Triple-negative breast cancer (TNBC) is a subtype of breast cancer with an aggressive phenotype which shows high metastatic capability and poor prognosis. The aggressive behavior of TNBC may involve aberrant EGFR expression and activation of its downstream PI3K/Akt/mTOR and RAF/ERK signaling which has received considerable attention as druggable targets in recent times. Currently, no targeted therapy has been approved for treating TNBC. In this study, we report for the first time that novel estrone analogs with modified cucurbitacin pharmacophores exhibited cytotoxic killing towards TNBC cells through multiple mechanisms, inhibition of cell proliferation and induction of mitochondrial apoptosis. Molecular docking studies were carried out with OpenEye software. The MTT cell viability assay was used to perform cytotoxicity studies. Apoptosis and cell cycle arrest were carried out by microscopy,  annexin v assay and flow cytometry. In-cell western assay was used to detect the protein expression levels. We demonstrated that MMA307 and MMA321 showed strong potency towards the EGFR kinase domain (pdb code: 1M17) <i>in silico</i> and exhibited cation-π and hydrogen bonding interactions towards key amino acid residues within the ATP binding site. MMA307 was over tenfold more cytotoxic than sorafenib-reference drug when dosed to MDA-MB-468 cells and MMA321 was almost twenty-fold more potent than sorafenib. Moreover, condensed nuclei with fragmented chromatin, phosphatidylserine flip and modulated expression of proteins within the intrinsic apoptosis pathway (Apaf1, cytochrome c, caspases 3 and 9) confirms mitochondrial apoptosis and suggest MMA307 and MMA321 induced apoptosis via caspase-dependent pathways. Also, MMA307 and MMA321 downregulated cyclin D1 expression levels contributing to G<sub>1</sub> phase cell cycle arrest. Furthermore, both compounds inhibited the expression of EGFR and activated EGFR (Y1068); Activated proteins within the RAF/ERK and AKT/mTOR pathways were downregulated upon MMA307 and MMA321 treatment. Particularly, pARaf, pERK1/2, pAKT, pmTOR and p70S6Kα were all suppressed. Taken together, we report for the first time that estrone analogs with modified cucurbitacin pharmacophores may be an effective therapy for TNBC, which urgently needs novel treatment options. Further studies are needed to develop these novel candidates as targeted agents for TNBC.

Novel cucurbitacin hybrid analogs exhibit cytotoxic effects against triple negative breast cancers (TNBC)

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.