Novel circulating tumor cell-based blood test for the assessment of PD-L1 protein expression in treatment-naïve, newly diagnosed patients with non-small cell lung cancer.

We evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I-IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis.

Novel SUMO-Protease SENP7S Regulates ß-catenin Signaling and Mammary Epithelial Cell Transformation.

SUMO post-translational modification of proteins or SUMOylation ensures normal cell function. Disruption of SUMO dynamics prompts various pathophysiological conditions, including cancer. The burden of deSUMOylating the large SUMO-proteome rests on 6 full-length mammalian SUMO-proteases or SENP. While multiple SENP isoforms exist, the function of these isoforms remains undefined. We now delineate the biological role of a novel SENP7 isoform SENP7S in mammary epithelial cells. SENP7S is the predominant SENP transcript in human mammary epithelia but is significantly reduced in precancerous ductal carcinoma in situ and all breast cancer subtypes. Like other SENP family members, SENP7S has SUMO isopeptidase activity but unlike full-length SENP7L, SENP7S is localized in the cytosol. In vivo, SUMOylated ß-catenin and Axin1 are both SENP7S-substrates. With knockdown of SENP7S in mammary epithelial cells, Axin1-ß-catenin interaction is lost and ß-catenin escapes ubiquitylation-dependent proteasomal degradation. SUMOylated ß-catenin accumulates at the chromatin and activates multiple oncogenes. Hence, non-tumorigenic MCF10-2A cells with reduced SENP7S exhibit greater cell proliferation and anchorage-dependent growth. SENP7S depletion directly potentiates tumorigenic properties of MCF10-2A cells with induction of anchorage-independent growth and self-renewal in 3D-spheroid conditions. Collectively, the results identify SENP7S as a novel mediator of ß-catenin signaling and normal mammary epithelial cell physiology.

SUMOylation of HP1α supports association with ncRNA to define responsiveness of breast cancer cells to chemotherapy.

Epigenetic reprogramming allows cancer cells to bypass normal checkpoints and potentiate aberrant proliferation. Several chromatin regulators are subject to reversible SUMO-modification but little is known about how SUMOylation of chromatin-remodelers modulates the cancer epigenome. Recently, we demonstrated that SUMO-protease SENP7L is upregulated in aggressive BCa and maintains hypoSUMOylated heterochromatin protein 1-α (HP1α). Canonical models define HP1α as a "reader" of repressive H3K9m3 marks that supports constitutive heterochromatin. It is unclear how SUMOylation affects HP1α function in BCa cells. This report shows HP1α SUMO-dynamics are closely regulated in a complex with SENP7L and SUMO-E3 Polycomb-2 (PC2/CBX4). This complex accumulates at H3K9m3 sites, hypoSUMOylates HP1α and PC2, and reduces PC2's SUMO-E3 activity. HyperSUMO conditions cause complex dissociation, SUMOylation of PC2 and HP1α, and recruitment of SUMOylated HP1α to multiple DNA-repair genes including Rad51C. SUMOylated HP1α's enrichment at euchromatin requires chromatin-bound non-coding RNA (ncRNA), reduces Rad51C protein, and increases DNA-breaks in BCa cells. Hence, HP1α SUMOylation and consistently low SENP7L increase efficacy of DNA-damaging chemotherapeutic agents. BCa patients on chemotherapy that express low SENP7L exhibit greater survival rates than patients with high SENP7L. Collectively, these studies suggest that SUMOylated HP1α is a critical epigenetic-regulator of DNA-repair in BCa that could define chemotherapy responsiveness.

Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

Three distinct modes of Mec1/ATR and Tel1/ATM activation illustrate differential checkpoint targeting during budding yeast early meiosis.

Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.

Differential expression of SUMO-specific protease 7 variants regulates epithelial-mesenchymal transition.

Two Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) variants are naturally expressed in breast epithelia. Breast cancer (BCa) onset down-regulates the short SENP7 splice variant (SENP7S) and enhances the long transcript (SENP7L). Here, we show that SENP7L induction promotes gene expression profiles that favor aberrant proliferation and initiate epithelial-mesenchymal transition (EMT). SENP7L exhibits an interaction domain for the epigenetic remodeler heterochromatin protein 1 α (HP1α) and isopeptidase activity against SUMO-modified HP1α. Loss of this interaction domain, as observed with SENP7S, favors HP1α SUMOylation. SUMOylated HP1α is enriched at E2F-responsive and mesenchymal gene promoters, silences transcription of these genes, and promotes cellular senescence. Elevated SENP7L renders HP1α hypo-SUMOylated, which relieves transcriptional repression of the same genes and concurrently decreases transcription of epithelial-promoting genes via an HP1α-independent mechanism. Consequently, SENP7L levels correlate with EMT, motility, and invasiveness of BCa cells. Stable knockdown of elevated SENP7L levels lessens the dissemination of highly metastatic BCa cells to the lungs from primary implantation sites in in vivo studies. Thus, differential splicing of the SENP7 regulates either tumor suppression or progression.

Paxillin phosphorylation by JNK and p38 is required for NFAT activation.

Paxillin is an adaptor protein associated with focal adhesion complex, and is activated by tyrosine phosphorylation through focal adhesion kinase (FAK) and Src kinase. Recent studies reveal that serine phosphorylation of paxillin by JNK and p38 MAPK is essential for cell migration or neurite extension, but their cellular targets remain unclear. In this study, we examined the requirement of paxillin phosphorylation by p38 MAPK or JNK in T-cell motility and activation using paxillin mutants at the respective phosphorylation sites, Ser85, and Ser178. (S85A)-paxillin, (S178A)-paxillin, or (S85A/S178A)-paxillin inhibited the motility of NIH/3T3 fibroblasts, but did not interfere with T-cell migration and integrin-mediated T-cell adhesion. In contrast, activation of T cells was effectively suppressed by (S85A/S178A)-paxillin. Transgenic (S85A/S178A)-paxillin expression inhibited T-cell proliferation and reduced the production of IL-2, IFN-γ, and IL-4. In searching for signals modulated by (S85A/S178A)-paxillin, we found that NFAT activation was specifically blocked by (S85A/S178A)-paxillin. This could be partly attributed to diminished stromal interaction molecule 1 (STIM1) expression and attenuated TCR-induced Ca(2+) influx. Our results demonstrate that dual phosphorylation of paxillin by JNK and p38 MAPK is essential for T-cell activation and suggest that NFAT is a functional target of the JNK/p38 phosphorylated paxillin.

SUMO-specific protease 1 is critical for early lymphoid development through regulation of STAT5 activation.

Small ubiquitin-like modifier (SUMO) modification has emerged as an important regulatory mechanism during embryonic development. However, it is not known whether SUMOylation plays a role in the development of the immune system. Here, we show that SUMO-specific protease 1 (SENP1) is essential for the development of early T and B cells. STAT5, a key regulator of lymphoid development, is modified by SUMO-2 and is specifically regulated by SENP1. In the absence of SENP1, SUMO-2 modified STAT5 accumulates in early lymphoid precursors, resulting in a block in its acetylation and subsequent signaling. These results demonstrate a crucial role of SENP1 in the regulation of STAT5 activation during early lymphoid development.

Balancing act during development: lessons from a SUMO-less SF-1.

When a transcription factor is modified by small ubiquitin-like modifier (SUMO), this usually represses its transcriptional activity. In this issue of Developmental Cell, Lee et al. (2011) use a knockin mouse model to show that SUMO-less SF-1 binds and activates inappropriate targets, causing changes in cell fates and endocrine abnormalities.

Genetic requirements and meiotic function of phosphorylation of the yeast axial element protein Red1.

The synaptonemal complex (SC) is a meiosis-specific tripartite structure that forms between two homologous chromosomes; it consists of a central region and two parallel lateral elements. Lateral elements also are called axial elements prior to synapsis. In Saccharomyces cerevisiae, Red1, Hop1, and Mek1 are structural components of axial/lateral elements. The red1/mek1/hop1 mutants all exhibit reduced levels of interhomolog recombination and produce no viable spores. Red1 is a phosphoprotein. Several earlier reports proposed that phosphorylated Red1 plays important roles in meiosis, including in signaling meiotic DNA damage or in preventing exit from the pachytene chromosomes. We report here that the phosphorylation of Red1 is carried out in CDC28-dependent and CDC28-independent manners. In contrast to previous results, we found Red1 phosphorylation to be independent of meiotic DNA recombination, the Mec1/Tel1 DNA damage checkpoint kinases, and the Mek1 kinase. To functionally validate the phosphorylation of Red1, we mapped the phosphorylation sites on this protein. A red1(14A) mutant showing no detectable Red1 phosphorylation did not exhibit decreased sporulation efficiency, defects in viable spore production, or defects in meiotic DNA damage checkpoints. Thus, our results suggest that the phosphorylation of Red1 is not essential for its functions in meiosis.

Yeast axial-element protein, Red1, binds SUMO chains to promote meiotic interhomologue recombination and chromosome synapsis.

The synaptonemal complex (SC) is a tripartite protein structure consisting of two parallel axial elements (AEs) and a central region. During meiosis, the SC connects paired homologous chromosomes, promoting interhomologue (IH) recombination. Here, we report that, like the CE component Zip1, Saccharomyces cerevisiae axial-element structural protein, Red1, can bind small ubiquitin-like modifier (SUMO) polymeric chains. The Red1-SUMO chain interaction is dispensable for the initiation of meiotic DNA recombination, but it is essential for Tel1- and Mec1-dependent Hop1 phosphorylation, which ensures IH recombination by preventing the inter-sister chromatid DNA repair pathway. Our results also indicate that Red1 and Zip1 may directly sandwich the SUMO chains to mediate SC assembly. We suggest that Red1 and SUMO chains function together to couple homologous recombination and Mec1-Tel1 kinase activation with chromosome synapsis during yeast meiosis.

Temporal quantitative proteomics of Saccharomyces cerevisiae in response to a nonlethal concentration of furfural.

Furfural, one of the main inhibitory compounds in lignocellulosic hydrolytes, inhibits the growth and ethanol production rate of yeast. To get a global view of the dynamic expression pattern of proteins in Saccharomyces cerevisiae during the fermentation with the introduction of 8 g/L furfural, the protein samples were taken before the addition of furfural, during the initial phase of furfural conversion and immediately after the conversion of furfural for comparative proteomic analysis with iTRAQ on a LC-ESI-MS/MS instrument. A comparison of the temporal expression pattern of 107 proteins related to protein synthesis between the reference cultures and the furfural-treated cultures showed that a temporal downregulation of these proteins was retarded after the addition of furfural. The expression levels of 20 enzymes in glucose fermentation and 5 enzymes in the tricarboxylic acid cycle were reduced by furfural, with notably delayed temporal downregulations of Glk1p, Tdh1p, Eno1p and Aco1p, which is correlated to the reduced ethanol formation rate and glucose consumption rate by 66.7 and 60.4%, respectively. In the presence of furfural, proteins catalyzing the upper part of the super pathway of sulfur amino acid biosynthesis were repressed at all time points, which is related to the inhibited growth of furfural-treated yeast. The expressions of 18 proteins related to stress response showed increased trends, including several highly induced heat shock proteins and proteins related to cellular signaling pathways.

Comparative proteomic analysis of tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to furfural, a lignocellulosic inhibitory compound.

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, (18)O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD(+), and NADH/NAD(+) were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology.

The tumor suppressor death-associated protein kinase targets to TCR-stimulated NF-kappa B activation.

Death-associated protein kinase (DAPK) is a unique multidomain kinase acting both as a tumor suppressor and an apoptosis inducer. The molecular mechanism underlying the effector function of DAPK is not fully understood, while the role of DAPK in T lymphocyte activation is mostly unknown. DAPK was activated after TCR stimulation. Through the expression of a dominant-negative and a constitutively active form of DAPK in T cells, we found that DAPK negatively regulated T cell activation. DAPK markedly affected T cell proliferation and IL-2 production. We identified TCR-induced NF-kappaB activation as a target of DAPK. In contrast, IL-1beta- and TNF-alpha-triggered NF-kappaB activation was not affected by DAPK. We further found that DAPK selectively modulated the TCR-induced translocation of protein kinase Ctheta, Bcl-10, and IkappaB kinase into membrane rafts. Notably, the effect of DAPK on the raft entry was specific for the NF-kappaB pathway, as other raft-associated molecules, such as linker for activation of T cells, were not affected. Our results clearly demonstrate that DAPK is a novel regulator targeted to TCR-activated NF-kappaB and T cell activation.

Tying SUMO modifications to dynamic behaviors of chromosomes during meiotic prophase of Saccharomyces cerevisiae.

In budding yeast Saccharomyces cerevisiae, centromeres and telomeres are tethered to the nuclear envelope during premeiotic interphase. Immediately after cells enter meiotic prophase, chromosomes undergo global reorganization, including bouquet formation (telomere clustering), non-homologous centromere coupling, homologous pairing, and assembly/disassembly of synaptonemal complexes. These chromosome dynamics have been implicated in promoting pairing, synapsis, crossover DNA recombination and segregation between homologous chromosomes. This review discusses recent studies related to the role of small ubiquitin-like modifier (SUMO) modification in controlling the overall budding yeast chromosome dynamics during meiotic prophase.

Reactive oxygen species promote raft formation in T lymphocytes.

Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.

SUMO modifications control assembly of synaptonemal complex and polycomplex in meiosis of Saccharomyces cerevisiae.

The synaptonemal complex (SC) is a proteinaceous complex that apparently mediates synapsis between homologous chromosomes during meiotic prophase. In Saccharomyces cerevisiae, the Zip1 protein is the integral component of the SC. In the absence of a DNA double-strand break or the SC initiation protein Zip3, Zip1 proteins aggregate to form a polycomplex (PC). In addition, Zip1 is also responsible for DSB-independent nonhomologous centromere coupling at early meiotic prophase. We report here that Zip3 is a SUMO (small ubiquitin-related modifier) E3 ligase and that Zip1 is a binding protein for SUMO-conjugated products. Our results also suggest that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates (e.g., Top2) to promote nonhomologous centromere coupling. At and after mid-prophase, the Zip1 protein begins to associate with Zip3-dependent Smt3 conjugates (e.g., Red1) along meiotic chromosomes in the wild-type cell to form SCs and with Smt3 polymeric chains in the zip3 mutant to form PCs.

Initiation of RNA replication of cloned Taiwan-3 isolate of hepatitis delta virus genotype II in cultured cells.

Hepatitis delta virus (HDV) genotype II is the predominant genotype in Taiwan and is associated with less progressive disease than genotype I. Although the Taiwan-3 (T3) clone was the first genotype II HDV isolated in Taiwan, its replication in cultured cells has not previously been established. Here, we demonstrate that cloned T3 HDV is capable of replicating in cultured cells. Furthermore, we show that: (1). the replication level of T3 clones is 100-fold lower than that of a genotype I HDV prototype of Italian origin; (2). both forms of the genotype II T3 delta antigen are expressed; and (3). T3 HDV undergoes RNA editing during replication, with 4.8% of the T3 genomes showing evidence of editing. The low level of RNA replication may be related to the milder clinical outcomes of genotype II HDV infections.