Tom Keulers - Academia.edu (original) (raw)

Papers by Tom Keulers

Research paper thumbnail of Tumors Responsive to Autophagy-Inhibition: Identification and Biomarkers

Cancers, Aug 31, 2020

Although the principle of personalized medicine has been the focus of attention, many cancer ther... more Although the principle of personalized medicine has been the focus of attention, many cancer therapies are still based on a one-size-fits-all approach. The same holds true for targeting cancer cell survival mechanism that allows cancer cells to recycle their constituents (autophagy). In the past several indicators of elevated dependence of cancer cells on autophagy have been described. Addition of autophagy-inhibiting agents could be beneficial in treatment of these tumors. The biomarkers and mechanisms that lead to elevated dependence on autophagy are reviewed in the current manuscript.

Research paper thumbnail of PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS

Proceedings of the National Academy of Sciences of the United States of America, Mar 7, 2013

Research paper thumbnail of Autophagy-Dependent Secretion: Contribution to Tumor Progression

Frontiers in Oncology, Nov 25, 2016

Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular hom... more Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review, we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e., the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumor microenvironment (TME) and tumor progression. The autophagy-mediated secreted factors may stimulate cellular proliferation via auto-and paracrine signaling. The autophagy-mediated release of immune modulating proteins changes the immunosuppresive TME and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking, or alterations in homeostasis and/or autonomous cell signaling.

Research paper thumbnail of Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers

Cancers, Jan 29, 2019

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after trea... more Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio-and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis-and autophagy signalling cascades are listed.

Research paper thumbnail of EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival

Radiotherapy and Oncology, Sep 1, 2013

The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various hum... more The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.

Research paper thumbnail of ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer

Autophagy, Dec 14, 2021

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, r... more Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

Research paper thumbnail of Leukocyte Bim deficiency does not impact atherogenesis in ldlr (-/-) mice, despite a pronounced induction of autoimmune inflammation

Scientific reports, Jun 8, 2017

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and ac... more Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim (-/-) or wild type bone marrow transplanted ldlr (-/-) mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim (-/-) transplanted mice displayed splenomegaly and overt lymphocytosis. CD4(+) and CD8(+) T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim (-/-) mice. Bim (-/-) mice displayed massive intraplaque acc...

Research paper thumbnail of EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival

The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various hum... more The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.

Research paper thumbnail of Tumour hypoxia

People interested in the research are advised to contact the author for the final version of the ... more People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement:

Research paper thumbnail of ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer

Autophagy, 2021

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, r... more Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

Research paper thumbnail of Secretion of pro‐angiogenic extracellular vesicles during hypoxia is dependent on the autophagy‐related protein GABARAPL1

Journal of Extracellular Vesicles, 2021

Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, met... more Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro‐angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro‐angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+EVs with GABARAPL1 targeting antibodies results in blockade of pro‐angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

Research paper thumbnail of Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

EMBO reports, 2021

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC),... more The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4 À/À) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4 À/À ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

Research paper thumbnail of Tumors Responsive to Autophagy-Inhibition: Identification and Biomarkers

Cancers, 2020

Recent advances in cancer treatment modalities reveal the limitations of the prevalent “one-size-... more Recent advances in cancer treatment modalities reveal the limitations of the prevalent “one-size-fits-all” therapies and emphasize the necessity to develop personalized approaches. In this perspective, identification of predictive biomarkers and intrinsic vulnerabilities are an important advancement for further therapeutic strategies. Autophagy is an important lysosomal degradation and recycling pathway that provides energy and macromolecular precursors to maintain cellular homeostasis. Although all cells require autophagy, several genetic and/or cellular changes elevate the dependence of cancer cells on autophagy for their survival and indicates that autophagy inhibition in these tumors could provide a favorable addition to current therapies. In this context, we review the current literature on tumor (sub)types with elevated dependence on autophagy for their survival and highlight an exploitable vulnerability. We provide an inventory of microenvironmental factors, genetic alteratio...

Research paper thumbnail of Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers

Cancers, 2019

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after trea... more Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellul...

Research paper thumbnail of EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition

Autophagy, Jan 29, 2018

Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased ce... more Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIIIcells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIIItumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIIIcells. As hypoxic t...

Research paper thumbnail of Autophagy-Dependent Secretion: Contribution to Tumor Progression

Frontiers in Oncology, 2016

Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular hom... more Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review, we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e., the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumor microenvironment (TME) and tumor progression. The autophagy-mediated secreted factors may stimulate cellular proliferation via auto-and paracrine signaling. The autophagy-mediated release of immune modulating proteins changes the immunosuppresive TME and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking, or alterations in homeostasis and/or autonomous cell signaling.

Research paper thumbnail of LC3/GABARAP family proteins: autophagy‐(un)related functions

The FASEB Journal, 2016

From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis th... more From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.

Research paper thumbnail of PERK/eIF2a signaling protects therapy resistant hypoxic cells through induction of glutathione synth

Research paper thumbnail of JCI40027sd

Research paper thumbnail of GABARAPL1 is required for increased EGFR membrane expression during hypoxia

Radiotherapy and Oncology, 2015

Background and purpose: The epidermal growth factor receptor (EGFR) is overexpressed, amplified o... more Background and purpose: The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors and hypoxia is a common feature of solid tumors. Both EGFR and hypoxia are associated with therapy resistance and poor treatment outcome. To survive hypoxia, cells adapt by activation of hypoxia responsive pathways and expression of hypoxia-induced plasma membrane proteins. We observed that GABA A receptor associated protein like1 (GABARAPL1) and plasma membrane expression of EGFR were increased during hypoxia. Here we explored the role of the GABARAPL1 in EGFR membrane expression during hypoxia. Material and methods: Quantitative qPCR, immunoblot analysis, flow cytometry and cytochemistry were used to assess this interplay. Results: GABARAPL1 mRNA and protein levels are increased during hypoxia in vitro and correlate with tumor hypoxia in a panel of primary HNSCC xenografts. High GABARAPL1 mRNA is associated with poor outcome of HNSCC patients. During hypoxia, EGFR membrane expression is increased and GABARAPL1 and EGFR colocalize at the plasma membrane. GABARAPL1 knockdown inhibits EGFR membrane expression during hypoxia. Conclusion: GABARAPL1 is required for increased membrane expression of EGFR during hypoxia, suggesting a role for GABARAPL1 in the trafficking of these membrane proteins.

Research paper thumbnail of Tumors Responsive to Autophagy-Inhibition: Identification and Biomarkers

Cancers, Aug 31, 2020

Although the principle of personalized medicine has been the focus of attention, many cancer ther... more Although the principle of personalized medicine has been the focus of attention, many cancer therapies are still based on a one-size-fits-all approach. The same holds true for targeting cancer cell survival mechanism that allows cancer cells to recycle their constituents (autophagy). In the past several indicators of elevated dependence of cancer cells on autophagy have been described. Addition of autophagy-inhibiting agents could be beneficial in treatment of these tumors. The biomarkers and mechanisms that lead to elevated dependence on autophagy are reviewed in the current manuscript.

Research paper thumbnail of PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS

Proceedings of the National Academy of Sciences of the United States of America, Mar 7, 2013

Research paper thumbnail of Autophagy-Dependent Secretion: Contribution to Tumor Progression

Frontiers in Oncology, Nov 25, 2016

Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular hom... more Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review, we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e., the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumor microenvironment (TME) and tumor progression. The autophagy-mediated secreted factors may stimulate cellular proliferation via auto-and paracrine signaling. The autophagy-mediated release of immune modulating proteins changes the immunosuppresive TME and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking, or alterations in homeostasis and/or autonomous cell signaling.

Research paper thumbnail of Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers

Cancers, Jan 29, 2019

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after trea... more Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio-and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellular vesicles (EV). In this review, the evidence for transfer of a hypoxia tolerance phenotype between tumour cells via EV is discussed. In particular, proteins, mRNA and microRNA enriched in EV, derived from hypoxic cells, that impact HIF-1α-, UPR-, angiogenesis-and autophagy signalling cascades are listed.

Research paper thumbnail of EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival

Radiotherapy and Oncology, Sep 1, 2013

The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various hum... more The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.

Research paper thumbnail of ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer

Autophagy, Dec 14, 2021

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, r... more Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

Research paper thumbnail of Leukocyte Bim deficiency does not impact atherogenesis in ldlr (-/-) mice, despite a pronounced induction of autoimmune inflammation

Scientific reports, Jun 8, 2017

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and ac... more Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim (-/-) or wild type bone marrow transplanted ldlr (-/-) mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim (-/-) transplanted mice displayed splenomegaly and overt lymphocytosis. CD4(+) and CD8(+) T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim (-/-) mice. Bim (-/-) mice displayed massive intraplaque acc...

Research paper thumbnail of EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival

The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various hum... more The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.

Research paper thumbnail of Tumour hypoxia

People interested in the research are advised to contact the author for the final version of the ... more People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement:

Research paper thumbnail of ATG12 deficiency results in intracellular glutamine depletion, abrogation of tumor hypoxia and a favorable prognosis in cancer

Autophagy, 2021

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, r... more Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

Research paper thumbnail of Secretion of pro‐angiogenic extracellular vesicles during hypoxia is dependent on the autophagy‐related protein GABARAPL1

Journal of Extracellular Vesicles, 2021

Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, met... more Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro‐angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro‐angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+EVs with GABARAPL1 targeting antibodies results in blockade of pro‐angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

Research paper thumbnail of Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

EMBO reports, 2021

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC),... more The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4 À/À) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4 À/À ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

Research paper thumbnail of Tumors Responsive to Autophagy-Inhibition: Identification and Biomarkers

Cancers, 2020

Recent advances in cancer treatment modalities reveal the limitations of the prevalent “one-size-... more Recent advances in cancer treatment modalities reveal the limitations of the prevalent “one-size-fits-all” therapies and emphasize the necessity to develop personalized approaches. In this perspective, identification of predictive biomarkers and intrinsic vulnerabilities are an important advancement for further therapeutic strategies. Autophagy is an important lysosomal degradation and recycling pathway that provides energy and macromolecular precursors to maintain cellular homeostasis. Although all cells require autophagy, several genetic and/or cellular changes elevate the dependence of cancer cells on autophagy for their survival and indicates that autophagy inhibition in these tumors could provide a favorable addition to current therapies. In this context, we review the current literature on tumor (sub)types with elevated dependence on autophagy for their survival and highlight an exploitable vulnerability. We provide an inventory of microenvironmental factors, genetic alteratio...

Research paper thumbnail of Extracellular Vesicles as Transmitters of Hypoxia Tolerance in Solid Cancers

Cancers, 2019

Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after trea... more Tumour hypoxia is a common feature of solid tumours that contributes to poor prognosis after treatment. This is mainly due to increased resistance of hypoxic cells to radio- and chemotherapy and the association of hypoxic cells with increased metastasis development. It is therefore not surprising that an increased hypoxic tumour fraction is associated with poor patient survival. The extent of hypoxia within a tumour is influenced by the tolerance of individual tumor cells to hypoxia, a feature that differs considerably between tumors. High numbers of hypoxic cells may, therefore, be a direct consequence of enhanced cellular capability inactivation of hypoxia tolerance mechanisms. These include HIF-1α signaling, the unfolded protein response (UPR) and autophagy to prevent hypoxia-induced cell death. Recent evidence shows hypoxia tolerance can be modulated by distant cells that have experienced episodes of hypoxia and is mediated by the systemic release of factors, such as extracellul...

Research paper thumbnail of EGFRvIII expression triggers a metabolic dependency and therapeutic vulnerability sensitive to autophagy inhibition

Autophagy, Jan 29, 2018

Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased ce... more Expression of EGFRvIII is frequently observed in glioblastoma and is associated with increased cellular proliferation, enhanced tolerance to metabolic stresses, accelerated tumor growth, therapy resistance and poor prognosis. We observed that expression of EGFRvIII elevates the activation of macroautophagy/autophagy during starvation and hypoxia and explored the underlying mechanism and consequence. Autophagy was inhibited (genetically or pharmacologically) and its consequence for tolerance to metabolic stress and its therapeutic potential in (EGFRvIII) glioblastoma was assessed in cellular systems, (patient derived) tumor xenopgrafts and glioblastoma patients. Autophagy inhibition abrogated the enhanced proliferation and survival advantage of EGFRvIIIcells during stress conditions, decreased tumor hypoxia and delayed tumor growth in EGFRvIIItumors. These effects can be attributed to the supporting role of autophagy in meeting the high metabolic demand of EGFRvIIIcells. As hypoxic t...

Research paper thumbnail of Autophagy-Dependent Secretion: Contribution to Tumor Progression

Frontiers in Oncology, 2016

Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular hom... more Autophagy is best known as a lysosomal degradation and recycling pathway to maintain cellular homeostasis. During autophagy, cytoplasmic content is recognized and packed in autophagic vacuoles, or autophagosomes, and targeted for degradation. However, during the last years, it has become evident that the role of autophagy is not restricted to degradation alone but also mediates unconventional forms of secretion. Furthermore, cells with defects in autophagy apparently are able to reroute their cargo, like mitochondria, to the extracellular environment; effects that contribute to an array of pathologies. In this review, we discuss the current knowledge of the physiological roles of autophagy-dependent secretion, i.e., the effect on inflammation and insulin/ hormone secretion. Finally, we focus on the effects of autophagy-dependent secretion on the tumor microenvironment (TME) and tumor progression. The autophagy-mediated secreted factors may stimulate cellular proliferation via auto-and paracrine signaling. The autophagy-mediated release of immune modulating proteins changes the immunosuppresive TME and may promote an invasive phenotype. These effects may be either direct or indirect through facilitating formation of the mobilized vesicle, aid in anterograde trafficking, or alterations in homeostasis and/or autonomous cell signaling.

Research paper thumbnail of LC3/GABARAP family proteins: autophagy‐(un)related functions

The FASEB Journal, 2016

From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis th... more From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.

Research paper thumbnail of PERK/eIF2a signaling protects therapy resistant hypoxic cells through induction of glutathione synth

Research paper thumbnail of JCI40027sd

Research paper thumbnail of GABARAPL1 is required for increased EGFR membrane expression during hypoxia

Radiotherapy and Oncology, 2015

Background and purpose: The epidermal growth factor receptor (EGFR) is overexpressed, amplified o... more Background and purpose: The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors and hypoxia is a common feature of solid tumors. Both EGFR and hypoxia are associated with therapy resistance and poor treatment outcome. To survive hypoxia, cells adapt by activation of hypoxia responsive pathways and expression of hypoxia-induced plasma membrane proteins. We observed that GABA A receptor associated protein like1 (GABARAPL1) and plasma membrane expression of EGFR were increased during hypoxia. Here we explored the role of the GABARAPL1 in EGFR membrane expression during hypoxia. Material and methods: Quantitative qPCR, immunoblot analysis, flow cytometry and cytochemistry were used to assess this interplay. Results: GABARAPL1 mRNA and protein levels are increased during hypoxia in vitro and correlate with tumor hypoxia in a panel of primary HNSCC xenografts. High GABARAPL1 mRNA is associated with poor outcome of HNSCC patients. During hypoxia, EGFR membrane expression is increased and GABARAPL1 and EGFR colocalize at the plasma membrane. GABARAPL1 knockdown inhibits EGFR membrane expression during hypoxia. Conclusion: GABARAPL1 is required for increased membrane expression of EGFR during hypoxia, suggesting a role for GABARAPL1 in the trafficking of these membrane proteins.