Abstract 3003: Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling
Authors: Merati Aran , Nair Sindhu , Rajbhandari Rajani , Johnson Nicholas , Bredel Markus 
Published: March 2024

Abstract
Background: Glioblastoma (GBM) is one of the most lethal forms of cancer. The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which upon activation, are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling resulting in hyperactive RTK signaling. We demonstrated that the alternative splicing of the tumor suppressor Annexin A7 (ANXA7) supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. Exon six of ANXA7 can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2).
Methods: We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates first in U251 malignant glioma followed by stem-like brain tumor initiating cells (BTICs) originating from patient-derived xenografts (PDXs). U251s and primary BTICs (JX14) were transduced with lentivirus to overexpress ANXA7 and GFP for enrichment by flow cytometry. JX14 empty vector (EV; I2 only) and I1 (I2 and I1 expressing) cells were plated onto Geltrex and ligand starved overnight in serum-free media to maintain BTICs. We assessed EGFR protein levels following EGF stimulation in a time dependent manner. Immunofluorescence experiments were conducted to measure EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway, which included the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking.
Results: Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P&gt;0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (Rab11: R2=0.9267, P&lt;0.0001). Immunoblotting revealed a significant reduction in EGFR protein in I1 cells only. Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P&lt;0.0001). ANXA7 siRNA in EV cells did not alter EGFR trafficking, which implicates I2 as nonfunctional. On the other hand, ANXA7 KD in I1 cells impaired EGFR sorting via a 40% increase (P&lt;0.0010) in total EGFR protein and a 40% reduction (P&lt;0.0001) in colocalization with EEA1. Loss of I1 also shifted the degradation of EGFR back towards recycling.
Conclusion: GBMs express I2, which supports EGFR hyperactivity by favoring receptor recycling. I1 overexpression significantly reduced EGFR signaling by promoting degradation. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for BTICs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition.
Citation Format: Aran Merati, Sindhu Nair, Rajani Rajbhandari, Nicholas Johnson, Markus Bredel. Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3003.