ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma.

BACKGROUND: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. METHODS: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. RESULTS: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. CONCLUSIONS: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.

Promoter-associated proteins of EPAS1 identified by enChIP-MS - A putative role of HDX as a negative regulator.

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism.

The miR-17-92 microRNA cluster regulates multiple components of the TGF-ß pathway in neuroblastoma.

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-ß signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-ß signaling cascade as well as direct inhibition of TGF-ß-responsive genes.

The RNA-binding protein HuR inhibits expression of CCL5 and limits recruitment of macrophages into tumors.

The RNA-binding protein HuR promotes tumor growth by affecting proliferation, metastasis, apoptosis, and angiogenesis. Although immune cells, especially tumor-associated macrophages, are critical components of the tumor stroma, the influence of HuR in tumors on the recruitment of immune cells remains poorly understood. In the present study, we, therefore, aimed to elucidate the impact of tumor cell HuR on the interaction between tumor cells and macrophages. To this end, we stably depleted HuR in human MCF-7 breast cancer cells. We found that HuR-deficient cells not only showed reduced proliferation, they further expressed elevated levels of the chemokine CCL5. HuR-dependent repression of CCL5 was neither caused by altered CCL5 mRNA stability, nor by changes in CCL5 translation. Instead, loss of HuR augmented transcription of CCL5, which was mediated via an interferon-stimulated response element in the CCL5 promoter. Furthermore, HuR depletion enhanced macrophage recruitment into MCF-7 tumor spheroids, an effect which was completely lost upon neutralization of CCL5. HuR expression further negatively correlated with CCL5 expression and macrophage appearance in a cohort of breast tumors. Thus, while HuR is well-characterized to support various pro-tumorigenic features in tumor cells, we provide evidence that it limits the recruitment of macrophages into tumors by repressing CCL5. As macrophage infiltration is associated with poor prognosis, our findings underline the highly cell-type and context specific role of HuR in tumorigenesis.

Predominance of girls with cancer in families with multiple childhood cancer cases.

BACKGROUND: Recent studies indicate that one of four childhood cancers can be attributed to hereditary genetic abnormalities. METHODS: The Lund Childhood Cancer Genetic study includes newly diagnosed childhood cancer patients as well as childhood cancer survivors visiting the Department of Pediatrics or the Late Effect Clinic at Skåne University Hospital, Lund, Sweden. Questionnaires regarding family history of cancer and blood samples were provided. Reported data were validated and extended by use of the Swedish Population- and Cancer Registries. Demographics in families with one case of childhood cancer (FAM1) were investigated and compared to families with multiple cases of childhood cancer (FAM > 1) as well as to childhood cancer in the general population. RESULTS: Forty-one out of 528 families (7.8%) had more than one case of childhood cancer. In 23 families the affected children were relatives up to a 3rd degree (4.4%). In FAM > 1, 69.2% of the children with leukemia and 60% of those with tumors in the central nervous system (CNS) had a childhood relative with matching diagnosis, both significantly higher than expected. Significantly more female than male patients were observed in FAM > 1 compared to FAM1. This female predominance was most striking in childhood leukemia (77% female) and also, yet to a lesser extent, in CNS tumors (68% female). CONCLUSIONS: We conclude that the high proportion of children with leukemia or CNS tumors in FAM > 1 having a childhood relative with the same diagnosis suggests a hereditary background. Moreover, we report a female predominance in childhood leukemia and childhood CNS tumors in FAM > 1, which may indicate a hereditary gender-specific risk factor in these families.

Individual patient risk stratification of high-risk neuroblastomas using a two-gene score suited for clinical use.

Several gene expression-based prognostic signatures have been described in neuroblastoma, but none have successfully been applied in the clinic. Here we have developed a clinically applicable prognostic gene signature, both with regards to number of genes and analysis platform. Importantly, it does not require comparison between patients and is applicable amongst high-risk patients. The signature is based on a two-gene score (R-score) with prognostic power in high-stage tumours (stage 4 and/or MYCN-amplified diagnosed after 18 months of age). QPCR-based and array-based analyses of matched cDNAs confirmed cross platform (array-qPCR) transferability. We also defined a fixed cut-off value identifying prognostically differing subsets of high-risk patients on an individual patient basis. This gene expression signature independently contributes to the current neuroblastoma classification system, and if prospectively validated could provide further stratification of high-risk patients, and potential upfront identification of a group of patients that are in need of new/additional treatment regimens.

Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome.

Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leads to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma.

Tumors with nonfunctional retinoblastoma protein are killed by reduced γ-tubulin levels.

In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each other's expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.

Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression.

We show that the centrosome- and microtubule-regulating protein γ-tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ-Tubulin contains a C-terminal signal that results in its translocation to the nucleus during late G(1) to early S phase. γ-Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1-γ-tubulin complex is formed during the G(1)/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ-tubulin or by complex formation between γ-tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ-tubulin C terminus encodes a DNA-binding domain that interacts with E2F-regulated promoters, resulting in γ-tubulin-mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.

Prevalence of germline pathogenic variants in 22 cancer susceptibility genes in Swedish pediatric cancer patients.

Up to 10% of pediatric cancer patients harbor pathogenic germline variants in one or more cancer susceptibility genes. A recent study from the US reported pathogenic variants in 22 out of 60 analyzed autosomal dominant cancer susceptibility genes, implicating 8.5% of pediatric cancer patients. Here we aimed to assess the prevalence of germline pathogenic variants in these 22 genes in a population-based Swedish cohort and to compare the results to those described in other populations. We found pathogenic variants in 10 of the 22 genes covering 3.8% of these patients. The prevalence of TP53 mutations was significantly lower than described in previous studies, which can largely be attributed to differences in tumor diagnosis distributions across the three cohorts. Matched family history for relatives allowed assessment of familial cancer incidence, however, no significant difference in cancer incidence was found in families of children carrying pathogenic variants compared to those who did not.

The Irradiated Brain Microenvironment Supports Glioma Stemness and Survival via Astrocyte-Derived Transglutaminase 2.

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma. SIGNIFICANCE: These findings presented here indicate that radiotherapy can result in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2101/F1.large.jpg.

Increased Cancer Risk in Families with Pediatric Cancer Is Associated with Gender, Age, Diagnosis, and Degree of Relation to the Child.

BACKGROUND: Studies of cancer risk among relatives of children with cancer beyond parents and siblings are limited. We have investigated the cancer risk up to the third degree of relation in families with pediatric cancer to reveal patterns of inheritance. METHODS: A single-center cohort of 757 patients with pediatric cancer was linked to the Swedish National Population Register, resulting in 16,137 relatives up to the third degree of relation. All relatives were matched to the Swedish Cancer Register, and standard incidence ratios (SIR) were calculated to define relatives at risk. RESULTS: Children and adults up to the third degree of relation had increased cancer risk, with SIRs of 1.48 (P = 0.01) and 1.07 (P < 0.01), respectively. The SIRs for first- and third-degree adult relatives were 1.22 and 1.10, respectively, but no increased risk was observed in second-degree relatives. Male relatives had a higher risk than females, especially when related to a girl and when the child had leukemia. The risk was mainly increased for lung, prostate, and gastrointestinal cancer. When excluding 29 families of children with known pathogenic germline variants, the increased risk remained. CONCLUSIONS: Relatives to children with cancer up to third degree of relation have an increased cancer risk. Known pathogenic germline variants do not explain this increased risk. IMPACT: The overall increased cancer risk among relatives of children with cancer in this population-based cohort strengthens the importance of surveillance programs for families with pediatric cancer.

Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma.

The PI3K pathway is a major driver of cancer progression. However, clinical resistance to PI3K inhibition is common. IBL-302 is a novel highly specific triple PIM, PI3K, and mTOR inhibitor. Screening IBL-302 in over 700 cell lines representing 47 tumor types identified neuroblastoma as a strong candidate for PIM/PI3K/mTOR inhibition. IBL-302 was more effective than single PI3K inhibition in vitro, and IBL-302 treatment of neuroblastoma patient-derived xenograft (PDX) cells induced apoptosis, differentiated tumor cells, and decreased N-Myc protein levels. IBL-302 further enhanced the effect of the common cytotoxic chemotherapies cisplatin, doxorubicin, and etoposide. Global genome, proteome, and phospho-proteome analyses identified crucial biological processes, including cell motility and apoptosis, targeted by IBL-302 treatment. While IBL-302 treatment alone reduced tumor growth in vivo, combination therapy with low-dose cisplatin inhibited neuroblastoma PDX growth. Complementing conventional chemotherapy treatment with PIM/PI3K/mTOR inhibition has the potential to improve clinical outcomes and reduce severe late effects in children with high-risk neuroblastoma.

Refined control of cell stemness allowed animal evolution in the oxic realm.

Animal diversification on Earth has long been presumed to be associated with the increasing extent of oxic niches. Here, we challenge that view. We start with the fact that hypoxia (<1-3% O2) maintains cellular immaturity (stemness), whereas adult stem cells continuously-and paradoxically-regenerate animal tissue in oxygenated settings. Novel insights from tumour biology illuminate how cell stemness nevertheless can be achieved through the action of oxygen-sensing transcription factors in oxygenated, regenerating tissue. We suggest that these hypoxia-inducible transcription factors provided animals with unprecedented control over cell stemness that allowed them to cope with fluctuating oxygen concentrations. Thus, a refinement of the cellular hypoxia-response machinery enabled cell stemness at oxic conditions and, then, animals to evolve into the oxic realm. This view on the onset of animal diversification is consistent with geological evidence and provides a new perspective on the challenges and evolution of multicellular life.

Patient-Derived Xenograft Models Reveal Intratumor Heterogeneity and Temporal Stability in Neuroblastoma.

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain underexplored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX-Avatar studies into preclinical cancer research.Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. Cancer Res; 78(20); 5958-69. ©2018 AACR.

Neuroblastoma patient-derived xenograft cells cultured in stem-cell promoting medium retain tumorigenic and metastatic capacities but differentiate in serum.

Cultured cancer cells serve as important models for preclinical testing of anti-cancer compounds. However, the optimal conditions for retaining original tumor features during in vitro culturing of cancer cells have not been investigated in detail. Here we show that serum-free conditions are critical for maintaining an immature phenotype of neuroblastoma cells isolated from orthotopic patient-derived xenografts (PDXs). PDX cells could be grown either as spheres or adherent on laminin in serum-free conditions with retained patient-specific genomic aberrations as well as tumorigenic and metastatic capabilities. However, addition of serum led to morphological changes, neuronal differentiation and reduced cell proliferation. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were central for PDX cell proliferation and MYCN expression, and also hindered the serum-induced differentiation. Although serum induced a robust expression of neurotrophin receptors, stimulation with their cognate ligands did not induce further sympathetic differentiation, which likely reflects a block in PDX cell differentiation capacity coupled to their tumor genotype. Finally, PDX cells cultured as spheres or adherent on laminin responded similarly to various cytotoxic drugs, suggesting that both conditions are suitable in vitro screening models for neuroblastoma-targeting compounds.

Musculocontractural Ehlers-Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin.

Of all live births with congenital anomalies, approximately one-third exhibit deformities of the head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers-Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused by dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 in connective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1 is important for the generation of isolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial-mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers.

PI3K-mTORC2 but not PI3K-mTORC1 regulates transcription of HIF2A/EPAS1 and vascularization in neuroblastoma.

Hypoxia-inducible factor (HIF) is a master regulator of cellular responses to oxygen deprival with a critical role in mediating the angiogenic switch in solid tumors. Differential expression of the HIF subunits HIF1α and HIF2α occurs in many human tumor types, suggesting selective implications to biologic context. For example, high expression of HIF2α that occurs in neuroblastoma is associated with stem cell-like features, disseminated disease, and poor clinical outcomes, suggesting pivotal significance for HIF2 control in neuroblastoma biology. In this study, we provide novel insights into how HIF2α expression is transcriptionally controlled by hypoxia and how this control is abrogated by inhibition of insulin-like growth factor-1R/INSR-driven phosphoinositide 3-kinase (PI3K) signaling. Reducing PI3K activity was sufficient to decrease HIF2α mRNA and protein expression in a manner with smaller and less vascularized tumors in vivo. PI3K-regulated HIF2A mRNA expression was independent of Akt or mTORC1 signaling but relied upon mTORC2 signaling. HIF2A mRNA was induced by hypoxia in neuroblastoma cells isolated from metastatic patient-derived tumor xenografts, where HIF2A levels could be reduced by treatment with PI3K and mTORC2 inhibitors. Our results suggest that targeting PI3K and mTORC2 in aggressive neuroblastomas with an immature phenotype may improve therapeutic efficacy.