Wei-Xia Peng

Overexpressed fibroblast growth factor receptor 2 in the invasive front of colorectal cancer: A potential therapeutic target in colorectal cancer

Fibroblast growth factor receptor 2 (FGFR2) is considered a novel therapeutic target for various cancer. We used a silencing strategy to clarify the effect of reduced FGFR2 expression in human colorectal cancer (CRC) cells. The invasive front of cancer cells exhibited stronger FGFR2 expression than the surface area of the cancers. FGFR2 shRNA-transfected LoVo cells inhibited cell migration, invasion and tumor growth in vitro and in vivo. Thus, FGFR2 plays important roles in CRC progression in association with tumor cell migration, invasion and growth, and FGFR2 might be a novel therapeutic target for CRC.

Morphological and cytoskeletal changes of pancreatic cancer cells in three-dimensional spheroidal culture

Three-dimensional (3D) cell cultures are expected to mimic in vivo environments. We used a NanoCulture plate to determine the spheroid-forming ability of pancreatic ductal adenocarcinoma (PDAC) cell lines and compared the morphology and expression of cytoskeletal proteins of PDAC cells to those in two-dimensional (2D) cultures. All examined PDAC cells grew as monolayers in 2D culture. PANC-1 and KLM-1 formed spheroids in 3D culture, but PK-45H and MIAPaCa-2 did not. Strong expression of F-actin was observed in the cells attached to the surface of the plate, which formed cell projections in 3D culture. F-actin was detected on the grids of the NanoCulture plate in PANC-1 cells but not in PK-45H. The levels of tubulin expression in cells were higher in 3D culture than in 2D culture. The expression level of E-cadherin mRNA in PANC-1 and KLM-1 was higher than that in PK-45H and MIAPaCa-2. In conclusion, PDAC cells showed morphological changes, spheroid formation, and alterations of cytoskeletal proteins in 3D culture. E-cadherin might be one of the key molecules involved in spheroid formation of PDAC cells. The 3D spheroidal culture system was a useful method for cell imaging with contrast-phase microscopy and confocal microscopy.

“Wild type” GIST: Clinicopathological features and clinical practice

Gastrointestinal stromal tumor (GIST) is a mesenchymal tumor of the gastrointestinal tract. Mutation of KIT and PDGFRA genes is implicated in the tumorigenesis. Approximately 10% of GISTs do not harbor mutation of these genes, and they are designated as “wild type” GIST. They are classified into succinate dehydrogenase (SDH)‐deficient and non‐SDH‐deficient groups. SDH‐deficient group includes Carney triad and Carney Stratakis syndrome. The patients are young women. Tumors occur in the antrum of the stomach, and tumor cells are epithelioid. Lymph node metastasis is frequent. The non‐SDH‐deficient group includes neurofibromatosis (NF) type 1 and GISTs with mutations of BRAF, KRAS, and PIK3CA and with the ETV6‐NTRK3 fusion gene. GIST in NF occurs in the small intestine, and tumor cells are spindle shaped. GIST with BRAF mutation arises in the small intestine. Attention to the age, gender, family history and other neoplasms may raise the prediction of syndromic disease. Location of the tumor, morphology, and pleomorphism of the tumor cells are further informative. Lymphovascular invasion should be carefully evaluated. The determination of KIT expression is essential for the diagnosis. When wild type GIST is suspected, intensive genetic analysis is required. Further, a careful and long‐time observation is recommended.

Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.

Insulin-like growth factor 2 mRNA-binding protein-3 as a marker for distinguishing between cutaneous squamous cell carcinoma and keratoacanthoma

In the histopathological diagnosis of cutaneous tumors, the differential diagnosis of squamous cell carcinoma (SCC) with crateriform architecture and keratoacanthoma (KA) is often difficult so an accurate understanding of the biological features and the identification of reliable markers of SCC and KA are crucial issues. Insulin-like growth factor 2 mRNA-binding protein-3 (IGF2BP3, also known as IMP3) is thought of as a bona fide oncofetal protein, which is overexpressed and is involved in cell proliferation, migration, and invasion in several kinds of tumors. However, the role of IMP3 in cutaneous SCC and KA has not been well studied. Therefore, we focused on studying the biological functions of IMP3 in SCC and KA. In human skin SCC cell lines, HSC-1 and HSC-5, and the human keratinocyte cell line, HaCaT, IMP3 mRNA levels were significantly higher than that of normal human skin. The knockdown of IMP3 expression reduced the proliferation of HSC-1, and significantly reduced invasion by HSC-1 and HSC-5. In contrast, the knockdown of IMP3 did not significantly affect invasion by HaCaT cells. In immunohistochemical studies of SCC and KA tissues, the Ki-67 labeling index (LI) of the suprabasal cell layer was significantly higher in SCC, compared with KA tissues and the tumor-free margin (TFM) adjacent to SCC and KA. Most SCC tissues stained strongly positive for IMP3, but KA tissues and TFM were mostly negative for IMP3. The Ki-67 LI of the IMP3-positive group was significantly higher than that of the IMP3-negative group in the suprabasal cell layer of SCC. These results suggest that IMP3 plays an important role in proliferation and, more significantly, in the invasion of SCC, and may be a suitable marker for the histopathological diagnosis of SCC with a crateriform architecture and KA. Furthermore, IMP3 may potentially be a new therapeutic target for SCC.

Analysis of protein expression regulated by lumican in PANC‑1 cells using shotgun proteomics

Lumican, a member of the class II small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. We previously reported that lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. Lumican stimulates growth and inhibits the invasion of a PDAC cell line. In the present study, we performed a global shotgun proteomic analysis using lumican-overexpressing PANC‑1 cells and lumican downregulated PANC‑1 cells to identify candidate proteins that are regulated by lumican and related to cell growth and invasion in PDAC cells. A total of 448 proteins were identified from lumican-overexpressing PANC‑1 and control cells. Additionally, 451 proteins were identified from lumican-downregulated PANC‑1 cells and control cells. As a result of semi-quantification based on spectral counting, 174 differentially expressed proteins were identified by lumican upregulation, and 143 differentially expressed proteins were identified by lumican downregulation. The expression levels of 24 proteins, including apoptosis- and invasion-related proteins correlated with lumican expression levels. It is likely that the expression of these proteins is regulated by lumican, and that they are involved in apoptosis and invasion in PDAC. These findings suggest that lumican may be involved in cell growth and invasion through the regulation of these 24 proteins expressed in PDAC.

Increased expression of PDIA3 and its association with cancer cell proliferation and poor prognosis in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) is unfavorable following complete tumor resection. The aim of the present study was to identify a molecule able to predict HCC prognosis through comprehensive protein profiling and to elucidate its clinicopathological significance. Comprehensive protein profiling of HCC was performed by liquid chromatography-tandem mass spectrometry. Through the bioinformatic analysis of proteins expressed differentially in HCC and non-HCC tissues, protein disulfide-isomerase A3 (PDIA3) was identified as a candidate for the prediction of prognosis. PDIA3 expression was subsequently examined in 86 cases of HCC by immunostaining and associations between PDIA3 expression levels and clinicopathological characteristics were evaluated. The Ki-67 index and apoptotic cell death of carcinoma cells were examined by immunostaining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay in 24 cases. The results demonstrated that PDIA3 was expressed in all 86 HCC cases; 56 HCC cases (65%) exhibited high expression of PDIA3 and 30 (35%) exhibited low expression. The disease-free and overall survival times of HCC patients with high PDIA3 expression were significantly shorter than in HCC patients with low expression. Furthermore, increased expression of PDIA3 was associated with an elevated Ki-67 index, indicating increased cancer cell proliferation and a reduction in apoptotic cell death. Taken together, these results suggest that PDIA3 expression is associated with tumor proliferation and decreased apoptosis in HCC, and that increased expression of PDIA3 predicts poor prognosis. PDIA3 may therefore be a key molecule in the development of novel targeting therapies for patients with HCC.

Nodular fasciitis in the parotid gland: A case report and review of the literature

Nodular fasciitis (NF) is a benign, reactive lesion with a self‐limiting process. Because NF is rare in the parotid gland and has many cytological similarities to other benign or malignant tumors, cytological misinterpretation is common. The patient, a 30‐year‐old woman, had a painless mass in her right parotid gland. Fine needle aspiration cytology (FNAC) was performed. Spindle cells with basophilic and well‐demarcated cytoplasm were observed in a mucoid‐like background. The mucoid‐like substance was metachromatic and appeared to be the matrix of PA. Histopathologically, spindle‐shaped cells with intervening birefringent mature collagen were arranged in short irregular bundles. Prominent mucoid‐like matrixes as well as few infiltrating neutrophils and lymphocytes were found in the background. Lesional cells were positive for CD10 and β‐catenin in the cytoplasm, but negative for cytokeratin, the S‐100 protein, CD34, and neurofilament. Ultimately, this patient was diagnosed with NF. In FNAC of the parotid gland region, distinguishing NF from other real tumors is important for deciding treatment strategies. Diagn. Cytopathol. 2013;41:829–833.

Morphological and cytoskeletal alterations of nervous system tumor cells with different culturing methods.

Cell culture is one of the most important methods of research in molecular and cellular biology, and various culture systems have been developed, including two-dimensional (2D), three-dimensional (3D) and floating culture systems. In the present study, we examined morphological changes and different expression patterns of cytoskeletal proteins in three different types of nervous system tumor cells grown in 2D, 3D and floating cell cultures. A172, KG-1-C and IMR-32 cells showed marked morphological changes, depending on the cell culture methods. F-actin expression was clearly observed at the level of the cells nearest the plate surface in 2D and 3D cultures. On the other hand, expression of F-actin was weak in the floating culture system. α-tubulin was detected in the cytoplasm of cells in 2D culture, but in floating and 3D cultures, α-tubulin was expressed in the peripheral regions of spheres and spheroids. In conclusion, this study demonstrated that nervous system tumor cells showed different alterations in morphology, and different cytoskeletal protein expression patterns, depending on the culture methods.

Cystatin B as a potential diagnostic biomarker in ovarian clear cell carcinoma.

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.