Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. This study tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. Following 48 h exposure, we found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20-66% after NSL-YHJ-2-27 (5 μM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins.
KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) constitute a group of cancer therapy agents designed to specifically disrupt and suppress hyperactive G protein signaling, such as that triggered by RAS mutations. Here we determine the effects of PCAIs on the viability, G-proteins levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutant, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 µM, respectively. To determine the effect of the PCAIs on the levels of Mitogen-Activated Pathway Kinase (MAPK)-related enzymes, western blot analysis was performed. After 48 hours exposure to 5 μM of the compounds, 57 to 150% increases of BRAF, ERK1/2, MEK1/2, and p90RSK phosphorylation along with a 60 to 78% decrease of pCRAF were observed, indicating significant disruptions in RAS signaling. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed 52 and 42% reduction in cell migration after 24 h exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46, respectively. Western blot analysis was used to determine the effect PCAIs on caspase activation necessary for the initiation of apoptosis. After treatment with 5 μM of NSL-YHJ-2-27, full-length inactive caspase 3 and 7 levels dropped by 72 and 60%, while cleaved active caspase 3 and 7 levels increased by 274 and 130%, respectively. Treatment with 5 μM of NSL-YHJ-2-46 depleted full-length caspase 7 by 53% and increased cleaved active caspase 3 levels by 83%. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway, perhaps through the activation of proapoptotic isoforms of p90RSK. These results support the potential use of the PCAIs to serve as targeted therapies in cancers harboring RAS mutations. Citation Format: Matthew D. Gregory, Pablo E. Puente, Nadine L. Belony, Jassy Mary S. Lazarte, Nada Tawfeeq, Yong Huang, Ite A. Offringa, Nazarius S. Lamango. Treatment of a Black American lung cancer cell line harboring KRAS mutations with polyisoprenylated cysteinyl amide inhibitors reveals effects on the MAP kinase signaling pathway, cell migration and apoptosis [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C022.
Lung cancer is one of the top five deadliest cancers, and Black Americans have a higher mortality rate when compared to White Americans. Lung adenocarcinoma (LUAD) is the most common histological lung cancer subtype in all population groups including Blacks. Unfortunately, there are no in vitro model systems to study lung cancer and its treatments for Black subjects. Herein we aim to utilize three-dimensional (3D) printing to develop an in vitro three-dimensional (3D) alveolar model using a lung adenocarcinoma cell line from a Black patient (NCI-H23). The developed model is further utilized to study the therapeutic effects of potential LUAD therapeutic agents, polyisoprenylated cysteinyl amide inhibitors (PCAIs), which target and disrupt the RAS-mediated signaling pathway. KRAS is the most commonly mutated oncogene in LUAD in Blacks, and is mutated in NCI-H23 cells. For comparison, hAEC-FT-C7 cells, which are immortalized human alveolar epithelial cells and do not carry the KRAS mutation, will also be used separately to print the alveolar models and test the effects of PCAIs. The experimental results will be further compared to those using two-dimensional (2D) models to better understand the therapeutic effects of PCAIs on Black LUAD. The resulting knowledge will help design better treatments for ethnically/racially diverse patients in whom the RAS signaling pathway is frequently altered. Citation Format: Nadine L. Belony, Bing Ren, Phuc Pham, Matthew Gregory, Pablo E. Puente, Nazarius S. Lamango, Ite A. Offringa, Yong Huang. Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of Black American patients using 3D-printed alveolar models [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C024.
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