Oncogenesis Recapitulates Embryogenesis via the Hypoxia Pathway: Morphoproteomics and Biomedical Analytics Provide Proof of Concept and Therapeutic Options

Background Hypoxia (3 to 5% oxygen) is essential in maintaining the plasticity of embryonic stem cells and permitting their transformation via epithelial-mesenchymal transition(EMT) and mesenchymal-epithelial transition(MET) into tissues and organs of the developing fetus. Similarly, a relatively hypoxic microenvironment supports the development of tumor cells with stemness and epithelial-mesenchymal properties and capabilities. At the same time, such adaptation results in the tumor cells becoming relatively resistant to chemotherapy and radiation therapy and promotes intravasation into blood vessels with metastasis. In this context, current therapeutic strategies designed to target tumoral angiogenesis could promote stemness and EMT by rendering tumor cells more hypoxic, leading to chemoradioresistance and metastatic and recurrent disease. Objective The purpose of this report is to present a conceptual model that illustrates the impact of an hypoxic microenvironment on the signal transduction pathways involved in the hypoxia pathway. We will show the molecular connectivity and correlative association of these pathways with protein analytes in both embryogenesis and oncogenesis in order to strengthen our hypothesis that oncogenesis recapitulates embryogenesis. Finally, we propose to use the model as a basis for the construction of combinatorial, therapeutic options from existing pharmaceutical and nutraceutical agents that may obviate tumoral adaptation to hypoxia. Methods Morphoproteomics and biomedical analytics. Application and Results Archival data retrieved from morphoproteomic analysis of glioblastoma multiforme(GBM) cases revealed proteomic correlates of tumoral necrosis and associated hypoxia pathway signaling. Biomedical analytics using Ingenuity Pathway Analysis (IPA) showed comparative validation of the hypoxia pathway, as demonstrated by morphoproteomics in GBM, both with the hypoxia-induced genes in neuroblastoma and with the networks associated with embryogenesis. Additionally, therapeutic agents known to have activity against various components of the hypoxia pathway (identified by morphoproteomic analysis in GBM) were validated using UNIPROT identifiers entered into IPA and Path Designer. These therapies also connected with the hypoxia signature in neuroblastoma and embryogenesis. Conclusion The application of morphoproteomics to define the presence of an adaptive hypoxia pathway in GBM accords with biomedical analytics in the demonstration of concordant interaction with the hypoxia signature in neuroblastoma and embryogenesis, providing proof of concept that oncogenesis recapitulates embryogenesis. This approach also validates a new combinatorial therapeutic strategy targeting the hypoxia pathway and designed to prevent tumoral adaptation, chemoradioresistance and recurrent disease.     

Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion

Hypoxia and hypoxia-inducible factor-1 (HIF-1) play a critical role in glioblastoma multiforme (GBMs). CXCR4 is involved in angiogenesis and is upregulated by HIF-1alpha. CXCR4 is a chemokine receptor for stromal cell-derived factor-1 (SDF-1)alpha, also known as CXCL12. We hypothesized that CXCR4 would be upregulated by hypoxia in GBMs. First, we investigated the expression of HIF-1alpha and CXCR4 in GBMs. CXCR4 was consistently found colocalized with HIF-1alpha expression in pseudopalisading glioma cells around areas of necrosis. In addition, angiogenic tumor vessels were strongly positive for CXCR4. Next, we tested the in vitro effect of hypoxia and vascular endothelial growth factor (VEGF) on the expression of CXCR4 in glioma cell lines and in human brain microvascular endothelial cells (HBMECs). Exposure to hypoxia induced significant expression of CXCR4 and HIF-1alpha in glioma cells, whereas treatment with exogenous VEGF increased CXCR4 expression in HBMECs. We also transfected U87MG glioma cells with an HIF-1alpha construct and observed that CXCR4 was upregulated in these cells even in normoxic conditions. We then used a lentivirus-mediated shRNA expression vector directed against HIF-1alpha. When exposed to hypoxia, infected cells failed to show HIF-1alpha and CXCR4 upregulation. We performed migration assays under normoxic and hypoxic conditions in the presence or absence of AMD3100, a CXCR4 inhibitor. There was a significant increase in the migration of U87MG and LN308 glioma cells in hypoxic conditions, which was inhibited in the presence of AMD3100. These studies demonstrate the critical role played by hypoxia and CXCR4 in glioma cell migration. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at two levels. First, through HIF-1alpha in the pseudopalisading tumor cells themselves and, secondly, by the VEGF-stimulated angiogenic response in HBMECs. We believe this knowledge may lead to a potentially important two-pronged therapy against GBM progression using chemotherapy targeting CXCR4.     

The structural relationship between mesangial cells and basement membrane of the renal glomerulus

Summary It has been shown by many studies that mesangial cell contraction exerts considerable influences on glonerular filtration dynamics. However, experimental findings about the geometrical changes within the glomerular tuft going along with mesangial cell contractions are lacking. This study analyzes the geometry of mesangial cells and their relationship to glomerular capillaries, especially to the glomerular basement membrane (GBM).By applying a new staining technique of unosmicated specimens for TEM, the cellular outlines of glomerular cells (mesangial, endothelial and epithelial) and the distribution of extracellular matrices can be more easily studied than in conventionally osmicated specimens. It became obvious that mesangial cells and the GBM are extensively connected with each other, either by direct attachments or indirectly by microfibrils. These connections are especially prominent mesangial angles, i.e. at sites where the GBM deviates from its pericapillary course and covers the mesangium. Thereby, the GBM is not only coupled to the mesangium out—via mesangial cell processes—also to the GBM at the opposing mesangial angle. It seems possible that contraction of mesangial cells can bring the GBM from opposing mesangial angles closer together. Therefore we conclude that the GBM and the contractile mesangial cells together establish a biomechanical unit capable of developing wall tension in glomerular capillaries and of changing the geometry of glomerular capillaries following mesangial contraction or relaxation.Fellow of the Alexander von Humboldt Foundation     

Cancer stem cells may be mostly maintained by fluctuating hypoxia

We wonder if most cancer stem cells (CSCs) survive and are maintained in the region of fluctuating hypoxia, which protects them against differentiation. Fluctuating hypoxia, as an important and neglected factor, has been confirmed to induce malignant progression, confer to therapeutic resistance and exist extensively. The subsequent consequence is similar with the behavior of CSCs. Therefore, we cite some examples for our bases and hypothesize CSCs may be mostly maintained by fluctuating hypoxia.     

Human glioblastoma stem-like cells are more sensitive to allogeneic NK and T cell-mediated killing compared with serum-cultured glioblastoma cells

Glioblastoma multiforme (GBM) is the most dramatic primary brain cancer with a very poor prognosis because of inevitable disease recurrence. The median overall survival is less than 1 year after diagnosis. Cancer stem cells have recently been disclosed in GBM. GBM stem‐like cells (GSCs) exhibit resistance to radio/chemotherapeutic treatments and are therefore considered to play an important role in disease recurrence. GSCs are thus appealing targets for new treatments for GBM patients. In this study, we show that GBM cells with stem cell characteristics are resistant to lysis mediated by resting natural killer (NK) cells because of the expression of MHC class I molecules. However, GSCs are killed by lectin‐activated NK cells. Furthermore, in experiments using the therapeutic antibody CetuximAb, we show that GSCs are sensitive to antibody‐mediated cytotoxicity. We confirm the sensitivity of GSC to cytotoxicity carried out by IL2‐activated NK cells and tumor‐specific T cells. More importantly, we show that GSCs are more sensitive to NK and T cell‐mediated lysis relatively to their corresponding serum‐cultured GBM cells obtained from the same initial tumor specimen. Altogether, these results demonstrate the sensitivity of GSC to immune cell cytotoxicity and, therefore, strongly suggest that GSCs are suitable target cells for immunotherapy of GBM patients.     

慢性低氧小鼠肺组织糖蛋白的变化

目的 :研究在慢性常压低氧下的小鼠肺组织多糖、粘蛋白的适应性变化。方法 :低氧 (1 0 % )第 1、3、5、7天分别取左肺中部组织作冰冻切片 ,以高碘酸Schiff反应 (PAS)显示糖原及粘蛋白并进行光镜观察。结果 :与动脉平滑肌比较 ,低氧小鼠支气管平滑肌糖原合成一过性增强 ;静脉内皮细胞糖代谢的敏感性显著高于动脉 ;低氧能刺激支气管粘膜分泌细胞分泌粘蛋白 ,反应随低氧作用时间而变化。结论 :小鼠肺组织糖原、粘蛋白代谢在慢性低氧下产生适应性变化。杯状细胞与管壁内腺体的分泌物在上皮表面共同构成的粘液性屏障有利于小鼠的低氧适应     

Overexpression of ZNF217 in glioblastoma contributes to the maintenance of glioma stem cells regulated by hypoxia-inducible factors

Glioblastoma multiforme (GBM) is the most aggressive and common kind of primary brain tumor in adults, and is thought to be driven by a subpopulation of glioma stem cells (GSCs). GSCs reside in a specialized hypoxic niche, which can regulate the tumorigenic capacity of GSCs primarily through the hypoxia-inducible factors (HIFs), HIF1α and HIF2α. ZNF217 is an oncogene frequently amplified in many kinds of tumors. It is associated with aggressive tumor behavior and poor clinical prognosis, but its role in gliomas is poorly known. Gene expression and copy number analysis from TCGA data reveal that ZNF217 is amplified in 32% and overexpressed in 71.2% of GBMs. Quantitative RT-PCR and western blotting of a cohort of glioma samples showed that ZNF217 was highly expressed in gliomas and increased with tumor grade. Analysis of a molecular database demonstrated that ZNF217 expression correlated with poor survival of glioma patients. Investigation of ZNF217 expression in GSCs, non-GSCs and normal neural stem cells (NSCs) indicated that ZNF217 was more highly expressed in GSCs than in non-GSCs and NSCs. Knockdown of ZNF217 in GSCs by small-interfering RNA (siRNA) inhibited their growth and promoted their differentiation. Interestingly, ZNF217 was upregulated in GSCs and the GBM cell line U87 when exposed to the hypoxic environment of 1% oxygen. Knockdown of either HIF1α or HIF2α, which has a central role in the hypoxia-induced responses of these cells, inhibited ZNF217 expression. In addition, ZNF217 upregulation was compromised under hypoxia in U87 and GSCs when either HIF1α or HIF2α was targeted by siRNA. HIF2α knockdown inhibited ZNF217 expression more efficiently in both normoxia and hypoxia than HIF1α knockdown. Therefore, ZNF217 is overexpressed in GBMs and contributes to the maintenance of GSCs, which is regulated by HIFs released by the hypoxic environment of the tumor.     

Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma

Glioblastoma multiforme (GBM) is the most frequently occurring brain cancer. Although the existence of cancer stem cells (CSCs) in GBM has been established, there is little evidence to explain the link between CSCs and chemoresistance. In this study, we developed a dissociated cell system of human GBM cells, A172 and established GBM2 cells, that have shown resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). After exposure to a lethal dose of BCNU, the small population of GBM cancer cells survived and proliferated, as opposed to direct inhibition of the apoptosis and activation of the proliferation signal. Also, these cells contained subpopulations of stem-like cells, expressing CD133, CD117, CD90, CD71, and CD45 cell-surface markers, and had the capacity for multipotency. Moreover, we observed that BCNU-resistant subpopulations derived from GBM cancer cells can be grown to tumors when transplanted into severe combined immunodeficient (SCID) mouse brain. These results demonstrated that BCNU-resistant subpopulations derived from GBM have cancer stem-like cell properties. These findings provide further evidence that CSCs in GBM display chemotherapeutic drug resistance. Hopefully, it will be possible to improve the therapeutic outcome of GBM, leading to better anticancer strategies.     

Side population is not necessary or sufficient for a cancer stem cell phenotype in glioblastoma multiforme

There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stem-like cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP). We show that sphere formation in GBM cell lines and primary GBM cells enriches for a CSC-like phenotype of increased self-renewal gene expression in vitro and increased tumor initiation in vivo. However, the SP was absent from all sphere cultures. Direct isolation of the SP from the GBM lines did not enrich for stem-like activity in vitro, and tumor-initiating activity was lower in sorted SP compared with non-SP and parental cells. Transient exposure to doxorubicin enhanced both CSC and SP frequency. However, doxorubicin treatment altered the cytometric profile and obscured the SP demonstrating the difficulty of identifying SP in cells under stress. Doxorubicin-exposed cells showed a transient increase in SP, but the doxorubicin-SP cells were still not enriched for a stem-like self-renewal phenotype. These data demonstrate that the GBM SP does not necessarily contribute to self-renewal or tumor initiation, key properties of a CSC, and we advise against using SP to enumerate or isolate CSC.     

Synergistic effect of TRAIL and irradiation in elimination of glioblastoma stem-like cells

Glioblastoma multiforme (GBM) is the most common malignancy in central nervous system. A small subpopulation of GBM cells known as GBM stem-like cells (GSLCs) were supposed to be the most malignant cells among GBM cells as they are resistant to multiple therapies including radiotherapy. In this study, we set up two GSLCs cell lines from the two parental U87 and U251 glioma cell lines, and studied the expression of apoptosis-related genes alteration in GSLCs before and after irradiation. We found that one of the receptors of TNF-related apoptosis-inducing ligand (TRAIL), DR5, was dramatically up-regulated in GSLCs after irradiation (IR). Although GSLCs are resistant to both TRAIL and radiation treatment alone, the combined treatment with TRAIL and irradiation achieved maximum killing effect of GSLCs due to inducing the expression of DR5 and inhibiting the expression of cFLIP. Therefore, TRAIL and IR combined treatment would be a simple but practical therapeutic strategy for clinical application.     

Ablation of vagal preganglionic neurons innervating the extra-thoracic trachea affects ventilatory responses to hypercapnia and hypoxia

This study tested the hypothesis that during hypercapnia or hypoxia, airway-related vagal preganglionic neurons (AVPNs) of the nucleus ambiguus (NA) release acetylcholine (ACh), which in a paracrine fashion, activates ACh receptors expressed by inspiratory rhythm generating cells. AVPNs in the NA were ablated by injecting a saporin- (SA) cholera toxin b subunit (CTb-SA) conjugate into the extra-thoracic trachea (n=6). Control animals were injected with free CTb (n=6). In CTb treated rats, baseline ventilation and ventilatory responses to hypercapnia (5 and 12% CO(2) in O(2)) or hypoxia (8% O(2) in N(2)) were similar (p>0.05) prior to and 5 days after injection. CTb-SA injected rats maintained rhythmic breathing patterns 5 days post injection, however, tachypneic responses to hypercapnia or hypoxia were significantly reduced. The number of choline acetyltransferase (ChAT) immunoreactive cells in the NA was much lower (p<0.05) in CTb-SA rats as compared to animals receiving CTb only. These results suggest that AVPNs participate in the respiratory frequency response to hypercapnia or hypoxia.     

Angiogenesis in gliomas: biology and molecular pathophysiology

Glioblastoma multiforme (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression. The pathologic mechanisms driving this change and the biological behavior of gliomas remain unclear. One mechanism may involve cooption of native blood vessels by glioma cells inducing expression of angiopoietin-2 by endothelial cells. Subsequently, vascular apoptosis and involution leads to necrosis and hypoxia. This in turn induces angiogenesis that is associated with expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) in perinecrotic pseudopalisading glioma cells. Here we review the molecular and cellular mechanisms implicated in HIF-1-dependent and HIF-1-independent glioma-associated angiogenesis. In GBMs, both tumor hypoxia and genetic alterations commonly occur and act together to induce the expression of HIF-1. The angiogenic response of the tumor to HIF-1 is mediated by HIF-1-regulated target genes leading to the upregulation of several proangiogenic factors such as VEGF and other adaptive response molecules. Understanding the roles of these regulatory processes in tumor neovascularization, tumor growth and progression, and resistance to therapy will ultimately lead to the development of improved antiangiogenic therapies for GBMs.     

Cytoplasmic cyclin D1 regulates glioblastoma dissemination

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4–retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

TP53 deleted cells in de novo glioblastomas using fluorescence in situ hybridization

Glioblastoma (GBM) has been known to have two distinct genetic pathways of tumorigenesis. Secondary GBM shows frequent TP53 mutation, but de novo (primary) GBM is usually independent of TP53 alteration. However, the subpopulation of TP53 altered cells in the latter tumor is obscure. In order to assess TP53 deleted cells in de novo GBM quantitatively, we performed dual color fluorescence in situ hybridization (FISH) for TP53 and centromere 17 in nine cases of de novo GBM with frozen surgical materials. Single TP53 signal cells indicating TP53 deletion were recognized in 8.7-35.6% (mean, 21.3%) among the nine cases. In addition, immunohistochemistry was performed for the Ki-67 antigen (MIB-1) and p53 protein in all nine cases. Labeling indices (LI) of MIB-1 ranged from 2.8 to 46.9% (mean, 20.8%). Between the group with the more dense subpopulation of TP53 deleted cells (15% or more) by FISH and the group with less subpopulation than the former, these LI of MIB-1 demonstrated statistically significant difference (respective means, 28.2% and 6.1%; P < 0.05). Conversely, LI of p53 protein shown to be 0-50.9% (mean, 24.9%) had no correlation with the subpopulation of TP53 deleted cells by FISH. Four cases who had higher LI of p53 protein (mean, 39.7%) than the subpopulation of TP53 deleted cells (mean, 12.7%), respectively, indicated the presence of many p53 protein immunoreactive cells without TP53 deletion. These results suggest that: (i) de novo GBM also has subpopulation of TP53 deleted cells; (ii) TP53 alteration, which may not be a major event, participates in cell proliferation of de novo GBM; and (iii) de novo GBM tends to have accumulation of wild-type p53 protein.     

Delivery of Exogenous miR-124 to Glioblastoma Multiform Cells by Wharton’s Jelly Mesenchymal Stem Cells Decreases Cell Proliferation and Migration,and Confers Chemosensitivity

MicroRNAs (miRs) are potential therapeutic targets in glioblastoma multiforme (GBM), but the difficulties associated with their delivery to tumor target cells have hampered their widespread use. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers, including GBM and exert anti-tumor effects. In this study, it is shown that Wharton’s jelly-MSCs (WJ-MSCs) have the ability to deliver exogenous miRs to GBM cells and the functional impact of this delivery is characterized. It is found that the labeled miR-124, as an example for miR of interest, can be successfully delivered with WJ-MSCs to U87 GBM cells via dependent or exosome-independent processes. It is demonstrated that the delivered exogenous miR-124 significantly decreases the luciferase activity of the target gene CDK6. In addition, the delivered miR-124 enhances the chemosensitivity of GBM cells to temozolomide and decreases the migration of GBM cells. These results suggest that the use of exogenous miRNA delivery with the derived exosomes from WJ-MSCs may provide a novel approach for miRNA replacement therapy in GBM cancers.     

Ultrastructural organization of contractile proteins in rat glomerular mesangial cells.

Glomerular mesangial cells of the rat kidney contain actin, nonmuscle myosin, tropomyosin, and the muscular Z-line protein, alpha-actinin. This was shown for actin, myosin, and alpha-actinin by immunoblotting as well as by immunoelectron microscopy. Tropomyosin was localized in mesangial cells by immunofluorescence. In cultured mesangial cells, actin, myosin, and alpha-actinin constitute a considerable amount of the total cellular protein contents. In mesangial cells in situ actin, myosin and alpha-actinin were found to be colocalized within conspicuous microfilament bundles that traverse the cell body or major processes in various directions and project into either the tonguelike pericapillary processes, which run toward mesangial angles, or into the microvilluslike lateral extensions that abut on the perimesangial portion of the glomerular basement membrane (GBM). Thereby, the GBM of opposing mesangial angles as well as of opposing portions of the perimesangial GBM are regularly interconnected by filament bundles within mesangial cells that contain actin, myosin, and alpha-actinin. The authors suggest that the major function of actin-, myosin-, and alpha-actinin-containing filament bundles in mesangial cells is to create an isometric tension (or minute isotonic contractions) to counteract the distending forces of the rather high intracapillary hydraulic pressure and its resulting pressure gradients across the capillary wall and across the perimesangial GBM.