Publications by authors named "Sven Påhlman"

88 Publications

Therapeutic targeting of KSP in preclinical models of high-risk neuroblastoma.

Sci Transl Med 2020 09;12(562)

Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, 223 81 Lund, Sweden.

Neuroblastoma is a childhood malignancy with often dismal prognosis; relapse is common despite intense treatment. Here, we used human tumor organoids representing multiple -amplified high-risk neuroblastomas to perform a high-throughput drug screen with approved or emerging oncology drugs. Tumor-selective effects were calculated using drug sensitivity scores. Several drugs with previously unreported anti-neuroblastoma effects were identified by stringent selection criteria. ARRY-520, an inhibitor of kinesin spindle protein (KSP), was among those causing reduced viability. High expression of the KSP-encoding gene was associated with poor outcome in neuroblastoma. Genome-scale loss-of-function screens in hundreds of human cancer cell lines across 22 tumor types revealed that is particularly important for neuroblastoma cell viability. KSP inhibition in neuroblastoma patient-derived xenograft (PDX) cells resulted in the formation of abnormal monoastral spindles, mitotic arrest, up-regulation of mitosis-associated genes, and apoptosis. In vivo, KSP inhibition caused regression of -amplified neuroblastoma PDX tumors. Furthermore, treatment of mice harboring orthotopic neuroblastoma PDX tumors resulted in increased survival. Our results suggested that KSP inhibition could be a promising treatment strategy in children with high-risk neuroblastoma.
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http://dx.doi.org/10.1126/scitranslmed.aba4434DOI Listing
September 2020

Inflammatory macrophage derived TNFα downregulates estrogen receptor α via FOXO3a inactivation in human breast cancer cells.

Exp Cell Res 2020 05 4;390(1):111932. Epub 2020 Mar 4.

Cancer Immunology, Department of Translational Medicine, 214 28, Malmö, Lund University, Sweden. Electronic address:

Patients with estrogen receptor α positive (ERα) breast cancer can respond to endocrine therapy, but treatment resistance is common and associated with downregulation of ERα expression in the dormant residual cells. Here we show, using long-term NSG xenograft models of human breast cancer and primary human monocytes, in vitro primary cell cultures and tumors from breast cancer patients, that macrophage derived tumor necrosis factor alpha (TNFα) downregulates ERα in breast cancer cells via inactivation of the transcription factor Forkhead box O transcription factor 3a (FOXO3a). Moreover, presence of tumor associated macrophages in the primary tumor of breast cancer patients, was associated with ERα negativity, and with worse prognosis in patients with ERα tumors. We propose that pro-inflammatory macrophages, despite being tumoricidal, may have direct effects on tumor progression and endocrine resistance in breast cancer patients. Our findings suggest that TNFα antagonists should be evaluated for treatment of ERα breast cancer.
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http://dx.doi.org/10.1016/j.yexcr.2020.111932DOI Listing
May 2020

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

Exp Cell Res 2020 03 13;388(2):111845. Epub 2020 Jan 13.

Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, Lund, Sweden; Division of Pediatrics, Department of Clinical Sciences, Lund University, Lund, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2020.111845DOI Listing
March 2020

Integrative discovery of treatments for high-risk neuroblastoma.

Nat Commun 2020 01 3;11(1):71. Epub 2020 Jan 3.

Department of Immunology, Genetics and Pathology, Uppsala University, SE-751 85, Uppsala, Sweden.

Despite advances in the molecular exploration of paediatric cancers, approximately 50% of children with high-risk neuroblastoma lack effective treatment. To identify therapeutic options for this group of high-risk patients, we combine predictive data mining with experimental evaluation in patient-derived xenograft cells. Our proposed algorithm, TargetTranslator, integrates data from tumour biobanks, pharmacological databases, and cellular networks to predict how targeted interventions affect mRNA signatures associated with high patient risk or disease processes. We find more than 80 targets to be associated with neuroblastoma risk and differentiation signatures. Selected targets are evaluated in cell lines derived from high-risk patients to demonstrate reversal of risk signatures and malignant phenotypes. Using neuroblastoma xenograft models, we establish CNR2 and MAPK8 as promising candidates for the treatment of high-risk neuroblastoma. We expect that our method, available as a public tool (targettranslator.org), will enhance and expedite the discovery of risk-associated targets for paediatric and adult cancers.
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http://dx.doi.org/10.1038/s41467-019-13817-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941971PMC
January 2020

ALK positively regulates MYCN activity through repression of HBP1 expression.

Oncogene 2019 04 11;38(15):2690-2705. Epub 2018 Dec 11.

Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium.

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 PIK-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 PIK 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.
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http://dx.doi.org/10.1038/s41388-018-0595-3DOI Listing
April 2019

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

Cancer Res 2018 10 28;78(20):5958-5969. Epub 2018 Aug 28.

Department of Laboratory Medicine, Division of Translational Cancer Research, Lund University, Lund, Sweden.

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. These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0527DOI Listing
October 2018

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

Biochem Biophys Res Commun 2018 05 26;499(2):291-298. Epub 2018 Mar 26.

Department of Pediatrics, Clinical Sciences, Lund University, Lund, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2018.03.150DOI Listing
May 2018

A Novel Topical PPARγ Agonist Induces PPARγ Activity in Ulcerative Colitis Mucosa and Prevents and Reverses Inflammation in Induced Colitis Models.

Inflamm Bowel Dis 2018 03;24(4):792-805

Department of Surgery and Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.

Background: Peroxisome proliferator-activated receptor-gamma (PPARγ) exerts anti-inflammatory effects and is therefore a potential target in ulcerative colitis (UC). A novel PPARγ agonist (AS002) developed for local action was evaluated ex vivo in biopsies from UC patients and in vivo in mice with low-grade dextran sodium sulfate (DSS)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Methods: Colonic biopsies from UC patients (n = 18) and healthy controls (n = 6) were incubated with AS002 or rosiglitazone (positive control) to measure mRNA expression of the PPARγ-responsive gene ADIPOPHILIN and protein levels of UC-related cytokines (enzyme-linked immunosorbent assay). AS002 absorption was determined in the colonic mucosa of UC patients. DSS-colitis mice received PPARγ agonists or vehicle daily by intrarectal administration starting 2 days before induction of colitis (preventive) or from days 3 to 8 (curative). Myeloperoxidase (MPO) and cytokine levels in colonic mucosa were determined. In addition, AS002 effects were studied in TNBS colitis.

Results: AS002 displayed an absorption pattern of a lipophilic drug totally metabolized in the mucosa. AS002 and rosiglitazone increased ADIPOPHILIN mRNA expression (3-fold) and decreased TNF-α, IL-1β, and IL-13 levels in human UC biopsies. In DSS, in both preventive and curative treatment and in TNBS colitis, AS002 protected against macroscopic and histological damage and lowered MPO and TNF-α, IL-1β, and IL-13 levels.

Conclusions: AS002 triggers anti-inflammatory PPARγ activity in the human colonic mucosa of UC patients and prevents and reverses colitis in mice. Our data suggest that AS002 has potential for topical maintenance treatment of UC, which warrants further studies in vivo in patients.
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http://dx.doi.org/10.1093/ibd/izx079DOI Listing
March 2018

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

Nat Ecol Evol 2018 02 18;2(2):220-228. Epub 2018 Jan 18.

Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden.

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% O) 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.
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http://dx.doi.org/10.1038/s41559-017-0410-5DOI Listing
February 2018

No reason to reconsider HIF-2 as an oncogene in neuroblastoma and other cancer forms.

Proc Natl Acad Sci U S A 2017 12 12;114(51):E10856-E10858. Epub 2017 Dec 12.

Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, 22381 Lund, Sweden

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http://dx.doi.org/10.1073/pnas.1716644115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754808PMC
December 2017

Hypoxia and hypoxia-inducible factors in neuroblastoma.

Cell Tissue Res 2018 05 14;372(2):269-275. Epub 2017 Oct 14.

Pediatric Oncology & Hematology, Department of Clinical Sciences, Lund University, Lund, Sweden.

Hypoxia (i.e., low oxygen levels) is a known feature of aggressive tumors. Cells, including tumor cells, respond to conditions of insufficient oxygen by activating a transcriptional program mainly driven by hypoxia-inducible factors (HIF)-1 and HIF-2. Both HIF-1α and HIF-2α expression levels have been shown to correlate to patient outcome in various tumor forms and in neuroblastoma, a solid childhood tumor of the sympathetic nervous system, in particular, HIF-2α marks a subpopulation of immature neural crest-like perivascularly located cells and associates with aggressive disease and distant metastasis. It has for long been recognized that the HIF-α subunits are oxygen-dependently regulated at the post-translational level, via ubiquitination and proteasomal degradation. Evidence of oxygen-independent mechanisms of regulation, transcriptional control of EPAS1/HIF2A and possible cytoplasmic activities of HIF-2α has also emerged during recent years. In this review, we discuss these non-conventional actions of HIF-2α, its putative role as a therapeutic target and the constraints it carries, as well as the importance of HIF-2 activity in a vascularized setting, the so-called pseudo-hypoxic state.
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http://dx.doi.org/10.1007/s00441-017-2701-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915502PMC
May 2018

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

Sci Rep 2017 08 31;7(1):10274. Epub 2017 Aug 31.

Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1038/s41598-017-09662-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579187PMC
August 2017

Myc-induced glutaminolysis bypasses HIF-driven glycolysis in hypoxic small cell lung carcinoma cells.

Oncotarget 2017 Jul;8(30):48983-48995

Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.

We previously demonstrated that small cell lung carcinoma (SCLC) cells lack HIF-2α protein expression, whereas HIF-1α in these cells is expressed at both acute and prolonged hypoxia. Here we show that low HIF2A expression correlates with high expression of MYC genes. Knockdown of HIF1A expression had no or limited effect on cell survival and growth in vitro. Unexpectedly, hypoxic ATP levels were not affected by HIF-1α knockdown and SCLC cell viability did not decrease upon glucose deprivation. In line with these in vitro data, xenograft tumor-take and growth were not significantly affected by repressed HIF1A expression. Glutamine withdrawal drastically decreased SCLC cell proliferation and increased cell death at normoxia and hypoxia in a HIF-independent fashion and the dependence on glutaminolysis was linked to amplification of either MYC or MYCL. Downregulation of GLS expression, regulating the first step of the glutaminolysis pathway, in MYC/MYCL overexpressing SCLC cells resulted in both impaired growth and increased cell death. Our results suggest that MYC/MYCL overexpression in SCLC cells overrides the need of HIF-1 activity in response to hypoxia by inducing glutaminolysis and lipogenesis. Targeting the glutaminolysis pathway might hence be a novel approach to selectively kill MYC amplified SCLC cells in vivo.
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http://dx.doi.org/10.18632/oncotarget.16904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564742PMC
July 2017

Hypoxia, pseudohypoxia and cellular differentiation.

Exp Cell Res 2017 07 8;356(2):192-196. Epub 2017 Mar 8.

Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, Lund, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2017.03.007DOI Listing
July 2017

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

Nat Commun 2016 10 11;7:13050. Epub 2016 Oct 11.

Department of Translational Medicine, Cancer Immunology, Lund University, Malmö 205 02, Sweden.

Triple-negative (TN) breast cancers (ERPRHER2) 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.
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http://dx.doi.org/10.1038/ncomms13050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062608PMC
October 2016

Extracellular matrix composition defines an ultra-high-risk group of neuroblastoma within the high-risk patient cohort.

Br J Cancer 2016 08 14;115(4):480-9. Epub 2016 Jul 14.

Department of Pathology, Medical School, University of Valencia - INCLIVA, Avda. Blasco Ibañez 15, 46010 Valencia, Spain.

Background: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die.

Methods: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival.

Results: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgroup with 5-year survival rate <15%. Specifically, tumours with irregularly shaped blood vessels, large sinusoid-like vessels, smaller and tortuous venules and arterioles and with large areas of reticulin fibres forming large, crosslinking, branching and haphazardly arranged networks were linked to the ultra-high-risk phenotype.

Conclusions: We demonstrate that quantification of tumour stroma components by morphometric techniques has the potential to improve risk stratification of neuroblastoma patients.
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http://dx.doi.org/10.1038/bjc.2016.210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985353PMC
August 2016

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

Pharmacol Ther 2016 08 29;164:152-69. Epub 2016 Apr 29.

Translational Cancer Research, Medicon Village 404:C3, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1016/j.pharmthera.2016.04.009DOI Listing
August 2016

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

Cancer Lett 2016 Jun 18;375(2):384-389. Epub 2016 Mar 18.

Translational Cancer Research, Lund University, Lund, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.canlet.2016.02.046DOI Listing
June 2016

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

Oncotarget 2016 Mar;7(10):11238-50

Department of Laboratory Medicine, Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, Sweden.

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.
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http://dx.doi.org/10.18632/oncotarget.7167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905469PMC
March 2016

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

Cancer Res 2015 Nov 2;75(21):4617-28. Epub 2015 Oct 2.

Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0708DOI Listing
November 2015

Differential HIF-1α and HIF-2α Expression in Mammary Epithelial Cells during Fat Pad Invasion, Lactation, and Involution.

PLoS One 2015 8;10(5):e0125771. Epub 2015 May 8.

Department of Laboratory Medicine Lund, Translational Cancer Research, Lund University Cancer Center at Medicon Village, Lund University, Lund, Sweden.

The development and functional cycle of the mammary gland involves a number of processes that are caricatured by breast cancer cells during invasion and metastasis. Expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 has been associated with metastatic, poor prognosis, and high-grade breast cancers. Since hypoxia affects normal epithelial differentiation, we hypothesise that HIFs are important for normal breast epithelial development and regeneration as well as cancer initiation and progression. Here, we investigated the expression of the oxygen-sensitive HIF-alpha subunits during mouse mammary gland development, lactation, and involution. In breast epithelial cells, HIF-1α was expressed during early development, prior to cell polarisation. In contrast, expression of HIF-2α occurred later and was restricted to a subpopulation of luminal epithelial cells in the lactating gland. Mammary gland involution is a developmental stage that involves extensive tissue remodelling with cell death but survival of tissue stem/progenitor cells. At this stage, HIF-2α, but little HIF-1α, was expressed in CK14-positive epithelial cells. The temporal but differential expression of the HIF-alpha subunits during the mammary gland life cycle indicates that their expression is controlled by additional factors to hypoxia. Further functional studies of the roles of these proteins in the mammary gland and breast cancer are warranted.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425677PMC
February 2016

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

Biochem Biophys Res Commun 2015 Jun 23;461(3):560-7. Epub 2015 Apr 23.

Lund University, Translational Cancer Research, Lund University Cancer Center at Medicon Village, Building 406, SE-223 81 Lund, Sweden. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2015.04.083DOI Listing
June 2015

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

Int J Cancer 2015 Aug 20;137(4):868-77. Epub 2015 Feb 20.

Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1002/ijc.29461DOI Listing
August 2015

Neuroblastoma patient-derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours.

Int J Cancer 2015 Mar 7;136(5):E252-61. Epub 2014 Oct 7.

Translational Cancer Research, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1002/ijc.29217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299502PMC
March 2015

Induction of mitochondrial dysfunction as a strategy for targeting tumour cells in metabolically compromised microenvironments.

Nat Commun 2014 ;5:3295

1] Department of Oncology-Pathology, Karolinska Institute, Stockholm S-171 76, Sweden [2] Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University, Uppsala S-751 85, Sweden.

Abnormal vascularization of solid tumours results in the development of microenvironments deprived of oxygen and nutrients that harbour slowly growing and metabolically stressed cells. Such cells display enhanced resistance to standard chemotherapeutic agents and repopulate tumours after therapy. Here we identify the small molecule VLX600 as a drug that is preferentially active against quiescent cells in colon cancer 3-D microtissues. The anticancer activity is associated with reduced mitochondrial respiration, leading to bioenergetic catastrophe and tumour cell death. VLX600 shows enhanced cytotoxic activity under conditions of nutrient starvation. Importantly, VLX600 displays tumour growth inhibition in vivo. Our findings suggest that tumour cells in metabolically compromised microenvironments have a limited ability to respond to decreased mitochondrial function, and suggest a strategy for targeting the quiescent populations of tumour cells for improved cancer treatment.
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http://dx.doi.org/10.1038/ncomms4295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929804PMC
November 2015

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

Biochem Biophys Res Commun 2014 Feb 3;445(1):163-9. Epub 2014 Feb 3.

Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund, Sweden; CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2014.01.165DOI Listing
February 2014

snoRNPs Regulate Telomerase Activity in Neuroblastoma and Are Associated with Poor Prognosis.

Transl Oncol 2013 Aug 1;6(4):447-57. Epub 2013 Aug 1.

Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden.

Amplification of the MYCN oncogene is strongly associated with poor prognosis in neuroblastoma (NB). In addition to MYCN amplification, many studies have focused on identifying patients with a poor prognosis based on gene expression profiling. The majority of prognostic signatures today are comprised of large gene lists limiting their clinical application. In addition, although of prognostic significance, most of these signatures fail to identify cellular processes that can explain their relation to prognosis. Here, we determined prognostically predictive genes in a data set containing 251 NBs. Gene Ontology analysis was performed on significant genes with a positive hazard ratio to search for cellular processes associated with poor prognosis. An enrichment in ribonucleoproteins (RNPs) was found. Genes involved in the stabilization and formation of the central small nucleolar RNP (snoRNP) complex were scrutinized using a backward conditional Cox regression resulting in an snoRNP signature consisting of three genes: DKC1, NHP2, and GAR1. The snoRNP signature significantly and independently predicted prognosis when compared to the established clinical risk factors. Association of snoRNP protein expression and prognosis was confirmed using tissue micro-arrays. Knockdown of snoRNP expression in NB cell lines resulted in reduced telomerase activity and an increase in anaphase bridge frequency. In addition, in patient material, expression of the snoRNP complex was significantly associated with telomerase activity, occurrence of segmental aberrations, and expression-based measurements of chromosomal instability. Together, these results underscore the prognostic value of snoRNP complex expression in NB and suggest a role for snoRNPs in telomere maintenance and genomic stability.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730020PMC
http://dx.doi.org/10.1593/tlo.13112DOI Listing
August 2013

Quantitative proteomics profiling of primary lung adenocarcinoma tumors reveals functional perturbations in tumor metabolism.

J Proteome Res 2013 Sep 13;12(9):3934-43. Epub 2013 Aug 13.

Cancer Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.

In this study, we have analyzed human primary lung adenocarcinoma tumors using global mass spectrometry to elucidate the biological mechanisms behind relapse post surgery. In total, we identified over 3000 proteins with high confidence. Supervised multivariate analysis was used to select 132 proteins separating the prognostic groups. Based on in-depth bioinformatics analysis, we hypothesized that the tumors with poor prognosis had a higher glycolytic activity and HIF activation. By measuring the bioenergetic cellular index of the tumors, we could detect a higher dependency of glycolysis among the tumors with poor prognosis. Further, we could also detect an up-regulation of HIF1α mRNA expression in tumors with early relapse. Finally, we selected three proteins that were upregulated in the poor prognosis group (cathepsin D, ENO1, and VDAC1) to confirm that the proteins indeed originated from the tumor and not from a stromal or inflammatory component. Overall, these findings show how in-depth analysis of clinical material can lead to an increased understanding of the molecular mechanisms behind tumor progression.
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http://dx.doi.org/10.1021/pr4002096DOI Listing
September 2013

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells.

Proc Natl Acad Sci U S A 2013 Jun 3;110(25):10258-63. Epub 2013 Jun 3.

Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors.
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http://dx.doi.org/10.1073/pnas.1222404110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690852PMC
June 2013

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling.

Cell Signal 2013 Sep 21;25(9):1852-60. Epub 2013 May 21.

Experimental Clinical Chemistry, Department of Laboratory Medicine, Wallenberg Laboratory, Lund University, Skåne University Hospital, 20502 Malmö, Sweden.

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in the context of regulating Src family kinases. Here we present an additional role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.
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http://dx.doi.org/10.1016/j.cellsig.2013.05.016DOI Listing
September 2013