MODELING EPITHELIAL-MESENCHYMAL TRANSITION-INDUCED ERLOTINIB RESISTANCE IN NON-SMALL CELL LUNG CANCER

Lung cancer is the most common cause of cancer mortality throughout the world with an overall survival rate of around 17%. Adenocarcinoma is the most common histologic subtype of lung cancer, and a majority of human lung adenocarcinomas have somatic mutations in genes encoding members of the EGFR/KRAS/BRAF signaling pathway. Combining this knowledge with animal models of the pathway indicate that this signaling axis is of central importance to lung adenocarcinoma development. Non-small cell lung cancers with EGFR mutations, while initially responding well to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib, develop acquired resistance within 10-16 months. A relatively uncharacterized mechanism of resistance is lung cancer cells that have undergone a morphological change, termed epithelial-mesenchymal transition (EMT). Twist1 is one of the inducers of EMT and we have created a transgenic autochthonous lung cancer mouse model expressing EGFRL858R/Twist1. Using this novel in vivo model, as well as in vitro models, we have shown that overexpression of Twist1 can confer resistance to erlotinib in EGFR mutant lung cancers. Using these novel model systems we are trying to determine the downstream effects of Twist1 overexpression that lead to erlotinib resistance. To complement our work on Twist1-mediated erlotinib resistance, a bioinformatic-chemical screen was performed to identify potential pharmacological inhibitors of Twist1. From that screen, a promising class of agents, harmala alkaloids, was identified. Using human lung cancer cell lines to validate these agents has shown that several of the compounds have growth inhibitory effects. We tested one of the most promising compounds, harmine, in vivo with a transgenic KrasG12D/Twist1 mouse model of autochthonous lung adenocarcinomas. When mice were treated with harmine, tumor growth delay was observed by computed tomography imaging in harmine versus vehicle control mice. We are currently in the process of examining the molecular and cellular mechanisms of action of harmine using novel in vitro and in vivo model systems. These results are promising and provide a foundation for future experiments to determine if harmine, by inhibiting Twist1, can re-sensitize EGFR mutant lung cancers to erlotinib

Structure-function studies of the bHLH phosphorylation domain of TWIST1 in prostate cancer cells

The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice

A PWWP Domain-Containing Protein Targets the NuA3 Acetyltransferase Complex via Histone H3 Lysine 36 trimethylation to Coordinate Transcriptional Elongation at Coding Regions

Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5′-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with identifier PXD001156

Concurrent versus Sequential Sorafenib Therapy in Combination with Radiation for Hepatocellular Carcinoma

<div><p>Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both <i>in vitro</i> and <i>in vivo</i>. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines <i>in vitro</i> by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed <i>in vivo</i> by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. <i>In vitro</i>, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G₁-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. <i>In vivo</i>, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both <i>in vitro</i> and <i>in vivo</i>. These results may have implications for clinical decision-making and prospective trial design.</p></div

Active clinical trials currently registered on ClinicalTrials.gov that involve the administration of sorafenib and radiation for hepatocellular carcinoma (HCC).

<p>If available, the treatment schedule is specified in the right-most column; all sequential schedules consisted of radiotherapy first followed by sorafenib.</p

A sequential radiation-sorafenib regimen is most efficacious against HCC <i>in vivo</i>.

<p>A HepG2 hind-flank xenograft model was utilized to measure the efficacy of (A) 5 different treatment arms: control (sham injection of vehicle control on days 1–5), sorafenib alone (SOR; injection of 6 mg/mL sorafenib on days 1–5), radiation alone (RT; irradiation at a dose of 3 Gy on days 1–3), concurrent radiation-sorafenib (CONC; sorafenib injection on days 1–5 and irradiation at a dose of 3 Gy on days 2–4), and sequential radiation-sorafenib (SEQ; irradiation at a dose of 3 Gy on days 1–3 and sorafenib injection on days 4–8). The number of tumors per arm was: <i>n</i> = 13 for control, <i>n</i> = 12 for SOR, <i>n</i> = 15 for RT, <i>n</i> = 17 for CONC, and <i>n</i> = 19 for SEQ. Data for each arm are plotted as (B) tumor volume ratio over time (<i>left</i>) and as Kaplan-Meier curves with attainment of quadruple the pre-treatment tumor volume as the event of interest (<i>right</i>). Using two methods of statistical analysis (Mann-Whitney U-test for <i>left</i> and log-rank test for <i>right</i>), SEQ was shown to achieve a significantly longer time to quadruple the pre-treatment tumor volume than any of the other treatment arms. The CONC and RT arms were not significantly different from one another. (C–F) Immunofluorescence staining from xenografts harvested from all treatment arms show significantly more downregulation of vascularity (CD31) (C – <i>left column</i>, D) and decreased mitotic index (Ki-67) (C – <i>right column</i>, E) in arms that received sorafenib treatment, with the most pronounced reductions occurring in the SEQ arm. Immunohistochemical staining for γ-H2AX (C – <i>middle column</i>, F), however, revealed a significantly greater percent of nuclei with high or moderate numbers of foci, as well as a lower percent of nuclei with no foci, for the SEQ and RT arms compared to the CONC arm, similar to our <i>in vitro</i> results above. Column graphs summarizing the data for CD31, γ-H2AX, and Ki-67 are shown in D-F. Asterisks represent significant differences between columns ascertained by Student's <i>t-</i>test (CD31) or by Fisher's exact test (γ-H2AX and Ki-67) as indicated by the accompanying brackets.</p

Mechanism of sorafenib-mediated radioprotection <i>in vitro</i>.

<p>HepG2 cells were synchronized then re-fed with complete medium (10% serum) either containing 5 µM sorafenib (SOR) or vehicle control (DMSO). (A) Percent of cells in G₁, S, and G₂ phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G₁-S delay and cell cycle slowing in synchronized HepG2 cells, causing more cells to be in G₁-S versus G₂-M when radiation would be delivered 24 h after beginning incubation with SOR. (B & C) Unsynchronized Hep3b and HCC-4-4 cells were exposed to SOR or vehicle control for 24 h and then fixed with ethanol for cell cycle analysis. Percent of cells in G₁, S, and G₂ phases with SEM is plotted for control and SOR arms, with corresponding histograms generated from flow cytometry data analysis shown below. Treatment with SOR caused a G₁-S delay in both cell lines and reduced the number of cells in G₂-M when radiation would be delivered at 24 h after beginning incubation with SOR. Asterisks denote significant differences between corresponding columns in the control and SOR arms for each cell line by Student's <i>t-</i>test. Data for the HuH7 cell line is not shown because it was found to exhibit polyploidy; these data are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065726#pone.0065726.s002" target="_blank">Figure S2</a>. Data for unsynchronized HepG2 cells are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065726#pone.0065726.s002" target="_blank">Figure S2</a>. All experiments were done in triplicate and repeated. (D) Immunoblotting for phospho-p53 and p21 after treatment of HepG2 cells with each of the 5 different treatment arms (control—incubation with DMSO for 12 hours; SOR—incubation with 5-µM sorafenib for 12 hours; RT—incubation with DMSO for 12 hours with irradiation at 6-hour midpoint; CONC—incubation with 5-µM sorafenib for 12 hours with irradiation at 6-hour midpoint; SEQ—incubation with DMSO for 6 hours, irradiation, followed by incubation with 5-µM sorafenib for 6 hours). All irradiation doses were single fractions of 6 Gy. Corresponding immunoblot data for the remaining 3 cell lines can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065726#pone.0065726.s002" target="_blank">Figure S2C</a>.</p