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PDF file - 1004K, S1. The Twist box domain is required for full Twist1 transcriptional activity. S2. Quantification of immunofluorescence from isogenic prostate cancer cell lines stably expressing Twist1 and Twist1-F191G. S3. The Twist box domain is required for full Twist1-induced EMT marker phenotypes of prostate cancer cells. S4. Twist1 overexpression induces temporal changes in the material properties of prostate cancer cells during their migration in a wound healing assay. S5. The overexpression of Twist1 or Twist1-F191G does not increase cellular proliferation of prostate cancer cells in vitro. S6. The Twist box domain is required for full Twist1-induced cellular migration in PC3 cells. S7. Twist1 overexpression increases cell traction forces of individual androgenindependent PC3 prostate cancer cells. S8. Twist1 overexpression confers radioresistance to prostate cancer cells which is attenuated by mutation of the Twist box domain. S9. The Twist box domain is required for Twist1-induced soft agar anchorageindependent growth of 22Rv1 prostate cancer cells. S10. Twist1 overexpression does not confer prostate cancer cells increased primary tumorigenicity and slows primary tumor cell growth in vivo. S11. The Twist box domain is required for full Twist1-induced expression of Hoxa9/HOXA9.

PDF file - 116K, Supplemental Table S2 - Myc-CaP + Twist1-F191G versus Myc-CaP + Vector Differentially.

PDF file - 384K, Supplemental Table S1 - Myc-CaP + Twist1 versus Myc-CaP + Vector Differentially.

Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.

Supplementary Figure Legends from Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Supplementary Figure 1 from Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Supplementary Figure 2 from Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes, and treatment options are limited because of the lack of signature molecules and heterogeneous properties of cancer. COL8A1 expression is higher in breast cancer than in normal tissues and is strongly correlated with worse overall survival in patients with breast cancer. However, the biological function of COL8A1 on cancer progression is not fully understood. In this study, we investigated the biological function of COL8A1 on TNBC progression.COL8A1-deficient cells were generated using the CRISPR-Cas9 system. The tumor growth and metastasis of TNBC cells were evaluated using three-dimensional culture (3D) methods and xenograft mouse models. The activation of focal adhesion kinase (FAK)/Src by COL8A1 in TNBC cells was evaluated by immunoblotting.COL8A1 expression was primarily distributed into TNBC cell lines. Further, relapse-free survival in TNBC patients with the MSL subtype was strongly associated with the COL8A1 expression. MDA-MB-231 and Hs578T cells, classified as the MSL subtype, strongly express COL8A1, and COL8A1 protein expression was induced by hypoxia in both cell lines. Loss of COL8A1 expression inhibited spheroid /tumor growth and metastasis in vitro and in vivo. Further, exogenous COL8A1 promoted TNBC growth via the FAK/Src activation. Finally, the spheroid growth of MDA-MB-231 and Hs578T cells was inhibited by defactinib, a FAK inhibitor, without cytotoxicity.These results indicate that COL8A1-mediated FAK/Src activation produces a more aggressive phenotype in TNBC, and its target inhibition may be an efficacious treatment for TNBC.

A 253 J with 26 ns at 0.2 Hz laser performance was demonstrated using a LD pumped cryogenically cooled Yb:YAG ceramics laser amplifier. A high energy storage of 344 J was achieved with a stored energy density of 0.58 J/cm3 using a 1 kJ output multidirectional-pumping system. High energy-extraction efficiency of 56.5% was achieved with high energy fluence of 4.63 J /cm2. To the best of our knowledge, this is the highest output energy obtained with a repetitive nanosecond pulse by LD pumped solid-state laser. This paper presented a design of 1 kJ amplifier based on experimentally proven numerical data.

Objectives: This study aims to investigate whether endocan levels in the peripheral blood change after the removal of primary breast tumors and allow predicting postoperative prognosis for breast cancer. Significance: An early prediction of postoperative recurrence and metastases with liquid biopsy will lead to better management and quality of life of breast cancer patients. Background: Cancer recurrence comes down to a poor prognosis in breast cancer patients. Although removal of primary breast cancer can achieve a successful outcome, metastasis and local recurrence remain a critical clinical problem in later years. Although the overall survival of breast cancer patients varies among molecular subtypes, the individual patients have different prognostic outcomes; some patients relapse within a few months, and others have not seen any recurrence for several years. Our previous study indicated that endocan had the potential to be a prognostic biomarker for triple-negative breast cancer. It is critical to show changes in endocan levels in the peripheral blood after the surgical excision of the primary tumor, which, in turn, will corroborate our hypothesis that plasma endocan is a beneficial biomarker for a postoperative prognosis for breast cancer patients. We included all the breast cancer subtypes in this study as the information on endocan levels in the peripheral blood from breast cancer patients has been limited thus far. Methods: To detect metastatic recurrence of breast cancer cells noninvasively, MDA-MB-231BR was transfected with the mVenus-Akaluc gene. MDA-MB-231BR spontaneously overexpresses the ESM1 gene. MDA-MB-231BR/mVenus-Akaluc was inoculated into the mammary fat pad of female athymic nu/nu mice and NSG mice. Once tumors top 200 mm3, they were surgically removed. Metastatic recurrence was visualized by the IVIS imager. Blood was withdrawn from the tail of each mouse before and after the resection of the primary tumors in a timely manner, and plasma samples were stored at -80°C until usage. Endocan in plasma samples was quantitated with a commercial ELISA kit. Tumor burden was estimated from luminescence signals of images obtained by IVIS. We obtained IRB approval from both institutions for the following research. Blood samples were collected from breast cancer patients who had given informed consent at the Breast Cancer, Showa University Hospital, and the plasma samples have been stored at -80°C until usage. Endocan in plasma sample was determined with a commercial ELISA kit. Results and discussion: Before removing primary breast tumors, the median endocan level in plasma was 0.73 ng/mL (range, 0.38-1.54 ng/mL) in nude mice. After the removal, endocan remained below the detection limits even though small metastases were detected by IVIS imager at later time points. These data indicate that loss of primary tumors results in an out-of-detection range in endocan levels in the blood. In contrast, NSG mice displayed inconsistent results: out of eight mice, three mice showed a decrease in endocan, three had an increase in endocan, and two had no change three days postoperatively. IVIS images of NSG mice indicated that distant metastases were already detected even when some mice underwent tumor resection. In NSG mice, the occurrence of tumor metastases was much earlier, and metastatic tumor burdens were much more significant than in nude mice, which might lead to a different outcome. Endocan levels in plasma from breast cancer patients at the postoperative period were not always dropped, which was similar to the results obtained from NSG mice. The rationale behind the inconsistent kinetics of endocan remains to be clarified. Conclusion: Our results from NSG mice and clinical data in patients’ plasma may have more relevance to prognostic outcomes in breast cancer patients. Citation Format: Kentaro Daiki, Yoko Kanada, Aya Nagata, Seigo Nakamura, Yoshinori Kato. Kinetics of endocan in the peripheral blood before and after the resection of primary breast tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-05-12.

Direct-acting antivirals, asunaprevir (ASV), daclatasvir (DCV), and beclabuvir (BCV) are known to be mainly metabolized by CYP3A enzymes; however, the differences in the detailed metabolic activities of CYP3A4 and CYP3A5 on these drugs are not well clarified. The aim of the present study was to elucidate the relative contributions of CYP3A4 and CYP3A5 to the metabolism of ASV, DCV, and BCV, as well as the effect of CYP3A5*3 genetic variant in vitro. The amount of each drug and their major metabolites were determined using LC-MS/MS. Recombinant CYP3As and CYP3A5*3-genotyped human liver microsomes (CYP3A5 expressers or non-expressers) were used for the determination of their metabolic activities. The contribution of CYP3A5 to ASV metabolism was considerable compared to that of CYP3A4. Consistently, ASV metabolic activity in CYP3A5 expressers was higher than those in CYP3A5 non-expresser. Moreover, CYP3A5 expression level was significantly correlated with ASV metabolism. In contrast, these observations were not found in DCV and BCV metabolism. To our knowledge, this is the first study to directly demonstrate the effect of CYP3A5*3 genetic variants on the metabolism of ASV. The findings of the present study may provide basic information on ASV, DCV, and BCV metabolisms.

Purpose The cytochrome P450 (CYP) 3A family of enzymes metabolize the majority of clinically used drugs. CYP3A4 and CYP3A5 are the two major CYP3A isoforms, but exhinbit different substrate specificity. The aim of this study was to establish a simple screening method to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro. Methods A screening method was developed based on competitive inhibition using luciferin-PPXE (L-PPXE), a luminogenic CYP3A substrate. CYP3cide, tacrolimus, and midazolam were selected as standard compounds metabolized by CYP3A4 or CYP3A5. Nine clinically-used drugs were evaluated for their abilities to inhibit luminescence resulting from L-PPXE metabolism. Appropriate reaction conditions for the screening method were determined using recombinant CYP3A4 and CYP3A5. Results A significant decrease in luminescence resulting from L-PPXE metabolism by CYP3A4 and CYP3A5 was observed only for drugs reported to be metabolized by CYP3As. The substrate specificities of CYP3A4 or CYP3A5 for the proposed CYP3A substrates using our screening method were consistent with those of previous reports or available drug information from pharmaceutical companies. The reaction volume for this method was 50 μL, and the time required for the entire procedure was 70 min. Furthermore, this screening can be performed using a single tube with minimal training. Conclusions Through the establishment of our screening method in the present study, we are sure it is useful to determine the relative contributions of CYP3A4 and CYP3A5 to drug metabolism in vitro.

The large vertical ground motion during the 2008 Iwate-Miyagi Nairiku earthquake showed the importance of not only the horizontal ground motion but also the vertical motion in strong ground motion prediction and response analysis. In this study, considerations about the interaction behavior between the horizontal motion and the vertical motion during a strong earthquake were carried out by one-dimensional elasto-plastic earthquake analyses and cyclic shear box tests. It could be concluded from the results that the change of confining pressure caused by vertical motion affects the horizontal behavior, and strong horizontal motion produced vertical motion in the soil layer above the groundwater level.

Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir. The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3. The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5. CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level. This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy.

The injection and engagement of pellets using laser beam irradiation is one of the key technologies to realize a laser-driven inertial fusion energy (IFE) reactor. We irradiated ultra-intense laser...

A 11.5 J at 40 ns output has been obtained from a diode-pumped cryo-cooled Yb:YAG ceramics active-mirror laser amplifier system. The system consists of two amplifier heads which has four Yb:YAG ceramic disks and two pump LD modules. The Yb:YAG ceramics are cooled by conventional cryostat from rear side and are pumped by LD modules from front-side. A pump pulse is delivered to Yb:YAG ceramics coaxially with a seed pulse to reduce damage risk at a dielectric coating of Yb:YAG ceramics due to simplified coating design. To realize this system design, a LD module has been developed to keep a rectangle pattern with side length of around 37 mm among imaging depth of about 10cm at working distance of about 410 mm. As an experimental result of two pass amplification, a 11.5 J pulse energy was obtained with input energy of 1.0 J and total pump energy of 90.2 J. Then, an optical-to-optical conversion efficiency was 11.6% and an extraction efficiency was estimated to be 42%. In our knowledge, this is the highest output energy with nano second pulse duration in cryo-cooled Yb:YAG active-mirror laser amplification scheme. A repetition rate of 0.05 Hz depends on a limitation of a repetition rate of the seed pulse. A dependence of small-signal-gain on pumping repetition rate of the active-mirror laser head was experimentally evaluated. From the experimental result, we have estimated a feasible repetition rate of over 5 Hz. A 10 Hz operation will be demonstrated to reduce a thermal resistance between Yb:YAG ceramics and cryostat. Finally, this laser amplifier system is installed to a 100-J class laser system as preamplifier.

A 100 J class diode-pumped solid-state laser system has been developed as a platform for acceleration of laser processing application. A 117 J output of pulse energy has been demonstrated with the main amplifier.

Stem cell therapies are being developed for radiotherapy-induced brain injuries (RIBI). Magnetic resonance imaging (MRI) offers advantages for imaging transplanted stem cells. However, most MRI cell-tracking techniques employ superparamagnetic iron oxide particles (SPIOs), which are difficult to distinguish from hemorrhage. In current preclinical RIBI models, hemorrhage occurs concurrently with other injury markers. This makes the evaluation of the recruitment of transplanted SPIO-labeled stem cells to injury sites difficult. Here, we developed a RIBI model, with early injury markers reflective of hippocampal dysfunction, which can be detected noninvasively with MRI and behavioral tests. Lesions were generated by sub-hemispheric irradiation of mouse hippocampi with single X-ray beams of 80 Gy. Lesion formation was monitored with anatomical and contrast-enhanced MRI and changes in memory and learning were assessed with fear-conditioning tests. Early injury markers were detected 2 weeks after irradiation. These included an increase in the permeability of the blood-brain barrier, demonstrated by a 92 ± 20 % contrast enhancement of the irradiated versus the non-irradiated brain hemispheres, within 15 min of the administration of an MRI contrast agent. A change in short-term memory was also detected, as demonstrated by a 40.88 ± 5.03 % decrease in the freezing time measured during the short-term memory context test at this time point, compared to that before irradiation. SPIO-labeled stem cells transplanted contralateral to the lesion migrated toward the lesion at this time point. No hemorrhage was detected up to 10 weeks after irradiation. This model can be used to evaluate SPIO-based stem cell-tracking agents, short-term.

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.

Linezolid (LZD) is an antimicrobial that is commonly used for treatment of vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus infections. However, the development of thrombocytopenia, one of the most frequent adverse side effects of this antimicrobial, can lead to discontinuation of LZD treatment. While clinical studies indicate that risk factors for the development of LZD-induced thrombocytopenia include treatment for >14 consecutive days, renal dysfunction, and chronic liver disease, the fundamental mechanism governing the pathogenesis of this disorder remains unclear. In this study, we aimed to elucidate the mechanism of LZD-induced thrombocytopenia by investigating the impact of LZD treatment on platelet destruction and production using rat platelet-rich plasma (PRP) and human immortalized cell lines, respectively. Compared to the control population, an increase in lactate dehydrogenase release was not detected upon exposure of rat PRP to varying concentrations of LZD, indicating that this compound is not cytotoxic towards platelets. Meanwhile, LZD treatment resulted in a significant dose-dependent increase in the proliferation of HEL human erythroleukemia and MEG-01 human megakaryoblast cells in vitro, but did not influence the differentiation of these cell lines. Lastly, LZD treatment yielded elevated levels of phosphorylation of myosin light chain 2 (MLC2), which regulates platelet release, in MEG-01 cells. Based on these results, we speculate that LZD induces thrombocytopenia by promoting MLC2 phosphorylation and thereby suppressing the release of platelets from mature megakaryocytes. These findings provide the first insight into the mechanism of LZD-mediated thrombocytopenia and may facilitate the development of strategies to treat and/or prevent this disease.

We demonstrated 117-J laser output in a nanosecond regime with a diode-pumped Yb:YAG ceramic laser amplifier. The repetition rate was 0.05 Hz, and the pulse duration was around 40 ns. 5-J input energy was amplified by two cryogenically cooled Yb:YAG ceramic laser heads. Six Yb:YAG ceramic disks in each laser head were cooled by cryogenic helium gas with a temperature of 175 K. The output energy of the first and second laser heads was about 28 and 117 J.

We report a directly modulated laser diode system capable of generating picosecond-pulse bursts and nanosecond pulses simultaneously. A generated pulse shape can be arbitrary controlled with a temporal resolution of 1 ns and wavelength of 1064 nm. A two-stage Nd:YVO4 amplifier boosts the pulse energy to hundreds of microjoules to process JIS 304 stainless steel. Characterization of processed holes irradiated by different pulse durations and shapes reveals that the ablation efficiencies with the nanosecond pulses are two times higher than those with the picosecond-pulse bursts. A clear hole with a taper angle of 1.5° is realized by the picosecond-pulse bursts with a 10-ns pulse interval. The combination of pulses with large differences in timescales offers an efficient production line with a single laser system.

Recently, natural d-glucose was suggested as a potential biodegradable contrast agent. The feasibility of using d-glucose for dynamic perfusion imaging was explored to detect malignant brain tumors based on blood brain barrier breakdown.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays roles in both energy maintenance, and stress signaling by forming a protein complex with seven in absentia homolog 1 (Siah1). Mechanisms to coordinate its glycolytic and stress cascades are likely to be very important for survival and homeostatic control of any living organism. Here we report that apoptosis signal-regulating kinase 1 (ASK1), a representative stress kinase, interacts with both GAPDH and Siah1 and is likely able to phosphorylate Siah1 at specific amino acid residues (Thr-70/Thr-74 and Thr-235/Thr-239). Phosphorylation of Siah1 by ASK1 triggers GAPDH-Siah1 stress signaling and activates a key downstream target, p300 acetyltransferase in the nucleus. This novel mechanism, together with the established S-nitrosylation/oxidation of GAPDH at Cys-150, provides evidence of how the stress signaling involving GAPDH is finely regulated. In addition, the present results imply crosstalk between the ASK1 and GAPDH-Siah1 stress cascades.

Exogenous direct cell labeling with superparamagnetic iron oxide nanoparticles (SPIONs) is currently the most employed cell-labeling technique for tracking transplanted cells using magnetic resonance imaging (MRI). Although SPION-based cell labeling is effective for monitoring cell delivery and migration, monitoring cell survival is still a challenge. This unit describes an MRI technique that permits detection of the delivery, migration, and death of transplanted cells. This dual-contrast technique involves labeling cells with two different classes of MRI contrast agents, possessing different diffusion coefficients: SPIONs (T2/T2* contrast agents, with lower diffusion coefficients) and gadolinium chelates (T1 contrast agents, with higher diffusion coefficients). In live cells, where both agents are in close proximity, the T2/T2* contrast predominates and the T1 contrast is quenched. In dead cells, where the cell membrane is breached, gadolinium chelates diffuse from the SPIONs and generate a signature T1 contrast enhancement in the vicinity of dead cells. © 2017 by John Wiley & Sons, Inc. Keywords: Cellular MRI; direct cell labeling; cell death detection; superparamagnetic iron oxide nanoparticles; MRI dual-contrast technique

Mesenchymal stem cells (MSCs) have been explored as a "live" carrier of cytokines for targeted cancer therapy, but, in earlier reports in the literature, the secretion process of therapeutic cytokines was not regulated. The purpose of this study was to generate MSCs to conditionally secrete the melanoma differentiation-associated gene-7 (MDA-7) tumor-suppressor protein. To control the secretion of MDA-7 from MSCs, a well-established tetracycline-controlled transcriptional activation system was incorporated into MDA-7 plasmid. MDA-7 gene expression was induced in the engineered MSCs only in the presence of doxycycline, as characterized by quantitative reverse transcription (qRT)-PCR. Enzyme-linked immunosorbent assay (ELISA) also revealed that the MDA-7 protein was secreted from the engineered MSCs only after the cells had been exposed to doxycycline. Both recombinant human MDA-7 protein and the conditioned medium from the engineered MSCs in the presence of doxycycline significantly inhibited tube formation of human umbilical vascular endothelial cells (HUVECs), indicating that our system could be used for targeted, antiangiogenic therapy. Overall, this study provides useful information on the potential use of engineered MSCs for the controlled secretion of therapeutic proteins, in this case MDA-7, for targeted cancer therapy.

A 64J at 10 ns output was demonstrated by diode-pumped cryogenically cooled Yb:YAG ceramic laser amplifier. An extraction efficiency was evaluated 43.3% at stored energy of 148J with small-signal-gain of 20.4.

A compact 40kW at 10Hz output LD module has been developed for cryogenically cooled Yb:YAG ceramics active-mirror laser amplifiers for 10J at 10Hz output laser system. Pumping intensity is 2.5kW/cm2 and footprint of the LD module is 18cm × 77cm.

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.