NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 521.

HIF1A signaling selectively supports proliferation of breast cancer in the brain.

Blood-borne metastasis to the brain is a major complication of breast cancer, but cellular pathways that enable cancer cells to selectively grow in the brain microenvironment are poorly understood. We find that cultured circulating tumor cells (CTCs), derived from blood samples of women with advanced breast cancer and directly inoculated into the mouse frontal lobe, exhibit striking differences in proliferative potential in the brain. Derivative cell lines generated by serial intracranial injections acquire selectively increased proliferative competency in the brain, with reduced orthotopic tumor growth. Increased Hypoxia Inducible Factor 1A (HIF1A)-associated signaling correlates with enhanced proliferation in the brain, and shRNA-mediated suppression of HIF1A or drug inhibition of HIF-associated glycolytic pathways selectively impairs brain tumor growth while minimally impacting mammary tumor growth. In clinical specimens, brain metastases have elevated HIF1A protein expression, compared with matched primary breast tumors, and in patients with brain metastases, hypoxic signaling within CTCs predicts decreased overall survival. The selective activation of hypoxic signaling by metastatic breast cancer in the brain may have therapeutic implications.

Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.

Blood-based monitoring identifies acquired and targetable driver HER2 mutations in endocrine-resistant metastatic breast cancer.

Plasma genotyping identifies potentially actionable mutations at variable mutant allele frequencies, often admixed with multiple subclonal variants, highlighting the need for their clinical and functional validation. We prospectively monitored plasma genotypes in 143 women with endocrine-resistant metastatic breast cancer (MBC), identifying multiple novel mutations including HER2 mutations (8.4%), albeit at different frequencies highlighting clinical heterogeneity. To evaluate functional significance, we established ex vivo culture from circulating tumor cells (CTCs) from a patient with HER2-mutant MBC, which revealed resistance to multiple targeted therapies including endocrine and CDK 4/6 inhibitors, but high sensitivity to neratinib (IC50: 0.018 µM). Immunoblotting analysis of the HER2-mutant CTC culture line revealed high levels of HER2 expression at baseline were suppressed by neratinib, which also abrogated downstream signaling, highlighting oncogenic dependency with HER2 mutation. Furthermore, treatment of an index patient with HER2-mutant MBC with the irreversible HER2 inhibitor neratinib resulted in significant clinical response, with complete molecular resolution of two distinct clonal HER2 mutations, with persistence of other passenger subclones, confirming HER2 alteration as a driver mutation. Thus, driver HER2 mutant alleles that emerge during blood-based monitoring of endocrine-resistant MBC confer novel therapeutic vulnerability, and ex vivo expansion of viable CTCs from the blood circulation may broadly complement plasma-based mutational analysis in MBC.