Treatment of Triple Negative Breast Cancer‐Derived Cells with Polyisoprenylated Cysteinyl Amide Inhibitors Activates Caspase 3/7 and Disrupts F‐actin Organization leading to Apoptosis and Diminished Cell Motility
Authors: Salako Olufisayo , Poku Rosemary , Nkembo Augustine , Amissah Felix , Ntantie Elizabeth , Lamango Nazarius 
Published: November 2023

PURPOSETriple negative breast cancer (TNBC) is the most aggressive and lethal form of breast cancer. Patients with TNBC do not benefit from currently available targeted therapies since it does not, as the name implies, express the estrogen (ER), progesterone (PR), or human epidermal growth factor type 2 (Her2/Neu) receptors. Human epidermal growth factor receptors (EGFR) are overexpressed in 92% of basal‐like TNBC cases and 60% of TNBC cases overall. However, anti‐EGFR therapies are ineffective possibly due to downstream drivers such mutant K‐Ras. Monomeric G‐proteins that regulate cell proliferation, survival, motility, and apoptosis operate downstream of EGFR and other signal transduction pathways. Polyisoprenylated methylated protein methyl esterase (PMPMEase) metabolizes monomeric G‐proteins, allowing them to function properly. PMPMEase is overexpressed in many cancers and may serve as a biomarker and therapeutic target. We have synthesized polyisoprenylated cysteinyl amide inhibitors that may inhibit PMPMEase and/or disrupt polyisoprenylated protein function. We hypothesize that the PCAIs will promote anti‐tumorigenic effects, inducing apoptosis, while inhibiting cell survival, proliferation, migration, and invasion. The purpose of this study was to test the effectiveness of PCAIs against the cancer biological hallmarks of TNBC.METHODSFour TNBC cell lines were treated with the PCAIs and analyzed for their effects on cell viability, colony survival, F‐actin organization, cell migration, invasion and apoptosis.RESULTSTreatment of TNBC cells with the PCAIs resulted in concentration‐dependent death of the TNBC cells with EC50 values ranging from 2.1 to 2.8 mM for NSL‐BA‐040 and NSL‐BA‐055 in media containing 5% FBS. The PCAIs inhibited 68.3% and 70.2% of MDA‐MB‐231 colony formation with 500 and 1000 cells plated, respectfully at 0.5 mM. AO/EB staining and florescent microscopy revealed chromatin condensation at 1 mM, while annexin V and propidium iodide staining followed by flow cytometry revealed 64.4% of cells in early and late apoptotic stages following 2 μM treatment of NSL‐BA‐040 for 48 h. The Caspatag caspase‐3/7 in situ assay revealed the activation of caspase 3/7 on MDA‐MB‐231 cells at 0.5 mM after 48 h treatment. At 2 mM, NSL‐BA‐040 inhibited the motility of MDA‐MB‐231 cells by 78.6% in the migration assay and completely disrupted F‐actin filament organization in MDA‐MB‐468 cells.CONCLUSIONThe PCAIs’ ability to induce apoptosis in TNBC‐derived cells with mutant K‐Ras while inhibiting cell motility by disrupting F‐actin filaments suggest potential therapeutic use against TNBC through mechanisms that involve inhibition of tumor growth, metastasis and angiogenesis, which are all essential hallmarks for breast cancer progression.Support or Funding InformationThis research is supported by the National Cancer Institute of National Institutes of Health (NIH) under Award Number SC1CA190505 and the National Institute of Minority Health Disparities (NIMHD) of National Institutes of Health (NIH) under award numbers G12MD007582 and 1P20MD006738‐01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.