Publications by authors named "Carl-Henrik Heldin"

185 Publications

The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor β signaling.

Oncogene 2021 May 4;40(21):3748-3765. Epub 2021 May 4.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, Uppsala, Sweden.

Activation of the transforming growth factor β (TGFβ) pathway modulates the expression of genes involved in cell growth arrest, motility, and embryogenesis. An expression screen for long noncoding RNAs indicated that TGFβ induced mir-100-let-7a-2-mir-125b-1 cluster host gene (MIR100HG) expression in diverse cancer types, thus confirming an earlier demonstration of TGFβ-mediated transcriptional induction of MIR100HG in pancreatic adenocarcinoma. MIR100HG depletion attenuated TGFβ signaling, expression of TGFβ-target genes, and TGFβ-mediated cell cycle arrest. Moreover, MIR100HG silencing inhibited both normal and cancer cell motility and enhanced the cytotoxicity of cytostatic drugs. MIR100HG overexpression had an inverse impact on TGFβ signaling responses. Screening for downstream effectors of MIR100HG identified the ligand TGFβ1. MIR100HG and TGFB1 mRNA formed ribonucleoprotein complexes with the RNA-binding protein HuR, promoting TGFβ1 cytokine secretion. In addition, TGFβ regulated let-7a-2-3p, miR-125b-5p, and miR-125b-1-3p expression, all encoded by MIR100HG intron-3. Certain intron-3 miRNAs may be involved in TGFβ/SMAD-mediated responses (let-7a-2-3p) and others (miR-100, miR-125b) in resistance to cytotoxic drugs mediated by MIR100HG. In support of a model whereby TGFβ induces MIR100HG, which then enhances TGFβ1 secretion, analysis of human carcinomas showed that MIR100HG expression correlated with expression of TGFB1 and its downstream extracellular target TGFBI. Thus, MIR100HG controls the magnitude of TGFβ signaling via TGFβ1 autoinduction and secretion in carcinomas.
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http://dx.doi.org/10.1038/s41388-021-01803-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154591PMC
May 2021

TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation.

Cancers (Basel) 2021 Mar 1;13(5). Epub 2021 Mar 1.

Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden.

The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFβ) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.
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http://dx.doi.org/10.3390/cancers13051021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957764PMC
March 2021

BMP signaling is a therapeutic target in ovarian cancer.

Cell Death Discov 2020 Dec 5;6(1):139. Epub 2020 Dec 5.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Uppsala University, SE-751 23, Uppsala, Sweden.

BMP signaling has been found to have tumor-promoting as well as tumor-suppressing effects in different types of tumors. In this study, we investigated the effects of BMP signaling and of BMP inhibitors on ovarian cancer (OC) cells in vitro and in vivo. High expression of BMP receptor 2 (BMPR2) correlated with poor overall survival of OC patients in the TCGA dataset. Both BMP2 and BMPR2 enhanced OC cell proliferation, whereas BMP receptor kinase inhibitors inhibited OC cell growth in cell culture as well as in a mouse model. BMP2 also augmented sphere formation, migration, and invasion of OC cells, and induced EMT. High BMP2 expression was observed after chemotherapy of OC patients in the GSE109934 dataset. In accordance, carboplatin, used for the treatment of OC patients, increased BMP2 secretion from OC cells, and induced EMT partially via activation of BMP signaling. Our data suggest that BMP signaling has tumor-promoting effects in OC, and that BMP inhibitors might be useful therapeutic agents for OC patients. Considering that carboplatin treatment augmented BMP2 secretion, the possibility to use a combination of BMP inhibitors and carboplatin in the treatment of OC patients, would be worth exploring.
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http://dx.doi.org/10.1038/s41420-020-00377-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719168PMC
December 2020

Smad7 Enhances TGF-β-Induced Transcription of c-Jun and HDAC6 Promoting Invasion of Prostate Cancer Cells.

iScience 2020 Sep 3:101470. Epub 2020 Sep 3.

Ludwig Institute for Cancer Research, Ltd., Science for Life Laboratory, Uppsala University, Box 595, 751 24 Uppsala, Sweden; Department of Medical Biosciences, Umeå University, 901 87 Umeå, Sweden. Electronic address:

Transforming growth factor β (TGF-β) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-β to control TGF-β signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-β induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information.
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http://dx.doi.org/10.1016/j.isci.2020.101470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520897PMC
September 2020

Erratum: Specific targeting of PDGFRβ in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression: Erratum.

Theranostics 2020;10(16):7406-7408. Epub 2020 Jun 11.

Department of Pharmaceutical Biosciences, Uppsala University, Sweden.

[This corrects the article DOI: 10.7150/thno.37851.].
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http://dx.doi.org/10.7150/thno.48039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330841PMC
June 2020

Platelet-Specific PDGFB Ablation Impairs Tumor Vessel Integrity and Promotes Metastasis.

Cancer Res 2020 08 25;80(16):3345-3358. Epub 2020 Jun 25.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Biomedical Center, Uppsala, Sweden.

Platelet-derived growth factor B (PDGFB) plays a crucial role in recruitment of PDGF receptor β-positive pericytes to blood vessels. The endothelium is an essential source of PDGFB in this process. Platelets constitute a major reservoir of PDGFB and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. Here, we show that tumor vascular function, as well as pericyte coverage is significantly impaired in mice with conditional knockout of PDGFB in platelets. A lack of PDGFB in platelets led to enhanced hypoxia and epithelial-to-mesenchymal transition in the primary tumors, elevated levels of circulating tumor cells, and increased spontaneous metastasis to the liver or lungs in two mouse models. These findings establish a previously unknown role for platelet-derived PDGFB, whereby it promotes and maintains vascular integrity in the tumor microenvironment by contributing to the recruitment of pericytes. SIGNIFICANCE: Conditional knockout of PDGFB in platelets demonstrates its previously unknown role in the maintenance of tumor vascular integrity and host protection against metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3533DOI Listing
August 2020

Bone morphogenetic protein receptors: Structure, function and targeting by selective small molecule kinase inhibitors.

Bone 2020 09 6;138:115472. Epub 2020 Jun 6.

Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands; Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Uppsala University, SE-751 23 Uppsala, Sweden; Oncode Institute, Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Electronic address:

Bone morphogenetic proteins (BMPs) are secreted cytokines that control the fate and function of many different cell types. They exert their cellular responses via heteromeric complexes of specific BMP type I and type II serine/threonine kinase receptors, e.g. BMPRIA and BMPRII. Three type II and four type I receptors, also termed activin receptor-like kinases (ALKs), have been identified. The constitutively active type II kinase phosphorylates the type I receptor, which upon activation initiates intracellular signaling by phosphorylating SMAD effectors. Auxiliary cell surface receptors without intrinsic enzymatic motifs, such as Endoglin and Repulsive guidance molecules (RGM), can fine-tune signaling by regulating the interaction of the BMP ligands with the BMPRs. The functional annotation of the BMPR encoding genes has helped to understand underlying mechanisms of diseases in which these genes are mutated. Loss of function mutations in BMPRII, Endoglin or RGMc are causally linked to pulmonary arterial hypertension, hereditary hemorrhagic telangiectasia and juvenile hemochromatosis, respectively. In contrast, gain of function mutations in ACVR1, encoding ALK2, are linked to Fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. Here, we discuss BMPR identification, structure and function in health and disease. Moreover, we highlight the therapeutic promise of small chemical compounds that act as selective BMPR kinase inhibitors to normalize overactive BMPR signaling.
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http://dx.doi.org/10.1016/j.bone.2020.115472DOI Listing
September 2020

TGFβ and EGF signaling orchestrates the AP-1- and p63 transcriptional regulation of breast cancer invasiveness.

Oncogene 2020 05 29;39(22):4436-4449. Epub 2020 Apr 29.

Department of Cell and Chemical Biology, Oncode Institute, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands.

Activator protein (AP)-1 transcription factors are essential elements of the pro-oncogenic functions of transforming growth factor-β (TGFβ)-SMAD signaling. Here we show that in multiple HER2+ and/or EGFR+ breast cancer cell lines these AP-1-dependent tumorigenic properties of TGFβ critically rely on epidermal growth factor receptor (EGFR) activation and expression of the ΔN isoform of transcriptional regulator p63. EGFR and ΔNp63 enabled and/or potentiated the activation of a subset of TGFβ-inducible invasion/migration-associated genes, e.g., ITGA2, LAMB3, and WNT7A/B, and enhanced the recruitment of SMAD2/3 to these genes. The TGFβ- and EGF-induced binding of SMAD2/3 and JUNB to these gene loci was accompanied by p63-SMAD2/3 and p63-JUNB complex formation. p63 and EGFR were also found to strongly potentiate TGFβ induction of AP-1 proteins and, in particular, FOS family members. Ectopic overexpression of FOS could counteract the decrease in TGFβ-induced gene activation after p63 depletion. p63 is also involved in the transcriptional regulation of heparin binding (HB)-EGF and EGFR genes, thereby establishing a self-amplification loop that facilitates and empowers the pro-invasive functions of TGFβ. These cooperative pro-oncogenic functions of EGFR, AP-1, p63, and TGFβ were efficiently inhibited by clinically relevant chemical inhibitors. Our findings may, therefore, be of importance for therapy of patients with breast cancers with an activated EGFR-RAS-RAF pathway.
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http://dx.doi.org/10.1038/s41388-020-1299-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253358PMC
May 2020

Specific targeting of PDGFRβ in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression.

Theranostics 2020 1;10(3):1122-1135. Epub 2020 Jan 1.

Department of Pharmaceutical Biosciences, Uppsala University, Sweden.

PDGF-BB/PDGFRβ signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFRβ signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFRβ ATP binding site that allows specific targeting of PDGFRβ using the compound 1-NaPP1. To evaluate the impact of selective PDGFRβ inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFRβ expression, . Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). : We found that specific impairment of PDGFRβ kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, LLC and B16/PDGF-BB, while the clinically used PDGFRβ kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFRβ by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFRβ by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. analysis of PDGFRβ ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the data. : Specific targeting of PDGFRβ signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFRβ in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.
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http://dx.doi.org/10.7150/thno.37851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956815PMC
April 2021

JNK-Dependent cJun Phosphorylation Mitigates TGFβ- and EGF-Induced Pre-Malignant Breast Cancer Cell Invasion by Suppressing AP-1-Mediated Transcriptional Responses.

Cells 2019 11 21;8(12). Epub 2019 Nov 21.

Department of Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

Transforming growth factor-β (TGFβ) has both tumor-suppressive and tumor-promoting effects in breast cancer. These functions are partly mediated through Smads, intracellular transcriptional effectors of TGFβ. Smads form complexes with other DNA-binding transcription factors to elicit cell-type-dependent responses. Previously, we found that the collagen invasion and migration of pre-malignant breast cancer cells in response to TGFβ and epidermal growth factor (EGF) critically depend on multiple Jun and Fos components of the activator protein (AP)-1 transcription factor complex. Here we report that the same process is negatively regulated by Jun N-terminal kinase (JNK)-dependent cJun phosphorylation. This was demonstrated by analysis of phospho-deficient, phospho-mimicking, and dimer-specific cJun mutants, and experiments employing a mutant version of the phosphatase MKP1 that specifically inhibits JNK. Hyper-phosphorylation of cJun by JNK strongly inhibited its ability to induce several Jun/Fos-regulated genes and to promote migration and invasion. These results show that MEK-AP-1 and JNK-phospho-cJun exhibit distinct pro- and anti-invasive functions, respectively, through differential regulation of Smad- and AP-1-dependent TGFβ target genes. Our findings are of importance for personalized cancer therapy, such as for patients suffering from specific types of breast tumors with activated EGF receptor-Ras or inactivated JNK pathways.
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http://dx.doi.org/10.3390/cells8121481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952832PMC
November 2019

The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension.

Sci Signal 2019 11 12;12(607). Epub 2019 Nov 12.

Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.
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http://dx.doi.org/10.1126/scisignal.aay4430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908447PMC
November 2019

Structure-based discovery of novel small molecule inhibitors of platelet-derived growth factor-B.

Bioorg Chem 2020 01 24;94:103374. Epub 2019 Oct 24.

Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address:

Platelet-derived growth factor (PDGF) is a family of growth factors with mitogenic and chemotactic activity. However, uncontrolled and overactivated PDGF signaling has been implicated in a variety of diseases, such as cancers and atherosclerosis. In this context, inhibition of PDGF-PDGFR signaling is of paramount importance in progression of such diseases. The purpose of the current study was to identify novel PDGF-B inhibitors using virtual screening methods. To this end, a combination of molecular modeling techniques such as molecular docking and dynamics simulation, as well as drug likeness filtering criteria, was applied to select anti-PDGF peptidomimetic candidates based on crystallography solved structure of an anti-PDGF-B monoclonal antibody named, MOR8457. In vitro biological assays of the selected compounds revealed two of them being active at micromolar IC concentrations. The presented work can provide a framework for systematic peptidomimetic identification for anti-PDGF-B agents from large chemical libraries.
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http://dx.doi.org/10.1016/j.bioorg.2019.103374DOI Listing
January 2020

Involvement of hyaluronan and CD44 in cancer and viral infections.

Cell Signal 2020 01 22;65:109427. Epub 2019 Oct 22.

Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, SE-751 23 Uppsala, Sweden. Electronic address:

Hyaluronan and its major receptor CD44 are ubiquitously distributed. They have important structural as well as signaling roles, regulating tissue homeostasis, and their expression levels are tightly regulated. In addition to signaling initiated by the interaction of the intracellular domain of CD44 with cytoplasmic signaling molecules, CD44 has important roles as a co-receptor for different types of receptors of growth factors and cytokines. Dysregulation of hyaluronan-CD44 interactions is seen in diseases, such as inflammation and cancer. In the present communication, we discuss the mechanism of hyaluronan-induced signaling via CD44, as well as the involvement of hyaluronan-engaged CD44 in malignancies and in viral infections.
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http://dx.doi.org/10.1016/j.cellsig.2019.109427DOI Listing
January 2020

The TGFB2-AS1 lncRNA Regulates TGF-β Signaling by Modulating Corepressor Activity.

Cell Rep 2019 Sep;28(12):3182-3198.e11

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, and Ludwig Cancer Research Box 582, Biomedical Center, Uppsala University, 751 23 Uppsala, Sweden. Electronic address:

Molecular processes involving lncRNAs regulate cell function. By applying transcriptomics, we identify lncRNAs whose expression is regulated by transforming growth factor β (TGF-β). Upon silencing individual lncRNAs, we identify several that regulate TGF-β signaling. Among these lncRNAs, TGFB2-antisense RNA1 (TGFB2-AS1) is induced by TGF-β through Smad and protein kinase pathways and resides in the nucleus. Depleting TGFB2-AS1 enhances TGF-β/Smad-mediated transcription and expression of hallmark TGF-β-target genes. Increased dose of TGFB2-AS1 reduces expression of these genes, attenuates TGF-β-induced cell growth arrest, and alters BMP and Wnt pathway gene profiles. Mechanistically, TGFB2-AS1, mainly via its 3' terminal region, binds to the EED adaptor of the Polycomb repressor complex 2 (PRC2), promoting repressive histone H3K27me modifications at TGF-β-target gene promoters. Silencing EED or inhibiting PRC2 methylation activity partially rescues TGFB2-AS1-mediated gene repression. Thus, the TGF-β-induced TGFB2-AS1 lncRNA exerts inhibitory functions on TGF-β/BMP signaling output, supporting auto-regulatory negative feedback that balances TGF-β/BMP-mediated responses.
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http://dx.doi.org/10.1016/j.celrep.2019.08.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859500PMC
September 2019

High levels of serum hyaluronan is an early predictor of dengue warning signs and perturbs vascular integrity.

EBioMedicine 2019 Oct 13;48:425-441. Epub 2019 Sep 13.

Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden. Electronic address:

Background: A main pathological feature of severe dengue virus infection is endothelial hyper-permeability. The dengue virus nonstructural protein 1 (NS1) has been implicated in the vascular leakage that characterizes severe dengue virus infection, however, the molecular mechanisms involved are not known.

Methods: A cohort of 250 dengue patients has been followed from the onset of symptoms to the recovery phase. Serum hyaluronan levels and several other clinical parameters were recorded. The effect of NS1 treatment of cultured fibroblasts and endothelial cells on the expressions of hyaluronan synthetic and catabolic enzymes and the hyaluronan receptor CD44, were determined, as have the effects on the formation of hyaluronan-rich matrices and endothelial permeability.

Findings: Elevated serum hyaluronan levels (≥70 ng/ml) during early infection was found to be an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells cultured in vitro recruited CD44-expressing macrophage-like cells, suggesting a mechanism for enhancement of inflammation. In cultured endothelial cells, perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity.

Interpretation: Pharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during moderate-to-severe dengue virus infection. FUND: This work was supported in part by grants from the Swedish Cancer Society (2018/337; 2016/445), the Swedish Research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106-2314-B-037-088- and 106-2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3 T05) and Academy of Finland. The funders played no role in the design, interpretation or writing of the manuscript.
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http://dx.doi.org/10.1016/j.ebiom.2019.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838418PMC
October 2019

Dual specificity phosphatase (DUSP)-4 is induced by platelet-derived growth factor -BB in an Erk1/2-, STAT3- and p53-dependent manner.

Biochem Biophys Res Commun 2019 11 13;519(3):469-474. Epub 2019 Sep 13.

Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden; Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, Box 591, SE-751 24, Uppsala, Sweden.

Dual specificity phosphatase (DUSP) 4 has been described as a negative regulator of MAP kinase signaling, in particular for the ERK1/2 and JNK pathways. We found that DUSP4 expression was upregulated in response to prolonged platelet-derived growth factor (PDGF)-BB stimulation. The PDGF-BB-induced DUSP4 expression was dependent on ERK1/2, STAT3 and p53. We found that inhibition of ERK1/2 effectively reduced DUSP4 mRNA levels, whereas STAT3 was necessary for maintaining p53 expression. p53 has binding sites in the DUSP4 promoter and was found to promote DUSP4 expression.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.014DOI Listing
November 2019

LXRα limits TGFβ-dependent hepatocellular carcinoma associated fibroblast differentiation.

Oncogenesis 2019 May 16;8(6):36. Epub 2019 May 16.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Box 582, Biomedical Center, SE-751 23, Uppsala, Sweden.

Transforming growth factor β (TGFβ) is deposited in the extracellular space of diverse tissues. Resident fibroblasts respond to TGFβ and undergo myofibroblastic differentiation during tissue wound healing and cancer progression. Cancer-associated fibroblasts (CAFs) communicate with tumor cells during cancer progression, under the guidance of TGFβ signaling. We report that agonist-activated liver X receptors (LXR) limit the expression of key components of myofibroblast differentiation, including the α-smooth muscle actin (αSMA) gene in liver cancer cells. CAFs derived from hepatocellular carcinoma (HCC) express high αSMA and low LXRα levels, whereas hepatocarcinoma cells exhibit an inverse expression pattern. All hepatoma cells analyzed responded to the LXRα agonist T0901317 by inducing fatty acid synthase (FASN) expression. On the other hand, T0901317 antagonized TGFβ-induced fibroblastic marker responses, such as fibronectin and calponin, in a subset of hepatoma cells and all CAFs analyzed. Mechanistically, LXRα antagonized TGFβ signaling at the transcriptional level. Smad3 and LXRα were recruited to adjacent DNA motifs of the ACTA2 promoter. Upon cloning the human ACTA2 promoter, we confirmed its transcriptional induction by TGFβ stimulation, and LXRα overexpression repressed the promoter activity. Hepatosphere formation by HCC cells was enhanced upon co-culturing with CAFs. T0901317 suppressed the positive effects exerted on hepatosphere growth by CAFs. Taken together, the data suggest that LXRα agonists limit TGFβ-dependent CAF differentiation, potentially limiting primary HCC growth.
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http://dx.doi.org/10.1038/s41389-019-0140-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522550PMC
May 2019

Transforming growth factor β (TGFβ) induces NUAK kinase expression to fine-tune its signaling output.

J Biol Chem 2019 03 8;294(11):4119-4136. Epub 2019 Jan 8.

the Ludwig Institute for Cancer Research, Science for Life Laboratory, Box 595 Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden

TGFβ signaling via SMAD proteins and protein kinase pathways up- or down-regulates the expression of many genes and thus affects physiological processes, such as differentiation, migration, cell cycle arrest, and apoptosis, during developmental or adult tissue homeostasis. We here report that NUAK family kinase 1 () and are two TGFβ target genes. NUAK1/2 belong to the AMP-activated protein kinase (AMPK) family, whose members control central and protein metabolism, polarity, and overall cellular homeostasis. We found that TGFβ-mediated transcriptional induction of and requires SMAD family members 2, 3, and 4 (SMAD2/3/4) and mitogen-activated protein kinase (MAPK) activities, which provided immediate and early signals for the transient expression of these two kinases. Genomic mapping identified an enhancer element within the first intron of the gene that can recruit SMAD proteins, which, when cloned, could confer induction by TGFβ. Furthermore, NUAK2 formed protein complexes with SMAD3 and the TGFβ type I receptor. Functionally, NUAK1 suppressed and NUAK2 induced TGFβ signaling. This was evident during TGFβ-induced epithelial cytostasis, mesenchymal differentiation, and myofibroblast contractility, in which NUAK1 or NUAK2 silencing enhanced or inhibited these responses, respectively. In conclusion, we have identified a bifurcating loop during TGFβ signaling, whereby transcriptional induction of NUAK1 serves as a negative checkpoint and NUAK2 induction positively contributes to signaling and terminal differentiation responses to TGFβ activity.
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http://dx.doi.org/10.1074/jbc.RA118.004984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422081PMC
March 2019

Has2 natural antisense RNA and Hmga2 promote Has2 expression during TGFβ-induced EMT in breast cancer.

Matrix Biol 2019 07 5;80:29-45. Epub 2018 Sep 5.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden; Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Biomedical Center, Box 595, SE-751 24 Uppsala, Sweden. Electronic address:

The glycosaminoglycan hyaluronan has a crucial role in tissue organization and cell signaling. Hyaluronan accumulates in conjunction with rapid tissue remodeling during embryogenesis, as well as in inflammatory conditions and cancer. We report a negative correlation between the expression of genes encoding hyaluronan synthase HAS2, its natural antisense transcript HAS2-AS, the chromatin modulating factor HMGA2 and transforming growth factor-β (TGFβ), and survival of patients with invasive breast carcinomas. In mouse mammary epithelial cells, TGFβ activates Smad and non-Smad signaling pathways, resulting in the transcriptional induction of Has2, Has2as (the mouse ortholog of HAS2-AS) and Hmga2, as well as epithelial-mesenchymal transition (EMT)-promoting transcription factors, such as Snail. Importantly, Has2as abrogation suppressed the TGFβ induction of EMT markers, including Snai1, Hmga2, Fn1, and suppressed the mesenchymal phenotype. TGFβ induction of Hmga2, Has2as and Has2, and synthesis of hyaluronan were accompanied with activation of Akt and Erk1/2 MAP-kinase signaling and were required for breast cancer cell motility. Importantly, the hyaluronan receptor Cd44, but not Hmmr, was required for TGFβ-mediated EMT phenotype. Interestingly, Has2as was found to contribute to the maintenance of stem cell factors and breast cancer stemness. Our findings show that Has2as has a key role in TGFβ- and HAS2-induced breast cancer EMT, migration and acquisition of stemness.
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http://dx.doi.org/10.1016/j.matbio.2018.09.002DOI Listing
July 2019

Targeting PDGF-mediated recruitment of pericytes blocks vascular mimicry and tumor growth.

J Pathol 2018 12 30;246(4):447-458. Epub 2018 Oct 30.

Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.

Aggressive tumor cells can adopt an endothelial cell-like phenotype and contribute to the formation of a tumor vasculature, independent of tumor angiogenesis. This adoptive mechanism is referred to as vascular mimicry and it is associated with poor survival in cancer patients. To what extent tumor cells capable of vascular mimicry phenocopy the angiogenic cascade is still poorly explored. Here, we identify pericytes as important players in vascular mimicry. We found that pericytes are recruited by vascular mimicry-positive tumor cells in order to facilitate sprouting and to provide structural support of the vascular-like networks. The pericyte recruitment is mediated through platelet-derived growth factor (PDGF)-B. Consequently, preventing PDGF-B signaling by blocking the PDGF receptors with either the small tyrosine kinase inhibitor imatinib or blocking antibodies inhibits vascular mimicry and tumor growth. Collectively, the current study identifies an important role for pericytes in the formation of vascular-like structures by tumor cells. Moreover, the mechanism that controls the pericyte recruitment provides therapeutic opportunities for patients with aggressive vascular mimicry-positive cancer types. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587443PMC
December 2018

Genome-wide binding of transcription factor ZEB1 in triple-negative breast cancer cells.

J Cell Physiol 2018 10 10;233(10):7113-7127. Epub 2018 May 10.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, and Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcriptional regulator involved in embryonic development and cancer progression. ZEB1 induces epithelial-mesenchymal transition (EMT). Triple-negative human breast cancers express high ZEB1 mRNA levels and exhibit features of EMT. In the human triple-negative breast cancer cell model Hs578T, ZEB1 associates with almost 2,000 genes, representing many cellular functions, including cell polarity regulation (DLG2 and FAT3). By introducing a CRISPR-Cas9-mediated 30 bp deletion into the ZEB1 second exon, we observed reduced migratory and anchorage-independent growth capacity of these tumor cells. Transcriptomic analysis of control and ZEB1 knockout cells, revealed 1,372 differentially expressed genes. The TIMP metallopeptidase inhibitor 3 and the teneurin transmembrane protein 2 genes showed increased expression upon loss of ZEB1, possibly mediating pro-tumorigenic actions of ZEB1. This work provides a resource for regulators of cancer progression that function under the transcriptional control of ZEB1. The data confirm that removing a single EMT transcription factor, such as ZEB1, is not sufficient for reverting the triple-negative mesenchymal breast cancer cells into more differentiated, epithelial-like clones, but can reduce tumorigenic potential, suggesting that not all pro-tumorigenic actions of ZEB1 are linked to the EMT.
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http://dx.doi.org/10.1002/jcp.26634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055758PMC
October 2018

Genomewide binding of transcription factor Snail1 in triple-negative breast cancer cells.

Mol Oncol 2018 06 21;12(7):1153-1174. Epub 2018 May 21.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Ludwig Institute for Cancer Research, Uppsala University, Sweden.

Transcriptional regulation mediated by the zinc finger protein Snail1 controls early embryogenesis. By binding to the epithelial tumor suppressor CDH1 gene, Snail1 initiates the epithelial-mesenchymal transition (EMT). The EMT generates stem-like cells and promotes invasiveness during cancer progression. Accordingly, Snail1 mRNA and protein is abundantly expressed in triple-negative breast cancers with enhanced metastatic potential and phenotypic signs of the EMT. Such high endogenous Snail1 protein levels permit quantitative chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. Snail1 associated with 185 genes at cis regulatory regions in the Hs578T triple-negative breast cancer cell model. These genes include morphogenetic regulators and signaling components that control polarized differentiation. Using the CRISPR/Cas9 system in Hs578T cells, a double deletion of 10 bp each was engineered into the first exon and into the second exon-intron junction of Snail1, suppressing Snail1 expression and causing misregulation of several hundred genes. Specific attention to regulators of chromatin organization provides a possible link to new phenotypes uncovered by the Snail1 loss-of-function mutation. On the other hand, genetic inactivation of Snail1 was not sufficient to establish a full epithelial transition to these tumor cells. Thus, Snail1 contributes to the malignant phenotype of breast cancer cells via diverse new mechanisms.
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http://dx.doi.org/10.1002/1878-0261.12317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026864PMC
June 2018

PDGFRβ translocates to the nucleus and regulates chromatin remodeling via TATA element-modifying factor 1.

J Cell Biol 2018 05 15;217(5):1701-1717. Epub 2018 Mar 15.

Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Translocation of full-length or fragments of receptors to the nucleus has been reported for several tyrosine kinase receptors. In this paper, we show that a fraction of full-length cell surface platelet-derived growth factor (PDGF) receptor β (PDGFRβ) accumulates in the nucleus at the chromatin and the nuclear matrix after ligand stimulation. Nuclear translocation of PDGFRβ was dependent on PDGF-BB-induced receptor dimerization, clathrin-mediated endocytosis, β-importin, and intact Golgi, occurring in both normal and cancer cells. In the nucleus, PDGFRβ formed ligand-inducible complexes with the tyrosine kinase Fer and its substrate, TATA element-modifying factor 1 (TMF-1). PDGF-BB stimulation decreased TMF-1 binding to the transcriptional regulator Brahma-related gene 1 (Brg-1) and released Brg-1 from the SWI-SNF chromatin remodeling complex. Moreover, knockdown of TMF-1 by small interfering RNA decreased nuclear translocation of PDGFRβ and caused significant up-regulation of the Brg-1/p53-regulated cell cycle inhibitor (encoding p21) without affecting PDGFRβ-inducible immediate-early genes. In conclusion, nuclear interactions of PDGFRβ control proliferation by chromatin remodeling and regulation of p21 levels.
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http://dx.doi.org/10.1083/jcb.201706118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940298PMC
May 2018

The protein kinase SIK downregulates the polarity protein Par3.

Oncotarget 2018 Jan 31;9(5):5716-5735. Epub 2017 Dec 31.

Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

The multifunctional cytokine transforming growth factor β (TGFβ) controls homeostasis and disease during embryonic and adult life. TGFβ alters epithelial cell differentiation by inducing epithelial-mesenchymal transition (EMT), which involves downregulation of several cell-cell junctional constituents. Little is understood about the mechanism of tight junction disassembly by TGFβ. We found that one of the newly identified gene targets of TGFβ, encoding the serine/threonine kinase salt-inducible kinase 1 (SIK), controls tight junction dynamics. We provide bioinformatic and biochemical evidence that SIK can potentially phosphorylate the polarity complex protein Par3, an established regulator of tight junction assembly. SIK associates with Par3, and induces degradation of Par3 that can be prevented by proteasomal and lysosomal inhibition or by mutation of Ser885, a putative phosphorylation site on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3 protein. High mRNA expression also correlates with lower chance for survival in various carcinomas. In specific human breast cancer samples, aneuploidy of tumor cells best correlated with cytoplasmic SIK distribution, and SIK expression correlated with TGFβ/Smad signaling activity and low or undetectable expression of Par3. Our model suggests that SIK can act directly on the polarity protein Par3 to regulate tight junction assembly.
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http://dx.doi.org/10.18632/oncotarget.23788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814169PMC
January 2018

Snail regulates BMP and TGFβ pathways to control the differentiation status of glioma-initiating cells.

Oncogene 2018 05 16;37(19):2515-2531. Epub 2018 Feb 16.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, SE-75123, Uppsala, Sweden.

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.
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http://dx.doi.org/10.1038/s41388-018-0136-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945579PMC
May 2018

TUFT1 interacts with RABGAP1 and regulates mTORC1 signaling.

Cell Discov 2018 9;4. Epub 2018 Jan 9.

1Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033 Japan.

The mammalian target of rapamycin (mTOR) pathway is commonly activated in human cancers. The activity of mTOR complex 1 (mTORC1) signaling is supported by the intracellular positioning of cellular compartments and vesicle trafficking, regulated by Rab GTPases. Here we showed that tuftelin 1 (TUFT1) was involved in the activation of mTORC1 through modulating the Rab GTPase-regulated process. TUFT1 promoted tumor growth and metastasis. Consistently, the expression of TUFT1 correlated with poor prognosis in lung, breast and gastric cancers. Mechanistically, TUFT1 physically interacted with RABGAP1, thereby modulating intracellular lysosomal positioning and vesicular trafficking, and promoted mTORC1 signaling. In addition, expression of predicted sensitivity to perifosine, an alkylphospholipid that alters the composition of lipid rafts. Perifosine treatment altered the positioning and trafficking of cellular compartments to inhibit mTORC1. Our observations indicate that TUFT1 is a key regulator of the mTORC1 pathway and suggest that it is a promising therapeutic target or a biomarker for tumor progression.
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http://dx.doi.org/10.1038/s41421-017-0001-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798889PMC
January 2018

The TGFβ superfamily in Lisbon: navigating through development and disease.

Development 2017 12;144(24):4476-4480

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Uppsala University, SE-751 23 Uppsala, Sweden.

The 10th FASEB meeting 'The TGFβ Superfamily: Signaling in Development and Disease' took place in Lisbon, Portugal, in July 2017. As we review here, the findings presented at the meeting highlighted the important contributions of TGFβ family signaling to normal development, adult homeostasis and disease, and also revealed novel mechanisms by which TGFβ signals are transduced.
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http://dx.doi.org/10.1242/dev.159756DOI Listing
December 2017

Snail mediates crosstalk between TGFβ and LXRα in hepatocellular carcinoma.

Cell Death Differ 2018 05 11;25(5):885-903. Epub 2017 Dec 11.

Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, SE-75123, Uppsala, Sweden.

Understanding the complexity of changes in differentiation and cell survival in hepatocellular carcinoma (HCC) is essential for the design of new diagnostic tools and therapeutic modalities. In this context, we have analyzed the crosstalk between transforming growth factor β (TGFβ) and liver X receptor α (LXRα) pathways. TGFβ is known to promote cytostatic and pro-apoptotic responses in HCC, and to facilitate mesenchymal differentiation. We here demonstrate that stimulation of the nuclear LXRα receptor system by physiological and clinically useful agonists controls the HCC response to TGFβ. Specifically, LXRα activation antagonizes the mesenchymal, reactive oxygen species and pro-apoptotic responses to TGFβ and the mesenchymal transcription factor Snail mediates this crosstalk. In contrast, LXRα activation and TGFβ cooperate in enforcing cytostasis in HCC, which preserves their epithelial features. LXRα influences Snail expression transcriptionally, acting on the Snail promoter. These findings propose that clinically used LXR agonists may find further application to the treatment of aggressive, mesenchymal HCCs, whose progression is chronically dependent on autocrine or paracrine TGFβ.
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http://dx.doi.org/10.1038/s41418-017-0021-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943406PMC
May 2018

Pro-invasive properties of Snail1 are regulated by sumoylation in response to TGFβ stimulation in cancer.

Oncotarget 2017 Nov 9;8(58):97703-97726. Epub 2017 Aug 9.

Department of Medical Biosciences, Umeå University, Umeå, Sweden.

Transforming growth factor β (TGFβ) is a key regulator of epithelial-to-mesenchymal transition (EMT) during embryogenesis and in tumors. The effect of TGFβ, on ΕΜΤ, is conveyed by induction of the pro-invasive transcription factor Snail1. In this study, we report that TGFβ stimulates Snail1 sumoylation in aggressive prostate, breast and lung cancer cells. Sumoylation of Snail1 lysine residue 234 confers its transcriptional activity, inducing the expression of classical EMT genes, as well as TGFβ receptor I (TβRI) and the transcriptional repressor Hes1. Mutation of Snail1 lysine residue 234 to arginine (K234R) abolished sumoylation of Snail1, as well as its migratory and invasive properties in human prostate cancer cells. An increased immunohistochemical expression of Snail1, Sumo1, TβRI, Hes1, and c-Jun was observed in aggressive prostate cancer tissues, consistent with their functional roles in tumorigenesis.
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http://dx.doi.org/10.18632/oncotarget.20097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716685PMC
November 2017

JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion.

Nucleic Acids Res 2018 02;46(3):1180-1195

Ludwig Cancer Research, Science for Life Laboratory, Box 595, Biomedical Center, Uppsala University, SE-751 24 Uppsala, Sweden.

It is well established that transforming growth factor-β (TGFβ) switches its function from being a tumor suppressor to a tumor promoter during the course of tumorigenesis, which involves both cell-intrinsic and environment-mediated mechanisms. We are interested in breast cancer cells, in which SMAD mutations are rare and interactions between SMAD and other transcription factors define pro-oncogenic events. Here, we have performed chromatin immunoprecipitation (ChIP)-sequencing analyses which indicate that the genome-wide landscape of SMAD2/3 binding is altered after prolonged TGFβ stimulation. De novo motif analyses of the SMAD2/3 binding regions predict enrichment of binding motifs for activator protein (AP)1 in addition to SMAD motifs. TGFβ-induced expression of the AP1 component JUNB was required for expression of many late invasion-mediating genes, creating a feed-forward regulatory network. Moreover, we found that several components in the WNT pathway were enriched among the late TGFβ-target genes, including the invasion-inducing WNT7 proteins. Consistently, overexpression of WNT7A or WNT7B enhanced and potentiated TGFβ-induced breast cancer cell invasion, while inhibition of the WNT pathway reduced this process. Our study thereby helps to explain how accumulation of pro-oncogenic stimuli switches and stabilizes TGFβ-induced cellular phenotypes of epithelial cells.
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http://dx.doi.org/10.1093/nar/gkx1190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814809PMC
February 2018