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.

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.

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.

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation.

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα(+)) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

Increased gene copy number of KIT and VEGFR2 at 4q12 in primary breast cancer is related to an aggressive phenotype and impaired prognosis.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and no targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and KIT, have shown some promising results for patients with TNBC. The aim of the study was to investigate whether gains and/or amplifications of VEGFR2 and KIT, located at 4q12, occur in TNBC. Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and KIT in 83 primary human breast cancers including 31 TNBCs. Gains were defined as ≥ 4 gene copies in >40% of the cancer cells, whereas amplification was defined as CEP >2 in more than 10% of the cancer cells. A tumor was considered FISH positive for KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and KIT in nine (18%) cases for both genes, but no significant difference between TNBCs and non-TNBCs was found. FISH positivity for VEGFR2 and KIT was significantly correlated (χ(2) test, P < 0.001), and significantly related to ER negativity and high Nottingham histological grade (NHG). A significantly worse 5-year breast cancer specific survival (BCSS) was seen for FISH positive cases. Increased copy number of VEGFR2 and KIT thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category.

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.

Acetylation-dependent oncogenic activity of metastasis-associated protein 1 co-regulator.

High expression of metastasis-associated protein 1 co-regulator (MTA1), a component of the nuclear remodelling and histone deacetylase complex, has been associated with human tumours. However, the precise role of MTA1 in tumorigenesis remains unknown. In this study, we show that induced levels of MTA1 are sufficient to transform Rat1 fibroblasts and that the transforming potential of MTA1 is dependent on its acetylation at Lys626. Underlying mechanisms of MTA1-mediated transformation include activation of the Ras-Raf pathway by MTA1 but not by acetylation-inactive MTA1; this was due to the repression of Galphai2 transcription, which negatively influences Ras activation. We observed that acetylated MTA1-histone deacetylase (HDAC) interaction was required for the recruitment of the MTA1-HDAC complex to the Galphai2 regulatory element and consequently for the repression of Galphai2 transcription and expression leading to activation of the Ras-Raf pathway. The findings presented in this study provide for the first time--to the best of our knowledge--evidence of acetylation-dependent oncogenic activity of a cancer-relevant gene product.

Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.

p27Kip1 is a predictive factor for tamoxifen treatment response but not a prognostic marker in premenopausal breast cancer patients.

The cell-cycle regulating protein p27(Kip1) (p27) has dual roles by acting as both a cdk inhibitor and as an assembly factor for different cdk complexes. Loss of p27 has been linked to malignant features in tumours; however, the exact role of p27 deregulation in breast cancer regarding prognostic and treatment predictive information has not been fully clarified. We have evaluated p27 expression in 328 primary, Stage II breast cancers from premenopausal patients who had been randomised to either tamoxifen treatment or no adjuvant treatment after surgery. p27 was associated with the oestrogen receptor and cyclin D1, and p27 downregulation was associated with high proliferation. There was no association between recurrence-free survival (RFS) and p27 (HR = 0.800, 95% CI 0.523-1.222, p = 0.300), indicating that p27 is not a prognostic marker. The predictive value of p27 was analysed by comparing RFS in tamoxifen-treated and untreated patients in subgroups of low and high p27 expression (HR = 0.747, 95% CI 0.335-1.664, p = 0.474 and HR = 0.401, 95% CI 0.240-0.670, p < 0.001, respectively). Only patients with p27-high tumours benefited from tamoxifen (multivariate interaction analysis p = 0.034). Our study suggests that p27 downregulation is associated with tamoxifen resistance in premenopausal breast cancer but is not linked to impaired prognosis.

ALK positively regulates MYCN activity through repression of HBP1 expression.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PI3K-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PI3K antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.

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.

Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer.

Insufficient tissue oxygenation, or hypoxia, contributes to tumor aggressiveness and has a profound impact on clinical outcomes in cancer patients. At decreased oxygen tensions, hypoxia-inducible factors (HIFs) 1 and 2 are stabilized and mediate a hypoxic response, primarily by acting as transcription factors. HIFs exert differential effects on tumor growth and affect important cancer hallmarks including cell proliferation, apoptosis, differentiation, vascularization/angiogenesis, genetic instability, tumor metabolism, tumor immune responses, and invasion and metastasis. As a consequence, HIFs mediate resistance to chemo- and radiotherapy and are associated with poor prognosis in cancer patients. Intriguingly, perivascular tumor cells can also express HIF-2α, thereby forming a "pseudohypoxic" phenotype that further contributes to tumor aggressiveness. Therefore, therapeutic targeting of HIFs in cancer has the potential to improve treatment efficacy. Different strategies to target hypoxic cancer cells and/or HIFs include hypoxia-activated prodrugs and inhibition of HIF dimerization, mRNA or protein expression, DNA binding capacity, and transcriptional activity. Here we review the functions of HIFs in the progression and treatment of malignant solid tumors. We also highlight how HIFs may be targeted to improve the management of patients with therapy-resistant and metastatic cancer.

HIF2α contributes to antiestrogen resistance via positive bilateral crosstalk with EGFR in breast cancer cells.

The majority of breast cancers express estrogen receptor α (ERα), and most patients with ERα-positive breast cancer benefit from antiestrogen therapy. The ERα-modulator tamoxifen and ERα-downregulator fulvestrant are commonly employed antiestrogens. Antiestrogen resistance remains a clinical challenge, with few effective treatments available for patients with antiestrogen-resistant breast cancer. Hypoxia, which is intrinsic to most tumors, promotes aggressive disease, with the hypoxia-inducible transcription factors HIF1 and HIF2 regulating cellular responses to hypoxia. Here, we show that the ERα-expressing breast cancer cells MCF-7, CAMA-1, and T47D are less sensitive to antiestrogens when hypoxic. Furthermore, protein and mRNA levels of HIF2α/HIF2A were increased in a panel of antiestrogen-resistant cells, and antiestrogen-exposure further increased HIF2α expression. Ectopic expression of HIF2α in MCF-7 cells significantly decreased sensitivity to antiestrogens, further implicating HIF2α in antiestrogen resistance. EGFR is known to contribute to antiestrogen resistance: we further show that HIF2α drives hypoxic induction of EGFR and that EGFR induces HIF2α expression. Downregulation or inhibition of EGFR led to decreased HIF2α levels. This positive and bilateral HIF2-EGFR regulatory crosstalk promotes antiestrogen resistance and, where intrinsic hypoxic resistance exists, therapy itself may exacerbate the problem. Finally, inhibition of HIFs by FM19G11 restores antiestrogen sensitivity in resistant cells. Targeting HIF2 may be useful for counteracting antiestrogen resistance in the clinic.

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.

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.

Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue --- optimization for genome wide array analyses.

BACKGROUND: Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses. FINDINGS: We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g® (Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g® gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM. CONCLUSIONS: LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g® WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling.

Estrogen receptor-alpha phosphorylation at serine-118 and tamoxifen response in breast cancer.

Although estrogen receptor-alpha (ER) [corrected] is a marker used to identify breast cancer patients most likely to benefit from endocrine therapy, approximately 50% of ER-positive [corrected] breast carcinomas are resistant to tamoxifen. Preclinical studies have shown that phosphorylation of ER [corrected] at serine-118 (ER alpha S118-P) is required for tamoxifen-mediated inhibition of ER-induced [corrected] gene expression. We evaluated the association between recurrence-free survival after tamoxifen treatment and ER alpha S118-P expression by use of Cox proportional hazards regression. Data were from 239 premenopausal patients with breast cancer who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment vs no systemic treatment. ER alpha S118-P expression was assessed by immunohistochemistry and categorized by use of the Allred score (low expression = score of 0-6; high expression = score of 7-8). All statistical tests were two-sided. Compared with systemically untreated patients, we found evidence of a benefit from adjuvant tamoxifen among patients whose tumors had high ER alpha S118-P expression (23.7 recurrences per 1000 person-years versus 72.2 recurrences per 1000 person-years, hazard ratio [HR] of recurrence = 0.36, 95% confidence interval [CI] = 0.20 to 0.65) but not among patients whose tumors had low expression (51.0 recurrences per 1000 person-years versus 57.0 recurrences per 1000 person-years, HR of recurrence = 0.87, 95% CI = 0.51 to 1.48), a statistically significant difference (P for interaction = .037). ER alpha 118-P was not associated with recurrence-free survival among untreated patients. Thus, ER alpha S118-P expression appears to be associated with response to tamoxifen. [corrected]