Radiotherapy and Oncology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Original article
GABARAPL1 is required for increased EGFR membrane expression during hypoxia Tom G. Keulers a,1, Marco B.E. Schaaf a,1, Hanneke J.M. Peeters a, Kim G.M. Savelkouls a, Marc A. Vooijs a, Johan Bussink b, Barry Jutten a, Kasper M.A. Rouschop a,⇑ a Maastricht Radiation Oncology (MaastRO) lab, GROW – School for Oncology and Developmental Biology, Maastricht University; and b Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, The Netherlands
a r t i c l e
i n f o
Article history: Received 17 April 2015 Received in revised form 16 June 2015 Accepted 17 June 2015 Available online xxxx Keywords: EGFR GABARAPL1 Autophagy Hypoxia UPR Receptor trafficking
a b s t r a c t 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 GABAA 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. Ó 2015 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2015) xxx–xxx
Solid tumors are characterized by the presence of abnormal and immature vasculature that leads to extreme homogeneities in oxygenation. Oxygenation in these regions varies from normal (approx. 5% O2) to severe hypoxia and even anoxia [1,2]. Tumor cells within hypoxic regions contribute to increased invasion [3], metastatic spread [4] and therapy resistance [5]. For survival, cancer cells adapt to hypoxia by activation of several signaling pathways including the hypoxia inducible factor (HIF) pathway and the unfolded protein response (UPR). During normoxic conditions, the HIF-1a protein is ubiquitinated and rapidly degraded. HIF-1a is stabilized during hypoxia and modifies the microenvironment by upregulating expression of several proteins, for example, through increasing vascular endothelial growth factor (VEGF) expression to induce angiogenesis [6]. Other HIF-1 transcriptional targets are carbonic anhydrase 9 (CA9) and Glucose Transporter 1 (GLUT1). These proteins are respectively ⇑ Corresponding author at: Maastricht University Medical Center+, Universiteitssingel 50, Room 3.318, 6200MD Maastricht, The Netherlands. E-mail address: [email protected] (K.M.A. Rouschop). 1 Contributed equally.
involved in deacidification and increased glucose uptake during hypoxia [7]. The UPR is activated during hypoxia as a consequence of increased misfolded or unfolded proteins [8]. The UPR constitutes of three parallel signaling cascades initiated by PERK (PKR-like ER kinase), IRE1 (inositol-requiring protein 1) and ATF6 (activating transcription factor 6). PERK is a kinase that phosphorylates eIF2a to inhibit translation initiation. The blockade in overall protein translation allows selective translation of the transcription factor, ATF4 [9], and subsequently CHOP [10]. The UPR is important for inhibition of mRNA translation [11], promote ER homeostasis [12], increasing the cell’s antioxidant capacity [13] and induce expression of several autophagy-related genes [14] [15] during hypoxia. Epidermal growth factor receptor (EGFR) signaling is increased in many cancer types due to overexpression, amplification or mutation. Activation of the receptor leads to a cascade of signaling pathways that control growth, migration and differentiation and acquisition of an EMT-like phenotype [16]. EGFR is upregulated during hypoxia [17] and combined targeting of EGFR and hypoxia modification is expected to increase tumor responsiveness to radiotherapy [18].
http://dx.doi.org/10.1016/j.radonc.2015.06.023 0167-8140/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Keulers TG et al. GABARAPL1 is required for increased EGFR membrane expression during hypoxia. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.06.023
2
GABARAPL1 mediates EGFR expression
After production, membrane proteins, including EGFR, require active transportation to the plasma membrane. GABARAPL1 is a member of the GABARAP protein family, which consists of 3 members, GABARAP, GABARAPL1 and gate-16/GABARAPL2. Although their function is largely unknown, they are implicated in several vesicular mechanisms as autophagy and receptor trafficking. For example, GABARAPL1 promotes cell surface expression of the Kappa Opioid Receptor (KOR) by facilitating its trafficking along the secretory pathway. Accordingly, knockdown of GABARAPL1 decreases the surface expression of KOR [19]. Binding of GABARAPL1 to the KOR is based on hydrophobic interactions, and the N-terminal region of GABARAPL1 is important for binding to tubulin and surface expression of the KOR [20]. In addition, the tubulin binding region is important for GABAA receptor clustering [21]. We observed that EGFR membrane expression and GABARAPL1 production are increased during hypoxia. We therefore hypothesized that GABARAPl1 mediates increased EGFR membrane-expression.
Scientific) that allows maintaining a stable O2 concentration over time due to continuous gas influx. Immunoblot Cells were lysed and processed as described previously [11]. Proteins were probed with antibodies against GABARAPL1 (Proteintech group, 11010-1-AP), EGFR (SC03, Santa Cruz), eIF4E, p-ERK (Cell signaling) and Beta-Actin (MP Biomedicals). Bound antibodies were visualized using HRP-linked antibodies (Cell Signaling). Membrane isolation Cells were exposed to anoxia (O2 < 0.02%), trypsinized and resuspended in hypotonic swelling buffer (10 mM Tris, 1 mM b-mercaptoethanol, 100 lm EDTA and HEPES, pH 7.3) for 1 h on ice. Cell ghosts were ultracentrifuged at 145,000g for 30 min and analyzed by western blot. Clinical study
Material and methods Cell culture Colorectal adenocarcinoma (HT29, HCT116), mammary adenocarcinoma (MCF7), glioblastoma astrocytoma (U87, U373) cells and large cell lung cancer (H460) were maintained as described by ATCC. Doxycycline-inducible (1 lg/ml) shRNA targeting GABARAPL1 [TTACCTTACTTCATACTTGCCC] or scrambled control [CGAGGGCGACTTAACCTTAGG] was achieved through lentiviral pTripZ (Thermo Scientific) expression as described previously [14]. Knockdown of UPR components (siRNAs) and HIF1a (shRNA expression) was done as described previously [15]. Hypoxia exposure was done using a MACS VA500 workstation (Don Whitley
GABARAPL1 mRNA expression was determined by quantitative PCR in 86 head and neck cancer patients [22]) and used to median-dichotomize the patient cohort. Differences between patient characteristics (Suppl. Table 1) were assessed with a v2-test or student’s t-test. Differences in recurrence-free survival were determined using Kaplan–Meier survival analysis and Log-Rank (Mantel-Cox) test. Immunofluorescence Cells were fixed with 4% paraformaldehyde, permeabilized, blocked and probed with anti-GABARAPL1 (Aviva Systems Biology, ARP55398_P050) and anti-EGFR (Oregon Green-labeled
Fig. 1. Increased GABARAPL1 expression is PERK/ATF4 dependent. (A) Transcriptional regulation of GABARAPL1 in HT29, MCF7, Du145 and U87 cancer cells during anoxia (O2 < 0.02%). (B) HIF1a knockdown (shHIF1a) in HT29 and U373 cells does not affect GABARAPL1 mRNA expression. (C) Transcriptional upregulation of GABARAPL1 during hypoxia is dependent on PERK/ATF4 signaling. A–C, n = 3, mean ± SEM.
Please cite this article in press as: Keulers TG et al. GABARAPL1 is required for increased EGFR membrane expression during hypoxia. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.06.023
T.G. Keulers et al. / Radiotherapy and Oncology xxx (2015) xxx–xxx
Cetuximab). Anti-GABARAPL1 was visualized anti-rabbit Alexa-555 (Invitrogen, A-21428).
using
goat
3
using a repeated measures ANOVA with a Bonferroni post hoc test. P-values
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Original article
GABARAPL1 is required for increased EGFR membrane expression during hypoxia Tom G. Keulers a,1, Marco B.E. Schaaf a,1, Hanneke J.M. Peeters a, Kim G.M. Savelkouls a, Marc A. Vooijs a, Johan Bussink b, Barry Jutten a, Kasper M.A. Rouschop a,⇑ a Maastricht Radiation Oncology (MaastRO) lab, GROW – School for Oncology and Developmental Biology, Maastricht University; and b Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, The Netherlands
a r t i c l e
i n f o
Article history: Received 17 April 2015 Received in revised form 16 June 2015 Accepted 17 June 2015 Available online xxxx Keywords: EGFR GABARAPL1 Autophagy Hypoxia UPR Receptor trafficking
a b s t r a c t 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 GABAA 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. Ó 2015 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2015) xxx–xxx
Solid tumors are characterized by the presence of abnormal and immature vasculature that leads to extreme homogeneities in oxygenation. Oxygenation in these regions varies from normal (approx. 5% O2) to severe hypoxia and even anoxia [1,2]. Tumor cells within hypoxic regions contribute to increased invasion [3], metastatic spread [4] and therapy resistance [5]. For survival, cancer cells adapt to hypoxia by activation of several signaling pathways including the hypoxia inducible factor (HIF) pathway and the unfolded protein response (UPR). During normoxic conditions, the HIF-1a protein is ubiquitinated and rapidly degraded. HIF-1a is stabilized during hypoxia and modifies the microenvironment by upregulating expression of several proteins, for example, through increasing vascular endothelial growth factor (VEGF) expression to induce angiogenesis [6]. Other HIF-1 transcriptional targets are carbonic anhydrase 9 (CA9) and Glucose Transporter 1 (GLUT1). These proteins are respectively ⇑ Corresponding author at: Maastricht University Medical Center+, Universiteitssingel 50, Room 3.318, 6200MD Maastricht, The Netherlands. E-mail address: [email protected] (K.M.A. Rouschop). 1 Contributed equally.
involved in deacidification and increased glucose uptake during hypoxia [7]. The UPR is activated during hypoxia as a consequence of increased misfolded or unfolded proteins [8]. The UPR constitutes of three parallel signaling cascades initiated by PERK (PKR-like ER kinase), IRE1 (inositol-requiring protein 1) and ATF6 (activating transcription factor 6). PERK is a kinase that phosphorylates eIF2a to inhibit translation initiation. The blockade in overall protein translation allows selective translation of the transcription factor, ATF4 [9], and subsequently CHOP [10]. The UPR is important for inhibition of mRNA translation [11], promote ER homeostasis [12], increasing the cell’s antioxidant capacity [13] and induce expression of several autophagy-related genes [14] [15] during hypoxia. Epidermal growth factor receptor (EGFR) signaling is increased in many cancer types due to overexpression, amplification or mutation. Activation of the receptor leads to a cascade of signaling pathways that control growth, migration and differentiation and acquisition of an EMT-like phenotype [16]. EGFR is upregulated during hypoxia [17] and combined targeting of EGFR and hypoxia modification is expected to increase tumor responsiveness to radiotherapy [18].
http://dx.doi.org/10.1016/j.radonc.2015.06.023 0167-8140/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Keulers TG et al. GABARAPL1 is required for increased EGFR membrane expression during hypoxia. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.06.023
2
GABARAPL1 mediates EGFR expression
After production, membrane proteins, including EGFR, require active transportation to the plasma membrane. GABARAPL1 is a member of the GABARAP protein family, which consists of 3 members, GABARAP, GABARAPL1 and gate-16/GABARAPL2. Although their function is largely unknown, they are implicated in several vesicular mechanisms as autophagy and receptor trafficking. For example, GABARAPL1 promotes cell surface expression of the Kappa Opioid Receptor (KOR) by facilitating its trafficking along the secretory pathway. Accordingly, knockdown of GABARAPL1 decreases the surface expression of KOR [19]. Binding of GABARAPL1 to the KOR is based on hydrophobic interactions, and the N-terminal region of GABARAPL1 is important for binding to tubulin and surface expression of the KOR [20]. In addition, the tubulin binding region is important for GABAA receptor clustering [21]. We observed that EGFR membrane expression and GABARAPL1 production are increased during hypoxia. We therefore hypothesized that GABARAPl1 mediates increased EGFR membrane-expression.
Scientific) that allows maintaining a stable O2 concentration over time due to continuous gas influx. Immunoblot Cells were lysed and processed as described previously [11]. Proteins were probed with antibodies against GABARAPL1 (Proteintech group, 11010-1-AP), EGFR (SC03, Santa Cruz), eIF4E, p-ERK (Cell signaling) and Beta-Actin (MP Biomedicals). Bound antibodies were visualized using HRP-linked antibodies (Cell Signaling). Membrane isolation Cells were exposed to anoxia (O2 < 0.02%), trypsinized and resuspended in hypotonic swelling buffer (10 mM Tris, 1 mM b-mercaptoethanol, 100 lm EDTA and HEPES, pH 7.3) for 1 h on ice. Cell ghosts were ultracentrifuged at 145,000g for 30 min and analyzed by western blot. Clinical study
Material and methods Cell culture Colorectal adenocarcinoma (HT29, HCT116), mammary adenocarcinoma (MCF7), glioblastoma astrocytoma (U87, U373) cells and large cell lung cancer (H460) were maintained as described by ATCC. Doxycycline-inducible (1 lg/ml) shRNA targeting GABARAPL1 [TTACCTTACTTCATACTTGCCC] or scrambled control [CGAGGGCGACTTAACCTTAGG] was achieved through lentiviral pTripZ (Thermo Scientific) expression as described previously [14]. Knockdown of UPR components (siRNAs) and HIF1a (shRNA expression) was done as described previously [15]. Hypoxia exposure was done using a MACS VA500 workstation (Don Whitley
GABARAPL1 mRNA expression was determined by quantitative PCR in 86 head and neck cancer patients [22]) and used to median-dichotomize the patient cohort. Differences between patient characteristics (Suppl. Table 1) were assessed with a v2-test or student’s t-test. Differences in recurrence-free survival were determined using Kaplan–Meier survival analysis and Log-Rank (Mantel-Cox) test. Immunofluorescence Cells were fixed with 4% paraformaldehyde, permeabilized, blocked and probed with anti-GABARAPL1 (Aviva Systems Biology, ARP55398_P050) and anti-EGFR (Oregon Green-labeled
Fig. 1. Increased GABARAPL1 expression is PERK/ATF4 dependent. (A) Transcriptional regulation of GABARAPL1 in HT29, MCF7, Du145 and U87 cancer cells during anoxia (O2 < 0.02%). (B) HIF1a knockdown (shHIF1a) in HT29 and U373 cells does not affect GABARAPL1 mRNA expression. (C) Transcriptional upregulation of GABARAPL1 during hypoxia is dependent on PERK/ATF4 signaling. A–C, n = 3, mean ± SEM.
Please cite this article in press as: Keulers TG et al. GABARAPL1 is required for increased EGFR membrane expression during hypoxia. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.06.023
T.G. Keulers et al. / Radiotherapy and Oncology xxx (2015) xxx–xxx
Cetuximab). Anti-GABARAPL1 was visualized anti-rabbit Alexa-555 (Invitrogen, A-21428).
using
goat
3
using a repeated measures ANOVA with a Bonferroni post hoc test. P-values
