Siv Beckman

Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo

Loss of the tumor suppressor gene von Hippel-Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21 Cip1 and/or p27 Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes.

BackgroundNeuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands.ResultsExpression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis.ConclusionThis unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.

HIF-2 alpha maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells

High hypoxia-inducible factor-2α (HIF-2α) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2α as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2α reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2α-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2α maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2α is an attractive target for neuroblastoma therapy.

HIF-1{alpha} and HIF-2{alpha} Are Differentially Regulated In vivo in Neuroblastoma: High HIF-1{alpha} Correlates Negatively to Advanced Clinical Stage and Tumor Vascularization.

Purpose: Hypoxia is considered to be a major driving force behind tumor angiogenesis. The stabilization and activation at hypoxia of the hypoxia-inducible factors HIF-1α and HIF-2α and the concomitant induction of expression of vascular endothelial growth factor (VEGF) and other proangiogenic factors provide a molecular frame for hypoxia-driven tumor angiogenesis. This study has investigated how HIF and VEGF protein levels relate to each other with regard to vascularization, tumor stage, and overall survival in neuroblastoma. Experimental Design: Tissue cores taken from tumor specimens representing 93 children with neuroblastoma were arranged on a microarray and stained for HIF-1α, HIF-2α, VEGF, and CD31 proteins. Both fraction of positive cells and staining intensity were evaluated and protein levels were correlated with each other and with clinical variables. Results: Although high levels of both HIF-1α (P < 0.001) and HIF-2α (P < 0.001) correlated positively to VEGF expression, they did not fully correlate with each other. Moreover, HIF-1α (P = 0.002) and VEGF (P < 0.001), but not HIF-2α, correlated negatively to vascularization as determined by CD31 staining abundance. VEGF expression or degree of vascularization did not correlate with tumor stage or overall survival. High HIF-1α levels correlated with low tumor stage (P < 0.001) and were associated with a favorable patient prognosis (P = 0.08). Conclusions: The discordant results on expression of HIF-1α and HIF-2α suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression/stabilization mechanisms. The association between HIF-1α and favorable outcome stresses the importance of discriminating HIF-2α from HIF-1α expression and has implications for using HIFs as treatment targets. (Clin Cancer Res 2009;15(23):7130–6)

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.

HIF-1alpha induces MXI1 by alternate promoter usage in human neuroblastoma cells.

Adaptation to low oxygen conditions is essential for maintaining homeostasis and viability in oxygen-consuming multi-cellular tissues, including solid tumors. Central in these processes are the hypoxia-inducible transcription factors, HIF-1 and HIF-2, controlling genes involved in e.g. glucose metabolism and neovascularization. Tumor hypoxia and HIF expression have also been associated with a dedifferentiated phenotype and increased aggressiveness. In this report we show that the MAX interactor-1 (MXI1) gene is directly regulated by HIF proteins in neuroblastoma and breast cancer cells. HIF-binding and transactivation were detected within MXI1 gene regulatory sequences in the vicinity of the MXI1-0 promoter, leading to rapid induction of the alternate MXI1-0 isoform followed by a long-term induction of both the MXI1-0 and MXI1 isoforms. Importantly, knock-down of MXI1 had limited effect on MYC/MYCN activity under hypoxia, an observation that might be related to the different functional attributes of the two MXI1 isoforms.

Hypoxic Conditions Induce a Cancer-Like Phenotype in Human Breast Epithelial Cells

Introduction Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. Methods Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot. Results In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state.

Arsenic trioxide-induced neuroblastoma cell death is accompanied by proteolytic activation of nuclear Bax.

Arsenic trioxide (As2O3) is toxic to multidrug-resistant neuroblastoma cells in vivo and in vitro. In neuroblastoma, As2O3 does not exert its cell death-promoting effects via a classical apoptotic pathway. A death mechanism involving proteolytic cleavage of Bax to a p18 form seems to be of importance, because inhibition of Bax cleavage coincides with diminished cell death. As existing models of cell death implicate Bax in the intrinsic apoptotic pathway, triggering death after Bax translocation to the mitochondria, we investigated the cellular localization of p18 Bax by subcellular fractionation. After As2O3 treatment, p18 Bax was only present in nuclei-enriched, mitochondria-depleted fractions. Cytoplasmic p21 Bax levels decreased, whereas total (p21 and p18) nuclear Bax increased. Overexpressed p21 Bax localized to the cytoplasm and nuclei, whereas overexpressed p18 Bax localized to extra-nuclear structures only. The inability of overexpressed p18 Bax to locate to the nucleus, and the As2O3-induced reduction of p21 Bax in the cytosol, suggest an As2O3-induced mechanism where p18 Bax gets cleaved and ‘trapped’ in the nucleus. This model is strengthened by the observation that calpain, the protease responsible for p18 Bax generation, is present in the nuclei, and that nuclear calpain is induced by increasing As2O3 and Ca2+ levels.

Neuroblastoma patient-derived orthotopic xenografts reflect the microenvironmental hallmarks of aggressive patient tumours

Treatment of high-risk childhood neuroblastoma is a clinical challenge which has been hampered by a lack of reliable neuroblastoma mouse models for preclinical drug testing. We have previously established invasive and metastasising patient-derived orthotopic xenografts (PDXs) from high-risk neuroblastomas that retained the genotypes and phenotypes of patient tumours. Given the important role of the tumour microenvironment in tumour progression, metastasis, and treatment responses, here we analysed the tumour microenvironment of five neuroblastoma PDXs in detail. The PDXs resembled their parent tumours and retained important stromal hallmarks of aggressive lesions including rich blood and lymphatic vascularisation, pericyte coverage, high numbers of cancer-associated fibroblasts, tumour-associated macrophages, and extracellular matrix components. Patient-derived tumour endothelial cells occasionally formed blood vessels in PDXs; however, tumour stroma was, overall, of murine origin. Lymphoid cells and lymphatic endothelial cells were found in athymic nude mice but not in NSG mice; thus, the choice of mouse strain dictates tumour microenvironmental components. The murine tumour microenvironment of orthotopic neuroblastoma PDXs reflects important hallmarks of aggressive and metastatic clinical neuroblastomas. Neuroblastoma PDXs are clinically relevant models for preclinical drug testing.

HIF-2α Expression Is Suppressed in SCLC Cells, Which Survive in Moderate and Severe Hypoxia When HIF-1α Is Repressed.

Small cell lung carcinoma (SCLC) is extremely aggressive and frequently metastasizes widely in its early stage. Because tumor hypoxia is related to aggressive tumor behavior and the hypoxic adaptation of SCLC is poorly documented, we stained SCLC tumors arranged in a tissue microarray for hypoxia-inducible factor (HIF)-1α and HIF-2α proteins. We found an overall lack of HIF-2α protein expression, which was confirmed in large tumor sections. HIF-1α protein was strongly expressed in most tumors, frequently adjacent to necrotic regions. In concordance, cultured SCLC but not non-small cell lung carcinoma cells showed no or extremely low levels of HIF-2α mRNA and no HIF-2α protein at hypoxia. HIF-1α was stabilized after 4 hours at hypoxia, and its accumulation increased up to 96 hours. SCLC cells survived well and showed net proliferation and low cell death in modest (1% oxygen) and severe (0.1% oxygen) hypoxia. HIF-1α repression virtually did not influence cell death or viability despite reduced levels of hypoxia-inducible genes, such as BNIP3 and BNIP3L. At 1% oxygen no increased autophagy (LC3B-II activation) or NF-κB signaling were detected, whereas the unfolded protein response was activated at severe hypoxia. Our data indicate that HIFs are not exclusively required for SCLC cell survival at modest or severe hypoxia and that additional, yet uncharacterized, hypoxia-driven adaptation pathways may become activated.