Publications by authors named "Fritz Aberger"

65 Publications

Casein Kinase 1D Encodes a Novel Drug Target in Hedgehog-GLI-Driven Cancers and Tumor-Initiating Cells Resistant to SMO Inhibition.

Cancers (Basel) 2021 Aug 23;13(16). Epub 2021 Aug 23.

Department of Bioscience, Cancer Cluster Salzburg, Paris-Lodron University Salzburg, 5020 Salzburg, Austria.

(1) Background: Aberrant activation of the hedgehog (HH)-GLI pathway in stem-like tumor-initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH-GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH-GLI-driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity, and monitored the oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO inhibitor-sensitive and -resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent TIC in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor-sensitive and -resistant tumors.
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http://dx.doi.org/10.3390/cancers13164227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394935PMC
August 2021

The life in a gradient: calcium, the lncRNA SPRR2C and mir542/mir196a meet in the epidermis to regulate the aging process.

Aging (Albany NY) 2021 08 2;13(15):19127-19144. Epub 2021 Aug 2.

Department of Biosciences, Paris-Lodron University Salzburg, Salzburg, Austria.

The turnover of the epidermis beginning with the progenitor cells in the basal layer to the fully differentiated corneocytes is tightly regulated by calcium. Calcium more than anything else promotes the differentiation of keratinocytes which implies the need for a calcium gradient with low concentrations in the stratum basale and high concentrations in the stratum granulosum. One of the hallmarks of skin aging is a collapse of this gradient that has a direct impact on the epidermal fitness. The rise of calcium in the stratum basale reduces cell proliferation, whereas the drop of calcium in the stratum granulosum leads to a changed composition of the cornified envelope. We showed that keratinocytes respond to the calcium induced block of cell division by a large increase of the expression of several miRNAs (hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p). The pitfall of this rescue mechanism is a dramatic change in gene expression which causes a further impairment of the epidermal barrier. This effect is attenuated by a pseudogene (SPRR2C) that gives rise to a lncRNA. SPRR2C specifically resides in the stratum granulosum/corneum thus acting as a sponge for miRNAs.
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http://dx.doi.org/10.18632/aging.203385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386546PMC
August 2021

Context-dependent modulation of aggressiveness of pediatric tumors by individual oncogenic RAS isoforms.

Oncogene 2021 Aug 25;40(31):4955-4966. Epub 2021 Jun 25.

Department of Human Genetics, University Medical Center Goettingen, Goettingen, Germany.

A prototypic pediatric cancer that frequently shows activation of RAS signaling is embryonal rhabdomyosarcoma (ERMS). ERMS also show aberrant Hedgehog (HH)/GLI signaling activity and can be driven by germline mutations in this pathway. We show, that in ERMS cell lines derived from sporadic tumors i.e. from tumors not caused by an inherited genetic variant, HH/GLI signaling plays a subordinate role, because oncogenic mutations in HRAS, KRAS, or NRAS (collectively named oncRAS) inhibit the main HH target GLI1 via the MEK/ERK-axis, but simultaneously increase proliferation and tumorigenicity. oncRAS also modulate expression of stem cell markers in an isoform- and context-dependent manner. In Hh-driven murine ERMS that are caused by a Patched mutation, oncHRAS and mainly oncKRAS accelerate tumor development, whereas oncNRAS induces a more differentiated phenotype. These features occur when the oncRAS mutations are induced at the ERMS precursor stage, but not when induced in already established tumors. Moreover, in contrast to what is seen in human cell lines, oncRAS mutations do not alter Hh signaling activity and marginally affect expression of stem cell markers. Together, all three oncRAS mutations seem to be advantageous for ERMS cell lines despite inhibition of HH signaling and isoform-specific modulation of stem cell markers. In contrast, oncRAS mutations do not inhibit Hh-signaling in Hh-driven ERMS. In this model, oncRAS mutations seem to be advantageous for specific ERMS populations that occur within a specific time window during ERMS development. In addition, this window may be different for individual oncRAS isoforms, at least in the mouse.
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http://dx.doi.org/10.1038/s41388-021-01904-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8342309PMC
August 2021

Opioids drive breast cancer metastasis through the δ-opioid receptor and oncogenic STAT3.

Neoplasia 2021 02 16;23(2):270-279. Epub 2021 Jan 16.

Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Vienna, Austria. Electronic address:

The opioid crisis of pain medication bears risks from addiction to cancer progression, but little experimental evidence exists. Expression of δ-opioid receptors (DORs) correlates with poor prognosis for breast cancer patients, but mechanistic insights into oncogenic signaling mechanisms of opioid-triggered cancer progression are lacking. We show that orthotopic transplant models using human or murine breast cancer cells displayed enhanced metastasis upon opioid-induced DOR stimulation. Interestingly, opioid-exposed breast cancer cells showed enhanced migration and strong STAT3 activation, which was efficiently blocked by a DOR-antagonist. Furthermore, opioid treatment resulted in down-regulation of E-Cadherin and increased expression of epithelial-mesenchymal transition markers. Notably, STAT3 knockdown or upstream inhibition through the JAK1/2 kinase inhibitor ruxolitinib prevented opioid-induced breast cancer cell metastasis and migration in vitro and in vivo. We conclude on a novel mechanism whereby opioid-triggered breast cancer metastasis occurs via oncogenic JAK1/2-STAT3 signaling to promote epithelial-mesenchymal transition. These findings emphasize the importance of selective and restricted opioid use, as well as the need for safer pain medication that does not activate these oncogenic pathways.
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http://dx.doi.org/10.1016/j.neo.2020.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815495PMC
February 2021

STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pathway.

Oncogene 2021 02 15;40(6):1091-1105. Epub 2020 Dec 15.

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

Metastatic melanoma is hallmarked by its ability of phenotype switching to more slowly proliferating, but highly invasive cells. Here, we tested the impact of signal transducer and activator of transcription 3 (STAT3) on melanoma progression in association with melanocyte inducing transcription factor (MITF) expression levels. We established a mouse melanoma model for deleting Stat3 in melanocytes with specific expression of human hyperactive NRAS in an Ink4a-deficient background, two frequent driver mutations in human melanoma. Mice devoid of Stat3 showed early disease onset with higher proliferation in primary tumors, but displayed significantly diminished lung, brain, and liver metastases. Whole-genome expression profiling of tumor-derived cells also showed a reduced invasion phenotype, which was further corroborated by 3D melanoma model analysis. Notably, loss or knockdown of STAT3 in mouse or human cells resulted in the upregulation of MITF and induction of cell proliferation. Mechanistically we show that STAT3-induced CAAT Box Enhancer Binding Protein (CEBP) expression was sufficient to suppress MITF transcription. Epigenetic analysis by ATAC-seq confirmed that CEBPa/b binding to the MITF enhancer region silenced the MITF locus. Finally, by classification of patient-derived melanoma samples, we show that STAT3 and MITF act antagonistically and hence contribute differentially to melanoma progression. We conclude that STAT3 is a driver of the metastatic process in melanoma and able to antagonize MITF via direct induction of CEBP family member transcription.
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http://dx.doi.org/10.1038/s41388-020-01584-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116782PMC
February 2021

CD44 engagement enhances acute myeloid leukemia cell adhesion to the bone marrow microenvironment by increasing VLA-4 avidity.

Haematologica 2021 08 1;106(8):2102-2113. Epub 2021 Aug 1.

3rd Medical Department, SCRI-LIMCR, Paracelsus Medical University, Cancer Cluster Salzburg.

Adhesive properties of leukemia cells shape the degree of organ infiltration and the extent of leukocytosis. CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer, are important factors of progenitor cell adhesion in bone marrow (BM). Here, we report their cooperation in acute myeloid leukemia (AML) by a novel non-classical CD44-mediated way of inside-out VLA-4 activation. In primary AML BM samples from patients and the OCI-AML3 cell line, CD44 engagement by hyaluronan induced inside-out activation of VLA-4 resulting in enhanced leukemia cell adhesion on VCAM-1. This was independent from VLA-4 affinity regulation but based on ligand-induced integrin clustering on the cell surface. CD44-induced VLA-4 activation could be inhibited by the Src family kinase inhibitor PP2 and the multikinase inhibitor midostaurin. In further consequence, the increased adhesion on VCAM-1 allowed AML cells to strongly bind stromal cells. Thereby VLA-4/VCAM-1 interaction promoted activation of Akt, MAPK, NF-kB and mTOR signaling and decreased AML cell apoptosis. Collectively, our investigations provide a mechanistic description of an unusual CD44 function in regulating VLA-4 avidity in AML, supporting AML cell retention in the supportive BM microenvironment.
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http://dx.doi.org/10.3324/haematol.2019.231944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8327716PMC
August 2021

Epidermal activation of Hedgehog signaling establishes an immunosuppressive microenvironment in basal cell carcinoma by modulating skin immunity.

Mol Oncol 2020 09 21;14(9):1930-1946. Epub 2020 Jul 21.

Department of Biosciences, Cancer Cluster Salzburg, Paris-Lodron University Salzburg, Austria.

Genetic activation of hedgehog/glioma-associated oncogene homolog (HH/GLI) signaling causes basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer. Small molecule targeting of the essential HH effector Smoothened (SMO) has proven an effective therapy of BCC, though the frequent development of drug resistance poses major challenges to anti-HH treatments. In light of recent breakthroughs in cancer immunotherapy, we analyzed the possible immunosuppressive mechanisms in HH/GLI-induced BCC in detail. Using a genetic mouse model of BCC, we identified profound differences in the infiltration of BCC lesions with cells of the adaptive and innate immune system. Epidermal activation of Hh/Gli signaling led to an accumulation of immunosuppressive regulatory T cells, and to an increased expression of immune checkpoint molecules including programmed death (PD)-1/PD-ligand 1. Anti-PD-1 monotherapy, however, did not reduce tumor growth, presumably due to the lack of immunogenic mutations in common BCC mouse models, as shown by whole-exome sequencing. BCC lesions also displayed a marked infiltration with neutrophils, the depletion of which unexpectedly promoted BCC growth. The study provides a comprehensive survey of and novel insights into the immune status of murine BCC and serves as a basis for the design of efficacious rational combination treatments. This study also underlines the need for predictive immunogenic mouse models of BCC to evaluate the efficacy of immunotherapeutic strategies in vivo.
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http://dx.doi.org/10.1002/1878-0261.12758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463314PMC
September 2020

Phosphoproteomics of short-term hedgehog signaling in human medulloblastoma cells.

Cell Commun Signal 2020 06 23;18(1):99. Epub 2020 Jun 23.

Department of Biosciences, Bioanalytical Research Laboratories and Molecular Cancer Research and Tumor Immunology, Cancer Cluster Salzburg, University of Salzburg, Hellbrunner Straße 34, 5020, Salzburg, Austria.

Background: Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 β controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation.

Methods: We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway.

Results: Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment.

Conclusions: This study provides evidence that phosphorylation triggered in response to SMO modulation dictates the localization of hedgehog pathway components within the primary cilium and affects the regulation of the SMO-SUFU-GLI axis. The data are relevant for the development of targeted therapies of HH-associated cancers including sonic HH-type medulloblastoma. A deeper understanding of the mechanisms of action of SMO inhibitors such as vismodegib may lead to the development of compounds causing fewer adverse effects and lower frequencies of drug resistance. Video Abstract.
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http://dx.doi.org/10.1186/s12964-020-00591-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310537PMC
June 2020

Proteins and Molecular Pathways Relevant for the Malignant Properties of Tumor-Initiating Pancreatic Cancer Cells.

Cells 2020 06 3;9(6). Epub 2020 Jun 3.

Department of Biosciences, Bioanalytical Research Labs, University of Salzburg, A-5020 Salzburg, Austria.

Cancer stem cells (CSCs), a small subset of the tumor bulk with highly malignant properties, are deemed responsible for tumor initiation, growth, metastasis, and relapse. In order to reveal molecular markers and determinants of their tumor-initiating properties, we enriched rare stem-like pancreatic tumor-initiating cells (TICs) by harnessing their clonogenic growth capacity in three-dimensional multicellular spheroid cultures. We compared pancreatic TICs isolated from three-dimensional tumor spheroid cultures with nontumor-initiating cells (non-TICs) enriched in planar cultures. Employing differential proteomics (PTX), we identified more than 400 proteins with significantly different expression in pancreatic TICs and the non-TIC population. By combining the unbiased PTX with mRNA expression analysis and literature-based predictions of pro-malignant functions, we nominated the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14) as well as galactin-3-binding protein LGALS3BP (MAC-2-BP) as putative determinants of pancreatic TICs. In silico pathway analysis followed by candidate-based RNA interference mediated loss-of-function analysis revealed a critical role of S100A8, S100A9, and LGALS3BP as molecular determinants of TIC proliferation, migration, and in vivo tumor growth. Our study highlights the power of combining unbiased proteomics with focused gene expression and functional analyses for the identification of novel key regulators of TICs, an approach that warrants further application to identify proteins and pathways amenable to drug targeting.
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http://dx.doi.org/10.3390/cells9061397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349116PMC
June 2020

STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Mol Syst Biol 2020 04;16(4):e9247

Department of Analytical Chemistry, University of Vienna, Vienna, Austria.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.
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http://dx.doi.org/10.15252/msb.20199247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178451PMC
April 2020

Hedgehog/GLI signaling in tumor immunity - new therapeutic opportunities and clinical implications.

Cell Commun Signal 2019 12 26;17(1):172. Epub 2019 Dec 26.

Department of Biosciences, Cancer Cluster Salzburg, University of Salzburg, Hellbrunner Strasse, 34, 5020, Salzburg, Austria.

Uncontrolled activation of the Hedgehog/Glioma-associated oncogene (HH/GLI) pathway is a potent oncogenic driver signal promoting numerous cancer hallmarks such as proliferation, survival, angiogenesis, metastasis and metabolic rewiring. Several HH pathway inhibitors have already been approved for medical therapy of advanced and metastatic basal cell carcinoma and acute myeloid leukemia with partially impressive therapeutic activity. However, de novo and acquired resistance as well as severe side effects and unexplained lack of therapeutic efficacy are major challenges that urgently call for improved treatment options with more durable responses. The recent breakthroughs in cancer immunotherapy have changed our current understanding of targeted therapy and opened up promising therapeutic opportunities including combinations of selective cancer pathway and immune checkpoint inhibitors. Although HH/GLI signaling has been intensely studied with respect to the classical hallmarks of cancer, its role in the modulation of the anti-tumoral immune response has only become evident in recent studies. These have uncovered HH/GLI regulated immunosuppressive mechanisms such as enhanced regulatory T-cell formation and production of immunosuppressive cytokines. In light of these exciting novel data on oncogenic HH/GLI signaling in immune cross-talk and modulation, we summarize and connect in this review the existing knowledge from different HH-related cancers and chronic inflammatory diseases. This is to provide a basis for the investigation and evaluation of novel treatments combining immunotherapeutic strategies with approved as well as next-generation HH/GLI inhibitors. Further, we also critically discuss recent studies demonstrating a possible negative impact of current HH/GLI pathway inhibitors on the anti-tumoral immune response, which may explain some of the disappointing results of several oncological trials with anti-HH drugs. Additional file 1Video abstract. (9500 kb).
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http://dx.doi.org/10.1186/s12964-019-0459-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933925PMC
December 2019

Next-Generation Hedgehog/GLI Pathway Inhibitors for Cancer Therapy.

Cancers (Basel) 2019 Apr 15;11(4). Epub 2019 Apr 15.

Department of Biosciences, Paris-Lodron University of Salzburg, Cancer Cluster Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria.

The Hedgehog/Glioma-associated oncogene homolog (HH/GLI) signaling pathway regulates self-renewal of rare and highly malignant cancer stem cells (CSC), which have been shown to account for the initiation and maintenance of tumor growth as well as for drug resistance, metastatic spread and relapse. Efficacious therapeutic approaches targeting CSC pathways, such as HH/GLI signaling in combination with chemo, radiation or immunotherapy are, therefore, of high medical need. Pharmacological inhibition of HH/GLI pathway activity represents a promising approach to eliminate malignant CSC. Clinically approved HH/GLI pathway inhibitors target the essential pathway effector Smoothened (SMO) with striking therapeutic efficacy in skin and brain cancer patients. However, multiple genetic and molecular mechanisms resulting in de novo and acquired resistance to SMO inhibitors pose major limitations to anti-HH/GLI therapies and, thus, the eradication of CSC. In this review, we summarize reasons for clinical failure of SMO inhibitors, including mechanisms caused by genetic alterations in HH pathway effectors or triggered by additional oncogenic signals activating GLI transcription factors in a noncanonical manner. We then discuss emerging novel and rationale-based approaches to overcome SMO-inhibitor resistance, focusing on pharmacological perturbations of enzymatic modifiers of GLI activity and on compounds either directly targeting oncogenic GLI factors or interfering with synergistic crosstalk signals known to boost the oncogenicity of HH/GLI signaling.
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http://dx.doi.org/10.3390/cancers11040538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520835PMC
April 2019

Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma.

Leukemia 2019 03 21;33(3):696-709. Epub 2018 Aug 21.

Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore, Singapore.

TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1, a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.
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http://dx.doi.org/10.1038/s41375-018-0239-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076043PMC
March 2019

Synergistic cross-talk of hedgehog and interleukin-6 signaling drives growth of basal cell carcinoma.

Int J Cancer 2018 12 1;143(11):2943-2954. Epub 2018 Oct 1.

Department of Biosciences, Cancer Cluster Salzburg, Paris-Lodron University of Salzburg, Salzburg, Austria.

Persistent activation of hedgehog (HH)/GLI signaling accounts for the development of basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer with rising incidence. Targeting HH/GLI signaling by approved pathway inhibitors can provide significant therapeutic benefit to BCC patients. However, limited response rates, development of drug resistance, and severe side effects of HH pathway inhibitors call for improved treatment strategies such as rational combination therapies simultaneously inhibiting HH/GLI and cooperative signals promoting the oncogenic activity of HH/GLI. In this study, we identified the interleukin-6 (IL6) pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. Mechanistically, we provide evidence that signal integration of IL6 and HH/GLI occurs at the level of cis-regulatory sequences by co-binding of GLI and STAT3 to common HH-IL6 target gene promoters. Genetic inactivation of Il6 signaling in a mouse model of BCC significantly reduced in vivo tumor growth by interfering with HH/GLI-driven BCC proliferation. Our genetic and pharmacologic data suggest that combinatorial HH-IL6 pathway blockade is a promising approach to efficiently arrest cancer growth in BCC patients.
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http://dx.doi.org/10.1002/ijc.31724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282712PMC
December 2018

Microenvironment-induced CD44v6 promotes early disease progression in chronic lymphocytic leukemia.

Blood 2018 03 19;131(12):1337-1349. Epub 2018 Jan 19.

Third Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases, and Rheumatology, Oncologic Center, Paracelsus Medical University, Salzburg, Austria.

Chronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell-specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation.
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http://dx.doi.org/10.1182/blood-2017-08-802462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865232PMC
March 2018

Targeting class I histone deacetylases by the novel small molecule inhibitor 4SC-202 blocks oncogenic hedgehog-GLI signaling and overcomes smoothened inhibitor resistance.

Int J Cancer 2018 03 6;142(5):968-975. Epub 2017 Nov 6.

Department of Molecular Biology, Cancer Cluster Salzburg, Paris-Lodron University of Salzburg, Salzburg, 5020, Austria.

Aberrant activation of Hedgehog (HH)/GLI signaling is causally involved in numerous human malignancies, including basal cell carcinoma (BCC) and medulloblastoma. HH pathway antagonists targeting smoothened (SMO), an essential effector of canonical HH/GLI signaling, show significant clinical success in BCC patients and have recently been approved for the treatment of advanced and metastatic BCC. However, rapid and frequent development of drug resistance to SMO inhibitors (SMOi) together with severe side effects caused by prolonged SMOi treatment call for alternative treatment strategies targeting HH/GLI signaling downstream of SMO. In this study, we report that 4SC-202, a novel clinically validated inhibitor of class I histone deacetylases (HDACs), efficiently blocks HH/GLI signaling. Notably, 4SC-202 treatment abrogates GLI activation and HH target gene expression in both SMOi-sensitive and -resistant cells. Mechanistically, we propose that the inhibition of HDACs 1/2/3 is crucial for targeting oncogenic HH/GLI signaling, and that class I HDAC inhibitors either in combination with SMOi or as second-line therapy may improve the treatment options for HH-associated malignancies with SMOi resistance.
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http://dx.doi.org/10.1002/ijc.31117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813224PMC
March 2018

The sound of tumor cell-microenvironment communication - composed by the Cancer Cluster Salzburg research network.

Cell Commun Signal 2017 06 2;15(1):20. Epub 2017 Jun 2.

Cancer Cluster Salzburg, Salzburg Cancer Research Institute (SCRI) - Laboratory for Immunological and Molecular Cancer Research (LIMCR), A-5020, Salzburg, Austria.

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http://dx.doi.org/10.1186/s12964-017-0176-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457625PMC
June 2017

From inflammation to gastric cancer - the importance of Hedgehog/GLI signaling in Helicobacter pylori-induced chronic inflammatory and neoplastic diseases.

Cell Commun Signal 2017 04 20;15(1):15. Epub 2017 Apr 20.

Division of Molecular Tumor Biology, Cancer Cluster Salzburg, Department of Molecular Biology, Paris-Lodron University of Salzburg, Hellbrunner Strasse 34, A-5020, Salzburg, Austria.

Infections with the human pathogen Helicobacter pylori (H. pylori) are closely associated with the development of inflammatory disorders and neoplastic transformation of the gastric epithelium. Drastic changes in the micromilieu involve a complex network of H. pylori-regulated signal transduction pathways leading to the release of proinflammatory cytokines, gut hormones and a wide range of signaling molecules. Besides controlling embryonic development, the Hedgehog/GLI signaling pathway also plays important roles in epithelial proliferation, differentiation, and regeneration of the gastric physiology, but also in the induction and progression of inflammation and neoplastic transformation in H. pylori infections. Here, we summarize recent findings of H. pylori-associated Hedgehog/GLI signaling in gastric homeostasis, malignant development and the modulation of the gastric tumor microenvironment.
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http://dx.doi.org/10.1186/s12964-017-0171-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397778PMC
April 2017

Understanding cell signaling in cancer stem cells for targeted therapy - can phosphoproteomics help to reveal the secrets?

Cell Commun Signal 2017 Mar 29;15(1):12. Epub 2017 Mar 29.

Department of Molecular Biology, Cancer Cluster Salzburg, Paris-Lodron University of Salzburg, Hellbrunner Strasse 34, 5020, Salzburg, Austria.

Background: Cancer represents heterogeneous and aberrantly proliferative manifestations composed of (epi)genetically and phenotypically distinct cells with a common clonal origin. Cancer stem cells (CSC) make up a rare subpopulation with the remarkable capacity to initiate, propagate and spread a malignant disease. Furthermore, CSC show increased therapy resistance, thereby contributing to disease relapse. Elimination of CSC, therefore, is a crucial aim to design efficacious treatments for long-term survival of cancer patients. In this article, we highlight the nature of CSC and propose that phosphoproteomics based on unbiased high-performance liquid chromatography-mass spectrometry provides a powerful tool to decipher the molecular CSC programs. Detailed knowledge about the regulation of signaling processes in CSC is a prerequisite for the development of patient-tailored multi-modal treatments including the elimination of rare CSC.

Main Body: Phosphorylation is a crucial post-translational modification regulating a plethora of both intra- and intercellular communication processes in normal and malignant cells. Small-molecule targeting of kinases has proven successful in the therapy, but the high rates of relapse and failure to stem malignant spread suggest that these kinase inhibitors largely spare CSC. Studying the kinetics of global phosphorylation patterns in an unbiased manner is, therefore, required to improve strategies and successful treatments within multi-modal therapeutic regimens by targeting the malignant behavior of CSC. The phosphoproteome comprises all phosphoproteins within a cell population that can be analyzed by phosphoproteomics, allowing the investigation of thousands of phosphorylation events. One major aspect is the perception of events underlying the activation and deactivation of kinases and phosphatases in oncogenic signaling pathways. Thus, not only can this tool be harnessed to better understand cellular processes such as those controlling CSC, but also applied to identify novel drug targets for targeted anti-CSC therapy.

Conclusion: State-of-the-art phosphoproteomics approaches focusing on single cell analysis have the potential to better understand oncogenic signaling in heterogeneous cell populations including rare, yet highly malignant CSC. By eliminating the influence of heterogeneity of populations, single-cell studies will reveal novel insights also into the inter- and intratumoral communication processes controlling malignant CSC and disease progression, laying the basis for improved rational combination treatments.
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http://dx.doi.org/10.1186/s12964-017-0166-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372284PMC
March 2017

Acute myeloid leukemia - strategies and challenges for targeting oncogenic Hedgehog/GLI signaling.

Cell Commun Signal 2017 01 25;15(1). Epub 2017 Jan 25.

Cancer Cluster Salzburg, Salzburg Cancer Research Institute (SCRI) - Laboratory for Immunological and Molecular Cancer Research (LIMCR), 5020, Salzburg, Austria.

Treatment of acute myeloid leukemia (AML), an aggressive and heterogeneous hematological malignancy, remains a challenge. Despite advances in our understanding of the complex genetics and biology of AML pathophysiology, these findings have been translated to the clinic with only limited success, and poor outcomes persist for the majority of patients. Thus, novel treatment strategies are clearly needed for achieving deeper and prolonged remissions and for avoiding the development of resistance. Due to its profound role in (cancer) stem cell biology and differentiation, the Hedgehog (HH)/Glioma-associated Oncogene Homolog (GLI) signaling pathway may be an attractive novel therapeutic target in AML. In this review, we aim to provide a critical and concise overview of the currently known potential and challenges of HH/GLI targeting. We describe the biological role of the HH/GLI pathway in AML pathophysiology. We specifically focus on ways of targeting non-canonical HH/GLI signaling in AML, particularly in combination with standard treatment regimens, which may overcome some hurdles observed with approved HH pathway inhibitors in solid tumors.
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http://dx.doi.org/10.1186/s12964-017-0163-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267446PMC
January 2017

A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray.

Microarrays (Basel) 2016 Feb 23;5(1). Epub 2016 Feb 23.

Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria.

A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.
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http://dx.doi.org/10.3390/microarrays5010005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003450PMC
February 2016

A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells.

Biosens Bioelectron 2016 Dec 8;86:20-26. Epub 2016 Jun 8.

Center for Advanced Bioanalysis GmbH, Gruberstrasse 40-42, 4020 Linz, Austria. Electronic address:

Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties - e.g. low sample dilution, low contamination - that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.
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http://dx.doi.org/10.1016/j.bios.2016.06.019DOI Listing
December 2016

The ratio of STAT1 to STAT3 expression is a determinant of colorectal cancer growth.

Oncotarget 2016 Aug;7(32):51096-51106

Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria.

The role of STAT1 and STAT3 for colorectal carcinoma (CRC) development and progression is controversial. We evaluated 414 CRC patient samples on tissue microarrays for differential expression of STAT1 and STAT3 protein levels and correlated ratios with clinical parameters. Concomitant absence of nuclear STAT1 and STAT3 expression was associated with significantly reduced median survival by ≥33 months (p=0.003). To gain insight into underlying mechanisms, we generated four CRC cell lines with STAT3 knockdown. The cell lines harbor different known mutational drivers and were xenografted into SCID mice to analyze the influence of STAT3 on their tumor growth behavior. Experimental downregulation of STAT3 expression had differential, cell-line specific effects on STAT1 expression levels. STAT1 consistently showed nuclear localization irrespective of its tyrosine phosphorylation status. Two characteristic STAT1/3 expression patterns with opposite growth behavior could be distinguished: cell lines with a low STAT1/high STAT3 ratio showed faster tumor growth in xenografts. In contrast, xenografts of cell lines showing high STAT1 and low STAT3 levels grew slower. Importantly, these ratios reflected clinical outcome in CRC patients as well. We conclude that the ratio of STAT1 to STAT3 expression is a key determinant of CRC progression and that STAT1 counteracts pro-tumorigenic STAT3 signaling. Thus, we suggest that the STAT3/STAT1 ratios are better clinical predictors in CRC as compared to STAT3 or STAT1 levels alone.
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http://dx.doi.org/10.18632/oncotarget.9315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239461PMC
August 2016

ILK Induction in Lymphoid Organs by a TNFα-NF-κB-Regulated Pathway Promotes the Development of Chronic Lymphocytic Leukemia.

Cancer Res 2016 04 2;76(8):2186-96. Epub 2016 Feb 2.

Third Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases, and Rheumatology, Laboratory for Immunological and Molecular Cancer Research, Oncologic Center, Paracelsus Medical University, Salzburg, Austria. Salzburg Cancer Research Institute, Salzburg, Austria.

The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell-cycle progression, but is poorly characterized in hematologic malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high-risk markers such as unmutated IGHV genes, high Zap70, or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes from CLL patients. Using coculture conditions mimicking the proliferative lymph node microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein colocalized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B-cell-specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα-NF-κB-mediated mechanism by which ILK expression is induced in the lymph node microenvironment and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation. Cancer Res; 76(8); 2186-96. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3379DOI Listing
April 2016

DYRK1B as therapeutic target in Hedgehog/GLI-dependent cancer cells with Smoothened inhibitor resistance.

Oncotarget 2016 Feb;7(6):7134-48

Cancer Cluster Salzburg, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds.Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy.
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http://dx.doi.org/10.18632/oncotarget.6910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872774PMC
February 2016

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Nat Commun 2015 Jul 22;6:7736. Epub 2015 Jul 22.

Department of Pathology, University of Cambridge, CB2 0QQ Cambridge, UK.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.
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http://dx.doi.org/10.1038/ncomms8736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4525303PMC
July 2015

Lung Adenocarcinomas and Lung Cancer Cell Lines Show Association of MMP-1 Expression With STAT3 Activation.

Transl Oncol 2015 Apr;8(2):97-105

Institute of Biochemistry II, Jena University Hospital, Jena, Germany. Electronic address:

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in the majority of lung cancer. This study aims at defining connections between STAT3 function and the malignant properties of non-small cell lung carcinoma (NSCLC) cells. To address possible mechanisms by which STAT3 influences invasiveness, the expression of matrix metalloproteinase-1 (MMP-1) was analyzed and correlated with the STAT3 activity status. Studies on both surgical biopsies and on lung cancer cell lines revealed a coincidence of STAT3 activation and strong expression of MMP-1. MMP-1 and tyrosine-phosphorylated activated STAT3 were found co-localized in cancer tissues, most pronounced in tumor fronts, and in particular in adenocarcinomas. STAT3 activity was constitutive, although to different degrees, in the lung cancer cell lines investigated. Three cell lines (BEN, KNS62, and A549) were identified in which STAT3 activitation was inducible by Interleukin-6 (IL-6). In A549 cells, STAT3 activity enhanced the level of MMP-1 mRNA and stimulated transcription from the MMP-1 promoter in IL-6-stimulated A549 cells. STAT3 specificity of this effect was confirmed by STAT3 knockdown through RNA interference. Our results link aberrant activity of STAT3 in lung cancer cells to malignant tumor progression through up-regulation of expression of invasiveness-associated MMPs.
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http://dx.doi.org/10.1016/j.tranon.2015.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415137PMC
April 2015
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