CSPG4 Is a Potential Therapeutic Target in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting ∼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.

Sphingosine Kinase 1 Regulates the Survival of Breast Cancer Stem Cells and Non-stem Breast Cancer Cells by Suppression of STAT1.

Cancer stem cells (CSCs) represent rare tumor cell populations capable of self-renewal, differentiation, and tumor initiation and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches that can effectively target CSCs and tumor cells could be the key to efficient tumor treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibited cell proliferation and induced apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhanced breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signaling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrated that SPHK1 inhibitors, FTY720 and PF543, synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies.

Predicting peptide presentation by major histocompatibility complex class I: an improved machine learning approach to the immunopeptidome.

BACKGROUND: To further our understanding of immunopeptidomics, improved tools are needed to identify peptides presented by major histocompatibility complex class I (MHC-I). Many existing tools are limited by their reliance upon chemical affinity data, which is less biologically relevant than sampling by mass spectrometry, and other tools are limited by incomplete exploration of machine learning approaches. Herein, we assemble publicly available data describing human peptides discovered by sampling the MHC-I immunopeptidome with mass spectrometry and use this database to train random forest classifiers (ForestMHC) to predict presentation by MHC-I. RESULTS: As measured by precision in the top 1% of predictions, our method outperforms NetMHC and NetMHCpan on test sets, and it outperforms both these methods and MixMHCpred on new data from an ovarian carcinoma cell line. We also find that random forest scores correlate monotonically, but not linearly, with known chemical binding affinities, and an information-based analysis of classifier features shows the importance of anchor positions for our classification. The random-forest approach also outperforms a deep neural network and a convolutional neural network trained on identical data. Finally, we use our large database to confirm that gene expression partially determines peptide presentation. CONCLUSIONS: ForestMHC is a promising method to identify peptides bound by MHC-I. We have demonstrated the utility of random forest-based approaches in predicting peptide presentation by MHC-I, assembled the largest known database of MS binding data, and mined this database to show the effect of gene expression on peptide presentation. ForestMHC has potential applicability to basic immunology, rational vaccine design, and neoantigen binding prediction for cancer immunotherapy. This method is publicly available for applications and further validation.

Robust, Reproducible, and Economical Phosphopeptide Enrichment Using Calcium Titanate.

Mass-spectrometry-based phosphoproteomics has revolutionized phosphoprotein analysis and enhanced our understanding of diverse and fundamental cellular processes important for human health and disease. Because of their relative scarcity, phosphopeptides must be enriched before analysis. Many different enrichment methods and materials have been described, and many reports have made claims about the advantages of particular materials and methodological variations. We demonstrate an effective and highly reproducible single-step enrichment method using an off-the-shelf preparation of calcium titanate. Using two different cell lines and replicate analysis, we show that our method achieves a purity and depth of analysis comparable or superior to a widely used TiO2-based method at a reduced cost and effort. This method provides a new and immediately available tool for expanding the reach of phosphoproteomic inquiry.

Jerantinine A induces tumor-specific cell death through modulation of splicing factor 3b subunit 1 (SF3B1).

Precursor mRNA (pre-mRNA) splicing is catalyzed by a large ribonucleoprotein complex known as the spliceosome. Numerous studies have indicated that aberrant splicing patterns or mutations in spliceosome components, including the splicing factor 3b subunit 1 (SF3B1), are associated with hallmark cancer phenotypes. This has led to the identification and development of small molecules with spliceosome-modulating activity as potential anticancer agents. Jerantinine A (JA) is a novel indole alkaloid which displays potent anti-proliferative activities against human cancer cell lines by inhibiting tubulin polymerization and inducing G2/M cell cycle arrest. Using a combined pooled-genome wide shRNA library screen and global proteomic profiling, we showed that JA targets the spliceosome by up-regulating SF3B1 and SF3B3 protein in breast cancer cells. Notably, JA induced significant tumor-specific cell death and a significant increase in unspliced pre-mRNAs. In contrast, depletion of endogenous SF3B1 abrogated the apoptotic effects, but not the G2/M cell cycle arrest induced by JA. Further analyses showed that JA stabilizes endogenous SF3B1 protein in breast cancer cells and induced dissociation of the protein from the nucleosome complex. Together, these results demonstrate that JA exerts its antitumor activity by targeting SF3B1 and SF3B3 in addition to its reported targeting of tubulin polymerization.

In vitro anticancer properties and biological evaluation of novel natural alkaloid jerantinine B.

Natural products play a pivotal role in medicine especially in the cancer arena. Many drugs that are currently used in cancer chemotherapy originated from or were inspired by nature. Jerantinine B (JB) is one of seven novel Aspidosperma indole alkaloids isolated from the leaf extract of Tabernaemontana corymbosa. Preliminary antiproliferative assays revealed that JB and JB acetate significantly inhibited growth and colony formation, accompanied by time- and dose-dependent apoptosis induction in human cancer cell lines. JB significantly arrested cells at the G2/M cell cycle phase, potently inhibiting tubulin polymerisation. Polo-like kinase 1 (PLK1; an early trigger for the G2/M transition) was also dose-dependently inhibited by JB (IC50 1.5 µM). Furthermore, JB provoked significant increases in reactive oxygen species (ROS). Annexin V+ cell populations, dose-dependent accumulation of cleaved-PARP and caspase 3/7 activation, and reduced Bcl-2 and Mcl-1 expression confirm apoptosis induction. Preclinical in silico biopharmaceutical assessment of JB calculated rapid absorption and bioavailability >70%. Doses of 8-16 mg/kg JB were predicted to maintain unbound plasma concentrations >GI50 values in mice during efficacy studies. These findings advocate continued development of JB as a potential chemotherapeutic agent.

Impact of Chronic Total Occlusions and Coronary Revascularization on All-Cause Mortality and the Incidence of Ventricular Arrhythmias in Patients With Ischemic Cardiomyopathy.

Coronary chronic total occlusions (CTOs) have been associated with higher mortality in patients with ischemic cardiomyopathy and implantable cardioverter defibrillators (ICDs); yet the impact of CTO revascularization on subsequent clinical outcomes has not been studied. We evaluated the clinical characteristics and outcomes of patients with ischemic cardiomyopathy who also received an ICD for primary prevention of sudden death at the Dallas VA Medical Center from January 2002 to December 2013. On the basis of coronary angiography performed before device implantation, patients were divided into 3 groups: no CTOs, revascularized CTOs (with percutaneous coronary intervention or surgery), and unrevascularized CTOs. Primary and secondary outcomes were all-cause mortality and appropriate ICD therapy for sustained ventricular arrhythmias. A total of 307 patients (mean age 64.3 ± 8.1 years, 100% men) were included in the study. At least 1 CTO was present in 213 patients (69%) and was revascularized in 99 patients (32%). During a median follow-up of 4.1 years, 51 patients (17%) died and 98 (32%) had at least 1 episode of sustained ventricular arrhythmia. Mortality and incidence of ventricular arrhythmias were similar in the 3 study groups in both univariate and multivariate analyses. In conclusion, CTOs are commonly found in patients with ischemic cardiomyopathy. In contrast to previous studies, the presence of a CTO was not associated with higher mortality or incidence of ventricular arrhythmias. In addition, revascularization of CTOs was not associated with improved outcomes in this high-risk cohort.

Ibogan, tacaman, and cytotoxic bisindole alkaloids from tabernaemontana. Cononusine, an iboga alkaloid with unusual incorporation of a pyrrolidone moiety.

Six new indole alkaloids, viz., cononusine (1, a rare example of an iboga-pyrrolidone conjugate), ervaluteine (2), vincamajicine (3), tacamonidine (4), 6-oxoibogaine (5), and N(4)-chloromethylnorfluorocurarine chloride (6), and two new vobasinyl-iboga bisindole alkaloids, ervatensines A (7) and B (8), in addition to other known alkaloids, were isolated from the stem-bark extract of the Malayan Tabernaemontana corymbosa. The structures of these alkaloids were established on the basis of NMR and MS analyses and, in one instance (7), confirmed by X-ray diffraction analysis. Vincamajicine (3) showed appreciable activity in reversing multidrug resistance in vincristine-resistant KB cells (IC50 2.62 µM), while ervatensines A (7) and B (8) and two other known bisindoles displayed pronounced in vitro growth inhibitory activity against human KB cells (IC50 < 2 µM). Compounds 7 and 8 also showed good growth inhibitory activity against A549, MCF-7, MDA-468, HCT-116, and HT-29 cells (IC50 0.70-4.19 µM). Cell cycle and annexin V-FITC apoptosis assays indicated that compounds 7 and 8 inhibited proliferation of HCT-116 and MDA-468 cells, evoking apoptotic and necrotic cell death.

Prevalence and treatment of "balloon-uncrossable" coronary chronic total occlusions.

BACKGROUND: The frequency and outcomes of "balloon-uncrossable" coronary chronic total occlusions (CTOs) have received limited study. METHODS: We retrospectively examined 373 consecutive CTO percutaneous coronary interventions (PCIs) performed at our institution between 2005 and 2013 to determine the frequency and treatment of balloon-uncrossable CTOs. RESULTS: Mean age was 63.7 ± 8.3 years and 98.9% of the patients were men. Twenty-four patients (6.4%, 95% confidence intervals 4.2% to 9.4%) were found to have a balloon-uncrossable CTO. Compared to the other CTO PCI patients, those with balloon-uncrossable CTOs had similar clinical and angiographic characteristics. Successful crossing of the balloon-uncrossable CTO was achieved in 22 of 24 patients (91.7%) using a variety of techniques, such as successive balloon inflations (43.5%), microcatheter advancement (21.7%), laser (8.7%), techniques that increase guide catheter support (13.0%), and subintimal lesion crossing (13.0%). Patients with balloon-uncrossable CTOs had longer procedure time (184.5 ± 77.9 vs 134.0 ± 69.0 min, P<.01), fluoroscopy time (55.2 ± 24.9 vs 37.9 ± 20.8 min, P<.01), and received high contrast volume (404.4 ± 137.9 vs 351.7 ± 138.5 mL, P=.08), but had similar incidence of major complications (8.3% vs 3.2%, P=.25) as compared with patients who did not have balloon-uncrossable CTOs. CONCLUSION: Balloon-uncrossable CTOs are encountered in 6.4% of contemporary CTO PCIs and can be successfully treated in most patients using a variety of techniques.

Novel antitumour indole alkaloid, Jerantinine A, evokes potent G2/M cell cycle arrest targeting microtubules.

Natural products play a pivotal role in the treatment of cancer; identification of compounds such as taxanes and the vinca alkaloids were seminal landmarks in natural product drug discovery. Jerantinine A, a novel Aspidosperma alkaloid isolated from plant species Tabernaemontana corymbosa, was previously reported to possess cytotoxic activity against vincristine-resistant nasopharyngeal carcinoma cells and is therefore an ideal candidate for biological investigation. Furthermore, Tabernaemontana corymbosa, has been placed in the endangered list of threatened species by the International Union for Conservation of Nature thus making it a priority to elucidate the biological activity of this alkaloid. Herein, we report detailed biological evaluation of jerantinine A on various human-derived carcinoma cell lines. Our preliminary screens showed that significant inhibition of cell growth and colony formation accompanied time- and dose-dependent induction of apoptosis in human cancer cell lines after treatment with jerantinine A. Dose-dependent accumulations of cleaved PARP and caspase 3 further confirmed apoptosis. Profound G2/M cell cycle arrest was observed 24 h after treatment in all cell lines. Characteristics of mitotic arrest including inhibition of tubulin polymerisation, microtubule disruption, aneuploidy, and cyclin B1 down-regulation were clearly observed. The potent anti-proliferative, pro-apoptotic, and tubulin-destabilising activities of jerantinine A warrant further development of this molecule as a potential chemotherapeutic agent.

Use of the twin-pass catheter for wiring a jailed side branch.

Wiring through stent struts into a jailed side branch can be challenging. We describe a case of double-barrel left main coronary artery stenting into the first obtuse marginal branch that resulted in significant ostial circumflex coronary artery stenosis. We were unable to wire the jailed large distal circumflex artery. We introduced the Twin-Pass catheter over the first obtuse marginal wire, placing the proximal lumen tip over the distal circumflex ostium. The distal circumflex artery was successfully wired, allowing balloon dilatation and successful completion of the case.

Use of the venture wire control catheter for the treatment of coronary artery chronic total occlusions.

BACKGROUND: The Venture catheter (St Jude, Minneapolis, MN) has a deflectable tip for facilitating wire steering and a stiff body. Both properties can be useful in percutaneous coronary interventions (PCI) of coronary chronic total occlusions (CTOs). METHODS: We reviewed 26 consecutive patients in whom the Venture catheter was utilized during coronary CTO PCI at our institution between May 2008 and September 2009. RESULTS: Mean age was 63 ± 9 years and 96% of the patients were men. The CTO target lesion was located in the right coronary artery (35%), left anterior descending artery (27%), circumflex (27%) or a saphenous vein graft (4%). A prior attempt for CTO PCI had been done in 19%. The primary CTO PCI approach was antegrade in 92% and retrograde in 8%, but a retrograde approach was used in an additional 27% of the patients after antegrade approach failed. The Venture catheter was used to overcome vessel tortuosity (73%), for CTOs with side branch at the occlusion site (15%), to facilitate collateral branch wiring during retrograde PCI (8%), and to provide extra support (4%). The overall CTO PCI success rate was 77% and was 92% in patients with upfront Venture catheter use and in 64% of patients in whom the Venture was used after PCI attempts using other equipment failed. Procedural failure was due to inability to cross the lesion in all cases. CONCLUSIONS: The Venture catheter can facilitate CTO PCI, especially in patients with marked coronary tortuosity or when additional support is required.