Proteomic Profiling of Medullary Thyroid Cancer Identifies CAPN1 as a Key Regulator of NF1 and RET Fueled Growth

Background: Medullary thyroid cancer (MTC) is a frequently metastatic tumor of the thyroid that develops from the malignant transformation of C-cells. These tumors most commonly have activating mutations within the RET or RAS proto-oncogenes. Germline mutations within RET result in C-cell hyperplasia, and cause the MTC pre-disposition disorder, multiple endocrine neoplasia, type 2A (MEN2A). Single-agent therapies for MTC, including vandetanib (VAN) and cabozantinib for all MTCs and selpercatinib (SEL) for RET-mutated MTC, lead to partial responses but are not curative. Methods: To identify new therapeutic targets for MTC, we conducted proteomic profiling of normal C-cells, MTC cells, pre-malignant MEN2A patient samples, and MTC tumors. Results: From this analysis, we identified CAPN1, a member of the CALPAIN (CAPN) family endopeptidases, as widely upregulated in MTC samples. We found that short hairpin RNA-mediated depletion of CAPN1 or inhibitors of CAPN1 significantly reduced MTC cell growth, colony formation, and xenograft tumor growth in vivo. In addition, we show that CAPN1 inhibitors synergize with VAN and SEL in vitro, maximizing apoptosis. Mechanistic experiments implicate CAPN1 in inhibiting neurofibromin, encoded by NF1, and CAPN1 inhibitors stabilize NF1 protein levels and diminish downstream RAS/RET activation of AKT and ERK. Conclusions: Our data suggest that increased CAPN1 levels support RET and RAS-fueled growth by reducing NF1 levels. We find that combinatorial therapies between CAPN1 inhibitors and VAN or SEL show maximal efficacy in MTC cells.