Analysis of Gene Expression Heterogeneity Reveals Therapeutic Targets and Novel Regulators of Metastasis

Tumor cell heterogeneity has been implicated in metastatic progression of solid tumors such as triple-negative breast cancer (TNBC), leading to resistance and recurrence. We hypothesize that genes with low cell-to-cell transcriptional variability may be effective therapeutic targets, and that analysis of variability may facilitate identification of new metastatic regulators. Here we demonstrate, using single cell RNA sequencing, that the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) reduced overall transcriptional variability in TNBC xenograft tumors. Focusing on genes with reduced variability in response to RKIP, we identified targetable gene sets such as oxidative phosphorylation and showed that metformin could inhibit RKIP-expressing but not control tumor growth. We also found many regulators of cancer progression including a novel epigenetic metastasis suppressor, KMT5C. These studies demonstrate that a metastatic regulator can alter transcriptional variability in tumors and reveal the importance of genes involved in heterogeneity as potential therapeutic targets and regulators of metastatic progression in cancer. Overall design: RKIP vs CTRL mouse study: 1 × 10^6 BM1-VC or BM1-RKIP cells were injected orthotopically into the mammary fat pad of athymic nude mice (Harlan Envigo). The mice were sacrificed three weeks after tumor cell implantation. Primary tumors were collected for single-cell isolation and single-cell RNA-seq. Three replicate experiments were performed totalling three RKIP tumors and three CTRL tumors. For BM1 KMT5C primary tumor growth experiments: 1 × 10^6 BM1-VC or BM1-KMT5C cells were injected orthotopically into the mammary fat pad of athymic nude mice (Charles River). The mice were euthanized four weeks post tumor cell implantation. Primary tumors from five KMT5C mice and five CTRL mice were collected for RNA sequencing.