Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.

Evaluation of the RAS signaling network in response to MEK inhibition using organoids derived from a familial adenomatous polyposis patient.

RAS signaling is a promising target for colorectal cancer (CRC) therapy, and a variety of selective inhibitors have been developed. However, their use has often failed to demonstrate a significant benefit in CRC patients. Here, we used patient-derived organoids (PDOs) derived from a familial adenomatous polyposis (FAP) patient to analyze the response to chemotherapeutic agents targeting EGFR, BRAF and MEK. We found that PDOs carrying KRAS mutations were resistant to MEK inhibition, while those harboring the BRAF class 3 mutation were hypersensitive. We used a systematic approach to examine the phosphorylation of RAS effectors using reverse-phase protein array (RPPA) and found increased phosphorylation of MEK induced by binimetinib. A high basal level of ERK phosphorylation and its rebound activation after MEK inhibition were detected in KRAS-mutant PDOs. Notably, the phosphorylation of EGFR and AKT was more closely correlated with that of MEK than that of ERK. Transcriptome analysis identified MYC-mediated transcription and IFN signaling as significantly correlated gene sets in MEK inhibition. Our experiments demonstrated that RPPA analysis of PDOs, in combination with the genome and transcriptome, is a useful preclinical research platform to understand RAS signaling and provides clues for the development of chemotherapeutic strategies.

Utility of a Reverse Phase Protein Array to Evaluate Multiple Biomarkers in Diffuse Large B-Cell Lymphoma.

PURPOSE: Diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is a heterogeneous lymphoma with different clinical manifestations and molecular alterations, and several markers are currently being measured routinely for its diagnosis, subtyping, or prognostication by immunohistochemistry (IHC). Here, the utility of a reverse-phase-protein-array (RPPA) as a novel supportive tool to measure multiple biomarkers for DLBCL diagnosis is validated. EXPERIMENTAL DESIGN: The expression of seven markers (CD5, CD10, BCL2, BCL6, MUM1, Ki-67, and C-MYC) is analyzed by RPPA and IHC using 37 DLBCL tissues, and the correlation between the two methods is determined. To normalize tumor content ratio in the tissues, the raw RPPA values of each marker are adjusted by that of CD20 or PAX-5. RESULTS: The CD20-adjusted data for CD5, MUM1, BCL2, Ki-67, and C-MYC has better correlation with IHC results than PAX-5-adjusted data. Receiver operating characteristic (ROC) analysis reveals that CD5, MUM1, BCL2, and C-MYC exhibit a better sensitivity and specificity >0.750. Furthermore, the CD20-adjusted C-MYC value strongly correlates with that of IHC, and has a particularly high specificity (0.882). CONCLUSIONS AND CLINICAL RELEVANCE: Although further investigation using a large number of DLBCL specimens needs to be conducted, these results suggest that RPPA could be applicable as a supportive tool for determining lymphoma prognosis.

Herbivore-induced indirect defense across bean cultivars is independent of their degree of direct resistance.

We tested the extent to which resistance of common bean (Phaseolus vulgaris) cultivars to the spider mite Tetranychus urticae parallels the extent to which these plants display indirect defenses via the induced attraction of the predatory mite Phytoseiulus persimilis. First, via field and greenhouse trials on 19 commercial bean cultivars, we selected two spider mite-resistant (Naz and Ks41128) and two susceptible (Akthar and G11867) cultivars and measured the spider mite-induced volatiles and the subsequently induced attraction of predatory mites via olfactory choice assays. The two major volatiles, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and (Z)-3-hexenyl-acetate, were induced in the resistant but not in the susceptible cultivars. However, uninfested susceptible cultivars emitted these volatiles at levels similar to those of mite-infested resistant cultivars. Significant induction of several minor components was observed for all four cultivars except for the infested-susceptible cultivar G11867. Both, the spider mite-resistant cultivar Naz and the susceptible cultivar G11867, attracted more predatory mites when they were infested. In contrast, spider mites induced increased emission of two major and five minor volatiles in Ks41128, but predatory mites did not discriminate between infested and uninfested plants. Overall, the attraction of predatory mites appeared to correlate positively with the presence of TMTT and (Z)-3-hexenyl acetate and negatively with ß-caryophyllene and α-pinene in the bean headspace. Taken together, our data suggest that resistance and attraction of natural enemies via induced volatiles are independent traits. We argue that it should be possible to cross predator-attraction promoting traits into resistant cultivars that lack sufficiently inducible indirect defenses.

Temperature-dependent, behavioural, and transcriptional variability of a tritrophic interaction consisting of bean, herbivorous mite, and predator.

Different organisms compensate for, and adapt to, environmental changes in different ways. In this way, environmental changes affect animal-plant interactions. In this study, we assessed the effect of temperature on a tritrophic system of the lima bean, the herbivorous spider mite Tetranychus urticae and the predatory mite Phytoseiulus persimilis. In this system, the plant defends itself against T. urticae by emitting volatiles that attract P. persimilis. Over 20-40 °C, the emission of volatiles by infested plants and the subsequent attraction of P. persimilis peaked at 30 °C, but the number of eggs laid by T. urticae adults and the number of eggs consumed by P. persimilis peaked at 35 °C. This indicates that the spider mites and predatory mites performed best at a higher temperature than that at which most volatile attractants were produced. Our data from transcriptome pyrosequencing of the mites found that P. persimilis up-regulated gene families for heat shock proteins (HSPs) and ubiquitin-associated proteins, whereas T. urticae did not. RNA interference-mediated gene suppression in P. persimilis revealed differences in temperature responses. Predation on T. urticae eggs by P. persimilis that had been fed PpHsp70-1 dsRNA was low at 35 °C but not at 25 °C when PpHsp70-1 expression was very high. Overall, our molecular and behavioural approaches revealed that the mode and tolerance of lima bean, T. urticae and P. persimilis are distinctly affected by temperature variability, thereby making their tritrophic interactions temperature dependent.

Acquired immunity of transgenic torenia plants overexpressing agmatine coumaroyltransferase to pathogens and herbivore pests.

We investigated the ability of transgenic torenia (Scrophulariaceae) plants to resist fungi and arthropod herbivores. Torenia hybrida cv. Summerwave Blue was manipulated to produce Arabidopsis agmatine coumaroyltransferase (AtACT). This catalyses the last step in the biosynthesis of hydroxycinnamic acid amides (HCAAs) which function in defence. Transgenic plants accumulated substantial HCAAs, predominantly p-coumaroylagmatine, and the HCAAs were isomerized from the trans-form to the cis-form in planta. The transgenic line, accumulated the highest amount of endogenous HCAAs (CouAgm at 32.2 µM and total HCAAs at 47.5 µM) and this line was resistant to the necrotrophic fungus, Botrytis cinerea. There was no resistance, however, in their wild-type progenitors or in other transgenic lines accumulating low HCAA amounts. In contrast, the transformants were not significantly resistant to three representative herbivores, Frankliniella occidentalis, Aphis gossypii, and Tetranychus ludeni.

The composite effect of transgenic plant volatiles for acquired immunity to herbivory caused by inter-plant communications.

A blend of volatile organic compounds (VOCs) emitted from plants induced by herbivory enables the priming of defensive responses in neighboring plants. These effects may provide insights useful for pest control achieved with transgenic-plant-emitted volatiles. We therefore investigated, under both laboratory and greenhouse conditions, the priming of defense responses in plants (lima bean and corn) by exposing them to transgenic-plant-volatiles (VOCos) including (E)-ß-ocimene, emitted from transgenic tobacco plants (NtOS2) that were constitutively overexpressing (E)-ß-ocimene synthase. When lima bean plants that had previously been placed downwind of NtOS2 in an open-flow tunnel were infested by spider mites, they were more defensive to spider mites and more attractive to predatory mites, in comparison to the infested plants that had been placed downwind of wild-type tobacco plants. This was similarly observed when the NtOS2-downwind maize plants were infested with Mythimna separata larvae, resulting in reduced larval growth and greater attraction of parasitic wasps (Cotesia kariyai). In a greenhouse experiment, we also found that lima bean plants (VOCos-receiver plants) placed near NtOS2 were more attractive when damaged by spider mites, in comparison to the infested plants that had been placed near the wild-type plants. More intriguingly, VOCs emitted from infested VOCos-receiver plants affected their conspecific neighboring plants to prime indirect defenses in response to herbivory. Altogether, these data suggest that transgenic-plant-emitted volatiles can enhance the ability to prime indirect defenses via both plant-plant and plant-plant-plant communications.

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling.

BACKGROUND: Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable. RESULTS: To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response. CONCLUSIONS: These results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.

Polyamines and jasmonic acid induce plasma membrane potential variations in Lima bean.

Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone. JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex. Here, we report that a plasma membrane potential (V(m)) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter V(m) but the perfusion of JA and the polyamines significantly increased Cad and Put V(m) depolarization. When JA was perfused with polyamines, a negative correlation was found between V(m) depolarization and the number of amino group of the polyamines tested.

Exogenous polyamines elicit herbivore-induced volatiles in lima bean leaves: involvement of calcium, H2O2 and Jasmonic acid.

We investigated the role of polyamines (PAs) in lima bean (Phaseolus lunatus) leaves on the production of herbivorous mite (Tetranychus urticae)-induced plant volatiles that attract carnivorous natural enemies of the herbivores. To do this, we focused on the effects of the exogenous PAs [cadaverine, putrescine, spermidine and spermine (Spm)] on the production of volatiles, H(2)O(2) and jasmonic acid (JA) and the levels of defensive genes, cytosolic calcium and reactive oxygen species (ROS). Among the tested PAs, Spm was the most active in inducing the production of volatile terpenoids known to be induced by T. urticae. An increase in JA levels was also found after Spm treatment, indicating that Spm induces the biosynthesis of JA, which has been shown elsewhere to regulate the production of some volatile terpenoids. Further, treatment with JA and Spm together resulted in greater volatile emission than that with JA alone. In a Y-tube olfactometer, leaves treated with Spm + JA attracted more predatory mites (Phytoseiulus persimilis) than those treated with JA alone. After treatment with Spm + JA, no effects were found on the enzyme activity of polyamine oxidase and copper amine oxidase. However, induction of calcium influx and ROS production, and increased enzyme activities and gene expression for NADPH oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase were found after treatment with Spm + JA. These results indicate that Spm plays an important role in the production of T. urticae-induced lima bean leaf volatiles.

Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana.

Hydroxycinnamic acid amides (HCAAs) are secondary metabolites involved in the defense of plants against pathogens. Here, we report the first identification of HCAAs, p-coumaroylagmatine, feruloylagmatine, p-coumaroylputrescine and feruloylputrescine, in Arabidopsis thaliana rosette leaves infected with Alternaria brassicicola and the assignment of At5g61160 as the agmatine coumaroyltransferase (AtACT) that catalyzes the last reaction in the biosynthesis of the HCAAs. Feeding experiments with putative labeled precursors revealed that the four HCAAs were synthesized from hydroxycinnamic acids and agmatine or putrescine. AtACT gene function was identified from an analysis of a mutant that did not accumulate HCAAs. In wild-type Arabidopsis, AtACT transcripts markedly increased in response to A. brassicicola infection. Enzymatic activity that catalyzes the synthesis of the HCAAs was confirmed in vitro by using a recombinant AtACT expressed in Escherichia coli. The Atact mutant was susceptible to infection by A. brassicicola, indicating that HCAAs are responsible for defense against pathogens in A. thaliana.

Biochemical and spectroscopic properties of cyanide-insensitive quinol oxidase from Gluconobacter oxydans.

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.