Invasive lobular carcinoma: Integrated multi-omics analysis reveals silencing of Arginosuccinate synthase and upregulation of nucleotide biosynthesis in tamoxifen resistance

Invasive Lobular Carcinoma (ILC), a distinct subtype of breast cancer is hallmarked by E-Cadherin loss, slow proliferation, and strong hormone receptor positivity. ILC faces significant challenges in clinical management due to advanced stage at diagnosis, late recurrence, and development of resistance to endocrine therapy - a cornerstone of ILC treatment. To elucidate the mechanisms underlying endocrine resistance in ILC, ILC cell lines (MDA-MB-134-VI, SUM44PE) were generated to be resistant to tamoxifen, a selective estrogen receptor modulator. The tamoxifen-resistant (TAMR) cells exhibit a 2-fold increase tamoxifen IC50 relative to parental cells. Metabolomics and RNA-sequencing revealed deregulation of alanine, aspartate, and glutamate metabolism, purine metabolism, and arginine and proline metabolism in TAMR cells. Among the fifteen commonly dysregulated genes in these pathways, low ASS1 expression was identified in the TAMR cells and was significantly correlated with poor outcome in ILC patients, specifically in the context of endocrine therapy. Our study reveals methylation mediated silencing of ASS1 in TAMR cells as a likely mechanism of downregulation. Demethylation restored ASS1 expression and correspondingly reduced tamoxifen IC50 toward parental levels. Nucleic acid biosynthesis is augmented in TAMR cells, evidenced by increase in nucleotide intermediates. Both TAMR cell lines demonstrated increased expression of several nucleic acid biosynthesis enzymes, including PAICS, PRPS1, ADSS2, CAD, and DHODH. Furthermore, CAD, the key multifunctional protein of de novo pyrimidine biosynthesis pathway is differentially activated in TAMR cells. Treating TAMR cell with Decitabine, a demethylating agent, or Farudodstat, a pyrimidine biosynthesis inhibitor, markedly augmented efficacy of tamoxifen. Collectively, our study unveils ASS1 downregulation as a novel mechanism underlying acquired tamoxifen resistance in ILC and establishes a metabolic link between ASS1 and nucleic acid biosynthesis. Restoring ASS1 expression or inhibiting pyrimidine biosynthesis restored tamoxifen sensitivity. ASS1 could be a potential biomarker and therapeutic target in tamoxifen resistant ILC patients, warranting further investigation.