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Abstract
Metastasis of cancer cells is the primary cause of death for cancer patients. Current treatments are ineffective at inhibiting metastasis in solid cancers, such as triple-negative breast cancer (TNBC) because some tumor cells evade therapy and cause recurrence later. Tumor cell heterogeneity has been linked to metastatic progression, drug resistance, and survival of cancer patients. We hypothesize that regulators of metastatic progression may also modulate tumor cell heterogeneity. Using a metastasis suppressor, Raf kinase inhibitory protein (RKIP), and a pro-metastatic transcription factor, BACH1, we compared heterogeneity differences in TNBC tumors at different metastatic states. We performed single-cell RNA sequencing to study gene expression variability. In RKIP-overexpressing cells which were non-metastatic, overall expression variability was reduced compared to the highly metastatic control cells, and analyses of genes with reduced variability revealed therapeutic targets. In addition, we showed that KMT5C, a gene less variable and up-regulated in non-metastatic tumor cells, is a novel metastasis suppressor in TNBC. In BACH1-overexpressing cells, overall expression variability increased compared to the control cells, despite BACH1 itself being less variable, consistent with its pro-metastatic role. BACH1 reduced the variability of genes involved in invasion, metastasis, epithelial-mesenchymal transition, and stem cell pluripotency. However, depletion of BACH1 by shRNA acted as a noise generator, increasing overall transcriptional variability despite inhibition of metastasis. These results indicate that regulators of metastasis can impact gene expression variability, but increased heterogeneity alone is insufficient to drive metastasis. Our findings also show that analyzing both gene expression mean and variability changes between metastatic states can reveal novel anti-metastatic drug targets and regulators of metastasis.