To determine direct DDR binding partners and to further conn
To determine direct DDR1-binding partners and to further connect to Akt and mTOR signaling, we performed mass spectrometry on DDR1 immunoprecipitates and found various candidates of the Akt and mTOR pathway such as LARP1 (La-related protein 1) and RPS6 (40S ribosomal protein S6/ S6). Most intriguingly, we also identified 14-3-3 theta to be bound to DDR1 and dephosphorylated upon DDR1 inhibition. 14-3-3 proteins are adaptor molecules that function in a variety of cellular processes by mediating protein-protein interactions (Zhao et al., 2011). Binding of 14-3-3 proteins to target proteins via serine and threonine motifs is thought to affect protein localization, conformation, stability, and activity (Zhao et al., 2011). As 14-3-3 proteins are involved in numerous signaling pathways that control diverse physiological and pathological processes, dysregulation of 14-3-3-to-target interactions has been show to fundamentally contribute to human diseases, including cancer (Zhao et al., 2011). In cancer cells, 14-3-3 was shown to regulate autophagy via binding to Beclin-1 and Akt (Wang et al., 2012). We consequently hypothesized that 14-3-3 connects DDR1 to the Akt and mTOR pathway. Indeed, our investigations exhibit a 14-3-3-Beclin-1-Akt1 complex bound to DDR1. Importantly, DDR1 contains the 14-3-3-binding motif R-X-X-S in its intracellular juxtamembrane and kinase domain. FRET analysis and exogenous expression of variants with mutations in the putative 14-3-3-binding sites proved direct binding and dependence of DDR1-regulated Akt and mTOR signaling on complex assembly.
As 14-3-3 theta was reported to regulate autophagy by modifying Akt phosphorylation and DDR1 inhibition failed to induce apoptosis, we reasoned that autophagy was regulated through the uncovered DDR1 and 14-3-3-Beclin-1-Akt1 complex. In clear 5-Azacytidine to the current paradigm of autophagy counteracting anti-cancer treatment, we observed the induction of a cytotoxic variant of autophagy, termed autophagic cell death, upon DDR1 inhibition (Fulda and Kögel, 2015, Galluzzi et al., 2018). This type of cell death is caspase independent and can be blocked by the pharmacological or genetic inhibition of autophagy, while apoptosis and necroptosis remain unaffected. Moreover, autophagy does not overlap with apoptosis or necroptosis genetically (Galluzzi et al., 2018). In GBM, the induction of autophagic cell death by TMZ, dasatinib, or the Bcl-2/Bcl-xl inhibitor (−)-Gossypol was found to cause caspase-independent cell death (Kanzawa et al., 2004, Milano et al., 2009, Voss et al., 2010). These findings are in line with the failure of autophagy-inhibiting agents like chloroquine or hydroxychloroquine in clinical trials when applied as monotherapy or in combination with irradiation and TMZ (Rangwala et al., 2014, Rosenfeld et al., 2014). Hence, new strategies to allow autophagy to occur for therapy sensitization are required. Accumulating evidence from preclinical and clinical studies is pointing to enforced overactivation of autophagy as an effective approach for autophagy-induced cell death (Galluzzi et al., 2017). Such an approach might be highly potent to augment the effectiveness of radiochemotherapy (Dou et al., 2016, Yang et al., 2015), similarly to the strategy we present here.
Acknowledgments We are grateful to Katrin Lamzus (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing GS-5 and GS-8 cells. The authors thank Inga Lange and Liane Stolz-Kieslich for excellent technical assistance. This work was supported by the Wilhelm Sander Foundation (2012.149.1 to N.C.), the EFRE Europäische Fonds für regionale Entwicklung, Europa fördert Sachsen (100066308 to N.C.), and the RKA Förderpool (Fl_35_2016 to J.-W.B.).
Introduction Esophageal squamous cell carcinoma (ESCC) is one of the most common types of cancer in East Asia and is a leading cause of cancer-related death worldwide.1, 2 Surgery is widely used for treatment in localized disease, where stolons can provide definitive management and potential cure. Treatment of metastatic disease is more problematic, and recent advances in multimodal therapy in the neoadjuvant setting have only modestly improved outcomes. Moreover, patients diagnosed with localized disease frequently develop distant metastases, which are typically refractory to systemic chemotherapy.