Sofie Mohlin

The HIF-2alpha-Driven Pseudo-Hypoxic Phenotype in Tumor Aggressiveness, Differentiation, and Vascularization.

Cellular adaptation to diminished tissue oxygen tensions, hypoxia, is largely governed by the hypoxia inducible transcription factors, HIF-1 and HIF-2. Tumor hypoxia and high HIF protein levels are frequently associated with aggressive disease. In recent years, high tumor cell levels of HIF-2 and the oxygen sensitive subunit HIF-2α have been associated with unfavorable disease and shown to be highly expressed in tumor stem/initiating cells originating from neuroblastoma and glioma, respectively. In these cells, HIF-2 is active under nonhypoxic conditions as well, creating a pseudo-hypoxic phenotype with clear influence on tumor behavior. Neuroblastoma tumor initiating cells are immature with a neural crest-like phenotype and downregulation of HIF-2α in these cells results in neuronal sympathetic differentiation and the cells become phenotypically similar to the bulk of neuroblastoma cells found in clinical specimens. Knockdown of HIF-2α in neuroblastoma and glioma tumor stem/initiating cells leads to reduced levels of VEGF and poorly vascularized, highly necrotic tumors. As high HIF-2α expression further correlates with disseminated disease as demonstrated in neuroblastoma, glioma, and breast carcinoma, we propose that targeting HIF-2α and/or the pseudo-hypoxic phenotype induced by HIF-2 under normoxic conditions has great clinical potential.

Neuroblastoma Patient-Derived Orthotopic Xenografts Retain Metastatic Patterns and Geno- and Phenotypes of Patient Tumours.

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient‐derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose–positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient‐specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high‐risk neuroblastoma in patients. PDX‐derived cells were cultured in serum‐free medium where they formed free‐floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour‐initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK‐N‐BE(2)c cell line‐derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high‐risk metastatic neuroblastoma.

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.

Cancer-associated fibroblast-secreted CXCL16 attracts monocytes to promote stroma activation in triple-negative breast cancers

Triple-negative (TN) breast cancers (ER−PR−HER2−) are highly metastatic and associated with poor prognosis. Within this subtype, invasive, stroma-rich tumours with infiltration of inflammatory cells are even more aggressive. The effect of myeloid cells on reactive stroma formation in TN breast cancer is largely unknown. Here, we show that primary human monocytes have a survival advantage, proliferate in vivo and develop into immunosuppressive myeloid cells expressing the myeloid-derived suppressor cell marker S100A9 only in a TN breast cancer environment. This results in activation of cancer-associated fibroblasts and expression of CXCL16, which we show to be a monocyte chemoattractant. We propose that this migratory feedback loop amplifies the formation of a reactive stroma, contributing to the aggressive phenotype of TN breast tumours. These insights could help select more suitable therapies targeting the stromal component of these tumours, and could aid prediction of drug resistance.

TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival

Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan–Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

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.

The PI3-kinase isoform p110δ is essential for cell transformation induced by the D816V mutant of c-Kit in a lipid-kinase-independent manner

PI3-kinase has a crucial role in transformation mediated by the oncogenic c-Kit mutant D816V. In this study, we demonstrate that the c-Kit/D816V-mediated cell survival is dependent on an intact direct binding of PI3-kinase to c-Kit. However, mutation of this binding site had little effect on the PI3-kinase activity in the cells, suggesting that c-Kit/D816V-mediated cell survival is dependent on PI3-kinase but not its kinase activity. Furthermore, inhibition of the lipid kinase activity of PI3-kinase led only to a slight inhibition of cell survival. Knockdown of the predominant PI3-kinase isoform p110δ in c-Kit/D816V-expressing Ba/F3 cells led to reduced cell transformation both in vitro and in vivo without affecting the overall PI3-kinase activity. This suggests that p110δ has a lipid-kinase-independent role in c-Kit/D816V-mediated cell transformation. We furthermore demonstrate that p110δ is phosphorylated at residues Y524 and S1039 and that phosphorylation requires an intact binding site for PI3-kinase in c-Kit/D816V. Overexpression of p110δ carrying the Y523F and S1038A mutations significantly reduced c-Kit/D816V-mediated cell survival and proliferation. Taken together, our results demonstrate an important lipid-kinase-independent role of p110δ in c-Kit/D816V-mediated cell transformation. This furthermore suggests that p110δ could be a potential diagnostic factor and selective therapeutic target for c-Kit/D816V-expressing malignancies.

Neuroblastoma aggressiveness in relation to sympathetic neuronal differentiation stage.

Neuroblastoma is a childhood malignancy of the sympathetic neuronal lineage. It is a rare disease, but since it is frequently diagnosed during infancy, neuroblastoma causes life-long medical follow up of those children that survive the disease. It was early recognized that a high tumor cell differentiation stage correlates to favorable clinical stage and positive clinical outcome. Today, highly differentiated tumors are surgically removed and not further treated. Cells of many established human neuroblastoma cell lines have the capacity to differentiate when stimulated properly, and these cell lines have been used as models for studying and understanding central concepts of tumor cell differentiation. One recent aspect of this issue is the observation that tumor cells can dedifferentiate and gain a stem cell-like phenotype during hypoxic conditions, which was first shown in neuroblastoma. Aberrant or blocked differentiation is a central aspect of neuroblastoma genesis. In this review we summarize known genetic and non-genetic events in neuroblastoma that might be coupled to an aberrant sympathetic neuronal differentiation and thereby indirectly influencing tumorigenesis and/or aggressive neuroblastoma behavior.

Tumor-Initiating Cells in Childhood Neuroblastoma—Letter

We report here that the neuroblastoma tumor-initiating cell (TIC) cultures (NB12, NB88, and NB122R) isolated from neuroblastoma patients and established in culture by Dr. David Kaplan and colleagues [reported by Hansford et al. ([1][1]) and Pietras et al. ([2][2])] are severely overgrown by Epstein-

Hypoxia, pseudohypoxia and cellular differentiation

Tumor hypoxia correlates to aggressive disease, and while this is explained by a variety of factors, one clue to understand this phenomena was the finding that hypoxia induces a de-differentiated, stem cell-like phenotype in neuroblastoma and breast tumor cells. The hypoxia inducible transcription factors (HIFs) are regulated at the translational level by fluctuating oxygen concentrations, but emerging data reveal that both HIF-1α and HIF-2α expression can be induced by aberrantly activated growth factor signaling independently of oxygen levels. Furthermore, HIF-2α is regulated by hypoxia also at the transcriptional level in neuroblastoma and glioma cells. In cultured tumor cells, HIF-2α is stabilized at physiological oxygen concentrations followed by induced expression of classical hypoxia-driven genes, resulting in a pseudohypoxic phenotype. In addition, in neuroblastoma and glioma specimens, a small subset of HIF-2α positive, HIF-1α negative, tumor cells is found adjacent to blood vessels, i.e. in areas with presumably adequate oxygenation. These tumor niches are thus pseudohypoxic, and the HIF-2α expressing cells present immature features. We have postulated that this niche in neuroblastomas encompass the tumor stem cells. Oncogenes or tumor suppressor genes associated with pseudohypoxia are frequently mutated or deleted in the germline, implicating that the pseudohypoxic phenotype indeed is tumorigenic. In summary, the hypoxic and pseudohypoxic phenotypes of solid tumors are attractive therapeutic targets.