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

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

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