Tumor dormancy has important implications for early detection and treatment of cancer. Lack of experimental models and limited clinical accessibility constitute major obstacles to the molecular characterization of dormant tumors. We have developed models in which human tumors remain dormant for a prolonged period of time (>120 days) until they switch to rapid growth and become strongly angiogenic. These angiogenic tumors retain their ability to grow fast once injected in new mice. We hypothesized that dormant tumors undergo a stable genetic reprogramming during their switch to the fast-growing phenotype. Genomewide transcriptional analysis was done to dissect the molecular mechanisms underlying the switch of dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors. A consensus expression signature distinguishing all four dormant versus switched fast-growing tumors was generated. In alignment with our phenotypic observation, the angiogenesis process was the most significantly affected functional gene category. The switch of dormant tumors was associated with down-regulation of angiogenesis inhibitor thrombospondin and decreased sensitivity of angiogenic tumors to angiostatin. The conversion of dormant tumors to exponentially growing tumors was also correlated with regulation and activation of pathways not hitherto linked to tumor dormancy process, such as endothelial cell-specific molecule-1, 5 ¶-ecto-nucleotidase, tissue inhibitor of metalloproteinase-3, epidermal growth factor receptor, insulin-like growth factor receptor, and phosphatidylinositol 3-kinase signaling. Further, novel dormancy-specific biomarkers such as H2BK and Eph receptor A5 (EphA5) were discovered. EphA5 plasma levels in mice and mRNA levels in tumor specimens of glioma patients correlated with diseases stage. These data will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for development of dormancy-promoting tumor therapy strategies. [Cancer Res 2009;69(3):836-44]

Transfer of cellular material via tunneling nanotubes (TNT) was recently discovered as a novel mechanism for intercellular communication. The role of intercellular exchange in communication of renal epithelium is not known. Here we report extensive spontaneous intercellular exchange of cargo vesicles and organelles between primary human proximal tubular epithelial cells (RPTEC). Cells were labeled with two different quantum dot nanocrystals (Qtracker 605 or 525) and intercellular exchange was quantified by high-throughput fluorescence imaging and FACS analysis. In co-culture, a substantial fraction of cells (67.5%) contained both dyes indicating high levels of spontaneous intercellular exchange in RPTEC. The double positive cells could be divided into three categories based on the preponderance of 605 Qtracker (46.30%), 525 Qtracker (48.3%) and approximately equal content of both Qtrackers (4.57%). The transfer of mitochondria between RPTECs was also detected using an organelle specific dye. Inhibition of TNT genesis by actin polymerization inhibitor (Latrunculin B) markedly reduced intercellular exchange (>60%) suggesting that intercellular exchange in RPTEC was in part mediated via TNT-like structures. In contrast, induction of cellular stress by Zeocin treatment increased tube-genesis in RPTEC. Our data indicates an unexpected dynamic of intercellular communication between RPTEC by exchange of cytosolic material, which may play an important role in renal physiology.

Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.

Tumor dormancy refers to a critical stage in cancer development in which tumor cells remain occult for a prolonged period of time until they eventually progress and become clinically apparent. We previously showed that the switch of dormant tumors to fast-growth is angiogenesis dependent and requires a stable transcriptional reprogramming in tumor cells. Considering microRNAs (miRs) as master regulators of transcriptome, we sought to investigate their role in the control of tumor dormancy. We report here the identification of a consensus set of 19 miRs that govern the phenotypic switch of human dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors to fast-growth. Loss of expression of dormancy-associated miRs (DmiRs, 16/19) was the prevailing regulation pattern correlating with the switch of dormant tumors to fast-growth. The expression pattern of two DmiRs (miR-580 and 190) was confirmed to correlate with disease stage in human glioma specimens. Reconstitution of a single DmiR (miR-580, 588 or 190) led to phenotypic reversal of fast-growing angiogenic tumors towards prolonged tumor dormancy. Of note, 60% of angiogenic glioblastoma and 100% of angiogenic osteosarcoma over-expressing miR190 remained dormant during the entire observation period of ∼ 120 days. Next, the ability of DmiRs to regulate angiogenesis and dormancy-associated genes was evaluated. Transcriptional reprogramming of tumors via DmiR-580, 588 or 190 over-expression resulted in downregulation of pro-angiogenic factors such as TIMP-3, bFGF and TGFalpha. In addition, a G-CSF independent downregulation of Bv8 was found as a common target of all three DmiRs and correlated with decreased tumor recruitment of bone marrow-derived CD11b+ Gr-1+ myeloid cells. In contrast, antiangiogenic and dormancy promoting pathways such as EphA5 and Angiomotin were upregulated in DmiR over-expressing tumors. This work suggests novel means to reverse the malignant tumor phenotype into an asymptomatic dormant state and may provide promising targets for early detection or prevention of cancer.

Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation.

Aging is the major determinant of cancer incidence, which, in turn, is likely dictated in large part by processes that influence the progression of early subclinical (occult) cancers. However, there is little understanding of how aging informs changes in aggregate host signaling that favor cancer progression. In this study, we provide direct evidence that aging can serve as an organizing axis to define cancer progression-modulating processes. As a model system to explore this concept, we employed adolescent (68 days), young adult (143 days), middle-aged (551 days), and old (736 days) C57BL/6 mice as syngeneic hosts for engraftment of Lewis lung cancer to identify signaling and functional processes varying with host age. Older hosts exhibited dysregulated angiogenesis, metabolism, and apoptosis, all of which are associated with cancer progression. TGFβ1, a central player in these systemic processes, was downregulated consistently in older hosts. Our findings directly supported the conclusion of a strong host age dependence in determining the host tumor control dynamic. Furthermore, our results offer initial mechanism-based insights into how aging modulates tumor progression in ways that may be actionable for therapy or prevention.

Transforming growth factor-β1 (TGF-β1) and -β2 are correlated with poorer prognosis in gastric cancer (GC), which act in both tumor and immune cells. However, their expressions in precancer and tumor-cell interactions with peripheral blood mononuclear cells (PBMCs) remain unclear. Protein levels of TGF-β1 and -β2 were analyzed by immunohistochemistry and corresponding mRNA levels were determined by quantitative real-time polymerase chain reaction in 93 surgical and biopsy specimens. Serum TGF-β concentration was detected by enzyme-linked immunosorbent assays. AGS and MKN45 cell lines were directly or indirectly cocultured with PBMCs in vitro. TGF-β and Smad molecules were detected after cocultures and the growths of GC cells and PBMCs were assessed by cell proliferation assay. The results showed positive staining for TGF-β1 was detected in 20% of control samples, 52.3% of precancer, 59.1% of early GC and 66.7% of advanced GC samples, correlated with lesion progression (χ2 = 9.487, P = 0.002). All tissues were positive for TGF-β2. TGF-β1 mRNA levels were increased in advanced cancers, while TGF-β2 increased earlier. TGF-β1 mRNA levels were higher in tumor than in peritumor, which positively correlated with Smad2 and Smad7. Serum TGF-β levels were significantly higher in patients with early and advanced cancers compared to controls (TGF-β1∶50.08±4.38 and 45.76±5.00 vs. 27.78±6.11 ng/mL; TGF-β2∶133.61±21.90 and 111.34±15.76 vs. 59.41±15.42 ng/mL, both P<0.05). The levels of TGF-β1 mRNA and cytokine secretion were higher in GC cells after direct coculture compared to indirect culture. TGF-β1 was decreased and TGF-β2 was increased in PBMCs after cocultures. Moreover, TGF-β1 inhibited the viability of PBMCs but not cancer cells. Collectively, neoplastic transformation may be an early event involving the increase of TGF-β1 in the general and local environment. TGF-β1 production is promoted by the direct interaction between GC cells and PBMCs, which might facilitate cancer development.

Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both in vitro and in vivo and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression.