Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.     

Interleukin-1 beta and tumor necrosis factor-alpha induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells

Mucin hypersecretion is commonly observed in many inflammatory diseases of the respiratory tract. MUC5AC is generally recognized to be a major airway mucin because MUC5AC is highly expressed in the goblet cells of human airway epithelium. Moreover, it is regulated by various inflammatory cytokines. However, the mechanisms by which the interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induce MUC5AC gene expression in normal nasal epithelial cells, and the signal molecules involved, especially in the downstream signaling of mitogen-activated protein (MAP) kinases, remain unclear. Here we show that pharmacologic or genetic inhibition of either ERK or p38 MAP kinase pathway abolished IL-1beta- and TNF-alpha-induced MUC5AC gene expression in normal human nasal epithelial cells. Our results also indicate that the activation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-response element-binding protein and cAMP-response element signaling cascades via ERK and p38 MAP kinases are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression. Taken together, these studies give additional insights into the molecular mechanism of IL-1beta- and TNF-alpha-induced MUC5AC gene expression and enhance our understanding on mucin hypersecretion during inflammation.     

山羊角膜缘干细胞荧光蛋白(Venus)细胞株的建立及角膜上皮植片构建

角膜缘干细胞是角膜上皮更新与修复的来源,角膜上皮受损严重常会导致角膜盲。尽管近几年通过角膜缘干细胞移植术(LSCT)治愈角膜上皮受损的临床应用已被推广,但是对于角膜缘干细胞移植受损机体后的修复机理并不明确。为了实现角膜缘干细胞移植后的活体追踪,使用G418筛选标记有Venus荧光蛋白的角膜缘干细胞株(GLSC-V),并以其为种子细胞接种于去上皮羊膜上,体外培养21d构建成荧光角膜上皮植片。荧光倒置显微镜下观察GLSC-V的细胞质和细胞核均有绿色荧光表达,在体外培养荧光至少持续3个月。免疫荧光检测GLSC-V细胞P63、Integrinβ1均呈阳性表达,对GLSC-V细胞及未转染的GLSCs进行半定量RT-PCR检测显示,两组细胞皆未表达终末分化角膜上皮细胞基因k3、k12,GLSC-V中p63及pcna较未转染组细胞略上调,venus强表达。经HE染色观察构建的人工角膜组织由5～6层上皮细胞组成,组织中上表皮细胞个数少、体积大且呈扁平状;基底部细胞密集、体积小且成立方状。经免疫荧光检测仅组织基底部最基层细胞表达P63,上表皮细胞不表达。该人工角膜与正常角膜上皮组织结构特性相似,可用于移植,为研究角膜缘干细胞修复严重受损角膜上皮机理奠定基础。     

The role of fibroblast growth factors on the differentiation of vaginal epithelium of neonatal mice

The uterus and upper 3/5 of the vagina originate from the Müllerian duct; however, these organs show quite distinct characteristics in morphology and function. To investigate factors controlling vaginal epithelial cell differentiation from a single layer of pseudostratified epithelium to a multi-layered stratified epithelium with keratin, we focused on fibroblast growth factors (Fgfs). Transformation related protein 63 (Trp63) expression, a marker of stratified epithelium, increased in the Müllerian vaginal epithelial cells from days 0 to 5, and keratin 14 (Krt14) was expressed from day 5, suggesting that Trp63-negative vaginal epithelial cells can differentiate into Trp63-positive cells after birth. Fgf7 and Fgf10 were localized in the vaginal stroma but their receptor, Fgf receptor 2IIIb (Fgfr2IIIb), was localized in the vaginal epithelium. Both Fgf9 and its receptor, Fgfr2IIIc, were localized in the vaginal epithelium. Vaginae cultured with FGF10 or anti-FGF9 antibody showed stratified epithelium with an intense Krt14 expression; however, an inhibitor of phosphorylation of mitogen-activated protein kinase 1/3 (MAPK1/3) canceled the effect of FGF10 and anti-FGF9 antibody. Thus, Fgf10 stimulates the differentiation of pseudostratified epithelial cells into stratified cells via MAPK1/3 pathway, and Fgf9 inhibits this differentiation in the neonatal mouse vagina.     

Roles of p63 in the diethylstilbestrol-induced cervicovaginal adenosis

Women exposed to diethylstilbestrol (DES) in utero develop abnormalities, including cervicovaginal adenosis that can lead to cancer. We report that transient disruption of developmental signals by DES permanently changes expression of p63, thereby altering the developmental fate of Müllerian duct epithelium. The cell fate of Müllerian epithelium to be columnar (uterine) or squamous (cervicovaginal) is determined by mesenchymal induction during the perinatal period. Cervicovaginal mesenchyme induced p63 in Müllerian duct epithelium and subsequent squamous differentiation. In p63(-/-) mice, cervicovaginal epithelium differentiated into uterine epithelium. Thus, p63 is an identity switch for Müllerian duct epithelium to be cervicovaginal versus uterine. P63 was also essential for uterine squamous metaplasia induced by DES-exposure. DES-exposure from postnatal day 1 to 5 inhibited induction of p63 in cervicovaginal epithelium via epithelial ERalpha. The inhibitory effect of DES was transient, and most cervicovaginal epithelial cells recovered expression of p63 by 2 days after discontinuation of DES-treatment. However, some cervicovaginal epithelial cells failed to express p63, remained columnar and persisted into adulthood as adenosis.     

The Function and Significance of SELENBP1 Downregulation in Human Bronchial Epithelial Carcinogenic Process

Background

Our quantitative proteomic study showed that selenium-binding protein 1 (SELENBP1) was progressively decreased in human bronchial epithelial carcinogenic process. However, there is little information on expression and function of SELENBP1 during human lung squamous cell cancer (LSCC) carcinogenesis.

Methods

iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in the human bronchial epithelial carcinogenic process. SELENBP1, member of selenoproteins family and progressively downregulated in this process, was selected to further study. Both Western blotting and immunohistochemistry were performed to detect SELENBP1 expression in independent sets of tissues of bronchial epithelial carcinogenesis, and ability of SELENBP1 for discriminating NBE (normal bronchial epithelium) from preneoplastic lesions from invasive LSCC was evaluated. The effects of SELENBP1 downregulation on the susceptibility of benzo(a)pyrene (B[a]P)-induced human bronchial epithelial cell transformation were determined.

Results

102 differentially expressed proteins were identified by quantitative proteomics, and SELENBP1 was found and confirmed being progressively decreased in the human bronchial epithelial carcinogenic process. The sensitivity and specificity of SELENBP1 were 80% and 79% in discriminating NBE from preneoplastic lesions, 79% and 82% in discriminating NBE from invasive LSCC, and 77% and 71% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, knockdown of SELENBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation.

Conclusions

The present data shows for the first time that decreased SELENBP1 is an early event in LSCC, increases B[a]P-induced human bronchial epithelial cell transformation, and might serve as a novel potential biomarker for early detection of LSCC.     

LCM纯化的人支气管上皮癌变各阶段组织的定量蛋白质组学研究

为分析支气管上皮癌变进程中的差异表达蛋白质,筛选肺鳞癌早期诊断标志物,以人支气管上皮癌变各阶段组织为研究对象,先采用激光捕获显微切割技术(LCM) 纯化人正常支气管上皮组织、鳞状化生、不典型增生、原位癌、浸润性肺鳞癌组织,再用同位素标记相对和绝对定量 (iTRAQ) 技术结合二维液相色谱串联质谱（2D LC-MS/MS）鉴定支气管上皮癌变进程中各阶段的差异表达蛋白质。结果共鉴定了1036个蛋白质,筛选出102个与人支气管上皮癌变相关的差异蛋白质,在这些差异蛋白质中,有的在支气管上皮癌变过程中进行性上调,有的在支气管上皮癌变过程中进行性下调,有的呈阶段特异性改变;功能分析表明,这些差异蛋白质涉及代谢、细胞凋亡、增殖、分化、信号传导、转录、翻译、细胞粘附、免疫反应与发育等。Western blotting 及免疫组织化学技术验证了其中 2个差异蛋白(S100A9和 CKB) 的表达,证实了定量蛋白质组学结果的可靠性。研究结果提示：这些差异表达蛋白质与支气管上皮癌变相关,并可成为肺鳞癌的早期诊断标志物,进一步研究差异蛋白的生物学功能,将有助于阐明支气管上皮的癌变机制,从而为肺鳞癌的早期诊断与发病机制研究提供新思路。     

Comparative proteomics of pulmonary tumors with neuroendocrine differentiation

We aimed to evaluate neuroendocrine pulmonary tumors (NEPT) by a novel method involving map tree construction by comparing all of the protein spots. We performed a proteomics analysis to assess the similarities in protein expression between neuroendocrine pulmonary tumors (NEPT), including typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine carcinomas (LCNEC) and small cell carcinomas (SCLC). Total protein lysates were obtained from seven histologically confirmed frozen NEPT tissues, including 1TC, 2 SCLC, and 4 cases ranging from AC to LCNEC. 2-DE demonstrated that TC was similar to normal lung. AC, LCNEC, and SCLC were similar to each other, forming a group separate from TC, however, SCLC at an early stage showed a similarity to TC. MALDI analysis detected 9 surrogate endpoint biomarkers, including eIF5A1, GST M3, cytokeratin 18 (CK 18), FK506-binding protein p59, p63, MAGE-D2, mitochondrial short-chain enoyl-coenzyme A hydratase 1, tranferrin and poly (rC) binding protein 1. Immunohistochemical staining revealed a gradual decrease in expression rate of p63 and CK 18 with poor differentiation of NEPT. Our results demonstrate that (1) the comparative proteomics of NEPT match the WHO classification except for AC and LCNEC; (2) SCLC show differences in their proteomics according to tumor stage; and (3) CK 18 and p63 may be useful as diagnostically and prognostically available markers.     

Accumulation of CD1a-positive Langerhans cells and mast cells in actinic cheilitis

LCs and MCs are known to be directly influenced by UV radiation. This study investigated the presence of Langerhans cells (LCs) and mast cells (MCs) in actinic cheilitis (AC) exhibiting epithelial dysplasia (ED). Using immunohistochemistry for CD1a and mast cell tryptase, LCs and MCs density was assessed in 35 cases of AC with different degrees of ED. LCs were found in 32 cases of AC whereas MCs were found in all cases. There was an increase in LCs density irrespective of degree of ED when the cases were compared to normal lip mucosa (P = 0.04343). No statistical difference in LCs density was observed regarding the different degrees of dysplasia (P > 0.05). Significant difference in MCs density between mild and moderate dysplasia and normal lip mucosa was found (P < 0.05). No significant correlation between LCs and MCs was seen (P = 0.1258). Although no correlation could be established between LCs and MCs and the different degrees of ED; it is possible that the accumulation of LCs plays an immunostimulatory and protective role in the defense against progression of dysplasia. Further studies are necessary to determine the role of MCs in the development of AC.     

Cigarette smoke induces growth differentiation factor 15 production in human lung epithelial cells: implication in mucin over-expression

Excessive mucus is a hallmark of chronic obstructive pulmonary disease (COPD). There is an emerging interest in the role of TGF-β signaling in the initiation and progression of COPD. Growth differentiation factor 15 (GDF15) is a divergent member of TGF-β superfamily. However, whether cigarette smoke induces airway epithelial GDF15 production and its functions in the airways have not been revealed. Therefore, we first analyzed GDF15 protein expression in airway epithelium of human COPD smokers versus normal non-smokers. We then examined the regulation and function of GDF15 in human airway epithelial cells in response to cigarette smoke exposure. We found increased GDF15 protein expression in airway epithelium (mainly in ciliated cells) of human COPD smokers compared with normal non-smokers. Furthermore, cigarette smoke exposure consistently up-regulated GDF15 expression in human airway epithelial cells. Moreover, GDF15 was shown to play a critical role in cigarette smoke-induced airway epithelial MUC5AC expression. Lastly, activation of phosphoinositide 3-kinase (PI3K) pathway was largely responsible for GDF15-induced airway epithelial MUC5AC expression. Our findings indicate that human airway epithelial cells can produce GDF15 during cigarette smoke exposure, which subsequently activates PI3K pathway to promote mucin (e.g. MUC5AC) expression. This highlights a novel role of GDF15 in regulating airway mucosal immunity (e.g. mucin) in cigarette smoke-exposed lungs.     

p63 in prostate biology and pathology

The identification of stem cells and differentiation programs regulating the development and maintenance of the normal prostate epithelium is essential for the identification of the cell type(s) and molecular alterations involved in the development and propagation of prostate cancer (CaP). The p53-homologue p63 is highly expressed in normal prostate basal cells and is a clinically useful biomarker for the diagnosis of CaP. Importantly, p63 has been shown to play a critical role in prostate development. Recent experimental evidence also suggests that this gene is essential for normal stem cell function in the prostate as well as other epithelial organs. Future studies aimed at better defining the role of p63 in the renewal of the adult prostate epithelium are likely to shed new light on the mechanisms involved in prostate carcinogenesis.     

The role of p63 in embryonic external genitalia outgrowth in mice

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh–Wnt/Ctnnb1–Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.     

Cytokeratin expression patterns in normal and malignant urothelium: a review of the biological and diagnostic implications

The cytokeratins are the intermediate filament proteins characteristic of epithelial cells. In human cells, some 20 different cytokeratin isotypes have been identified. Epithelial cells express between two and ten cytokeratin isotypes and the consequent profile which reflects both epithelial type and differentiation status may be useful in tumour diagnosis. The transitional epithelium or urothelium of the urinary tract shows alterations in the expression and configuration of cytokeratin isotypes related to stratification and differentiation. In transitional cell carcinoma, changes in cytokeratin profile may provide information of potential diagnostic and prognostic significance. The intensification of immunolabelling with some CK8 and CK18 antibodies may underly an active role in tumour invasion and foci of CK17-positive cells may represent proliferating populations. Loss of CK13 is a marker of grade and stage and de novo expression of CK14 is indicative of squamous differentiation and an unfavourable prognosis. However, perhaps the most important recent finding is the demonstration that a normal CK20 expression pattern is predictive of tumour non-recurrence and can be used to make an objective differential diagnosis between transitional cell papilloma and carcinoma. This review will consider cytokeratin expression in urothelium and discuss the application of cytokeratin typing to the diagnosis and prognosis of patients with TCC.     

P~（53） PROTEIN OVEREXPRESSION IN PREMALIGNANT AND MALIGNANT LESIONS OF ORAL MUCOSA：IMMUNOHISTOCHEMICAL OBSERVATION

Ｐ~（５３）　ＰＲＯＴＥＩＮ　ＯＶＥＲＥＸＰＲＥＳＳＩＯＮ　ＩＮ　ＰＲＥＭＡＬＩＧＮＡＮＴ　ＡＮＤ　ＭＡＬＩＧＮＡＮＴ　ＬＥＳＩＯＮＳ　ＯＦ　ＯＲＡＬ　ＭＵＣＯＳＡ：ＩＭＭＵＮＯＨＩＳＴＯＣＨＥＭＩＣＡＬ　ＯＢＳＥＲＶＡＴＩＯＮＰ~（５３）ＰＲＯＴＥＩ...     

Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study

Aim To investigate the immunohistochemical expression of Ki-67, p53 and p63 in Keratocyst Odontogenic Tumours (KOTs) in order to contribute to the biological profile of this tumor. Methods Immunohistochemical technique was performed using the EnVision™ System in 37 cases of KOTs. Results Ki-67- and p53-immunostained cells were mainly located in the suprabasal layers. p63-positive cells were found throughout the lining cystic epithelium. No difference in the immunostaining for these proteins was observed between primary and recurrent KOTs (Ki-67: P = 0.5591; p53: P = 0.9847; p63: P = 0.9127), or between KOTs associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and sporadic KOTs (Ki-67: P = 0.7013; p53: P = 0.3197; p63: P = 0.2427). Conclusions It is possible that biological behavior of KOTs may be related to suprabasal proliferative compartment in the cystic epithelium as observed by high levels of Ki-67, p53 and p63. In addition, p63 immunostaining may represent immaturity of keratinocytes in KOTs, and suggests that this protein may participate in the regulation of epithelial cell differentiation. Taken together, these data may favor tumorigenesis on KOTs.     

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.