Project Summary Lung cancer is the primary cause of cancer-related death worldwide, with lung squamous cell carcinoma (LUSC) being the second most common subtype. Although LUSC has been characterized by several well-defined driver mutations, including amplifications of chromosomes 3q26, targeted therapy approaches have been unsuccessful so far. Copy number gain or amplification of chromosome 3q occurs in more than 80% of LUSC, while progress has been slow in developing therapeutic strategies to target this specific LUSC subtype. We have identified several Golgi genes in this amplicon, including Golgi Integral Membrane Protein 4 (GOLIM4), which encodes a transmembrane Golgi protein that regulates endosome-to-Golgi trafficking, and ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1), which encodes a Golgi-resident Ca2+/Mn2+ pump that regulates Ca2+-dependent protein sorting and secretion. We found that GOLIM4 and ATP2C1 form a complex on the Golgi and play a fundamental role in LUSC growth and metastasis by promoting the secretion of pro-tumorigenic proteins. In addition, ATP2C1 regulates Mn2+ influx into the Golgi lumen, causing GOLIM4 degradation upon Mn2+ exposure. Mn2+ treatment inhibits the growth of chromosome 3q-amplified LUSC cells in vitro and in vivo. In the proposed work, we will explore how GOLIM4 and ATP2C1 cooperatively modulate the Golgi secretory pathway during LUSC progression and test whether GOLIM4 and ATP2C1 co-amplification creates a therapeutic vulnerability in chromosome 3q-amplified LUSC.