Chih-Yung Yang

Human cytomegalovirus preferentially infects the neoplastic epithelium of colorectal cancer: a quantitative and histological analysis.

BACKGROUND It has long been suggested that human cytomegalovirus (HCMV) might be involved in human oncogenesis. However, whether HCMV was associated with colorectal cancer (CRC) was still controversial. OBJECTIVE To clarify whether HCMV specifically infects the tumorous tissue of CRC. STUDY DESIGN Paired tumor and adjacent non-neoplastic CRC specimens were collected from 163 patients. HCMV DNA was detected and quantified through PCR and quantitative real-time PCR. Virus location was determined by in situ hybridization (ISH) of formalin-fixed paraffin-embedded tissue sections with an HCMV-specific probe. RESULTS By PCR, HCMV DNA was detected in 42.3% (69/163) of the tumor specimens, while only 5.6%(14/163) samples of adjacent non-neoplastic tissue were positive for HCMV (p<0.0001). Quantitative real-time PCR in 54 sample pairs revealed significantly higher viral copies in the tumor specimens than the adjacent non-neoplastic tissue specimens (p<0.001). By ISH, the nucleic acids of HCMV were detected in the cytoplasm of neoplastic epithelium. No hybridization was detected in the inflammatory infiltrates, submucosa, or other stromal tissues. CONCLUSIONS HCMV preferentially infects the tumor epithelium of CRC. How the virus subsists in and interacts with the microenvironment of tumor epithelium of CRC should be studied.

Generation of infectious virus particles by transient co-expression of human immunodeficiency virus type 1 gag mutants

We have demonstrated that COS7 cells transiently co-expressing myristylation-defective (Myr-) and protease-defective (PR-) human immunodeficiency virus (HIV) mutants can release infectious virions when co-transfected with an amphotropic murine leukaemia virus envelope protein expression plasmid (SV-A-MLV-env). In contrast, no infectious virions were detected when a PR-, noninfectious HIV gag mutant was co-expressed with the Myr- mutant, although the Myr- mutant could still process the immature core particles in trans. This result indicates that generation of functionally normal Gag proteins is required for virus infectivity in our complementation system. A mutant with a 56-amino-acid deletion in the N-terminal region of the capsid (CA) domain could still complement the PR- mutant to generate infectious virions, suggesting that the deletion mutant could provide a functional protease for processing in the PR- mutant. This result is consistent with the concept that mutations within the N-terminal region of the CA domain have no major effects on Gag-Pol incorporation into particles.

Dynamic Changes in Numbers and Properties of Circulating Tumor Cells and Their Potential Applications

Circulating tumor cells (CTCs) can be detected in the blood of different types of early or advanced cancer using immunology-based assays or nucleic acid methods. The detection and quantification of CTCs has significant clinical utility in the prognosis of metastatic breast, prostate, and colorectal cancers. CTCs are a heterogeneous population of cells and often different from those of their respective primary tumor. Understanding the biology of CTCs may provide useful predictive information for the selection of the most appropriate treatment. Therefore, CTC detection and characterization could become a valuable tool to refine prognosis and serve as a “real-time biopsy” and has the potential to guide precision cancer therapies, monitor cancer treatment, and investigate the process of metastasis.

Lysosomal targeting of phafin1 mediated by Rab7 induces autophagosome formation.

Autophagy orchestrates programmed cell death via crossroads of complex vesicle trafficking including autophagosome and lysosome interaction. Phafin1, an endosome proteins composed of Pleckstrin homology (PH) and Fab1-YotB-Vac1p-EEA1 (FYVE) domain membrane-binding domains, is involved in caspase-independent apoptosis. We report here that the increased expression of phafin1 and its FYVE domain caused the formation of enlarged endosomes. Phafin1 also modulates the membrane density of certain receptors and participates in endocytosis and autophagy processes. The PH-domain of phafin1 is dispensable for lysosomal targeting. Moreover, the tail-domain of phafin1 provides lysosomal targeting signature and the ability to induce autophagy that is mediated by Rab7 signaling. The results suggest that in addition to its role in endosome transport, phafin1 is also involved in lysosomal targeting and autophagosome formation.

Interleukin-17A modulates circulating tumor cells in tumor draining vein of colorectal cancers and affects metastases.

Purpose: Metastasis is the major cause of death in patients with colorectal cancer (CRC). Circulating tumor cells (CTC) are believed to cause metastasis and serve as a prognostic marker for mortality in clinical stage IV patients. However, most studies are conducted in late-stage cases when distant metastases have already occurred; thus, such results provide limited clinical use. This study focused on whether CTCs can predict the risk of metastasis after treatment of the primary tumor in early-stage patients with CRC. Experimental Design: CTCs were quantified using EpCAM-positive/CD45-negative immunoselection and flow cytometry in patients with CRC. A mouse model was used to investigate the mechanistic roles of CTCs and interleukin (IL)-17A in metastasis. Results: The number of mesenteric CTCs obtained from stage II patients was higher than that obtained from patients in stages I, III, and IV. In addition, following invasion of orthotopically implanted tumors in our mouse model, we found that CTCs exhibited an increase-then-decrease pattern, accompanied by corresponding changes in serum IL-17A levels and opposing changes in serum granulocyte macrophage colony-stimulating factor (GM-CSF) levels. Ablation of IL-17A and administration of rGM-CSF effectively suppressed the increase in CTCs and prevented metastasis in mice. Moreover, IL-17A promoted cancer cell motility, matrix digestion, and angiogenesis, whereas GM-CSF stimulated the elimination of CTCs by boosting host immunity. Notably, serum levels of IL-17A were also correlated with disease-free survival in patients with CRC. Conclusions: Our results showed that CTCs and IL-17A could serve as prognostic markers and therapeutic targets for CRC metastasis. Clin Cancer Res; 20(11); 2885–97. ©2014 AACR.

Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 Modulates Local ATP-Induced Calcium Signaling in Human HaCaT Keratinocytes

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

Magnetically triggered nanovehicles for controlled drug release as a colorectal cancer therapy

Magnetic silica core/shell nanovehicles presenting atherosclerotic plaque-specific peptide-1 (AP-1) as a targeting ligand (MPVA-AP1 nanovehicles) have been prepared through a double-emulsion method and surface modification. Amphiphilic poly(vinyl alcohol) was introduced as a polymer binder to encapsulate various drug molecules (hydrophobic, hydrophilic, polymeric) and magnetic iron oxide (Fe3O4) nanoparticles. Under a high-frequency magnetic field, magnetic carriers (diameter: ca. 50 nm) incorporating the anti-cancer drug doxorubicin collapsed, releasing approximately 80% of the drug payload, due to the heat generated by the rapidly rotating Fe3O4 nanoparticles, thereby realizing rapid and accurate controlled drug release. Simultaneously, the magnetic Fe3O4 themselves could also kill the tumor cells through a hyperthermia effect (inductive heating). Unlike their ungrafted congeners (MPVA nanovehicles), the AP1-grafted nanovehicles bound efficiently to colorectal cancer cells (CT26-IL4Rα), thereby displaying tumor-cell selectivity. The combination of remote control, targeted dosing, drug-loading flexibility, and thermotherapy and chemotherapy suggests that magnetic nanovehicles such as MPVA-AP1 have great potential for application in cancer therapy.

Circulating CD133+/ESA+ cells in colorectal cancer patients

BACKGROUND Tumor initiating cells are a small subset of cancer cells responsible for tumor growth and recurrence. The status of tumor initiating cells was measured using the surface markers CD133 (prominin-1) and ESA (epithelial-specific antigen). The aims of this study were to investigate the significance of CD133(+)/ESA(+) cells in mesenteric venous blood (MVB) and tumor mass (TM) for overall survival (OS) and disease-free survival (DFS) in colorectal cancer (CRC) patients undergoing curative resection. MATERIALS AND METHODS A total of 229 CRC patients undergoing curative resection were prospectively enrolled in the study. Using CD133 and ESA as surface markers, CD133(+)/ESA(+) cells were enumerated from MVB and TM using flow cytometry. RESULTS We analyzed the presence of CD133(+)/ESA(+) cells in TM from 158 patients and found no correlation to patient DFS, OS, or clinical stage. In 135 patients, an analysis of CD133(+)/ESA(+) cells in MVB showed an inverse correlation with both DFS and OS (P = 0.014 and P = 0.008, respectively). It exhibited an increase-then-decrease pattern with the peak in stage II patients. A multivariate Cox analysis demonstrated that the status of CD133(+)/ESA(+) cells in MVB, but not the TM, was a significant prognostic factor for DFS and OS (P = 0.003 and P = 0.011, respectively). CONCLUSIONS The status of CD133(+)/ESA(+) cells in MVB, but not in TM, could be a useful indicator for predicting tumor recurrence and a prognostic marker for CRC patients.

Single nucleotide polymorphisms associated with colorectal cancer susceptibility and loss of heterozygosity in a Taiwanese population.

Given the significant racial and ethnic diversity in genetic variation, we are intrigued to find out whether the single nucleotide polymorphisms (SNPs) identified in genome-wide association studies of colorectal cancer (CRC) susceptibility in East Asian populations are also relevant to the population of Taiwan. Moreover, loss of heterozygosity (LOH) may provide insight into how variants alter CRC risk and how regulatory elements control gene expression. To investigate the racial and ethnic diversity of CRC-susceptibility genetic variants and their relevance to the Taiwanese population, we genotyped 705 CRC cases and 1,802 healthy controls (Taiwan Biobank) for fifteen previously reported East Asian CRC-susceptibility SNPs and four novel genetic variants identified by whole-exome sequencing. We found that rs10795668 in FLJ3802842 and rs4631962 in CCND2 were significantly associated with CRC risk in the Taiwanese population. The previously unreported rs1338565 was associated with a significant increased risk of CRC. In addition, we also genotyped tumor tissue and paired adjacent normal tissues of these 705 CRC cases to search for LOH, as well as risk-associated and protective alleles. LOH analysis revealed preferential retention of three SNPs, rs12657484, rs3802842, and rs4444235, in tumor tissues. rs4444235 has been recently reported to be a cis-acting regulator of BMP4 gene; in this study, the C allele was preferentially retained in tumor tissues (p = 0.0023). rs4631962 and rs10795668 contribute to CRC risk in the Taiwanese and East Asian populations, and the newly identified rs1338565 was specifically associated with CRC, supporting the ethnic diversity of CRC-susceptibility SNPs. LOH analysis suggested that the three CRC risk variants, rs12657484, rs3802842, and rs4444235, exhibited somatic allele-specific imbalance and might be critical during neoplastic progression.

Response to stress in early tumor colonization modulates switching of CD133-positive and CD133-negative subpopulations in a human metastatic colon cancer cell line, SW620.

According to the cancer stem cell (CSC) model, higher CD133 expression in tumor tissue is associated with metastasis and poor prognosis in colon cancer. As such, the CD133-positive (CD133+) subpopulation of cancer cells is believed to play a central role in tumor development and metastatic progression. Although CD133+ cells are believed to display more CSC-like behavior and be solely responsible for tumor colonization, recent research indicates that CD133− cells from metastatic colon tumors not only also possess colonization capacity but also promote the growth of larger tumors in a mouse model than CD133+ cells, suggesting that an alternative mechanism of metastasis exists. This study investigated this possibility by examining the cell viability, tumorigenicity, and proliferation and growth capacity of the CD133+ and CD133− subpopulations of the SW620 cell line, a human metastatic colon cancer cell line, in both an in vitro cell model and an in vivo mouse model. While both SW620 CD133− and SW620CD133+ cells were found to engage in bidirectional cell-type switching in reaction to exposure to environmental stressors, including hypoxia, a cell adhesion-free environment, and extracellular matrix stimulation, both in vitro and in vivo, CD133− cells were found to have a growth advantage during early colonization due to their greater resistance to proliferation inhibition. Based on these findings, a hypothetical model in which colon cancer cells engage in cell-type switching in reaction to exposure to environmental stressors is proposed. Such switching may provide a survival advantage during early colonization, as well as that explain previous conflicting observations.