Acetylated KLF5 as a therapeutic target mediating the anti-metastatic effects of doxycycline and methacycline in prostate cancer bone metastasis

Abstract

Bone metastasis is a common and lethal complication of breast, prostate, and lung cancers. TGF-β in the bone microenvironment is pivotal for metastatic progression. We previously demonstrated that TGF-β induces acetylation of KLF5 at K369 and that acetylated KLF5 (Ac-KLF5) drives epithelial-mesenchymal transition, invasiveness, and bone metastasis, positioning Ac-KLF5 and its effectors as therapeutic targets. Tetracycline derivatives, including doxycycline, show anti-metastatic activity, yet their molecular targets remain unidentified, limiting their clinical repurposing. Therapeutic development targeting Ac-KLF5 and elucidating targets of tetracycline derivatives are important areas of research in cancer therapy. Surface plasmon resonance (SPR) screening of the FDA-approved drug library (1987 compounds) targeting purified KLF5 K369Q (KQ, Ac-KLF5 mimic) protein identified candidates that bound to KQ, and the binding selectivities were verified by SPR and circular dichroism with wild-type KLF5 and acetylation-deficient KLF5 K369R (KR) as controls. We assessed the anti-invasive activities of verified candidates in KQ-expressing prostate cancer cells using spheroid invasion and Transwell assays. In vivo therapeutic efficacy against bone metastasis was evaluated in tibial injection xenograft models using bioluminescence imaging, micro-CT, and histology. Mechanistic integration of RNA-seq and ChIP-seq, validated by qPCR, siRNA, and rescue experiments, elucidated downstream effectors and pathways. Doxycycline (D) and methacycline (M), but not their parent compound tetracycline (T), selectively bound to the KQ protein, with no binding to the KR acetylation-deficient KLF5 mutant. D and M, but not T, inhibited KQ-induced cell invasion, osteoclast differentiation, and osteolytic bone metastasis. RNA-seq and ChIP-seq analyses identified 22 genes whose transcription was regulated not only by KQ via direct promoter binding but also by D and M treatments. One of the genes, PGK1 , was further confirmed to be responsible for D and M’s suppressive effects in cell invasion, osteolytic differentiation, and bone metastasis. Doxycycline and methacycline are potential therapeutic agents for TGF-β-induced bone metastasis through selectively binding to Ac-KLF5.