Divergent iron-regulatory states contribute to heterogeneity in breast cancer aggressiveness.

Primary tumors with similar mutational profiles can progress to vastly different outcomes where transcriptional state, rather than mutational profile, predicts prognosis. A key challenge is to understand how distinct tumor cell states are induced and maintained. In triple negative breast cancer cells, invasive behaviors and aggressive transcriptional signatures linked to poor patient prognosis can emerge in response to contact with collagen type I. Herein, collagen-induced migration heterogeneity within a TNBC cell line was leveraged to identify transcriptional programs associated with invasive versus non-invasive phenotypes and implicate molecular switches. Phenotype-guided sequencing revealed that invasive cells upregulate iron uptake and utilization machinery, anapleurotic TCA cycle genes, actin polymerization promoters, and a distinct signature of Rho GTPase activity and contractility regulating genes. The non-invasive cell state is characterized by actin and iron sequestration modules along with glycolysis gene expression. These unique tumor cell states are evident in patient tumors and predict divergent outcomes for TNBC patients. Glucose tracing confirmed that non-invasive cells are more glycolytic than invasive cells, and functional studies in cell lines and PDO models demonstrated a causal relationship between phenotype and metabolic state. Mechanistically, the OXPHOS dependent invasive state resulted from transient HO-1 upregulation triggered by contact with dense collagen that reduced heme levels and mitochondrial chelatable iron levels. This induced expression of low cytoplasmic iron response genes regulated by ACO1/IRP1. Knockdown or inhibition of HO-1, ACO1/IRP1, MRCK, or OXPHOS abrogated invasion. These findings support an emerging theory that heme and iron flux serve as important regulators of TNBC aggressiveness.