Therapies for liver cancer particularly those including radiation are still inadequate. Inhibiting the stress response machinery is an appealing anti-cancer and radiosensitizing therapeutic strategy. Heat-shock-protein-90 (HSP90) is a molecular chaperone that is a prominent effector of the stress response machinery and is overexpressed in liver cancer cells. HSP90 client proteins include critical components of pathways implicated in liver cancer cell survival and radioresistance. The effects of a novel non-geldanamycin HSP90 inhibitor, ganetespib, combined with radiation were examined on 3 liver cancer cell lines, Hep3b, HepG2 and HUH7, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γH2AX foci kinetics and client protein expression in pathways important for liver cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined ganetespib-radiation treatment on tumor cell proliferation in a HepG2 hind-flank tumor graft model. Nanomolar levels of ganetespib alone exhibited liver cancer cell anti-cancer activity in vitro as shown by decreased clonogenic survival that was associated with increased apoptotic cell death, prominent G2-M arrest and marked changes in PI3K/AKT/mTOR and RAS/MAPK client protein activity. Ganetespib caused a supra-additive radiosensitization in all liver cancer cell lines at low nanomolar doses with enhancement ratios between 1.33-1.78. These results were confirmed in vivo, where the ganetespib-radiation combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in HepG2 tumor grafts. Our data suggest that combined ganetespib-radiation therapy exhibits promising activity against liver cancer cells, which should be investigated in clinical studies.
<p>Figure S1 shows basal centrosome amplification in cell lines. Figure S2 shows the effect of GF on cell viability. Figure S3 shows HSET expression in cell lines. Figure S4 shows the effect of GF on clonogenic potential after RT. Figure S5 shows the induction of micronuclei after GF and/or RT. Figure S6 shows the expression of IFN-β after GF and/or RT.</p>
Abstract Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer mortality. Although therapeutic advances have been made, resistance to treatments remain high and the overall survival is still dismal. The high expression of the transcription factor TWIST1 strongly correlates with invasive and metastatic cancers, and is generally attributed to the epithelial-to-mesenchymal transition phenotype. We have demonstrated that TWIST1 can antagonize the induction of fail-safe programs as oncogene (KrasG12D)-induced senescence (OIS) in primary NSCLC tumor. OIS suppression by TWIST1 required increased global O-GlcNAcylation, which perhaps can also impact DNA repair and radiation response. As TWIST1 is essential for development, deciphering the critical domains and downstream transcriptional targets required for pro-tumorigenicity and radioresistance may allow the identification of new therapeutic strategies by targeting TWIST1. We created a transactivation-null TWIST1 mutant, by mutation of phenylalanine 191 to glycine, genetically engineered mouse model (GEMM) utilizing the tetracycline-inducible gene expression system. In these GEMMs, doxycycline treatment allows a concomitant induction of KrasG12D oncogene (R) with TWIST1 (T) or with TWIST1F191G mutant (F) expression, specifically in the lung epithelium directed by CCSP promoter-rtTA (C) transgene. CRT mice presented a more aggressive tumor progression and a shorter survival (median= 15.6 weeks) compared to CR (31 weeks). TWIST1F191G expression in CRF abrogates these effects (26.7 weeks) suggesting that the TWIST1 transactivation domain is required for TWIST1-dependent accelerated tumorigenesis. CRT mice, HBEC, and H460 cells overexpressing TWIST1 showed radiation resistance. A second KrasG12D lung tumor GEMM with induction of TWIST1 prior to 15Gy lung tumor irradiation showed lung tumor stasis compared to regression without TWIST1 expression. Histological analysis showed a strong expression of TWIST1 in CRT lungs while CRF showed a progressive loss over time suggesting that TWIST1F191G was non-functional and conferred no selective advantage. CRT mice also had lung tumors with higher proliferation (by Ki67), reduced apoptosis (by cleaved caspase3) and a decreased cell cycle arrest (by p16) compared to CR lung tumors. In comparison, CRF mice lung tumors did not show any change in cell death but showed increased p16 cell cycle arrest marker suggesting that the transactivation domain of TWIST1 is important for the suppression of OIS. We are exploring similar in vitro phenotypes using a primary immortalized HBEC cell lines co-transfected with HRasG12V oncogene and TWIST1 versus TWIST1 transactivation mutant. In future work, we are investigating the role of the TWIST1 transactivation domain in the induction of O-GlcNAcylation and the stabilization and/or activation of critical targets for OIS suppression and radiation resistance, with the goal of identifying new therapeutic targets and radiosensitizers. Citation Format: Audrey Lafargue, Hailun Wang, Sivarajan T. Chettiar, Rajendra P. Gajula, Caleb Smack, Ismaeel Siddiqui, Kekoa Taparra, Christine Lam, Francesca Carrieri, Katriana Nugent, Natasha Zachara, Phuoc T. Tran. The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-067.
<p>This document contains supplemental details such as antibodies and primers, list of genes overrepresented in TWIST1 and TWIST1 mutants, IHC staining for TWIST1 in mouse prostate development, IHC staining for TWIST1 and HOXA9 in various mouse models of prostate cancer, correlation of TWIST1 and HOXA9 alteration with poor survival in human prostate cancer patients, IHC for TWIST1 and HOXA9 on primary tumors and bone metastasis from prostate cancer patients, IHC for HOXA9 on cell pellets expressing HOXA9 or control and on adult mouse prostate, data showing sufficiency of HOXA9 for some pro-metastatic behaviour in prostate cancer cells, data showing requirement of WDR5 and HOTTIP for TWIST1-dependent pro-metastatic behavior, ChIP data showing binding of TWIST1 and HOXA9 at the HOXA9 promoter, data showing effect of drugs that can inhibit HOXA9 on prostate cancer cell viability and pro metastatic behaviour.</p>
