SCAMP3-Driven Regulation of ERK1/2 and Autophagy Phosphoproteomics Signatures in Triple-Negative Breast Cancer

Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors show therapeutic potential in triple-negative breast Cancer (TNBC), but resistance through compensatory signaling limits their efficacy. We previously identified the secretory carrier membrane protein 3 (SCAMP3) as a regulator of TNBC progression and ERK1/2 activation. Here, we investigated the role of SCAMP3 in ERK1/2 signaling and therapeutic response using TMT-based LC-MS/MS phosphoproteomics of wild-type (WT) and SCAMP3 knockout (SC3KO) SUM-149 cells under basal conditions, after epidermal growth factor (EGF) stimulation, and during ERK1/2 inhibition with MK-8353. A total of 4408 phosphosites were quantified, with 1093 significantly changed. SC3KO abolished residual ERK activity under MK-8353 and affected the compensatory activation of oncogenic pathways observed in WT cells. SC3KO reduced the phosphorylation of ERK feedback regulators Raf proto-oncogene serine/threonine-protein kinase Raf-1 (S43) and the dual-specificity mitogen-activated protein kinase kinase 2 (MEK2) (T394), affected Other ERK targets, including nucleoporins, transcription factors, and metabolic Enzymes triosephosphate isomerase (TPI1) (S21) and ATP-citrate lyase (ACLY) (S455). SCAMP3 loss also impaired the mammalian target of rapamycin complex I (mTORC1) signaling and disrupted autophagic flux, evidenced by elevated sequestosome-1 (SQSTM1/p62) and microtubule-associated protein light chain 3 (LC3B-II) with reduced levels of the autophagosome lysosome maturation marker, Rab7A. Beyond ERK substrates, SC3KO affected phosphorylation events mediated by Other kinases. These findings position SCAMP3 as a central coordinator of ERK signaling and Autophagy. Our results support SCAMP3 as a potential therapeutic target to enhance ERK1/2 inhibitor clinical efficacy and overcome adaptive resistance mechanisms in TNBC.

ERK1/2; SCAMP3; autophagy; mTOR; phosphoproteomics; triple negative breast cancer.