Clinical applications of circulating biomarkers in non-small cell lung cancer.

Despite recent advances in cancer diagnostics and treatment, the mortality associated with lung cancer is still the highest in the world. Late-stage diagnosis, often accompanied by metastasis, is a major contributor to the high mortality rates, emphasizing the urgent need for reliable and readily accessible diagnostic tools that can detect biomarkers unique to lung cancer. Circulating factors, such as circulating tumor DNA and extracellular vesicles, from liquid biopsy have been recognized as diagnostic or prognostic markers in lung cancer. Numerous clinical studies are currently underway to investigate the potential of circulating tumor DNA, circulating tumor RNA, exosomes, and exosomal microRNA within the context of lung cancer. Those clinical studies aim to address the poor diagnostics and limited treatment options for lung cancer, with the ultimate goal of developing clinical markers and personalized therapies. In this review, we discuss the roles of each circulating factor, its current research status, and ongoing clinical studies of circulating factors in non-small cell lung cancer. Additionally, we discuss the circulating factors specifically found in lung cancer stem cells and examine approved diagnostic assays designed to detect circulating biomarkers in lung cancer patients.

Mitotic protein kinase-driven crosstalk of machineries for mitosis and metastasis.

Accumulating evidence indicates that mitotic protein kinases are involved in metastatic migration as well as tumorigenesis. Protein kinases and cytoskeletal proteins play a role in the efficient release of metastatic cells from a tumor mass in the tumor microenvironment, in addition to playing roles in mitosis. Mitotic protein kinases, including Polo-like kinase 1 (PLK1) and Aurora kinases, have been shown to be involved in metastasis in addition to cell proliferation and tumorigenesis, depending on the phosphorylation status and cellular context. Although the genetic programs underlying mitosis and metastasis are different, the same protein kinases and cytoskeletal proteins can participate in both mitosis and cell migration/invasion, resulting in migratory tumors. Cytoskeletal remodeling supports several cellular events, including cell division, movement, and migration. Thus, understanding the contributions of cytoskeletal proteins to the processes of cell division and metastatic motility is crucial for developing efficient therapeutic tools to treat cancer metastases. Here, we identify mitotic kinases that function in cancer metastasis as well as tumorigenesis. Several mitotic kinases, namely, PLK1, Aurora kinases, Rho-associated protein kinase 1, and integrin-linked kinase, are considered in this review, as an understanding of the shared machineries between mitosis and metastasis could be helpful for developing new strategies to treat cancer.

CRISPR-mediated promoter de/methylation technologies for gene regulation.

DNA methylation on cytosines of CpG dinucleotides is well established as a basis of epigenetic regulation in mammalian cells. Since aberrant regulation of DNA methylation in promoters of tumor suppressor genes or proto-oncogenes may contribute to the initiation and progression of various types of human cancer, sequence-specific methylation and demethylation technologies could have great clinical benefit. The CRISPR-Cas9 protein with a guide RNA can target DNA sequences regardless of the methylation status of the target site, making this system superb for precise methylation editing and gene regulation. Targeted methylation-editing technologies employing the dCas9 fusion proteins have been shown to be highly effective in gene regulation without altering the DNA sequence. In this review, we discuss epigenetic alterations in tumorigenesis as well as various dCas9 fusion technologies and their usages in site-specific methylation editing and gene regulation.

Phosphorus and Nitrogen Drive the Seasonal Dynamics of Bacterial Communities in Pinus Forest Rhizospheric Soil of the Qinling Mountains.

The temporal distribution patterns of bacterial communities, as an important group in mountain soil, are affected by various environmental factors. To improve knowledge regarding the successional seasonal dynamics of the mountain soil bacterial communities, the rhizospheric soil of a 30-year-old natural secondary Pinus tabulaeformis forest, located in the high-altitude (1900 m a.s.l.) of the temperate Qinling Mountains, was sampled and studied during four different seasons. The bacterial community composition and structure in the rhizospheric soil were studied using an Illumina MiSeq Sequencing platform. Furthermore, the edaphic properties and soil enzymatic activities (urease, phosphatase, and catalase) were measured in order to identify the main impact factors on the soil bacterial community. According to the results, all of the edaphic properties and soil enzymatic activities were significantly affected by the seasonal changes, except for the C/N ratio. Although the biomasses of soil bacterial communities increased during the summer and autumn (warm seasons), their Shannon diversity and Pielou's evenness were decreased. Proteobacteria, Acidobacteria, Actinobacteria, Planctomycetes, and Bacteroidetes were the predominant bacterial groups in all of the soil samples, and the genera of Ktedonobacter, Sphingobium as well as an unclassified member of the Ktedonobacteria were the keystone taxa. The composition and structure of soil bacterial communities were strongly impacted by the edaphic properties, especially the temperature, moisture, ammoniacal nitrogen, available phosphorus and total phosphorus which were the crucial factors to drive the temporal distribution of the soil bacterial community and diversity. In conclusion, the soil temperature, moisture and the nutrients N and P were the crucial edaphic factors for shaping the rhizospheric soil bacterial communities as season and climate change in a P. tabulaeformis forest of Qinling Mountains.

Roles of SALL2 in tumorigenesis.

The proteins p150Sal2 (product of SALL2) and p53 share growth arrest and pro-apoptotic functions by independently inducing p21Cip1/Waf1 and BAX, and both proteins are targeted by the human papilloma virus E6 protein, leading to blockage of growth arrest in infected cells. Loss of both p53 and Sall2 in mice causes significantly higher mortality and metastasis rates compared with p53 single mutant mice. Therefore, p150Sal2 seems to have strong potential as a novel cancer biomarker for early diagnosis and risk prediction. Loss of SALL2 expression is observed in many cases of human serous ovarian carcinoma, whereas normal ovarian epithelial cells maintain high levels of the p150Sal2 protein, supporting an important tumor suppressive role for p150Sal2 in the human ovary. In contrast, p150Sal2 is a transcription factor required to convert differentiated glioblastoma cells into stem-like tumor-propagating cells, suggesting that its functional roles are dependent on tissue types and cellular context. The function of p150Sal2 in normal and diseased cells and possible therapeutic approaches are discussed in this review.

Promoter methylation of the SALL2 tumor suppressor gene in ovarian cancers.

The SALL2 gene product and transcription factor p150 were first identified in a search for tumor suppressors targeted for inactivation by the oncogenic mouse polyoma virus. SALL2 has also been identified as a cellular quiescence factor, essential for cells to enter and remain in a state of growth arrest under conditions of serum deprivation. p150 is a transcriptional activator of p21(Cip1/Waf1) and BAX, sharing important growth arrest and proapoptotic properties with p53. It also acts as a repressor of c-myc. Restoration of SALL2 expression in cells derived from a human ovarian carcinoma (OVCA) suppresses growth of the cells in immunodeficient mice. Here we examine the pattern of p150 expression in the normal human ovary, in OVCA-derived cell lines and in primary ovarian carcinomas. Immunohistochemical staining showed that p150 is highly expressed in surface epithelial cells of the normal human ovary. Expression is exclusively from the P2 promoter governing the E1A splice variant of p150. The P2 promoter is CpG-rich and susceptible to methylation silencing. p150 expression was restored in OVCA cell lines following growth in the presence of 5-azacytidine. In a survey of 210 cases of OVCA, roughly 90% across major and minor histological types failed to show expression of the protein. Immunological and biochemical approaches were used to show hypermethylation of the SALL2 P2 promoter in OVCA-derived cell lines and in a majority of primary tumors. These results bring together molecular biological and clinical evidence in support of a role of SALL2 as a suppressor of ovarian cancers.

Nek1 and TAZ interact to maintain normal levels of polycystin 2.

Polycystic kidney disease (PKD) in mice can arise from defects in Nek kinases, which participate in ciliogenesis. PKD can also arise from loss of the protein TAZ, an adaptor protein in the E3 ubiquitin ligase complex that targets the ciliary protein polycystin 2 (PC2) for degradation, but whether Nek and TAZ contribute to the same biochemical pathway is unknown. Here, we report that the nimA-related protein kinase Nek1 phosphorylates TAZ at a site essential for the ubiquitination and proteasomal degradation of PC2. Loss of Nek1 leads to underphosphorylation of TAZ, thereby promoting the abnormal accumulation of PC2. Furthermore, TAZ targets Nek1 for degradation. These data suggest that TAZ and Nek1 constitute a negative feedback loop linked through phosphorylation and ubiquitination and that the interaction of Nek1 and TAZ maintain PC2 at the level needed for proper ciliogenesis.

DNA-binding and regulatory properties of the transcription factor and putative tumor suppressor p150(Sal2).

The product of the SALL2 protein p150(Sal2) is a multi-zinc finger transcription factor with growth arrest and proapoptotic functions that overlap those of p53. Its DNA-binding properties are unknown. We have used a modified SELEX procedure with purified p150(Sal2) and a pool of oligonucleotides of random sequence to identify those that are bound preferentially by p150(Sal2). The consensus sequence for optimal binding in vitro is GGG(T/C)GGG, placing p150(Sal2) among a large group of GC box-binding proteins including the Sp1 family of transcription factors. A triple zinc finger motif in p150(Sal2) similar to that in Sp1 is required for DNA binding. p150(Sal2) and Sp1 show evidence of co-operative binding in vitro and of interaction in vivo. p150(Sal2), a known activator of the CDK inhibitor p21(Cip1/Waf1) (p21), binds to regions of the human p21 promoter that contain variations of the consensus sequence in multiple copies. p150(Sal2) is also shown to bind to the BAX promoter with similar elements and to activate its expression following an apoptotic stimulus. These results demonstrate binding of p150(Sal2) to two natural promoters with GC elements related to the optimal binding sequence defined in vitro and whose regulation is important for suppression of tumor growth.

Transcriptional and post-translational regulation of the quiescence factor and putative tumor suppressor p150(Sal2).

The evolutionarily conserved SALL genes encode transcription factors with roles in embryonic development. The product of the SALL2 gene was first identified as a binding partner of the mouse polyoma virus large T antigen and later shown to possess tumor suppressor-like functions. Independent studies identified SALL2 as a factor regulating the quiescent state in human fibroblasts. Here, we investigate factors that regulate the expression of SALL2 and turnover of p150(Sal2) in growing vs. resting cells. The transcription factor AP4 increases along with SALL2 in quiescent cells and positively regulates SALL2 expression. TGFß effectively inhibits expression of SALL2 and its regulator AP4 when added to quiescent fibroblasts. TGFß repression of SALL2 and AP4 is independent of the induction of connective tissue growth factor (CTGF) by TGFß. p150(Sal2) disappears rapidly on restoration of serum. In both growing fibroblasts and established ovarian surface epithelial cells, p150(Sal2) undergoes polyubiquitination and proteosomal degradation. A CUL4/DDB1 E3 ligase containing RBBP7 as the p150(Sal2) receptor has been identified as mediating the destruction of p150(Sal2) as cells transition from a quiescent to an actively growing state.

Murine Polyomavirus encodes a microRNA that cleaves early RNA transcripts but is not essential for experimental infection.

MicroRNAs are small regulatory RNAs that post-transcriptionally regulate gene expression and can be encoded by viral as well as cellular genomes. The functions of most viral miRNAs are unknown and few have been studied in an in vivo context. Here we show that the murine polyomavirus (PyV) encodes a precursor microRNA that is processed into two mature microRNAs, both of which are active at directing the cleavage of the early PyV mRNAs. Furthermore, we identify a deletion mutant of polyomavirus that is defective in encoding the microRNAs. This mutant replicates normally and transforms cultured cells with efficiencies comparable to wildtype PyV. The miRNA mutant is competent to establish a transient infection of mice following parenteral inoculation, and is cleared post infection at approximately the same rate as the wildtype virus. In addition, under these laboratory conditions, we observe no differences in anti-viral CD8 T cell responses. These results indicate that PyV miRNA expression is not essential for infection of cultured cells or experimentally inoculated mice, and raise the possibility that its role in natural infection might involve aspects of acquisition or spread that are not recapitulated by experimental inoculation.

Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature.

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic potato plants with enhanced tolerance to environmental stress, the genes of both Cu/Zn superoxide dismutase and ascorbate peroxidase were expressed in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants). SSA plants showed enhanced tolerance to 250 microM methyl viologen, and visible damage in SSA plants was one-fourth that of non-transgenic (NT) plants that were almost destroyed. In addition, when SSA plants were treated with a high temperature of 42 degrees C for 20 h, the photosynthetic activity of SSA plants decreased by only 6%, whereas that of NT plants decreased by 29%. These results suggest that the manipulation of the antioxidative mechanism of the chloroplasts may be applied in the development of industrial transgenic crop plants with increased tolerance to multiple environmental stresses.

Mechanistic investigation and implications of photodynamic therapy induction of vascular endothelial growth factor in prostate cancer.

Photodynamic therapy (PDT) is now an approved therapeutic modality, and induction of vascular endothelial growth factor (VEGF) following subcurative PDT is of concern as VEGF may provide a survival stimulus to tumors. The processes that limit the efficacy of PDT warrant investigation so that mechanism-based interventions may be developed. This study investigates VEGF increase following subcurative PDT using the photosensitizer benzoporphyrin derivative (BPD) both in an in vitro and in an orthotopic model of prostate cancer using the human prostate cancer cell line LNCaP. The two subcurative doses used, 0.25 and 0.5 J/cm(2), mimicked subcurative PDT and elicited a 1.6- and 2.1-fold increase, respectively, in secreted VEGF 24 hours following PDT. Intracellular VEGF protein measurement and VEGF mRNA showed a 1.4- and 1.6-fold increase only at 0.5 J/cm(2). In vivo subcurative PDT showed an increase in VEGF by both immunohistochemistry and ELISA. In vitro analysis showed no activation of hypoxia-inducible factor-1alpha (HIF-1alpha) or cyclooxygenase-2 (COX-2) following subcurative PDT; furthermore, small interfering RNA inhibition of HIF-1alpha and COX-2 inhibitor treatment had no effect on PDT induction of VEGF. PDT in the presence of phosphatidylinositol 3-kinase/AKT inhibitor or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor still induced VEGF. However, subcurative PDT increased phosphorylated p38 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase. The p38 MAPK inhibitor abolished PDT induction of VEGF. The results establish the importance of VEGF in subcurative BPD-PDT of prostate cancer and suggest possible molecular pathways for its induction. These findings should provide the basis for the development of molecular-based interventions for enhancing PDT and merit further studies.