Therapeutic targets and novel anticancer agents for endocrine cancers

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

1. Identification of novel synergistic drug combination therapy for ACC 1.1 Status: Published [J Exp Clin Cancer Res 2022]. The synergy of MELK and CDK inhibitors targeting multiple clinically-relevant molecules in ACC. Clinical trial development is ongoing. 1.2 The novel synergistic combination of PI3K and HSP90 inhibitors in ACC Aim: To identify and validate the effective novel synergistic drug combinations using PI3K inhibitor and Heat-shock protein 90 inhibitors and to study the molecular mechanisms of synergy identified by the computerized drug combination matrix screening in ACC cells. We analyzed publicly available databases and found that ACC overexpressed several isoforms of HSP90 and its clients involved in ACC initiation and progression. We validated the synergy in two ACC cell lines in a cell proliferation assay. ACC 3D tumor spheroid assays showed increased efficacy in the combination treatment group as compared to the single-drug groups. Because PIK75 is not yet available for a clinical trial, we identified and validated the synergy between BGT226 and STA9090 or with HSP990 in monolayer culture. The combination of BGT226 and one of the HSP90 inhibitors was more effective in ACC 3D tumor spheroids, clonogenic assays, and invasion/migration assays than single-drug groups at clinically achievable concentrations. We discovered that the synergistic mechanisms of cell death were completely different between PIK75 and BGT226. PIK75 combination induced G2M cell cycle arrest, followed by caspase-3/7 dependent apoptosis. However, the combination of BGT226 and STA9090 did not cause cell cycle arrest nor it induced apoptosis. We performed RNA-seq and compare gene expression profiles of ACC cells treated with these combinations and found that BGT226 combination induced autophagy-related genes and pathways, which were validated at a protein level. We are currently determining whether the induction of autophagy is the mechanism of cell death or it is the resistance mechanism responding to treatments. The in vivo efficacy in ACC xenografts is in progress. 2. The identification of radiosensitizer and oxidative stress response in ACC. Aim: To evaluate the effects and mechanism of action of auranofin in combination with radiotherapy (RT) in ACC, Summary: Most patients with ACC develop locoregional recurrence after surgery. We identified auranofin from a quantitative high-throughput drug screening in two ACC cell lines as a novel radiosensitizer with a potent cytotoxic effect. Auranofin is an FDA-approved gold particle-based drug with low toxicity for rheumatoid arthritis. The analyses of the independent databases of ACC samples showed the mRNAs of several genes involved in oxidative stress response including the TXNRD pathway, targetable by auranofin, are differentially expressed in ACC.TXNRD is the pro-survival reducing enzyme of TXN. In addition, patients with low TXNRD1 mRNA expression in ACC had shorter overall survival and disease-free survival (OS and DFS). We showed downregulation of several anti-oxidative stress-related genes was inversely correlated with overexpression of MKi67 and CCNB1 (poor prognostic markers). We confirmed the synergistic activity of auranofin and RTin monolayer culture. The combination treatment of auranofin and RT was more effective than single treatments in 3D ACC spheroids and clonogenic assays in two cell lines at clinically achievable concentrations. After pilot studies to optimize the dosing scheme, the in vivo experiment to test the efficacy will start soon. We confirmed that the effective concentrations inhibited thioredoxin reductase activity in ACC. We confirmed that the combination treatments induced a higher level of gH2AX, activated p-CHK1, and induction of p-BRCA1 in both cell lines consistent with double-strand DNA break and repair, respectively. We observed that NCI-H295R (cortisol-producing cells with activating CTNNB1 mutation) was more sensitive to RT and auranofin than SW-13 (SMARCA4 mutations, non-steroid producing cell line). While we observed cleaved-PARP in NCI-H295R treated with the combination, we did not observe cleaved-caspase or cleaved PARP in SW13 treated with auranofin and RT. Auranofin and RT induced p21 and p27 in SW-13 cells, but not NCI-H295R. To further explore the mechanism of action in both cell lines, we found that SW-13 effectively overexpressed NRF2 and KEAP1 nuclear translocation as well as TXNRD1 and HMOX1 as compared to that of NCI-H295R, suggesting SW-13 had a more robust anti-oxidative stress response. Preliminary data suggest that the combination treatment caused mitotic catastrophe in NCI-H295R but not in SW-13. Because strong induction of HMOX1 and a reduction in GPX4, we assessed ferroptosis. We confirmed in both cell lines that auranofin and RT induced glutathione-oxidative stress, which was rescued with N-acetylcysteine. However, we did not observe an increased lipid peroxidation, a hallmark of ferroptosis, when NCI-H295R cells were treated with auranofin and RT but we confirmed that the combination treatment induced ferroptosis in SW-13. We excluded the role of corticosteroids in radiosensitivity in ACC using various treatments. The bulk RNA-sequencing to understand the genes and pathways involved in treatment response is in progress. 3. Mechanism of TNF-a-related changes in tumor microenvironment (TME) to enhance drug delivery Status: manuscript preparation is in-progress. Aim: to elucidate the mechanism of action of TNF-a in reducing tumor interstitial pressure leading to improved drug delivery efficiency. I. Summary: Because the effects of TNF-a on the expression and functions of several intratumoral extracellular matrix proteins and cytokines and the roles of these proteins and cytokines on intratumoral vascular permeability are not fully understood. Using ECM, human matrix metalloproteinase (MMP) arrays, and gene expression profiling of iodine-resistant thyroid cancer cells treated with TNF-a, we identified and validated that TNF-a down-regulated TGFb and LOX, and upregulated MMPs, TIMP1, and ITGB3 which have roles in vascular permeability in TME. siLOX-treated thyroid cancer xenograft recapitulated the intratumoral vascular leakage seen in xenografts similar to that caused by TNF-a. Next, we validated the effects of TNF-a on vascular endothelial cells and found that TNF-a treatment downregulated TGFb1, Lox, and VEGF-R (a key regulator of vascular permeability). To confirm the efficacy of the treatments using recombinant TNF-a and TGFb inhibition, we combined the TME treatments with paclitaxel in two iodine-resistant poorly differentiated (PDTC) (TPC1) and anaplastic thyroid cancer (ATC) (8505C) 3D tumor spheroids and found that the anti-tumor efficacy was higher in the combination treatment groups than single-treatment groups. Furthermore, we demonstrated the higher intratumoral penetration of fluorescent-labeled paclitaxel when thyroid cancer spheroids were treated with TGFb inhibitors and LOX inhibition (siLOX and BAPN which inhibits LOX enzymatic activity) and the intratumoral paclitaxel concentration was severalfold higher than the paclitaxel-only group. We validated the enhanced intratumoral paclitaxel delivery in vivo using HPLC by treating 8505C xenografts with nanogold particles carrying TNF-a and TGFb inhibitor. To investigate the role of TGFb and LOX in the increased tumor interstitial fluid pressure, we identified the correlation between these and the synthesis of hyaluronan (hyaluronan synthase 1 (HAS1) in TCGA thyroid cancer database and validated this finding in vitro by treating the thyroid cancer cells with TGFb inhibitor and siLOX. Similar improvements in drug delivery efficiency and treatment efficacy can be seen in much denser co-cultured thyroid cancer spheroids with cancer-associated fibroblasts.