Lung cancer remains the leading cause of cancer-related deaths in the United States and worldwide. Non-small cell-lung cancer (NSCLC) accounts for 85% of all lung cancer cases. Plant-derived compounds have historically led to some of our most useful cancer drugs (e.g., paclitaxel, vincristine, etc). Celastrol (CEL), a triterpenoid isolated from Tripterygium wilfordii, has attracted significant attention for its strong anticancer properties. CEL demonstrate time- and dose-dependent anti-proliferative activity against H1299 and A549 cells with IC50 0.63 and 1.8 µM, respectively and shows dose dependent inhibition of A549 colony formation. CEL has minimal effect against the normal bronchial epithelium Beas2b. We have also demonstrated that CEL loaded onto bovine milk-derived exosomes (ExoCEL) enhanced therapeutic response vs. the free CEL in tumor xenograft model and lacks systemic and immunotoxicity. Our data indicate that the wound healing and cell migration rate was significantly reduced in the CEL treatment. In fact, the effect was time and dose dependent as analyzed by Wimasis software. CEL is an inhibitor of TAK1 and has shown to suppress basal TGF-β1 and TNF-α induced NF-κB activity, however, the precise mechanisms remain to be identified. Here, we systemically explored the mechanism of CEL by which it inhibits the growth and metastasis of NSCLC using RNAseq analysis. RNAseq followed by differential expression of genes demonstrate a total of over 600 genes modulated by the CEL treatment of which 287 genes were upregulated while 314 genes were downregulated. Gene Ontology analysis, genome analysis along with reactome pathway enrichment analyses indicated that CEL-treated genes are involved in cell cycle, EMT, PI3K-AKT and p53 signaling and pathways related to lung cancer and its metastasis. The molecules of major pathways involved in the RNAseq analysis were analyzed by western blot. Our findings indicated significant decrease in TGFβ-induced migratory behavior of cancer cells and reversal of TGFβ-mediated changes in EMT proteins such as ZO1, Vimentin, β-catenin and claudin by CEL in H1299 lung cancer cells. Cancer cells exhibited up-regulated N-cadherin with loss of E-cadherin called the "cadherin switch" and downregulation of E-cadherin is one of the hallmarks of EMT. We also confirmed that CEL inhibit TGFβ-induced cMyc expression by decreasing PI3K and AKT phosphorylation via TGF-β/non-Smad pathway and show dose- dependent reduction of pAKT, PI3K, cMyc and Smad4 in the absence and presence of TGF-β. In summary, the analysis of biological functions of the most differentially expressed genes revealed possible mechanisms of CEL for its strong activity against lung cancer. Pathways identification provide information about the potential use of CEL to inhibit tumorigenesis, progression, and metastasis of lung cancer. (Supported by Research and Development Office, KSA Grant# RDO-2003).

Citation Format: Farrukh Aqil, Raghuram Kandimalla, Neha Tyagi, Mohd Saeed, Ramesh Gupta. Celastrol targets multiple pathways and suppress cell invasion in lung cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 8.