Tumor- and Fibroblast-Derived Cell-Free DNAs Differently Affect the Progression of B16 Melanoma In Vitro and In Vivo.

It is widely postulated that the majority of pathologically elevated extracellular or cell-free DNA (cfDNA) in cancer originates from tumor cells; however, evidence has emerged regarding the significant contributions of other cells from the tumor microenvironment. Here, the effect of cfDNA originating from murine B16 melanoma cells and L929 fibroblasts on B16 cells was investigated. It was found that cfDNAL929 increased the viability and migration properties of B16 cells in vitro and their invasiveness in vivo. In contrast, cfDNAB16 exhibited a negative effect on B16 cells, reducing their viability and migration in vitro, which in vivo led to decreased tumor size and metastasis number. It was shown that cell treatment with both cfDNAs resulted in an increase in the expression of genes encoding DNases and the oncogenes Braf, Kras, and Myc. cfDNAL929-treated cells were shown to experience oxidative stress. Gene expression changes in the case of cfDNAB16 treatment are well correlated with the observed decrease in proliferation and migration of B16 cells. The obtained data may indicate the possible involvement of fibroblast DNA in the tumor microenvironment in tumor progression and, potentially, in the formation of new tumor foci due to the transformation of normal cells.

Enrichment of FGF8-expressing cells from neurally induced human pluripotent stem cell cultures.

In early vertebrate development, organizer regions-groups of cells that signal to and thereby influence neighboring cells by secreted morphogens-play pivotal roles in the establishment and maintenance of cell identities within defined tissue territories. The midbrain-hindbrain organizer drives regionalization of neural tissue into midbrain and hindbrain territories with fibroblast growth factor 8 (FGF8) acting as a key morphogen. This organizer has been extensively studied in chicken, mouse, and zebrafish. Here, we demonstrate the enrichment of FGF8-expressing cells from human pluripotent stem cells (hPSCs), cultured as attached embryoid bodies using antibodies that recognize "Similar Expression to Fgf" (SEF) and Frizzled proteins. The arrangement of cells in embryoid body subsets of these cultures and the gene expression profile of the FGF8-expressing population show certain similarities to the midbrain-hindbrain organizer in animal models. In the embryonic chick brain, the enriched cell population induces formation of midbrain structures, consistent with FGF8-organizing capability.

The Effect of Cell-Free DNA from Blood Serum of Mice with Metastatic Melanoma on Enhancement of Oncogenic Properties of Melanoma Cells.

Currently, a significant increase in the levels of circulating cell-free DNA (cfDNA) in the blood of patients is considered as a generally recognized marker of the development of oncological diseases. Although the tumor-associated cfDNA has been well studied, its biological functions remain unclear. In this work, we investigated the effect of cfDNA isolated from the blood serum of the mice with B16-F10 metastatic melanoma on the properties of the B16-F10 melanoma cells in vitro. It was found that the profile of cfDNA isolated from the blood serum of mice with melanoma differs significantly from the cfDNA isolated from the blood serum of healthy mice, and is similar to the genomic DNA of B16 cells with regards to abundance of oncogenes and mobile genetic elements (MGE). It was shown that the cfDNA of mice with melanoma penetrated into B16 cells, resulting in the increase in abundance of oncogenes and MGE fragments, and caused 5-fold increase of the mRNA level of the secreted DNase Dnase1l3 and a slight increase of the mRNA level of the Jun, Fos, Ras, and Myc oncogenes. cfDNA of the healthy mice caused increase of the mRNA level of intracellular regulatory DNase EndoG and 4-fold increase of the mRNA level of Fos and Ras oncogenes, which are well-known triggers of a large number of signal cascades, from apoptosis inhibition to increased tumor cell proliferation. Thus, it is obvious that the circulating cfDNA of tumor origin is able to penetrate into the cells and, despite the fact that no changes were found in the level of viability and migration activity of the tumor cells, cfDNA, even with a single exposure, can cause changes at the cellular level that increase oncogenicity of the recipient cells.

Mutations in SMARCB1 and in other Coffin-Siris syndrome genes lead to various brain midline defects.

Mutations in genes encoding components of BAF (BRG1/BRM-associated factor) chromatin remodeling complexes cause neurodevelopmental disorders and tumors. The mechanisms leading to the development of these two disease entities alone or in combination remain unclear. We generated mice with a heterozygous nervous system-specific partial loss-of-function mutation in a BAF core component gene, Smarcb1. These Smarcb1 mutant mice show various brain midline abnormalities that are also found in individuals with Coffin-Siris syndrome (CSS) caused by SMARCB1, SMARCE1, and ARID1B mutations and in SMARCB1-related intellectual disability (ID) with choroid plexus hyperplasia (CPH). Analyses of the Smarcb1 mutant animals indicate that one prominent midline abnormality, corpus callosum agenesis, is due to midline glia aberrations. Our results establish a novel role of Smarcb1 in the development of the brain midline and have important clinical implications for BAF complex-related ID/neurodevelopmental disorders.

Methyl-CpG binding domain protein 1 regulates localization and activity of Tet1 in a CXXC3 domain-dependent manner.

Cytosine modifications diversify and structure the genome thereby controlling proper development and differentiation. Here, we focus on the interplay of the 5-methylcytosine reader Mbd1 and modifier Tet1 by analyzing their dynamic subcellular localization and the formation of the Tet oxidation product 5-hydroxymethylcytosine in mammalian cells. Our results demonstrate that Mbd1 enhances Tet1-mediated 5-methylcytosine oxidation. We show that this is due to enhancing the localization of Tet1, but not of Tet2 and Tet3 at heterochromatic DNA. We find that the recruitment of Tet1 and concomitantly its catalytic activity eventually leads to the displacement of Mbd1 from methylated DNA. Finally, we demonstrate that increased Tet1 heterochromatin localization and 5-methylcytosine oxidation are dependent on the CXXC3 domain of Mbd1, which recognizes unmethylated CpG dinucleotides. The Mbd1 CXXC3 domain deletion isoform, which retains only binding to methylated CpGs, on the other hand, blocks Tet1-mediated 5-methylcytosine to 5-hydroxymethylcytosine conversion, indicating opposite biological effects of Mbd1 isoforms. Our study provides new insights on how cytosine modifications, their modifiers and readers cross-regulate themselves.

Acidosis Acts through HSP90 in a PHD/VHL-Independent Manner to Promote HIF Function and Stem Cell Maintenance in Glioma.

Hypoxia is a common feature of solid tumors, which controls multiple aspects of cancer progression. One important function of hypoxia and the hypoxia-inducible factors (HIF) is the maintenance of cancer stem-like cells (CSC), a population of tumor cells that possess stem cell-like properties and drives tumor growth. Among the changes promoted by hypoxia is a metabolic shift resulting in acidification of the tumor microenvironment. Here, we show that glioma hypoxia and acidosis functionally cooperate in inducing HIF transcription factors and CSC maintenance. We found that these effects did not involve the classical PHD/VHL pathway for HIF upregulation, but instead involved the stress-induced chaperone protein HSP90. Genetic or pharmacologic inactivation of HSP90 inhibited the increase in HIF levels and abolished the self-renewal and tumorigenic properties of CSCs induced by acidosis. In clinical specimens of glioma, HSP90 was upregulated in the hypoxic niche and was correlated with a CSC phenotype. Our findings highlight the role of tumor acidification within the hypoxic niche in the regulation of HIF and CSC function through HSP90, with implications for therapeutic strategies to target CSC in gliomas and other hypoxic tumors. Cancer Res; 76(19); 5845-56. ©2016 AACR.

PHD3 regulates EGFR internalization and signalling in tumours.

Tumours exploit their hypoxic microenvironment to induce a more aggressive phenotype, while curtailing the growth-inhibitory effects of hypoxia through mechanisms that are poorly understood. The prolyl hydroxylase PHD3 is regulated by hypoxia and plays an important role in tumour progression. Here we identify PHD3 as a central regulator of epidermal growth factor receptor (EGFR) activity through the control of EGFR internalization to restrain tumour growth. PHD3 controls EGFR activity by acting as a scaffolding protein that associates with the endocytic adaptor Eps15 and promotes the internalization of EGFR. In consequence, loss of PHD3 in tumour cells suppresses EGFR internalization and hyperactivates EGFR signalling to enhance cell proliferation and survival. Our findings reveal that PHD3 inactivation provides a novel route of EGFR activation to sustain proliferative signalling in the hypoxic microenvironment.

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR.

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability.

The cancer stem cell niche(s): the crosstalk between glioma stem cells and their microenvironment.

BACKGROUND: The initiation and progression of various types of tumors, including glioma, are driven by a population of cells with stem cell properties. Glioma stem cells (GSCs) are located in specialized microenvironments (niches) within tumors. These niches represent the hallmarks of malignant gliomas (vascular proliferations, hypoxia/necrosis) and bear analogy to the microenvironments in which physiological stem cells in the brain are found. SCOPE OF THE REVIEW: Here we review the progress that has been made towards uncovering the function of the perivascular and the hypoxic niche and the molecular pathways that control the properties of GSCs within them. We propose models of how the different niches and GSC pools in them interact with each other. MAJOR CONCLUSIONS: GSCs are not merely passive residents of their niches, but actively contribute to the shaping of the niches through a complex crosstalk with different components of the microenvironment. For example, GSCs play a dominant role in promoting new blood vessel formation through a variety of mechanisms, including the hypoxia dependent stimulation of angiogenesis, recruitment of endothelial progenitor cells and direct transdifferentiation into endothelial cells. Recent work has also revealed that GSCs can recruit and modulate the function of various immune cells to suppress anti-tumor immune responses and to foster tumor-promoting inflammation, which in turn could support the maintenance of GSCs. GENERAL SIGNIFICANCE: These findings underscore the central role of the GSC microenvironment in driving glioma progression making the GSC niche a prime therapeutic target for the design of therapies aimed at eradicating GSCs. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation.

Glioblastoma multiforme, a highly aggressive tumor of the central nervous system, has a dismal prognosis that is due in part to its resistance to radio- and chemotherapy. The protein kinase C (PKC) family of serine threonine kinases has been implicated in the formation and proliferation of glioblastoma multiforme. Members of the protein kinase D (PKD) family, which consists of PKD1, -2 and, -3, are prominent downstream targets of PKCs and could play a major role in glioblastoma growth. PKD2 was highly expressed in both low-grade and high-grade human gliomas. The number of PKD2-positive tumor cells increased with glioma grading (P < .001). PKD2 was also expressed in CD133-positive glioblastoma stem cells and various glioblastoma cell lines in which the kinase was found to be constitutively active. Inhibition of PKDs by pharmacological inhibitors resulted in substantial inhibition of glioblastoma proliferation. Furthermore, specific depletion of PKD2 by siRNA resulted in a marked inhibition of anchorage-dependent and -independent proliferation and an accumulation of glioblastoma cells in G0/G1, accompanied by a down-regulation of cyclin D1 expression. In addition, PKD2-depleted glioblastoma cells exhibited substantially reduced tumor formation in vivo on chicken chorioallantoic membranes. These findings identify PKD2 as a novel mediator of glioblastoma cell growth in vitro and in vivo and thereby as a potential therapeutic target for this devastating disease.

Novel shuttling domain in a regulator (RSC1A1) of transporter SGLT1 steers cell cycle-dependent nuclear location.

The gene product of RSC1A1, RS1, participates in the regulation of the Na(+)-D-glucose cotransporter SGLT1. RS1 inhibits release of SGLT1 from the trans Golgi network. In subconfluent LLC-PK(1) cells, RS1 migrates into the nucleus and modulates transcription of SGLT1, whereas most confluent cells do not contain RS1 in the nuclei. We showed that confluence-dependent nuclear location of RS1 is because of different phases of the cell cycle and identified a RS1 nuclear shuttling domain (RNS) with an associated protein kinase C (PKC) phosphorylation site (RNS-PKC) that mediates cell cycle-dependent nuclear location. RNS-PKC contains a novel non-conventional nuclear localization signal interacting with importin beta1, a nuclear export signal mediating export via protein CRM1 and a Ca(2+)-dependent calmodulin binding site. PKC and calmodulin compete for binding to RNS-PKC. Mutagenesis experiments and analyses of the phosphorylation status suggest the following sequences of events. Subconfluent cells without and with synchronization to the G2/M phase contain non-phosphorylated RNS-PKC that mediates nuclear import of RS1 but not its export. During confluence or synchronization of subconfluent cells to the G2/M phase, phosphorylation of RNS-PKC mediates rapid nuclear export of RS1.