Mitsuhiro Kudo

Aim: Infants with fetal growth restriction (FGR) are at an increased risk of perinatal morbidity and mortality. The long noncoding RNA H19 gene is expressed abundantly in placental villi and recent studies suggest that it regulates FGR. However, the role of H19 in the FGR placenta remains unclear. This study aimed to clarify the relationship between H19 expression and FGR using normotensive placentas after 34 weeks of gestation.Methods: Formalin‐fixed paraffin‐embedded tissues from human placentas collected from pregnancies resulting in small for gestational age (SGA) and appropriate for gestational age (AGA) newborns were used. The histopathological features of placenta tissues, such as villous stromal fibrosis, the numbers of terminal villi, villous vessels and cytotrophoblasts were analyzed using hematoxylin and eosin, Masson's trichrome staining and immunostaining. The localization and expression of H19 in the placentas were demonstrated by in situ hybridization and reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR), respectively. Moreover, the expression levels of H19‐regulated molecules such as IGF2 and decorin (DCN) were measured by RT‐qPCR.Results: Histopathological features of the placental villous were not different between placentas associated with SGA and AGA. H19 localized to the villous stroma, endothelial cells and cytotrophoblasts. Moreover, the expression level of H19 in SGA placentas was significantly lower than that in AGA placentas. The expression levels of IGF2 and DCN in SGA placentas tended to be lower than those in AGA placentas similarly to H19.Conclusion: This study highlights the potential importance of regulatory events mediated by H19 in SGA placentas without histopathological abnormalities in late pregnancy.

Toll-like receptor 4 (TLR4), a key regulator of the innate immune system, is expressed not only in immune cells, but also in a number of cancer cells. A biological role for TLR4 in cutaneous squamous cell carcinoma (SCC), however, is unclear. In this study, we first examined TLR4 expression and localization in cases of SCC, actinic keratosis (AK) and Bowen's disease (BD) by immunohistochemistry. TLR4 expression was significantly higher in the SCC than in the AK or BD tissues. We then determined the TLR4 expression level in vivo, in 3 histological subtypes of SCC. TLR4 expression in poorly differentiated SCC was significantly lower compared with that of the moderately and well-differentiated type. In addition, the CD44 immunoreactivity tended to be high in the cell membrane of poorly differentiated SCC. Of note, poorly differentiated SCC is a risk factor of unfavorable outcomes in affected patients. We then assessed the biological role of TLR4 in HSC-1 and HSC-5 SCC cells and HaCaT human keratinocytes. TLR4 knockdown by transfection with siRNA accelerated HSC-1 and HaCaT cell migration and invasion compared to the control siRNA-transfected cells. TLR4 knockdown resulted in an increased CD44 expression and in an enhanced filopodia protrusion formation, particularly in HSC-1. On the whole, these results suggest that a reduced TLR4 expression enhances the malignant features in SCC cases and cultured SCC cell lines. TLR4 may thus play an anti-tumor role in cutaneous SCC.

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.

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.

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.

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Fibroblast growth factor receptor 2 (FGFR2) is activated in many cancers and considered as a potential therapeutic molecular target including for endometrial endometrioid carcinoma (EEC). Overexpression of FGFR2 isoform IIIc (FGFR2IIIc) has been shown to be associated with carcinogenesis in various cancers, but its expression in EEC has not been reported yet to the best of our knowledge. In this study, we identified roles for FGFR2IIIc in EEC carcinogenesis and demonstrated its diagnostic and prognostic values in EEC. FGFR2IIIc expression was compared between 10 normal endometrium, 10 atypical endometrial hyperplasias, and 47 EEC specimens using immunohistochemistry and quantitative real-time PCR. Atypical hyperplasia, Grade 1 (G1), and Grade 2 (G2) differentiated EEC tissues showed significantly higher FGFR2IIIc expression than normal endometrium tissue. However, as compared to G1 and G2 EECs, Grade 3 (G3) differentiated EEC tissue showed lower FGFR2IIIc expression (P<0.05). There was no significant association between FGFR2IIIc expression and patient age, lymph node metastasis, and EEC stage. These results suggest that altered FGFR2IIIc expression plays an important role in EEC carcinogenesis and may occur in precancerous tissues. However, FGFR2IIIc appears to be not related to EEC progression. Some G3 EECs may develop through different carcinogenic processes than G1 and G2 EECs.

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.

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.