Yoon Pyo Choi

CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells

Cancer stem cells (CSCs) have been identified in solid tumors and cancer cell lines. In this study, we isolated a series of cancer cell clones, which were heterogeneous in growth rate, cell cycle distribution and expression profile of genes and proteins, from ovarian tumor specimens of a patient and identified a sub-population enriched for ovarian CSCs defined by CD24 phenotype. Experiments in vitro demonstrated CD24+ sub-population possessed stem cell-like characteristics of remaining quiescence and more chemoresistant compared with CD24− fraction, as well as a specific capacity for self-renewal and differentiation. In addition, injection of 5 × 103 CD24+ cells was able to form tumor xenografts in nude mice, whereas equal number of CD24− cells remained nontumorigenic. We also found that CD24+ cells expressed higher mRNA levels of some ‘stemness’ genes, including Nestin, β-catenin, Bmi-1, Oct4, Oct3/4, Notch1 and Notch4 which were involved in modulating many functions of stem cells, and lower E-cadherin mRNA level than CD24− cells. Altogether, these observations suggest human ovarian tumor cells are organized as a hierarchy and CD24 demarcates an ovarian cancer-initiating cell population. These findings will have important clinical applications for developing effective therapeutic strategies to treat ovarian cancer.

Selective homocysteine turn-on fluorescent probes and their bioimaging applications

The design and development of new pyrene-based fluorescent probes, P-Hcy-1 and P-Hcy-2, which display selective fluorescence enhancements in response to homocysteine (Hcy), are described. The distinctly different fluorescence responses of P-Hcy-1 and P-Hcy-2 to Hcy vs. Cys are explained by theoretical calculations. Finally, the results of cell experiments show that these probes can be used to selectively detect Hcy in mammalian cells.

Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial–mesenchymal transition-like state in breast cancer cells in vitro

Fibroblasts were extracted from tissue in tumor burden zones, distal normal zones and interface zones between tumor and normal tissue of human breast carcinomas, and the corresponding fibroblasts were designated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). The crosstalk between three types of fibroblasts and breast cancer cells was evaluated using an in vitro direct co-culture model. We found that INFs grew faster and expressed higher levels of fibroblast activation protein than did NFs and CAFs. Compared with CAFs and NFs, INFs grown with breast cancer cells were significantly more effective in inducing an epithelial-mesenchymal transition (EMT) in cancer cells, as indicated by induction of vimentin and N-cadherin and downregulation of E-cadherin. This EMT process was also accompanied by activation of extracellular signal-regulated kinase (ERK) and modulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression. Additionally, INFs promoted breast cell migration to a larger extent compared with NFs and CAFs. Taken together, these findings indicate that INFs isolated from the tumor interface zone exhibited more robust biological modulatory activity than did NFs and CAFs isolated from normal and tumor zones of the same tumor tissue, suggesting that the interface zone of the tumor represents a dynamic region vital to tumor progression.

Molecular portraits of intratumoral heterogeneity in human ovarian cancer

One of the most common characteristic profiles of cancer is intratumoral heterogeneity (ITH). We aimed to clarify the molecular profiles and biological significance of ITH with relation to cancer stem cell (CSC). We analyzed five primary cultured clones generated from different spatial zones, front and rear zone, of a fresh-frozen ovarian tumor tissue, performing ATP-CRA, conventional RT-PCR, side population (SP) analysis, flow cytometry immunophenotyping, and cell proliferation assays. We also carried out array CGH and Ingenuity Pathways Analysis (IPA) between SP and non-SP (NSP) cells. Clones from tumor front zone showed phenotypically and genetically distinct subpopulations with relatively higher SP proportions, CD24(+) and CD117(+) expression, and chemotherapeutic resistance. We demonstrate that phenotype of SP cells in heterogeneous clones of human ovarian cancer was closely related to CD24(+), CD117(+), and combined CD117(+)/CD24(+) fractions. Chromosomal alterations in SP cells relative to NSP cells were closely related to the novel core networks of cancer stem cell-like cells (CSCs), such as cycle checkpoint regulation, notch, PTEN, wnt/β-catenin, PI3K/AKT, integrin, and cytokine and chemokine signaling. ITH could arise from clonal diversity closely related to CSC-like molecules, as evidenced by accumulated genetic, transcriptional and gene products alterations in SP.

Laminin-332-Rich Tumor Microenvironment for Tumor Invasion in the Interface Zone of Breast Cancer

Dense fibrosis, which is caused by desmoplastic reaction, is usually found in invasive ductal carcinoma and may represent the alteration of the tumor microenvironment preceding tumor invasion. Thus, the dense fibrotic zone around invasive ductal carcinoma can be considered to be the actual tissue site of tumor microenvironment, where the precedent alterations for tumor invasion occur. To characterize the dense fibrotic zone, we classified invasive ductal carcinoma tissue into a tumor zone, a normal zone, and the novel interface zone (IZ), which shows dense fibrosis. The postulated IZ is a 5-mm-wide belt that circles the tumor margin and overlaps with normal tissue. Of the extracellular matrix components, laminin-332 was specifically overexpressed in the IZ. Events that appear to be similar to the epithelial-mesenchymal transition, a novel source of myofibroblast formation from epithelial cells, were observed in the IZ, according to the following characteristics: overexpression of matrix metalloproteinase 3, membrane type 1-matrix metalloproteinase, snail, and zinc finger E-box-binding homeobox 1, and the gain of N-cadherin expression, as well as the down-regulation of miR200c. The myofibroblasts isolated from the IZ, which were designated interface zone-fibroblast, displayed laminin-332 and membrane type 1-matrix metalloproteinase overexpression, in contrast with both cancer-associated fibroblasts and normal breast fibroblasts. Taken together, our results suggest that the IZ, which shows dense fibrosis, may provide a specialized microenvironment for guiding tumor invasion: the fibrosis caused by laminin-332 overexpressing myofibroblast formation (interface zone-fibroblast) via epithelial-mesenchymal transition.

A synthetic ion transporter that disrupts autophagy and induces apoptosis by perturbing cellular chloride concentrations

Perturbations in cellular chloride concentrations can affect cellular pH and autophagy and lead to the onset of apoptosis. With this in mind, synthetic ion transporters have been used to disturb cellular ion homeostasis and thereby induce cell death; however, it is not clear whether synthetic ion transporters can also be used to disrupt autophagy. Here, we show that squaramide-based ion transporters enhance the transport of chloride anions in liposomal models and promote sodium chloride influx into the cytosol. Liposomal and cellular transport activity of the squaramides is shown to correlate with cell death activity, which is attributed to caspase-dependent apoptosis. One ion transporter was also shown to cause additional changes in lysosomal pH, which leads to impairment of lysosomal enzyme activity and disruption of autophagic processes. This disruption is independent of the initiation of apoptosis by the ion transporter. This study provides the first experimental evidence that synthetic ion transporters can disrupt both autophagy and induce apoptosis.

Panel of candidate biomarkers for renal cell carcinoma

The timely diagnosis and therapeutic monitoring of human renal cell carcinoma (RCC) is limited by the lack of specific biomarkers. To identify candidate RCC biomarkers, we used 2-DE gel electrophoresis with mass spectrometry and 2-DE spot intensity-based ROC analysis to analyze 18 sets of paired normal and RCC tumor tissue including conventional, papillary, and chromophobe subtypes. Validation was performed with RCC patient plasma samples and confirmed by clustergram, shRNA, and immunohistochemistry assays. Cardinal candidates were evaluated by ELISA. The leading candidate biomarker that was upregulated in RCC samples according to the clustergram and validation analysis was nicotinamide N-methyltransferase (NNMT) (13/15, P < 0.0001). Other upregulated candidate biomarkers that were identified by this method include ferritin, hNSE, NM23, secretagogin, and L-plastin. The upregulation of NNMT in RCC was confirmed by immunoblotting and immunohistochemistry. Analysis of fractionated membrane-associated proteins identified CAP-G, mitofillin, tubulin alpha, RBBP7, and HSP27. Of these, RBBP7 and HSP27 were highly expressed in the chromophobe subtype of RCC (3/3) but were absent from conventional RCC (0/3). The triple combination of the NNMT, FTL, and hNSE biomarkers had the highest predictive capacity of 0.993, while NNMT was the single, most powerful candidate diagnostic biomarker for all types of RCC.

In situ identification and localization of IGHA2 in the breast tumor microenvironment by mass spectrometry

Modifications in the tumor microenvironment (TME) play a major role in the establishment, progression, and metastasis of cancer. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique that enables the simultaneous identification and localization of biological compounds within tissues. To detect markers of early TME remodeling in invasive breast cancer, we used MALDI-MSI to compare the molecular profiles of tissues from the breast cancer interface zone, tumor zone, and normal-tissue zone. Using direct-tissue MALDI tandem mass spectrometry (MS/MS), we identified immunoglobulin heavy constant alpha 2 (IGHA2) as a new, zone-specific protein in the breast TME. The zone-specific expression of IGHA2 was verified by immunoblotting and immunohistochemical analysis. IGHA2 expression was consistently positive in tumor cells that were metastatic to regional nodes, with intense expression along the cytoplasmic borders. As a factor related to an increased percentage of nodes with tumor metastasis, IGHA2 expression was upregulated 3.745-fold in cases with an increased number of cancerous nodes (p = 0.0468). Our results provide the first evidence of IGHA2 as a marker of the early process of TME remodeling in invasive breast cancer. Furthermore, IGHA2 may be a novel marker for regional metastases in the lymph nodes of patients with breast cancer.

Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia.

The cell cycle inhibitor, p16INK4a may be a useful surrogate biomarker of cervical intraepithelial neoplasia (CIN); however, there is currently no consensus of p16INK4a genetic alterations throughout the multiple step process of CIN. Our goal was to identify the methylation frequency of p16INK4a in each step of CIN that is associated with human papillomavirus (HPV) infection, using several different detection methods of p16INK4a methylation to correlate the data. The present study included a total of 43 patients, including 38 with CIN, and 5 normal patients. Three different methods were used to detect hypermethylation of CpG islands, methylation-specific PCR (MSP) amplification of different primer sets of M1, M2, and M3, pyrosequencing of each forward primer region, and immunohistochemistry of p16INK4a. Analysis of MSP showed that 20 of the 38 CIN patients (52.6%) revealed hypermethylation in at least 1 primer set of the p16INK4a promoter. A complete loss of p16INK4a protein expression was observed in 11 cases (28.9%). There was no observed association of methylation of the p16INK4a gene with either CIN grading (P=0.0698) or HPV status (P=0.2811): specifically 42.9% (3/7) was found in CIN 1, 57.1% (8/14) in CIN 2, and 52.9% (9/17) in CIN 3. In concordance with immunohistochemistry results, hypermethylation of the p16INK4a promoter was significantly correlated with a lack of p16 protein expression (P=0.0145). All positive peaks from pyrosequencing matched the MSP results, which ranged from 6.3% to 24.5%. In conclusion, p16INK4a gene silencing during CIN was not determined to be a particularly rare event; however, it does not correlate with either HPV status or CIN grading.

Proteomic molecular portrait of interface zone in breast cancer.

Surgical tumor margins are intended to encompass residual tumor cells but may not always accurately delineate the boundary between tumor and normal tissue. Efforts to define tumor margins based on molecular analysis have achieved limited success. Furthermore, no clinical trials have addressed the scope of the tumor microenvironment. Here, we considered the tumor cell population and surrounding microenvironment in delineating tumor margins, classifying breast cancer into tumor and normal zones, and introducing the concept of an interface zone, the region between the invading tumor front and normal tissue, which develops during tumor invasion and metastasis through remodeling of the tumor microenvironment. Pathological signatures of invasion markers in tumor tissues are most dynamic within the invading tumor front. We compared protein profiles of tumor, normal, and interface zones using MALDI-MS. Proteins upregulated in the interface zone were identified by peptide mass fingerprinting and confirmed by database searching with chemically assisted MALDI-PSD spectra. Upregulation was confirmed for RhoGDIα, CAPG, WDR1, and CK8 by Western and immunohistochemical analyses. Our results demonstrate that the molecular profile of the interface zone is unique and suggest that upregulation of proteins here may be related to progression and metastasis of breast carcinomas.