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

Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris

Background: Candida auris is a multidrug resistant, emerging agent of fungemia in humans. Its actual global distribution remains obscure as the current commercial methods of clinical diagnosis misidentify it as C. haemulonii. Here we report the first draft genome of C. auris to explore the genomic basis of virulence and unique differences that could be employed for differential diagnosis. Results: More than 99.5 % of the C. auris genomic reads did not align to the current whole (or draft) genome sequences of Candida albicans, Candida lusitaniae, Candida glabrata and Saccharomyces cerevisiae; thereby indicating its divergence from the active Candida clade. The genome spans around 12.49 Mb with 8527 predicted genes. Functional annotation revealed that among the sequenced Candida species, it is closest to the hemiascomycete species Clavispora lusitaniae. Comparison with the well-studied species Candida albicans showed that it shares significant virulence attributes with other pathogenic Candida species such as oligopeptide transporters, mannosyl transfersases, secreted proteases and genes involved in biofilm formation. We also identified a plethora of transporters belonging to the ABC and major facilitator superfamily along with known MDR transcription factors which explained its high tolerance to antifungal drugs. Conclusions: Our study emphasizes an urgent need for accurate fungal screening methods such as PCR and electrophoretic karyotyping to ensure proper management of fungemia. Our work highlights the potential genetic mechanisms involved in virulence and pathogenicity of an important emerging human pathogen namely C. auris. Owing to its diversity at the genomic scale; we expect the genome sequence to be a useful resource to map species specific differences that will help develop accurate diagnostic markers and better drug targets

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

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