Phosphorylation and Stabilization of PIN1 by JNK Promote Intrahepatic Cholangiocarcinoma Growth.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of the c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and therefore the key downstream effectors of this pathway, remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signaling in ICC that could open up therapeutic opportunities. APPROACH AND RESULTS: Using loss-of-function and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumors, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 through all-trans retinoic acid, a Food and Drug Administration-approved drug, impairs the growth of both cultured and xenografted ICC cells. CONCLUSIONS: Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation through PIN1 inhibition.

The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth.

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 µM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.

Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography.

Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture.

Strategies in functional proteomics: Unveiling the pathways to precision oncology.

Personalised strategies in cancer care are required to overcome the therapeutic challenges posed by variability between patients and disease subsets. To this end, enhanced precision tools must be developed to describe the molecular drivers of malignant proliferation. Such tools must also identify druggable targets and biomarkers in order to provide essential information regarding drug development and therapeutic outcome. Here we discuss how proteomics-based approaches provide a set of viable methodologies capable of delivering quantitative information throughout the main stages of personalised oncology and a ratiometric platform that delivers systems-wide methods for drug evaluation.

Choline Kinase Alpha (CHKα) as a Therapeutic Target in Pancreatic Ductal Adenocarcinoma: Expression, Predictive Value, and Sensitivity to Inhibitors.

Choline kinase α (CHKα) plays a crucial role in the regulation of membrane phospholipid synthesis and has oncogenic properties in vitro. We have analyzed the expression of CHKα in cell lines derived from pancreatic ductal adenocarcinoma (PDAC) and have found increased CHKα expression, associated with differentiation. CHKα protein expression was directly correlated with sensitivity to MN58b, a CHKα inhibitor that reduced cell growth through the induction of apoptosis. Accordingly, CHKα knockdown led to reduced drug sensitivity. In addition, we found that gemcitabine-resistant PDAC cells displayed enhanced sensitivity to CHKα inhibition and, in vitro, MN58b had additive or synergistic effects with gemcitabine, 5-fluorouracil, and oxaliplatin, three active drugs in the treatment of PDAC. Using tissue microarrays, CHKα was found to be overexpressed in 90% of pancreatic tumors. While cytoplasmic CHKα did not relate to survival, nuclear CHKα distribution was observed in 43% of samples and was associated with longer survival, especially among patients with well/moderately differentiated tumors. To identify the mechanisms involved in resistance to CHKα inhibitors, we cultured IMIM-PC-2 cells with increasingly higher concentrations of MN58b and isolated a subline with a 30-fold higher IC50. RNA-Seq analysis identified upregulation of ABCB1 and ABCB4 multidrug resistance transporters, and functional studies confirmed that their upregulation is the main mechanism involved in resistance. Overall, our findings support the notion that CHKα inhibition merits further attention as a therapeutic option in patients with PDAC and that expression levels may predict response.

LMTK3 escapes tumour suppressor miRNAs via sequestration of DDX5.

Lemur tyrosine kinase-3 (LMTK3) plays an important role in cancer progression and is associated with breast, lung, gastric and colorectal cancer. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that typically repress target genes at post-transcriptional level and have an important role in tumorigenesis. By performing a miRNA expression profile, we identified a subset of miRNAs modulated by LMTK3. We show that LMTK3 induces miR-34a, miR-196-a2 and miR-182 levels by interacting with DEAD-box RNA helicase p68 (DDX5). LMTK3 binds via DDX5 to the pri-miRNA of these three mature miRNAs, thereby sequestrating them from further processing. Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressing cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration. Interestingly, miR-34a and miR-182 directly bind to the 3'UTR of LMTK3 mRNA and consequently inhibit both its stability and translation, acting as tumour suppressor-like miRNAs. In aggregate, we show that LMTK3 is involved in miRNA biogenesis through modulation of the Microprocessor complex, inducing miRNAs that target LMTK3 itself.

Employing in-vivo molecular imaging in simulating and validating tumor growth.

During the last decades, especially via the EU initiative related to the Virtual Physiological Human, significant progress has been made in advancing "in-silico" computational models to produce accurate and reliable tumor growth simulations. However, currently most attempts to validate the outcome of the models are either done in-vitro or ex-vivo after tumor resection. In this work, we incorporate information provided by fluorescence molecular tomography performed in-vivo into a mathematical model that describes tumor growth. The outcome is validated against tumor evolution snapshots captured in-vivo using advanced molecular probes in laboratory animals. The simulations are inline with the actual in-vivo growth and although alternative modeling parameters can lead to similar results challenging for additional microscopic information and imaging modalities to drive the in-silico models, they all show that hypoxia plays a dominant role in the evolution of the tumor under study.

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning.

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Tomographic imaging with polarized light.

We report three-dimensional tomographic reconstruction of optical parameters for the mesoscopic light scattering regime from experimentally obtained datasets by using polarized light. We present a numerically inexpensive approximation to the radiative transfer equation governing the polarized light transport. This approximation is employed in the reconstruction algorithm, which computes two optical parameters by using parallel and perpendicular polarizations of transmitted light. Datasets were obtained by imaging a scattering phantom embedding highly absorbing inclusions. Reconstruction results are presented and discussed.

Prehistological evaluation of benign and malignant pigmented skin lesions with optical computed tomography.

Discrimination of benign and malignant melanocytic lesions is a major issue in clinical dermatology. Assessment of the thickness of melanoma is critical for prognosis and treatment selection. We aimed to evaluate a novel optical computed tomography (optical-CT) system as a tool for three-dimensional (3-D) imaging of melanocytic lesions and its ability to discriminate benign from malignant melanocytic lesions while simultaneously determining the thickness of invasive melanoma. Seventeen melanocytic lesions, one hemangioma, and normal skin were assessed immediately after their excision by optical-CT and subsequently underwent histopathological examination. Tomographic reconstructions were performed with a back-propagation algorithm calculating a 3-D map of the total attenuation coefficient (AC). There was a statistically significant difference between melanomas, dysplastic nevi, and non-dysplastic nevi, as indicated by Kruskal-Wallis test. Median AC values were higher for melanomas compared with dysplastic and non-dysplastic nevi. No statistically significant difference was observed when thickness values obtained by optical-CT were compared with histological thickness using a Wilcoxon sighed rank test. Our results suggest that optical-CT can be important for the immediate prehistological evaluation of biopsies, assisting the physician for a rapid assessment of malignancy and of the thickness of a melanocytic lesion.

Spectroscopic detection improves multi-color quantification in fluorescence tomography.

Simultaneous detection of several biological processes in vivo is a common requirement in biomedical and biological applications, and in order to address this issue the use of multiple fluorophores is usually the method of choice. Existing methodologies however, do not provide quantitative feedback of multiple fluorophore concentrations in small animals in vivo when their spectra overlap, especially when imaging the whole body in 3D. Here we present an approach where a spectroscopic module has been implemented into a custom-built Fluorescence Molecular Tomography (FMT) system. In contrast with other multispectral approaches, this multimodal imaging system is capable of recording the fluorescence spectra from each illumination point during a tomographic measurement. In situ spectral information can thus be extracted and used to improve the separation of overlapping signals associated with different fluorophores. The results of this new approach tested on both in vitro and in vivo experiments are presented, proving that accurate recovery of fluorophore concentrations can be obtained from multispectral tomography data even in the presence of high autofluorescence.

Source intensity profile in noncontact optical tomography.

Noncontact optical tomography in reflection mode is often the only possible configuration when imaging the expression of green fluorescent protein (GFP) or other fluorescent proteins in live animals owing to the short penetration depth of visible light. When imaging in reflection mode using noncontact approaches (i.e., without the use of fibers coupled to tissue), correctly accounting for the intensity profile of the source at the surface is a difficult task, usually needing to fit for source positions and/or approximating these to point sources. In this Letter we present a rigorous theoretical approach that directly accounts for the source's intensity profile and verify it using in vivo data from GFP-expressing mice. We show how this approach improves image quality and resolution, while considerably simplifying the forward and inverse problems of the image reconstruction process.

Fluorescence spectroscopy and anisotropy in the analysis of DNA-protein interactions.

Fluorescence spectroscopy can be used as a sensitive non-destructive technique for the characterisation of protein-DNA interactions. A comparison of the intrinsic emission spectra obtained for a protein-DNA complex and for free protein can be informative about the environment of tryptophan and tyrosine residues in the two states. Often there is quenching of the fluorescence intensity of an intrinsic emission spectrum and/or a shift in the wavelength maximum on protein binding to DNA. A step-by-step protocol describes the determination of a DNA-binding curve by measurement of the quenching of the intrinsic protein fluorescence.Fluorescence anisotropy can also be used to obtain a DNA-binding curve if the molecular size of the protein-DNA complex is sufficiently different from the free fluorescing component. Typically an extrinsic fluorophore attached to one or both 5' ends of single-stranded or duplex DNA is used, for this increases the sensitivity of measurement.Fitting of the binding curves, assuming a model, can often yield the stoichiometry and association constant of the interaction. The approach is illustrated using the interaction of the DNA-binding domains (HMG boxes) of mouse Sox-5 and mammalian HMGB1 with short DNA duplexes.