NOVEL ASPECTS OF EGFR NON - CLATHRIN ENDOCYTOSIS REGULATION: CELL CONTEXT DEPENDENCY AND ROLE OF RNA AND RNA - BINDING PROTEINS.

The epidermal growth factor receptor (EGFR) plays a pivotal role in several physiological processes; however, its aberrant regulation can lead to the development and progression of cancer. Endocytosis is a critical regulator of the EGFR; indeed, its levels of activity and fate strongly depend on the internalization route in addition to its sorting at the endosomal station. We recently discovered a novel endocytic route of the EGFR, called non-clathrin endocytosis (NCE), which is activated only at high concentrations of the ligand EGF and targets the receptor to lysosomal degradation. In contrast, clathrin-mediated endocytosis (CME) of the EGFR is active at all EGF concentrations and recycles the receptor to maintain signaling. Some important aspects of the mechanism and regulation of EGFR-NCE remain to be elucidated: i) proteomics analysis of EGFR-NCE vesicles identified RNA-binding proteins (RBPs) as positive regulators of this endocytic mechanism; however, the mechanism of their involvement in EGFR-NCE and whether RNA species are also involved are unknown; ii) EGFR-NCE is known to be active in some cell lines but not in others, but its relevance in different physiological cellular contexts is unknown. Therefore, the overall goal of this thesis project was to investigate these two aspects of EGFR-NCE. To explore the involvement of RBPs in EGFR-NCE, we utilized the HeLa cervical cancer cell line, which has been extensively studied as regards this endocytic route. We first confirmed that the single knockdown (KD) of two RBPs, hnRNPA1and FUS, in these cells impairs the internalization of an EGFR-NCE specific cargo, CD147. In contrast, no effects of RBP KD were observed on the internalization of the CME specific cargo transferrin (Tf). In addition, using confocal immunofluorescence (IF) microscopy, we observed that upon high dose EGF stimulation, the cytosolic localization of hnRNPA1 is specifically increased and partially colocalizing with EGF, suggesting a possible recruitment of hnRNPA1 to endocytic vesicles. FUS levels are instead not regulated by EGF but a fraction of FUS also localized with EGF. Given the potential involvement of RBPs in EGFR-NCE, we next investigated whether RNA itself, bound or not to RBPs, might also have a role in this endocytic mechanism. We observed that degradation of cytosolic RNAs by RNaseA treatment partially inhibited CD147 internalization but not Tf-CME. Moreover, the cytoplasmic localization of hnRNPA1 was strongly reduced by RNAseA treatment. These data indicate that the observed reduction in CD147 internalization upon RNaseA treatment could be due to the reduction in cytoplasmic hnRNPA1 levels, which in turn depend on one or more RNA species. To identify RNA species potentially involved in EGFR-NCE, we generated an APEX-RTN3 fusion protein, where RTN3 is an ER-resident protein known to be associated with EGFR-NCE vesicles, and performed an APEX-seq experiment. The analysis led to the identification of 26 transcripts whose role in EGFR-NCE is currently being validated using a siRNA approach. Taken together, our data suggest a putative role of RNA in EGFR-NCE, and more specifically the possible involvement of RNA-hnRNPA1 complex. To investigate the relevance of EGFR-NCE in different cellular contexts, we generated isogenic cell models by differentiation of human induced pluripotent stem cells (hiPSCs) into fibroblasts, cardiomyocytes, hepatocytes and keratinocytes. We performed detailed characterizations of the resulting cell populations by analyzing lineage-specific markers expression and performing single-cell RNA-seq analysis. Our results show that hiPSCs derived-fibroblasts, -cardiomyocytes and -keratinocytes exhibited a high level of homogeneity in terms of lineage-specific marker expression, while, -hepatocytes exhibited greater heterogeneity. We then assessed the levels of expression of the EGFR and the transferrin receptor (TfR) in the various lineages, as well as in the parental hiPSCs. We discovered that EGFR expression is dynamically regulated during differentiation, being negligibly expressed in hiPSCs and hiPSC-derived cardiomyocytes, whereas in the other differentiated lineages increased expression levels were observed. In contrast, the transferrin receptor (TfR) is equally expressed in hiPSCs and the various differentiated lineages. We next set up the EGF and Tf internalization assays in hiPSCs and the derived cell lineages. Hepatocytes were found to be unsuitable for these assays and therefore were excluded from further investigations. Our results revealed that EGF internalization occurred in both hiPSC-derived keratinocytes and fibroblasts, while parental hiPSCs and hiPSC-derived cardiomyocytes did not internalize EGF, in agreement with their negligible EGFR expression levels. In contrast, Tf is internalized in parental hiPSCs and hiPSC-derived fibroblasts and cardiomyocytes but not in hiPSC-derived keratinocytes despite its similar expression of the receptor. This finding raises the possibility that the endocytic machinery mediating TfR-CME is not present or active in keratinocytes. Finally, we analyzed the relevance of EGFR-NCE in hiPSC-derived fibroblasts and keratinocytes by monitoring the internalization of CD147. This analysis revealed that in hiPSC-derived fibroblasts, CD147 internalization increases above basal levels only after prolonged EGF stimulation. Moreover, a low colocalization rate was observed between CD147 and EGF after internalization, suggesting that in this cellular context, CD147 is internalized through a mechanism that does not reflect the properties of EGFR-NCE. In contrast, in hiPSC-derived keratinocytes, CD147 internalization is induced after a short EGF stimulation. A high colocalization rate between internalized EGF and CD147 was also observed, suggesting that EGFR-NCE is active in this cellular context. These results provide an initial indication that endocytic routes can be differentially regulated in distinct cellular contexts. To further investigate this possibility, we will analyze the sc-RNAseq data from hiPSCs and the differentiated lineages, to explore whether specific endocytic regulatory proteins are differentially expressed in different cellular contexts. In conclusion, this thesis project has explored previously uncharted territories of endocytic control, shedding light on the roles of RBPs and RNAs as potential novel regulators of this fundamental cellular process. Additionally, it underscores the significance of considering the cellular context in the study of this field. Finally, the isogenic cell lineages generated during this study, will provide an invaluable tool for the continuation of this work as well as other projects in the lab.