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. 2023 Feb;30(2):267-276.
doi: 10.1038/s41417-022-00541-7. Epub 2022 Oct 17.

Sorting nexin-dependent therapeutic targeting of oncogenic epidermal growth factor receptor

Benjamin Atwell  1 , Cheng-Yu Chen  2 , Matthew Christofferson  1 , William R Montfort  1   2   3   4 , Joyce Schroeder  5   6   7
Affiliations

Sorting nexin-dependent therapeutic targeting of oncogenic epidermal growth factor receptor

Benjamin Atwell et al. Cancer Gene Ther. 2023 Feb.

Abstract

Overexpression and/or overactivation of the Epidermal Growth Factor Receptor (EGFR) is oncogenic in several tumor types yet targeting the kinase domain of wildtype EGFR has had limited success. EGFR has numerous kinase-independent roles, one of which is accomplished through the Sorting Nexin-dependent retrotranslocation of EGFR to the nucleus, which is observed in some metastatic cancers and therapeutically resistant disease. Here, we have utilized the BAR domain of Sorting Nexin 1 to create a peptide-based therapeutic (cSNX1.3) that promotes cell death in EGFR-expressing cancer. We evaluated the efficacy of cSNX1.3 in tumor-bearing WAP-TGFα transgenic mice (an EGFR-dependent model of breast cancer), where cSNX1.3 treatment resulted in significant tumor regression without observable toxicity. Evaluation of remaining tumor tissues found evidence of increased PARP cleavage, suggesting apoptotic tumor cell death. To evaluate the mechanism of action for cSNX1.3, we found that cSNX1.3 binds the C-terminus of the EGFR kinase domain at an interface site opposite the ATP binding domain with a Kd of ~4.0 µM. In vitro analysis found that cSNX1.3 inhibits the nuclear localization of EGFR. To determine specificity, we evaluated cancer cell lines expressing wildtype EGFR (MDA-MB-468, BT20 and A549), mutant EGFR (H1975) and non-transformed lines (CHO and MCF10A). Only transformed lines expressing wildtype EGFR responded to cSNX1.3, while mutant EGFR and normal cells responded better to an EGFR kinase inhibitor. Phenotypically, cSNX1.3 inhibits EGF-, NRG-, and HGF-dependent migration, but not HA-dependent migration. Together, these data indicate that targeting retrotranslocation of EGFR may be a potent therapeutic for RTK-active cancer.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. cSNX1.3 peptide inhibits cell viability in MDA-MB-468 cells.
A Peptide sequence alignment of modified and stabilized peptides (−) indicates conserved residues, (/) indicates a deleted residue. Modified residues used for staples are (S)-2-(((9H-flouren-9-yl) methoxy) caronylamino)-2-methyl-hept-6-enoic acid [5] and (R)-2-(((9H-flouren-9-yl) methoxy) caronylamino)-2-methyl-dec-9-enoic acid [8]. PTD4 = Protein Transduction Domain. Ac = acetylation of 5’ end and NH2 = amidation of 3’ end. B MDA-MB-468 cells were treated with 10 μM of the indicated peptide for 3 days. Cell viability was measured using an MTT assay. Vehicle control represents 100%. C. A predicted peptide structure of SNX1.3 was generated using SWISSMODEL with negative (blue) and positive (red) residues highlighted.
Fig. 2
Fig. 2. cSNX1.3 driven tumor regression in WAP-TGFα transgenic mice.
Mice were bred continuously to induce transgene expression and palpated weekly for tumor formation. Once tumors reached 100 mm3, mice were entered into the study and given 10 µg/g body weight intravenous injections of either cPTD4 or cSNX1.3 3X/week. A Changes in tumor size from entry into study until end of study are shown for each individual tumor, (*) indicate tumors that entered the study at size greater than 500 mm3, all other tumors entered the study at ~100 mm3. B a Kaplan-Meier survival curve was generated showing when mice were sacrificed by either reaching tumor burden (2000 mm3) or the end of the study (p = 0.0002). C Upon sacrifice tumors were harvested and fixed in 10% formalin and embedded in paraffin. FFPE blocks were sectioned at 4 μM and stained with hematoxylin and eosin and imaged at 20x. D Protein lysates were generated from tumors upon the sacrifice and probed for the indicated proteins.
Fig. 3
Fig. 3. cSNX1.3 competitively inhibits binding of EGFR to SNX1.
A Ribbon drawing of EGFR kinase domain (PDB ID 5CNO) and SNX1 Bar domain (PDB ID 4FZS). The proposed SNX1 binding site is well away from the kinase active site. B SDS page gels for purified SNX1 Bar domain (residues 301–522) and EGFR kinase domain (residues 672–998). C MST Binding curves of EGFR kinase domain (50 nM) with fluorescent dye attached through the His-tag titrated against the Bar domain, peptides SNX1.3 (capped), SNX1.3, and control PTD4, as well as control BSA. D Binding of SNX1.3 to the kinase domain was also measured by Bio-Layer Interferometry (Octet BLI), which yielded a similar dissociation constant to that measured by MST.
Fig. 4
Fig. 4. cSNX1.3 peptide inhibits EGFR driven cancer cell viability.
A–G Cells were plated in a 96-well plate at 2000 cells per well. The indicated concentration of cPTD4, cSNX1.3, Sapitinib, or Erlotinib were added to the cells on day 0 and incubated for 3 days. Cell viability was then measured with an MTT assay, viability of treated cells were compared with the vehicle control. H MDA-MB-468 cells were plated and incubated+/− IPTG for 2 days prior to drug treatment to induce expression of an EGFR-targeted shRNA. Cells were then additionally treated with cPTD4 or cSNX1.3 for 3 days, and then viability was measured using an MTT assay.
Fig. 5
Fig. 5. cSNX1.3 inhibits nuclear EGFR and survival signaling.
A MDA-MB-468 cells were serum starved overnight then incubated with 20 ng/mL EGF and either cPTD4 or cSNX1.3 for 2 h. Cells were then fractionated and verified by HSP90 cytosolic protein, Bap31 membrane protein, or HDAC nuclear protein. B BT20 mammospheres were grown for 1 week in the presence of either vehicle, cPTD4, or cSNX1.3 and trypsinized, counted and replated. After the second week, the number of secondary mammospheres were counted.
Fig. 6
Fig. 6. SNX1.3 inhibits RTK driven 2D cell migration.
BT20 triple negative breast cancer cells were treated with either cSNX1.3, PTD4 control or vehicle (water) and either EGF-, Neuregulin-1 (NRG), Hepatic Growth Factor (HGF), or hyaluronic acid (HA)-induced migration on plastic was allowed for 12 h. Area migrated was measured with ImageJ.
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