Mario Tschan | University of Bern (original) (raw)

Papers by Mario Tschan

Nature Communications, 2017

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the produ... more Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.

Molecular cancer therapeutics, Jan 20, 2017

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine... more Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNETs) is an unmet medical need. The anti-angiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. Additionally, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chlor...

Cell Death & Disease

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phospho... more The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity....

EMBO Molecular Medicine, 2021

KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically target... more KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically targeting KRAS-driven tumors remain elusive. Here, we show that combined treatment with fibroblast growth factor receptor 1 (FGFR1) and polo-like kinase 1 (PLK1) inhibitors evoke synergistic cytotoxicity in KRAS-mutant tumor models in vitro and in vivo. Pharmacological and genetic suppression of FGFR1 and PLK1 synergizes to enhance antiproliferative effects and cell death in KRAS-mutant lung and pancreatic but not colon nor KRAS wild-type cancer cells. Mechanistically, co-targeting FGFR1 and PLK1 upregulates reactive oxygen species (ROS), leading to oxidative stress-activated c-Jun Nterminal kinase (JNK)/p38 pathway and E2F1-induced apoptosis. We further delineate that autophagy protects from PLK1/FGFR1 inhibitor cytotoxicity and that antagonizing the compensation mechanism by clinically approved chloroquine fully realizes the therapeutic potential of PLK1 and FGFR1 targeting therapy, producing potent and durable responses in KRAS-mutant patientderived xenografts and a genetically engineered mouse model of Kras-induced lung adenocarcinoma. These results suggest a previously unappreciated role for FGFR1 and PLK1 in the surveillance of metabolic stress and demonstrate a synergistic drug combination for treating KRAS-mutant cancer.

Cells, 2021

In recent years autophagy has attracted the attention of researchers from many medical fields, in... more In recent years autophagy has attracted the attention of researchers from many medical fields, including cancer research, and certain anti-macroautophagy drugs in combination with cytotoxic or targeted therapies have entered clinical trials. In the present study, we focused on a less explored subtype of autophagy, i.e., chaperone-mediated autophagy (CMA), with the key proteins LAMP2A and HSPA8 (HSC70), and their immunohistochemical evaluation with previously extensively validated antibodies. We were interested in whether the marker expression is influenced by the antecedent therapy, and its correlation with survival on a cohort of patients with non-small cell lung cancer (NSCLC) after neoadjuvant therapy and matched primary resected tumors. In concordance with our previous study, we did not find any intratumoral heterogeneity, nor correlation between the two parameters, nor correlation between the markers and any included pathological parameters. Surprisingly, the expression of both...

Leukemia, 2021

Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules... more Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules. However, in recent studies, they have begun to emerge as crucial regulators of cell homeostasis. They are at the crossroads of catabolic and anabolic pathways and are intricately involved in cellular trafficking, nutrient signaling, energy metabolism, and immune regulation. Their involvement in such essential cellular functions has renewed clinical interest in targeting the lysosome as a novel way to treat disease, particularly cancer. Acute myeloid leukemia (AML) is an aggressive blood cancer with a low survival probability, particularly in older patients. The genomic landscape of AML has been extensively characterized but few targeted therapies (with the exception of differentiation therapy) can achieve a long-term cure. Therefore, there is an unmet need for less intensive and more tolerable therapeutic interventions. In this review, we will give an overview on the myriad of function...

Clinical Epigenetics, 2018

Background: In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and assoc... more Background: In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration. Methods: CDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation. Results: Sixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988-0.997)) as well as BRAF V600E , mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2-to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2. Conclusion: CDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/ protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.

Cancer Research, 2016

A global understanding of miRNA function in EGFR signaling pathways may provide insights into imp... more A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRAS G12V and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non-small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRAS G12V-transduced BEAS-2B cells, introduction of anti-miR-29b constructs increased the sensitivity to apoptosis, arguing that miR-29b mediated apoptotic resistance conferred by mutant KRAS. Mechanistic investigations traced this effect to the ability of miR-29b to target TNFAIP3/A20, a negative regulator of NF-kB signaling. Accordingly, overexpression of an miR-29b-refractory isoform of TNFAIP3 restored NF-kB and extrinsic apoptosis, confirming that TNFAIP3 is a functionally relevant target of miR-29b. We also noted that miR-29b could confer sensitivity to intrinsic apoptosis triggered by exposure to cisplatin, a drug used widely in lung cancer treatment. Thus, miR-29b expression may tilt cells from extrinsic to intrinsic mechanisms of apoptosis. Overall, our results reveal a complexity in cancer for miR-29b, which can act as either an oncogene or tumor suppressor gene depending on signaling context. Cancer Res; 76(14); 4160-9. Ó2016 AACR.

Journal of Thoracic Oncology, 2016

Background: In cancer, autophagy may promote tumor cell survival or cell death. Protein light cha... more Background: In cancer, autophagy may promote tumor cell survival or cell death. Protein light chain 3B (LC3B) and p62 are proteins associated with autophagosomal membranes that engulf cytoplasmic content during autophagy, which is subsequently degraded. We studied the expression of these two markers in stage I/II non-small cell lung cancer (NSCLC). Methods: Immunohistochemistry for LC3B and p62 was performed on a tissue microarray with 466 NSCLC: 202 Adenocarcinomas (AC), 220 squamous cell carcinomas (SCC), 35 large cell carcinomas. Dot-like cytoplasmic expression of LC3B and dot-like, cytoplasmic and nuclear staining for p62 was determined. Results were compared with clinical and pathological parameters. Results: LC3B expression correlated with all p62 staining patterns, as those correlated among each other (x 2 < 0.001 each). Low LC3B was more frequent in SCC (x 2 = 0.017). Low cytoplasmic/nuclear p62 was more frequent in AC (x 2 = 0.029/ x 2 < 0.001). There was no correlation with stage, age or gender. Regarding dot-like staining only, a combination of high LC3B/high p62 showed a trend to better outcome (p = 0.11). This marker combination has been suggested to reflect activated impaired autophagy. All other dot-like staining pattern combinations were similarly unfavorable. Interestingly, the best prognostic value was observed for low cytoplasmic/low nuclear p62 expression (regardless of dot-like staining). This implied a significantly better outcome in univariate analysis (p = 0.004) and in multivariate analysis (p = 0.006; HR = 1.96) in addition to tumor stage (p = 0.005; HR = 1.4). Conclusions: The autophagy markers LC3B and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role in these tumors. High LC3B levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior. Interestingly, p62 cytoplasmic/nuclear expression-which may not be linked to autophagy such as the dot-like pattern-had the highest prognostic value. Our results warrant further investigations concerning the link between expression data and functional autophagy states and a non-autophagy related role of p62 in NSCLC. Legal entity responsible for the study: Kantonale Ethikkomission Bern Funding: Bernische Krebsliga Disclosure: All authors have declared no conflicts of interest.

Cell Death and Disease, 2014

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human.... more Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O 6-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor jB (NF-jB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-jB is in part owing to downregulation of negative regulators of NF-jB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-jB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-jB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFa-and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-jB confirming that miR-125b is implicated in NF-jB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.

Cells, 2021

Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by ... more Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by the formation of double-membrane vesicles called autophagosomes. During this process, autophagosomes engulf and deliver their intracellular content to lysosomes, where they are degraded by hydrolytic enzymes. Thereby, autophagy provides energy and building blocks to maintain cellular homeostasis and represents a dynamic recycling mechanism. Importantly, the clearance of damaged organelles and aggregated molecules by autophagy in normal cells contributes to cancer prevention. Therefore, the dysfunction of autophagy has a major impact on the cell fate and can contribute to tumorigenesis. Breast cancer is the most common cancer in women and has the highest mortality rate among all cancers in women worldwide. Breast cancer patients often have a good short-term prognosis, but long-term survivors often experience aggressive recurrence. This phenomenon might be explained by the high heterogenei...

Cell Death and Disease, 2015

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance ... more While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-x L leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of antitumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

Molecular Cancer Research, 2009

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that ... more The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53 −/− hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses Q2 .

Blood, 2012

The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On t... more The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of...

Swiss Medical Weekly, 2010

Cancers

Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health... more Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health burden. Uncovering proteins and pathways required for tumor cell growth is necessary for the development of novel targeted therapies. Ajuba is a member of the LIM domain family of proteins whose expression is positively associated with numerous cancers. Our data shows that Ajuba is highly expressed in human colon cancer tissue and cell lines. Publicly available data from The Cancer Genome Atlas shows a negative correlation between survival and Ajuba expression in patients with colon cancer. To investigate its function, we transduced SW480 human colon cancer cells, with lentiviral constructs to knockdown or overexpress Ajuba protein. The transcriptome of the modified cell lines was analyzed by RNA sequencing. Among the pathways enriched in the differentially expressed genes, were cell proliferation, migration and differentiation. We confirmed our sequencing data with biological assays; c...

Cells

Tremendous efforts have been made these last decades to increase our knowledge of intracellular d... more Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy...

Blood

DAPK2 is a Ca++/CAM-regulated S/T kinase which when ectopically over-expressed in 293 cells, can ... more DAPK2 is a Ca++/CAM-regulated S/T kinase which when ectopically over-expressed in 293 cells, can induce membrane blebbing and apoptosis. In contrast to DAPK1 (its tumor-suppressor orthologue), DAPK2’s expression is tissue- restricted, and presently is shown to be markedly up-regulated in developing primary erythroblasts. To assess DAPK2 function in erythroid progenitor cells, wild-type and kinase-inactive K52A forms were expressed in UT7epo cells, and an siRNA lentivirus also was developed. In this Epo-dependent human erythroid progenitor cell model, wt-DAPK2 did not significantly induce apoptosis. Sensitivity to cell death due to exposure to cisplatin, however, was markedly potentiated, and altered cell morphologies were observed. These effects depended upon DAPK2’s kinase activity, and were not mediated by DAPK2-K52A. Furthermore, siRNA-mediated knock-down of DAPK2 proved to protect UT7epo erythroid progenitor cells from apoptosis due to cytokine-withdrawal. Knock-downs were confi...

Nature Communications, 2017

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the produ... more Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.

Molecular cancer therapeutics, Jan 20, 2017

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine... more Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNETs) is an unmet medical need. The anti-angiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. Additionally, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chlor...

Cell Death & Disease

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phospho... more The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity....

EMBO Molecular Medicine, 2021

KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically target... more KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically targeting KRAS-driven tumors remain elusive. Here, we show that combined treatment with fibroblast growth factor receptor 1 (FGFR1) and polo-like kinase 1 (PLK1) inhibitors evoke synergistic cytotoxicity in KRAS-mutant tumor models in vitro and in vivo. Pharmacological and genetic suppression of FGFR1 and PLK1 synergizes to enhance antiproliferative effects and cell death in KRAS-mutant lung and pancreatic but not colon nor KRAS wild-type cancer cells. Mechanistically, co-targeting FGFR1 and PLK1 upregulates reactive oxygen species (ROS), leading to oxidative stress-activated c-Jun Nterminal kinase (JNK)/p38 pathway and E2F1-induced apoptosis. We further delineate that autophagy protects from PLK1/FGFR1 inhibitor cytotoxicity and that antagonizing the compensation mechanism by clinically approved chloroquine fully realizes the therapeutic potential of PLK1 and FGFR1 targeting therapy, producing potent and durable responses in KRAS-mutant patientderived xenografts and a genetically engineered mouse model of Kras-induced lung adenocarcinoma. These results suggest a previously unappreciated role for FGFR1 and PLK1 in the surveillance of metabolic stress and demonstrate a synergistic drug combination for treating KRAS-mutant cancer.

Cells, 2021

In recent years autophagy has attracted the attention of researchers from many medical fields, in... more In recent years autophagy has attracted the attention of researchers from many medical fields, including cancer research, and certain anti-macroautophagy drugs in combination with cytotoxic or targeted therapies have entered clinical trials. In the present study, we focused on a less explored subtype of autophagy, i.e., chaperone-mediated autophagy (CMA), with the key proteins LAMP2A and HSPA8 (HSC70), and their immunohistochemical evaluation with previously extensively validated antibodies. We were interested in whether the marker expression is influenced by the antecedent therapy, and its correlation with survival on a cohort of patients with non-small cell lung cancer (NSCLC) after neoadjuvant therapy and matched primary resected tumors. In concordance with our previous study, we did not find any intratumoral heterogeneity, nor correlation between the two parameters, nor correlation between the markers and any included pathological parameters. Surprisingly, the expression of both...

Leukemia, 2021

Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules... more Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules. However, in recent studies, they have begun to emerge as crucial regulators of cell homeostasis. They are at the crossroads of catabolic and anabolic pathways and are intricately involved in cellular trafficking, nutrient signaling, energy metabolism, and immune regulation. Their involvement in such essential cellular functions has renewed clinical interest in targeting the lysosome as a novel way to treat disease, particularly cancer. Acute myeloid leukemia (AML) is an aggressive blood cancer with a low survival probability, particularly in older patients. The genomic landscape of AML has been extensively characterized but few targeted therapies (with the exception of differentiation therapy) can achieve a long-term cure. Therefore, there is an unmet need for less intensive and more tolerable therapeutic interventions. In this review, we will give an overview on the myriad of function...

Clinical Epigenetics, 2018

Background: In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and assoc... more Background: In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration. Methods: CDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation. Results: Sixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988-0.997)) as well as BRAF V600E , mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2-to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2. Conclusion: CDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/ protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.

Cancer Research, 2016

A global understanding of miRNA function in EGFR signaling pathways may provide insights into imp... more A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRAS G12V and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non-small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRAS G12V-transduced BEAS-2B cells, introduction of anti-miR-29b constructs increased the sensitivity to apoptosis, arguing that miR-29b mediated apoptotic resistance conferred by mutant KRAS. Mechanistic investigations traced this effect to the ability of miR-29b to target TNFAIP3/A20, a negative regulator of NF-kB signaling. Accordingly, overexpression of an miR-29b-refractory isoform of TNFAIP3 restored NF-kB and extrinsic apoptosis, confirming that TNFAIP3 is a functionally relevant target of miR-29b. We also noted that miR-29b could confer sensitivity to intrinsic apoptosis triggered by exposure to cisplatin, a drug used widely in lung cancer treatment. Thus, miR-29b expression may tilt cells from extrinsic to intrinsic mechanisms of apoptosis. Overall, our results reveal a complexity in cancer for miR-29b, which can act as either an oncogene or tumor suppressor gene depending on signaling context. Cancer Res; 76(14); 4160-9. Ó2016 AACR.

Journal of Thoracic Oncology, 2016

Background: In cancer, autophagy may promote tumor cell survival or cell death. Protein light cha... more Background: In cancer, autophagy may promote tumor cell survival or cell death. Protein light chain 3B (LC3B) and p62 are proteins associated with autophagosomal membranes that engulf cytoplasmic content during autophagy, which is subsequently degraded. We studied the expression of these two markers in stage I/II non-small cell lung cancer (NSCLC). Methods: Immunohistochemistry for LC3B and p62 was performed on a tissue microarray with 466 NSCLC: 202 Adenocarcinomas (AC), 220 squamous cell carcinomas (SCC), 35 large cell carcinomas. Dot-like cytoplasmic expression of LC3B and dot-like, cytoplasmic and nuclear staining for p62 was determined. Results were compared with clinical and pathological parameters. Results: LC3B expression correlated with all p62 staining patterns, as those correlated among each other (x 2 < 0.001 each). Low LC3B was more frequent in SCC (x 2 = 0.017). Low cytoplasmic/nuclear p62 was more frequent in AC (x 2 = 0.029/ x 2 < 0.001). There was no correlation with stage, age or gender. Regarding dot-like staining only, a combination of high LC3B/high p62 showed a trend to better outcome (p = 0.11). This marker combination has been suggested to reflect activated impaired autophagy. All other dot-like staining pattern combinations were similarly unfavorable. Interestingly, the best prognostic value was observed for low cytoplasmic/low nuclear p62 expression (regardless of dot-like staining). This implied a significantly better outcome in univariate analysis (p = 0.004) and in multivariate analysis (p = 0.006; HR = 1.96) in addition to tumor stage (p = 0.005; HR = 1.4). Conclusions: The autophagy markers LC3B and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role in these tumors. High LC3B levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior. Interestingly, p62 cytoplasmic/nuclear expression-which may not be linked to autophagy such as the dot-like pattern-had the highest prognostic value. Our results warrant further investigations concerning the link between expression data and functional autophagy states and a non-autophagy related role of p62 in NSCLC. Legal entity responsible for the study: Kantonale Ethikkomission Bern Funding: Bernische Krebsliga Disclosure: All authors have declared no conflicts of interest.

Cell Death and Disease, 2014

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human.... more Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O 6-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor jB (NF-jB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-jB is in part owing to downregulation of negative regulators of NF-jB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-jB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-jB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFa-and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-jB confirming that miR-125b is implicated in NF-jB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy.

Cells, 2021

Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by ... more Macroautophagy (herein referred to as autophagy) is a complex catabolic process characterized by the formation of double-membrane vesicles called autophagosomes. During this process, autophagosomes engulf and deliver their intracellular content to lysosomes, where they are degraded by hydrolytic enzymes. Thereby, autophagy provides energy and building blocks to maintain cellular homeostasis and represents a dynamic recycling mechanism. Importantly, the clearance of damaged organelles and aggregated molecules by autophagy in normal cells contributes to cancer prevention. Therefore, the dysfunction of autophagy has a major impact on the cell fate and can contribute to tumorigenesis. Breast cancer is the most common cancer in women and has the highest mortality rate among all cancers in women worldwide. Breast cancer patients often have a good short-term prognosis, but long-term survivors often experience aggressive recurrence. This phenomenon might be explained by the high heterogenei...

Cell Death and Disease, 2015

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance ... more While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-x L leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of antitumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

Molecular Cancer Research, 2009

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that ... more The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53 −/− hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses Q2 .

Blood, 2012

The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On t... more The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of...

Swiss Medical Weekly, 2010

Cancers

Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health... more Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health burden. Uncovering proteins and pathways required for tumor cell growth is necessary for the development of novel targeted therapies. Ajuba is a member of the LIM domain family of proteins whose expression is positively associated with numerous cancers. Our data shows that Ajuba is highly expressed in human colon cancer tissue and cell lines. Publicly available data from The Cancer Genome Atlas shows a negative correlation between survival and Ajuba expression in patients with colon cancer. To investigate its function, we transduced SW480 human colon cancer cells, with lentiviral constructs to knockdown or overexpress Ajuba protein. The transcriptome of the modified cell lines was analyzed by RNA sequencing. Among the pathways enriched in the differentially expressed genes, were cell proliferation, migration and differentiation. We confirmed our sequencing data with biological assays; c...

Cells

Tremendous efforts have been made these last decades to increase our knowledge of intracellular d... more Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy...

Blood

DAPK2 is a Ca++/CAM-regulated S/T kinase which when ectopically over-expressed in 293 cells, can ... more DAPK2 is a Ca++/CAM-regulated S/T kinase which when ectopically over-expressed in 293 cells, can induce membrane blebbing and apoptosis. In contrast to DAPK1 (its tumor-suppressor orthologue), DAPK2’s expression is tissue- restricted, and presently is shown to be markedly up-regulated in developing primary erythroblasts. To assess DAPK2 function in erythroid progenitor cells, wild-type and kinase-inactive K52A forms were expressed in UT7epo cells, and an siRNA lentivirus also was developed. In this Epo-dependent human erythroid progenitor cell model, wt-DAPK2 did not significantly induce apoptosis. Sensitivity to cell death due to exposure to cisplatin, however, was markedly potentiated, and altered cell morphologies were observed. These effects depended upon DAPK2’s kinase activity, and were not mediated by DAPK2-K52A. Furthermore, siRNA-mediated knock-down of DAPK2 proved to protect UT7epo erythroid progenitor cells from apoptosis due to cytokine-withdrawal. Knock-downs were confi...