Krüppel-like factor 9 (KLF9) links hormone dysregulation and circadian disruption to breast cancer pathogenesis

Circadian disruption is an emerging driver of breast cancer (BCa), with epidemiological studies linking shift work and chronic jet lag to increased BCa risk. Indeed, several clock genes participate in the gating of mitotic entry, regulation of DNA damage response, and epithelial-to-mesenchymal transition, thus impacting BCa etiology. Dysregulated estrogen (17β-estradiol, E2) and glucocorticoid (GC) signaling prevalent in BCa may further contribute to clock desynchrony by directly regulating the expression and cycling dynamics of genes comprising the local breast oscillator. In this study, we investigated the tumor suppressor gene, Krüppel-like factor 9 (KLF9), as an important point of crosstalk between hormone signaling and the circadian molecular network, and further examine its functional role in BCa.

Through meta-analysis of publicly available RNA- and ChIP-sequencing datasets from BCa tumor samples and cell lines, and gene expression analysis by RT-qPCR and enhancer- reporter assays, we elucidated the molecular mechanism behind the clock and hormone regulation of KLF9. Lentiviral knockdown and overexpression of KLF9 in three distinct breast epithelial cell lines (MCF10A, MCF7 and MDA-MB-231) was generated to demonstrate the role of KLF9 in orthogonal assays on breast epithelial survival, proliferation, apoptosis, and migration.

We determined that KLF9 is a direct GC receptor target in mammary epithelial cells, and that induction is likely mediated through coordinate transcriptional activation from multiple GC-responsive enhancers in the KLF9 locus. More interestingly, rhythmic expression of KLF9 in MCF10A cells was abolished in the highly aggressive MDA-MB-231 line. In turn, forced expression of KLF9 altered the baseline and GC/E2-responsive expression of several clock genes, indicating that KLF9 may function as a regulator of the core clock machinery. Characterization of the role of KLF9 using complementary cancer hallmark assays in the context of the hormone-circadian axis revealed that KLF9 plays a tumor-suppressive role in BCa regardless of molecular subtype. KLF9 potentiated the anti-tumorigenic effects of GC in E2 receptor + luminal MCF7 cells, while it restrained GC-enhanced oncogenicity in triple-negative MCF10A and MDA-MB-231 cells.

Taken together, our findings support that dysregulation of KLF9 expression and oscillation in BCa impinges on circadian network dynamics, thus ultimately affecting the BCa oncogenic landscape.