cells infiltration, PD-L1 expression, and their relationship with CD44+/CD133+ CSCs and disease progression in PC. CD8+ T cells infiltration was associated with better survival while PD-L1 expression was correlated with PC recurrence. Both the low CD8+ T cells infiltration/high PD-L1 expression group and the high CD8+ T cells infiltration/high PD-L1 expression group show high levels of CD44+/CD133+ CSCs, but patients with low CD8+ T cells infiltration/high PD-L1 expression had worse survival and higher recurrence risk than those with high CD8+ T cells infiltration/high PD-L1 expression. Moreover, high infiltration of CD8+ T cells could reduce unfavorable prognostic effect of high co-expression of PD-L1 and CD44/CD133. Our study highlights an interaction among CD8+ T cells infiltration, PD-L1 expression, and CD44+/CD133+ CSCs existence, which contributes to PC progression and immune evasion.

Significance of Glioma Stem-Like Cells in the Tumor Periphery That Express High Levels of CD44 in Tumor Invasion, Early Progression, and Poor Prognosis in Glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and a subpopulation of glioma stem-like cells (GSCs) is likely responsible for the invariable recurrence following maximum resection and chemoradiotherapy. As most GSCs that are located in the perivascular and perinecrotic niches should be removed during tumor resection, it is very important to know where surviving GSCs are localized. Here, we investigated the existence and functions of GSCs in the tumor periphery, which is considered to constitute the invasion niche for GSCs in GBM, by analyzing expression of stem cell markers and stem cell-related molecules and measuring particular activities of cultured GSCs. In addition, the relationship between GSCs expressing particular stem cell markers and pathological features on MRI and prognosis in GBM patients was analyzed. We showed that GSCs that express high levels of CD44 are present in the tumor periphery. We also found that vascular endothelial growth factor (VEGF) is characteristically expressed at a high level in the tumor periphery. Cultured GSCs obtained from the tumor periphery were highly invasive and have enhanced migration phenotype, both of which were markedly inhibited by CD44 knockdown. Higher expression of CD44 in the tumor periphery than in the core was correlated with a highly invasive feature on MRI and was associated with early tumor progression and worse survival, whereas lower expression of CD44 in the tumor periphery corresponded to low invasion and was associated with longer survival. The low invasion type on MRI tended to show high levels of VEGF expression in the tumor periphery, thus presenting the tumor with high proliferative activity. These results imply the significance of GSCs with high levels of CD44 expression in the tumor periphery compared to the core, not only in tumor invasion but also rapid tumor progression and short survival in patients with GBM.

Abstract LB-053: Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system

Abstract Breast cancer stem cells (BCSCs) display plasticity allowing them to transition between quiescent mesenchymal- (M) and proliferative epithelial-like (E) states. This dynamic equilibrium of BCSCs in distinct M- and E-like states coordinately drives tumorigenesis, metastasis and treatment resistance. Despite the functional significance of CSC plasticity in tumor development and progression, tracing of BCSC state transitions under physiologic conditions is hampered by the lack of a functional dual fluorescent reporter to monitor state dynamics of BCSCs at single cell resolution. To this end, we developed a dual fluorescent reporter to assess EMT-MET state transitions of BCSCs in real-time. As E-BCSCs express high levels of ALDH1A3, we generated an ALDH1A3 fluorescent reporter system utilizing Crisper-Cas 9 mediated knockin of mCherry downstream of the ALDH1A3 promotor. To trace M-BCSCs that express high levels of CD44 and vimentin, we constructed a lentiviral reporter expressing GFP driven by a portion of the vimentin promotor that is specifically expressed in mesenchymal CD44+ cells. Dual reporter cells were generated by infection of ALDH1A3_mCherry expressing cells with the lentivirus expressing Vimentin_GFP. To assess stem cell characteristics of these reporter cells, we first examined their sphere-forming capacity. Both GFP-mCherry+ (mCherry+) and GFP+mCherry- (GFP+) cells have significantly higher capacity to form tumor spheres than GFP-mCherry- (DN) cells while the GFP+mCherry+ (DP) cells harbor the highest sphere-forming ability. To determine the relationship between cells expressing these reporters, we generated tumor spheres from single cells and examined the fluorescent pattern of resultant tumor spheres. While spheres generated from DP cells display mixed phenotypes of cells expressing mCherry and/or GFP, spheres derived from mCherry+ cells maintain mCherry+. Interestingly, spheres derived from GFP+ cells generated three distinct phenotypes with some spheres expressing strong GFP and weak mCherry fluorescence while others expressing strong mCherry but weak or no GFP signal. These distinct phenotypes of tumor spheres derived from GFP+ cells suggest that M-BCSCs possess capacity to transition to a dual (GFP+mCherry+) or epithelial-like (GFP-mCherry+) state in order to form tumor spheres at non-attachment culture conditions that are normally associated with increased oxidative stress. These results suggest that the DP cells are the most primitive, with the greatest tumor sphere forming capacity and multistate differentiation potential followed by the Vimentin_GFP+ cells, ALDH1A3_mcherry+ cells and DN cells. By time-lapse microscopy, we further monitored how metabolic/oxidative stress induced by glycolytic inhibitor 2DG or H2O2 affects BCSC state dynamics at single cell resolution. We found that oxidative stress promotes the transition of GFP+mCherry- M-BCSCs to GFP+mCherry+ (bipotent) and GFP-mCherry+ (E-like) BCSCs. In contrast, the antioxidant N-acetyl cysteine promoted the transition of bipotent and E-like BCSCs to a GFP+mCherry- M-like state. These studies demonstrate that the redox state of the tumor microenvironment regulates state transitions of BCSCs. This dual fluorescent reporter system should prove useful in monitoring CSC state transitions under more physiologically relevant in vivo conditions. Citation Format: Ming Luo, Michael Brooks, Joseph Burnett, Nathan Truchan, Duxin Sun, Max S. Wicha. Tracing redox-regulated breast cancer stem cell state transition using a dual fluorescent reporter system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-053.

Brain proteomic differences between wild-type and CD44- mice induced by chronic Toxoplasma gondii infection.

Chronic clinical Toxoplasma gondii (T. gondii) infection is the primary disease state that causes severe encephalitis. CD44 is a member of the cell adhesion molecule family and plays an important role in T. gondii infection. However, proteomic changes in CD44 during chronic T. gondii infection have rarely been reported. Thus, an iTRAQ-based proteomic study coupled with 2D-LC-MS/MS analysis was performed to screen CD44-related proteins during chronic T. gondii infection. As a result, a total of 2612 proteins were reliably identified and quantified. Subsequently, 259, 106, and 249 differentially expressed proteins (DEPs) were compared between CD44- mice (A) vs wild-type mice (B), B vs wild-type mice infected with T. gondii (C), and C vs CD44- mice infected with T. gondii (D). Gene ontology, KEGG pathway, and protein-protein interaction analyses were performed on the DEPs. According to the results, immune-related proteins were altered significantly among the A vs B, B vs C, and C vs D comparisons, which might indicate that chronic T. gondiiinfection caused changes in the host immune response. Additionally, Ca2+- and metabolism-related proteins were upregulated in C vs D, which supported the hypothesis that CD44 mediated the production of host Ca2+ and IFN-γ and that the parasite preferentially invaded cells expressing high levels of CD44. The present findings validate and enable a more comprehensive knowledge of the role of CD44 in hosts chronically infected with T. gondii, thus providing new ideas for future studies on the specific functions of CD44 in latent toxoplasmosis.

Targeting CD44 by CRISPR-Cas9 in Multi-Drug Resistant Osteosarcoma Cells

Background/Aims: Drug resistance is the main difficulty for the current treatment for osteosarcoma. Cluster of differentiation 44 (CD44) is a receptor for hyaluronic acid (HA) and HA-binding has been proven to participate in various biological tumor activities, including tumor progression, metastasis and drug resistance. In this study, we aimed to determine the effects of CD44 on migration, invasion, proliferation, and the drug-sensitivity of osteosarcoma. Methods: 96 human osteosarcoma tissues from 56 patients were collected to evaluate the expression of CD44 in osteosarcoma tissue by immunohistochemistry. CRISPR-Cas9 system was used to specifically silence CD44 in drug-resistant cell lines (KHOSR2 and U-2OSR2). The migration and invasion activities of cells was demonstrated by wound healing and transwell invasion assay. The proliferation speed of the cells was detected under 3D cell culture condition. Drug resistance of cells was detected by MTT and drug uptake assay. Results: The immunohistochemistry results demonstrated that a high level of CD44 may predict poor survival and higher potential of metastasis, recurrence and drug resistance in patients with osteosarcoma. After knocking-out of CD44 by the CRISPR-Cas9 system, not only the migration and invasion activities of osteosarcoma cells were significantly inhibited, but the drug sensitivity was also enhanced. Conclusion: CD44 silencing could inhibit the development of osteosarcoma migration, invasion, proliferation and ameliorate drug resistance to current treatment in osteosarcoma. This study applies new strategy to target CD44, which may improve the prognosis of osteosarcoma.

CRISPR-Cas9-Mediated Silencing of CD44 in Human Highly Metastatic Osteosarcoma Cells

Background/Aims: Metastasis is the major cause of death in patients with osteosarcoma. There is an urgent need to identify molecular markers that promote metastasis. Cluster of differentiation 44 is a receptor for hyaluronic acid (HA) and HA-binding has been proven to participate in various biological tumor activities, including tumor progression and metastasis. Methods: We performed a meta-analysis to investigate the relationship between CD44 expression, survival, and metastasis in patients with osteosarcoma. We then utilized the CRISPR-Cas9 system to specifically silence CD44 in highly metastatic human osteosarcoma cells (MNNG/HOS and 143B) and further determined the functional effects of CD44 knockout in these cells. Results: The meta-analysis demonstrated that a high level of CD44 may predict poor survival and higher potential of metastasis in patients with osteosarcoma. The expression of CD44 in highly metastatic human osteosarcoma cell lines was efficiently blocked by CRISPR-Cas9. When CD44 was silenced, the proliferation and spheroid formation of these osteosarcoma cells was inhibited under 3-D culture conditions. Furthermore, the migratory and invasive functions were also impaired in these highly metastatic osteosarcoma cells. Conclusion: These results suggest that developing new strategies to target CD44 in osteosarcoma may prevent metastasis and improve the clinical outcome of osteosarcoma patients.

The survival of fetal and bone marrow monocyte-derived alveolar macrophages is promoted by CD44 and its interaction with hyaluronan.

Alveolar macrophages maintain lung homeostasis by performing important roles in immunosurveillance and lung surfactant catabolism. They express high levels of CD44 and are one of the few macrophage populations that constitutively bind hyaluronan, a ligand for CD44 and component of pericellular and extracellular matrices. Using adoptive transfer experiments and a mouse model of inflammation, we found that alveolar macrophages are initially depleted after an inflammatory insult then rapidly self-renew and return to original numbers after the resolution phase. Monocytes recruited to an inflamed lung differentiate and contribute to the alveolar macrophage pool, but this occurs over a much slower time frame than alveolar macrophage self-renewal. CD44 expression on both fetal and bone marrow-derived alveolar macrophages promoted their survival and provided a competitive advantage over CD44-deficient alveolar macrophages at homeostasis and after inflammation. CD44-mediated hyaluronan binding was induced by the alveolar environment, and this interaction promoted alveolar macrophage survival both ex vivo and in vivo. Without CD44, alveolar macrophages lacked a hyaluronan coat, were more susceptible to death, and were present at lower numbers in the alveolar space. This demonstrates a new role for CD44 and hyaluronan in promoting alveolar macrophage survival.

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs).

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown.MethodsWe used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6α, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3’-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay.ResultsEnriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6α upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs.ConclusionGemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.

T Cell-Derived IL-10 Impairs Host Resistance to Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is a leading cause of mortality and morbidity, causing ∼1.5 million deaths annually. CD4+ T cells and several cytokines, such as the Th1 cytokine IFN-γ, are critical in the control of this infection. Conversely, the immunosuppressive cytokine IL-10 has been shown to dampen Th1 cell responses to M. tuberculosis infection impairing bacterial clearance. However, the critical cellular source of IL-10 during M. tuberculosis infection is still unknown. Using IL-10 reporter mice, we show in this article that during the first 14 d of M. tuberculosis infection, the predominant cells expressing IL-10 in the lung were Ly6C+ monocytes. However, after day 21 postinfection, IL-10–expressing T cells were also highly represented. Notably, mice deficient in T cell–derived IL-10, but not mice deficient in monocyte-derived IL-10, showed a significant reduction in lung bacterial loads during chronic M. tuberculosis infection compared with fully IL-10–competent mice, indicating a major role for T cell–derived IL-10 in TB susceptibility. IL-10–expressing cells were detected among both CD4+ and CD8+ T cells, expressed high levels of CD44 and Tbet, and were able to coproduce IFN-γ and IL-10 upon ex vivo stimulation. Furthermore, during M. tuberculosis infection, Il10 expression in CD4+ T cells was partially regulated by both IL-27 and type I IFN signaling. Together, our data reveal that, despite the multiple immune sources of IL-10 during M. tuberculosis infection, activated effector T cells are the major source accounting for IL-10–induced TB susceptibility.

Abstract 1926: The effect of Beta-Catenin upon the tumor molecular biology and growth in HNSCC: role of cancer stem cells

Abstract Overview: Head and neck squamous cell carcinomas (HNSCC) are the sixth most prevalent type of malignancy worldwide. Recent reports have demonstrated that nearly every tumor contain a small subpopulation of cells called cancer stem-like cells (CSCs), which are responsible for tumor maintenance, metastasis relapse and therapeutic resistance. Recent studies demonstrate that even established HNSCC cell lines contain a definite sub-population of CSC providing us with the opportunity to discerning their roles in progression, treatment, and relapse of the disease. Background: We have previously demonstrated that a definite subpopulation of CSCs exists in most HNSCC cell lines. Significant differences were seen between the HPV+ and HPV- subtypes, confirmed by analyses of stem-ness genes and EMT markers. In vivo tumor xenograft formation has established the heightened tumorigenicity of CSCs. We investigated the molecular and functional roles of Beta-catenin in HNSCC CSCs by shRNA knock down and then subjecting the knocked down (KD) cells to various molecular analysis and in-vivo tumor formations in nude mice. We have also identified SOX2 as a down-stream effector gene that could exert the tumor promoting effects of Beta-catenin. The promoter region of the Sox2 gene was found to contain several putative Beta-catenin binding sites. Methods & Results: After identifying CSCs by in-vitro Sphere Forming (SFA) capabilities and molecular gene signatures, we have knocked down both Beta-catenin and Sox2 in both HPV+ and HPV- HNSCC cell lines (UMSCC47 and UMSCC6). SFA with the Beta-catenin KD-cells depicts a 50% decrease in sphere formation ability. Molecular analyses show lower expression of stemness genes: Sox2 (~ 3 fold) and Oct4 (~ 2 fold); metastatic genes: N-cadherin (~ 2.5 fold), Vimentin (~ 3 fold), Twist (~ 4 fold), Snail (~ 2.5 fold), Slug (~ 2.5 fold) and Hif-1alpha (~ 1.5 fold). Two tissue microarrays (TMA) were stained for Oct4, Sox2, and CD44 gene expression by multiplex RNA in situ hybridization. Nearly all tumor cells expressed high levels of CD44. Cells high in both Oct4 and Sox2 were quantified within duplicate cores and correlated with local control (TMA-1) and radiation response (TMA-2). Conclusions: Initial molecular analyses depicted that upon loss of Beta-catenin, the crucial stem-ness genes are negatively affected and the loss of stem-ness is also reinforced by decreased sphere formations. Both the metastatic and hypoxic gene signatures are markedly altered. We expect similar outcomes with the in-vivo tumor formations in nude mice. Citation Format: Austin Maas, Anirban Chatterjee, Lisa Sudmeier, Kwang Nickel, Randall J. Kimple. The effect of Beta-Catenin upon the tumor molecular biology and growth in HNSCC: role of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1926. doi:10.1158/1538-7445.AM2017-1926

Abstract 3865: Targeting the epigenetic modifier HMGA2 in lethal adult and pediatric gliomas inhibits invasion, growth and tumorigenicity

Abstract Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are highly infiltrative, incurable adult and pediatric brain tumors characterized by the presence of tumor stem-like cells. Although GBM and DIPG have different epigenetic drivers, we found increased expression of an epigenetic modifier and stem cell factor HMGA2 in both tumors by immunohistochemistry and western blotting. HMGA2 is a DNA-binding protein that regulates transcription during normal embryogenesis and in cancer stem cells. We hypothesized that HMGA2 contributes to high grade glioma tumorigenicity through its ability to alter the transcription of many genes. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 in multiple patient-derived GBM cell lines significantly reduced invasion in transwell assay (shHMGA2 vs. shControl, P<0.01). Similarly, shRNA mediated suppression of HMGA2 in DIPG cell lines reduced proliferation (BrdU incorporation), reduced invasion (transwell assay), inhibited clonogenicity (soft agar assay) and increased apoptosis (cleaved caspase-3 immunofluorescence, shHMGA2 vs. shControl, P<0.001). Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited invasion (transwell assay), decreased proliferation (BrdU incorporation) and increased apoptosis of DIPG neurospheres (P<0.01). Mice bearing orthotopic GBM xenografted cells transduced with HMGA2 shRNA lived significantly longer (108 days) compared to control shRNA (67.5 days, P<0.0001 by log-rank analysis). To investigate the mechanism of HMGA2-mediated malignancy, we discovered high levels of CD44 (a known mediator of invasion and stemness) that correlated with high HMGA2 expression in DIPG and GBM tumors. Suppression of HMGA2 led to decreased CD44 expression while ectopic expression of HMGA2 increased CD44 expression in GBM cell neurospheres. Knockdown of CD44 in HMGA2-high GBM neurospheres reduced invasion (P<0.01). Our studies suggest an oncogenic role of HMGA2 and its downstream effectors in gliomas and identify this important pathway as a potential therapeutic target. We conclude that minor groove binding agents similar to Netropsin may be effective agents for HMGA2-expressing pediatric and adult high grade gliomas. Citation Format: Harpreet Kaur, Sabeen Z. Ali, Huizi Guo, Sepehr Akhtarkhavari, Fausto Rodriguez, Charles G. Eberhart, Eric H. Raabe. Targeting the epigenetic modifier HMGA2 in lethal adult and pediatric gliomas inhibits invasion, growth and tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3865. doi:10.1158/1538-7445.AM2017-3865

CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect.

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor-binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases.

Mesenchymal subtype of glioblastomas with high DNA-PKcs expression is associated with better response to radiotherapy and temozolomide.

A better understanding of the relationship between glioblastomas molecular subtypes and radio-chemotherapy is needed for the development of individualized strategies. In this study, we aimed to assess whether non-homologous end-joining (NHEJ) protein expression is associated and could predict responses to treatment of mesenchymal (MES) and proneural (PN) subtypes. Tumors from 122 patients with a glioblastoma treated at the University Hospital of Poitiers between 2002-2013 by an association of radiotherapy and temozolomide were collected. Among these tumors, 80 were suitable for in situ analysis and were included in TissueMicroArray. The expression of DNA-PKcs, Ku70, Ku80 and CD44, Olig2 (respectively surrogate markers of MES and PN subtypes) were evaluated by immunohistochemistry. The median survival of patients with high and low CD44 expression was 11.9 months (95% CI 7.7-14) and 19.1 months (95% CI 15.2-22.4) respectively (p = 0.008). Median survival of patients with high and low DNA-PKcs levels was 20.0 months (95% CI 15.2-25.3) and 12.9 months (95% CI 9.9-19.5) respectively (p = 0.036). High levels of Olig2, Ku70 and Ku80 tended to be associated with better overall survival but no significant differences were found. Overall survival of class I patients (CD44+ and DNA-PKcs+) was longer than class II (CD44+ and DNA-PKcs- or CD44- and DNA-PKcs+) and class III (CD44- and DNA-PKcs-), (p = 0.005 and 0.003 respectively). High levels of CD44 and DNA-PK are associated with a better survival and better response to radiotherapy and temozolomide and could establish prognosis classes by predicting survival and response to therapy for GBMs patients.

Prognostic Value of Cancer Stem Cell Markers CD44 and ALDH1/2 in Gastric Cancer Cases

Purpose:To determine expression levels of CD44 and ALDH1/2, known cancer stem cell (CSC) markers, in stomach adenocarcinomas and assess relationships with clinicopathologic parameters and prognosis.Methods:Eighty patients diagnosed with gastric cancer between the years 2011-2015 were included in this study of clinicopathologic characteristics, postoperative prognostic indexes and stem cell marker CD44 and ALDH1/2 expression in paraffin-embedded tumour sections analyzed immunohistochemically. Clinicopathologic parameters were evaluated using the chi-square test and t-test. Survival analyses were conducted using Kaplan-Meier statistics.Results:We observed positive CD44 and ALDH1/2 staining in 45.0 % and 67.5% of tumour tissues, respectively, but not in normal gastric mucosa. Recurrence-free survival (RFS) was found to be shorter in cases with high levels of CD44 expression (p=0.004). Similarly, short RFS was observed in patients with high levels of CD44 and ALDH1/2 co-expression (p=0.004). Furthermore, tumour invasion depth was found to correlate with high CD44 and ALDH1/2 co-expression (p=0.028).Conclusion:The cancer stem cell markers CD44 and ALDH1/2 may indicate poor patient prognosis and play a role in tumour development and invasion.

PDTB-11. DISRUPTING THE EPIGENETIC MODIFIER HMGA2 IN LETHAL PEDIATRIC AND ADULT GLIOMAS INHIBITS INVASION, GROWTH AND TUMORIGENICITY

Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are highly infiltrative, incurable pediatric and adult brain tumors characterized by the presence of tumor stem-like cells. Although DIPG and GBM have different epigenetic drivers, we found increased expression of an epigenetic modifier and stem cell factor HMGA2 in both tumors by immunohistochemistry and western blotting. HMGA2 is a DNA-binding protein that regulates transcription in normal and cancer stem cells. We hypothesized that HMGA2 contributes to high grade glioma tumorigenicity through its ability to alter the transcription of many genes. Lentiviral short hairpin RNA (shRNA)-mediated reduction of HMGA2 in multiple patient-derived GBM cell lines significantly reduced invasion in transwell assay and colony formation in soft agar assay (shHMGA2 vs. shControl, P<0.01). Similarly, shRNA mediated suppression of HMGA2 in DIPG cell lines reduced proliferation (BrdU incorporation), reduced invasion (transwell assay) and increased apoptosis (cleaved caspase-3 immunofluorescence, shHMGA2 vs. shControl, P<0.001). Pharmacological inhibition of HMGA proteins using the DNA minor-groove binding drug Netropsin significantly inhibited proliferation and increased apoptosis of DIPG cell lines (P<0.01). Mice bearing orthotopic GBM xenografted cells transduced with HMGA2 shRNA lived significantly longer (108 days) compared to control shRNA (67.5 days, P<0.0001 by log-rank analysis). To investigate the mechanism of HMGA2-mediated malignancy, we discovered high levels of CD44 (a known mediator of invasion and stemness) that correlated with high HMGA2 expression in DIPG and GBM tumors. Suppression of HMGA2 led to decreased CD44 protein expression. Knockdown of CD44 in HMGA2-high DIPG and GBM neurospheres reduced invasion and proliferation (P<0.01). Our studies suggest an oncogenic role of HMGA2 and its downstream effectors in gliomas and identify this important pathway as a potential therapeutic target. We conclude that minor groove binding agents similar to Netropsin may be effective agents for HMGA2-expressing pediatric and adult high grade gliomas.