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A Novel Allosteric Inhibitor Targets PLK1 in Triple Negative Breast Cancer Cells.

ABSTRACT: While Polo-like kinase 1 (PLK1) inhibitors have shown promise in clinical settings for treating triple-negative breast cancer tumors and other solid tumors, they are limited by their ability to bind non-selectively to the ATP kinase domain. Therefore, we sought to develop a PLK1 allosteric inhibitor targeting the PLK1 T-loop (a switch responsible for activation) and evaluate its effects in triple-negative breast cancer cells. A novel compound, RK-10, was developed based on an in silico model, and its effects on specificity, viability, migration, and cell cycle regulation in MCF-10A and MDA-MB 231 cells were evaluated. When MDA-MB 231 cells were treated with 0−50 µg/mL RK-10, phospho-PLK1 (Thr-210) was decreased in cells cultured adherently and cells cultured as mammospheres. RK-10 significantly inhibited viability after 24 h; however, by 48 h, 25−50 µM RK-10 caused >50% reduction. RK-10 attenuated wound healing by up to 99.7% and caused S and G2/M cell cycle arrest, which was associated with increased p21 expression. We developed a novel allosteric inhibitor which mediates anti-proliferative and anti-migratory properties through targeting phospho-PLK1 (Thr-210) in mammospheres and causing S phase and G2/M cell cycle arrest. Further development of PLK1 allosteric inhibitors may be a promising approach for TNBC treatment.

SUBMITTER: Patel JR

PROVIDER: S-EPMC9024838 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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A Novel Allosteric Inhibitor Targets PLK1 in Triple Negative Breast Cancer Cells.

Patel Jankiben R JR Thangavelu Prasad P Terrell Renee M RM Israel Bridg'ette B Sarkar Arindam Basu AB Davidson A Michael AM Zhang Kun K Khupse Rahul R Tilghman Syreeta L SL

Biomolecules 20220331 4

While Polo-like kinase 1 (PLK1) inhibitors have shown promise in clinical settings for treating triple-negative breast cancer tumors and other solid tumors, they are limited by their ability to bind non-selectively to the ATP kinase domain. Therefore, we sought to develop a PLK1 allosteric inhibitor targeting the PLK1 T-loop (a switch responsible for activation) and evaluate its effects in triple-negative breast cancer cells. A novel compound, RK-10, was developed based on an in silico model, an ...[more]

PMID: 35454120

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Project description:Breast cancer is the most common cancer in women worldwide. Many breast cancers originate from the cells lining the milk duct and some become invasive. Breast cancer lacking estrogen, progesterone receptors (ER-, PR-) and epidermal growth factor receptor 2 (HER2-) amplification, termed "Triple negative" (TNBC) is reported to frequently affect Black women and younger women. TNBC is an invasive ductal carcinoma with limited treatment options. To this end, we opted to investigate drugs that could be repurposed because they offer advantages in bringing effective treatments faster to the clinic. We chose to study the effect of the drug, dapivirine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV because it could be detected in the breast milk of lactating women when treated for HIV and the drug could potentially target the cancer. Here we show the potent impact of dapivirine on MDA-MB-231 TNBC cells, while NNRTI like nevirapine showed marginal effects. When dapivirine was tested on other breast cell lines, MCF-7 and MCF 10A, the inhibition was at higher concentrations. Molecular studies with dapivirine in TNBC cells, revealed an increase in reactive oxygen species (ROS), apoptotic cells, and activation of caspases. Importantly, protein profiling and STRING analysis revealed deregulation of the key molecule, PCNA and impact on pivotal cell signaling circuits including extracellular matrix (ECM), angiogenesis, cell adhesion, and immunomodulation. Of note, is its potential effect on stem-like cells due to downregulation of the basic transcription factor 3 (BTF3) and proteasome activator complex subunit 3; the latter affecting their dependence on the proteasome pathway. Taken together, dapivirine exhibits the potential to be considered as a repurposed drug for TNBC as monotherapy/combination therapy. Notably, it could also potentially be a treatment for individuals with dual ailments, such as HIV and TNBC, if the clinical outcomes with dapivirine for TNBC become favorable.

Project description:BackgroundDespite advancements in treatment approaches, patients diagnosed with aggressive breast cancer (BC) subtypes typically face an unfavorable prognosis. Globally, these cancers continue to pose a significant threat to women's health, leading to substantial morbidity and mortality. Consequently, there has been a significant struggle to identify viable molecular targets for therapeutic intervention in these patients. Polo-like Kinase-1 (PLK1) represents one of these molecular targets currently undergoing rigorous scrutiny for the treatment of such tumors. Yet, its role in the pathogenesis of BC in Middle Eastern ethnicity remains unexplored.MethodsWe investigated the expression of PLK1 protein in a cohort of more than 1500 Middle Eastern ethnicity BC cases by immunohistochemistry. Association with clinicopathological parameters and prognosis were performed. In vitro studies were conducted using the PLK1 inhibitor volasertib and the PARP inhibitor olaparib, either alone or in combination, in PTC cell lines.ResultsOverexpression of PLK1 was detected in 27.4% of all BC cases, and this was notably correlated with aggressive clinicopathological markers. PLK1 was enriched in the triple-negative breast cancer (TNBC) subtype and exhibited poor overall survival (p = 0.0347). Notably, there was a positive correlation between PLK1 and PARP overexpression, with co-expression of PLK1 and PARP observed in 15.7% of cases and was associated with significantly poorer overall survival (OS) compared to the overexpression of either protein alone (p = 0.0050). In vitro, we studied the effect of PLK1 and PARP inhibitors either single or combined treatments in two BRCA mutated, and one BRCA proficient TNBC cell lines. We showed that combined inhibition significantly reduced cell survival and persuaded apoptosis in TNBC cell lines. Moreover, our findings indicate that inhibition of PLK1 can reinstate sensitivity in PARP inhibitor (PARPi) resistant TNBC cell lines.ConclusionOur results shed light on the role of PLK1 in the pathogenesis and prognosis of Middle Eastern BC and support the potential clinical development of combined inhibition of PLK1 and PARP, a strategy that could potentially broaden the use of PLK1 and PARP inhibitors beyond BC cases lacking BRCA.

Project description:IntroductionOf the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.MethodsTNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.ResultsPanobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.ConclusionsThis study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.

Project description:PurposeTriple-negative breast cancer (TNBC) is an aggressive tumor with poor prognosis, it has higher recurrence and metastatic rates than other breast cancer subtypes. This study aims to investigate biomarkers and potential targets for TNBC related to ferroptosis through data mining and bioinformatics analysis. The findings may provide new insights for treating TNBC.MethodsThe TNBC patients' data from the Cancer Genome Atlas (TCGA) database were extracted for differential expression and prognosis analysis. Consensus genes obtained by intersecting differential expressed and ferroptosis-related genes was used to establish the prognostic model by the univariate and multivariate Cox analyses. Besides, TNBC data from the Gene Expression Omnibus (GEO) database was used to confirm the reliability of the prognosis model. Moreover, clinical information was analyzed by multifactorial independent analysis to identify independent prognostic factors. The expression of genes constituting the prognostic model was further validated using the Human Protein Atlas (HPA) database. Finally, the Comparative Toxicogenomic Data (CTD) database was used to explore possible treatment drugs for TNBC.ResultsWe obtained 13,245 differential expressed genes, and 177 consensus genes. 98 genes with prognostic implication were obtained by univariable Cox. Then, a prognostic model including 12 ferroptosis-related genes was constructed by multivariable Cox. The area under curve (AUC) value of the prognostic model for TNBC was 0.82. The GEO database validated that the model (AUC = 0.77) could predict the patient outcomes. The staining results of 10 out of 12 prognostic model genes in HPA database showed that their expression was consistent with our predictions. Clinical risk analysis indicated that risk score of patients could act as an independent prognostic factor. Finally, six drugs that may have interaction with 12 ferroptosis-related genes were obtained using the CTD database.ConclusionThe prognostic model composed of 12 ferroptosis-related genes could predict the prognosis of TNBC patients, and seven genes (ASNS, LAMP2, CAV1, DPP4, HELLS, TF, ZFP69B) could be potential new therapeutic targets for TNBC, and two drugs (1-methyl-3-isobutylxanthine, rosiglitazone) could act as potential therapeutic drugs for the treatment of TNBC.

Dataset Information

A Novel Allosteric Inhibitor Targets PLK1 in Triple Negative Breast Cancer Cells.

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A Novel Allosteric Inhibitor Targets PLK1 in Triple Negative Breast Cancer Cells.

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