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The interplay between cancer cells and the extracellular matrix (ECM) remains a critical regulator of both normal tissue organization and cancer cell invasion. Proteins that function as ECM receptors function to link the cell with the ECM. Abberations in either the structure of the ECM or the expression of ECM receptors leads to disrupted interaction and downstream signaling effects. Dystroglyan (DG) is an ECM receptor that is expressed in a variety of tissue types and functions to mediate sarcolemma stability, epithelial polarity, and is critical in the early formation of basement membranes. However, DG has primarily been studied in muscle where loss of its function is linked to a host of muscular dystrophies. In the epithelium, the role of DG remains enigmatic. While DG has repeatedly been shown to lose function during cancer development and progression, the mechanism and functional consequence of its loss are currently unknown. In order to increase our understanding of DG in cancer development, we analyzed its expression and glycosylation, a functional requirement for DG, in a range of prostate cancer cell lines. Previous work has shown DG to be downregulated in prostate cancer, but the mechanism by which this occurs has remained largely unclear. We found that DG expression is maintained while its glycosylation was heterogeneous in the cell lines. Further investigation revealed that lines with hypoglycosylated DG strongly associated with the loss of expression of the glycosyltransferase LARGE2. Further this enzyme is frequently downregulated in human cancers and appears to serve as a required enzyme in DG glycosylation within prostate epithelium. This is the first work to demonstrate the functional requirement of LARGE2 for DG, and the only work to implicate loss-of-function of LARGE2 in cancer progression. To determine whether loss of LARGE2 is found in other tumor types, we analyzed human clear cell renal cell carcinoma (ccRCC) samples by immunohistochemistry and via in silico analysis with the Cancer Genome Atlas (TCGA). Our work demonstrated a frequent and significant downregulation of LARGE2 expression and its association with DG hypoglycosylation. Additionally, we found the loss of LARGE2 strongly associated with increased mortality. Thus, we again demonstrated a functional requirement of LARGE2 but also found a clinical correlate with increased mortality. Finally, we examined the functional outcome of DG hypoglycosylation or loss of expression in both a mouse model of prostate cancer and a variety of cell lines models. We found that while loss of DG expression does not increase prostate cancer growth or metastasis in one model of cancer, loss of its glycosylation does seem to mediate downstream metabolic changes within cells. The mechanism for this change remains unclear. In summary, these studies have contributed to our understanding of DG glycosylation and function in both prostate and renal carcinoma. Additionally, we have shown a novel mechanism by which DG glycosylation is lost with downregulation of LARGE2 expression. Finally, while we were unable to demonstrate a clear mechanism by which signaling changes arose, we were able to demonstrate a strong correlation between DG hypoglycosylation and increased mortality in ccRCC. These insights could be used to improve treatment of multiple cancer types as our understanding of DG function continues to improve.

Head and neck cancers account for 3% of all newly diagnosed cancers, of which 90% are squamous cell carcinomas (SCC). Improvements in surgery, radiotherapy and chemotherapy have done little to improve the mortality of this disease over the past 20 years while current clinicopathological predictors of disease outcome are sub-optimal. Identifying molecular targets of prognostic and therapeutic significance in head and neck squamous cell carcinomas (HNSCC) may help direct novel therapies to patients whom it is most likely to benefit. Accrued knowledge of the biology of HNSCC has highlighted specific aberrations in pRb and p53 pathways which warrant further study. An immunohistochemical analysis (IHC) in a cohort of 145 patients with SCC of the anterior tongue was performed. Protein expression of the pRb and p53 pathways and related molecules that directly or indirectly influence cell cycle progression at the G1/S phase checkpoint was assessed. We determined that over-expression of E2F-1 occurred in &gt35% of these cancers and associated with improved overall survival on univariate analysis. The strongest multivariate model included: regional lymph node status, tumour grade, p16INK4A, cyclin D1 and p14ARF. This is the first study to determine that p14ARF is an independent marker of both improved diseasefree survival and overall survival in a cohort of SCC of the anterior tongue. Unrecognized molecular heterogeneity is thought to account for the unpredictable clinical response to ZD1839, an EGFR tyrosine kinase inhibitor. We explored the anti-proliferative effects following ZD1839 treatment alone or in combination with radiotherapy in cyclin D1 and E2F-1 over-expressing SCC9 HNSCC cells. SCC9 cells over-expressing cyclin D1 or E2F-1 were highly resistant to ZD1839 treatment, while E2F-1 clones were also radioresistant. Combined therapy in SCC9 controls had a greater anti-proliferative effect than each individual treatment. These data showed that cyclin D1 and E2F-1 may have utility as markers of ZD1839 resistance. The data in this thesis contribute to our knowledge of the clinical behaviour and molecular pathology of HNSCC. Specifically the molecular data identifies novel markers of outcome in SCC of the anterior tongue such as p14ARF, and therapeutic response to ZD1839 such as cyclin D1 and E2F-1. This study addresses in part, the current issues and limitations of management in HNSCC and has the potential to contribute to strategies that may be developed to improve the outcome for patients who develop HNSCC in the future.

Basal-like breast cancer represents a subgroup of mammary cancers associated with a particularly poor prognosis, as they are refractory to current targeted therapies employed for the treatment of breast cancer. In this work I aimed to explore the therapeutic potential of selective estrogen receptor modulators (SERMs), a targeted breast cancer treatment, in combination with epigallocatechin gallate (EGCG), for the treatment of basal-like breast cancer, using MDA-MB-231 cell as an in vitro model of the disease. A significant reduction in MDA-MB-231 cell number and a significant increase in cytotoxicity was observed following treatment with 25 [mu]M of EGCG in combination with 1 [mu]M of 4-hydroxytamoxifen (4-OHT) (EGCG+4-OHT) or 4 [mu]M of raloxifene (EGCG+Ral) over a 36 h time course. However, these effects were not resolved in time, with an increase in G₁-phase cell cycle arrest. Changes in the metabolism of EGCG were dismissed as a possible mechanism through which the combination treatments may be eliciting the cytotoxicity. Changes in the expression and phosphorylation of various signaling proteins, important for the proliferation and survival of basal-like breast cancer, were investigated through Western blotting. Interestingly, the two combination treatments produced very similar results; reductions in the phosphorylation of EGFR and AKT occurred after 6, 12, and 18 h with EGCG+4-OHT and 6, 12, 18 and 24 h with EGCG+Ral, while a reduction in S6K phosphorylation was observed following 6, 12, 18 and 24 h of both combination treatments. Interestingly, both SERMs contributed significantly to the net reduction in S6K phosphorylation, induced by the combination treatments. Both combination treatments were also associated with a significant increase in the phosphorylation and total expression of stress activated protein kinases, p38 and JNK1/2 following 12, 18 and 24 h of treatment. As changes were observed at an intracellular signaling level, the effect of the combination treatments were investigated at the transcriptomic level after 18 h of treatment, using human oligonucleotide microarrays. This transcriptomic analysis revealed that both combination treatments reduced the transcript expression of five enzymes involved with cholesterol synthesis, which was confirmed through qRT-PCR. Cholesterol is an important component of the plasma membrane and is critical for the transduction of extracellular signals. Furthermore, both combination treatments induced the transcriptomic expression of the zinc coordinating metallothionein (MT) proteins. This was associated with an increased nuclear localization of MTF-1, the transcription factor responsible for MT expression, after 6, 12 and 18 h of both combination treatments. Finally, nuclear Western blotting of the NF-[kappa]B subunit, p65, revealed that both combination treatments reduced the nuclear localization of NF-[kappa]B following 6, 12 and 18 h. In collating this data, it appears that the combination treatments of EGCG+4-OHT and EGCG+Ral are inducing cytotoxicity through various mechanisms, including reduced cellular signaling through EGFR, AKT and S6K, increased stress signaling through JNK1/2 and p38 and altered gene expression of MTs and enzymes involved with cholesterol synthesis. Therefore, the combination treatment of EGCG+SERMs exhibits therapeutic potential in MDA-MB-231 cells, a model of basal-like breast cancer.

<p>The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.</p>

Introduction Avec plus de 7000 cas diagnostiqués par an en Belgique, le cancer bronchique est l’un des cancers les plus fréquents chez l'homme. Son pronostic est très réservé, la survie à 5 ans tous stades confondus étant inférieure à 15 %. Ce faible taux de guérison s’explique en grande partie par le fait que le diagnostic se réalise généralement à un stade avancé de la maladie. Une méthode de détection précoce, l’endoscopie en autofluorescence, exploite des variations de la fluorescence bronchique sous l'effet d'un laser et permet ainsi de dévoiler des lésions précancéreuses invisibles en lumière blanche. Réalisée à l’Institut Jules Bordet depuis 1996, la photodétection nous a permis de constituer une banque de biopsies d’épithélium bronchique à tous les stades de la carcinogenèse bronchique, conservées initialement en blocs paraffinés et, depuis 2003, par congélation. Hypothèse Nous avons émis l’hypothèse que la compréhension de la genèse du carcinome épidermoïde bronchique par la caractérisation de l’évolution des anomalies moléculaires dans les lésions précancéreuses et cancéreuses de l'arbre bronchique nous permettrait d’identifier de nouvelles cibles de détection et éventuellement de chimioprévention pour le cancer bronchique. L'objectif de nos travaux a donc été de caractériser les modifications moléculaires successives et/ou cumulatives sous-jacentes à la transition entre les différents stades histologiques de la carcinogenèse épidermoïde bronchique (épithélium bronchique normal du fumeur, hyperplasie, métaplasie, dysplasies légère, modérée et sévère, CIS et les carcinomes invasifs). Différentes techniques complémentaires ont été successivement utilisées à cet effet : 1) étude de l’expression protéique par immunohistochimie (IHC) et immunofluorescence (IF), 2) étude de l’expression des ARNs messagers par microdamiers, 3) étude de l’expression des microARNs par RT-PCR quantitative. Travaux réalisés Afin de sélectionner les protéines à étudier aux différents stades de la carcinogenèse épidermoïde bronchique, nous avons réalisé une étude méthodologique de la littérature chez les patients atteints de cancer bronchique non à petites cellules (CBNPC). En effet, d’une part, la petite taille des biopsies bronchiques que nous projetions d’étudier et, d’autre part, le faible taux de découvertes de lésions de haut grade lors de la réalisation de bronchoscopies en fluorescence limitent le nombre d’analyses réalisables. Les revues systématiques de la littérature, intégrant une analyse méthodologique des études publiées et une méta-analyse de leur impact pronostique en terme de survie, ont permis de choisir une série de marqueurs sur base de leur intérêt potentiel grâce à la puissance apportée par l’agrégation de grands nombres de cas. La mutation de KRAS identifiée par PCR constitue un facteur péjoratif pour la survie des patients atteints de CBNPC et plus spécifiquement des adénocarcinomes (ADC). Etant donné que notre projet est consacré à la genèse du carcinome épidermoïde bronchique (CEB), nous n’avons pas étudié ce facteur dans nos biopsies. Par contre, les revues systématiques ont montré un impact en terme de survie dans les CBNPC, y compris dans les CEB, pour l’angiogenèse, des récepteurs de facteurs de croissance épithéliale (EGFR et c-erbB-2) et des marqueurs de la prolifération cellulaire (Ki-67). Nous avons donc étudié l’expression de ces protéines par IHC dans les lésions précurseurs de CEB et dans les CEB. L’expression de Ki-67 est augmentée aux stades de dysplasie sévère et de CIS par rapport à ceux de dysplasies légère et modérée. La mesure de l’expression de Ki-67 dans ces biopsies aide donc à les classifier en lésions de bas et de haut grade. L’étude de la corrélation de l’expression de plusieurs protéines (EGFR, c-erbB-2 et Ki-67) dans les mêmes lésions bronchiques a montré que la majorité des biopsies de haut grade (dysplasies sévères et CIS) expriment anormalement EGFR et/ou Ki-67. Par contre, l’homologue d’EGFR, c-erbB-2, n’apparaît que plus tardivement, dans les carcinomes invasifs. Nous avons également caractérisé l’expression de protéines impliquées dans les voies de l'apoptose dans les lésions précurseurs de CEB. L’expression du facteur suppresseur de tumeurs p53 s’est avérée être un marqueur pronostique péjoratif important pour la survie des patients atteints de CNBPC et augmente dès les premières étapes de la tumorigenèse bronchique et même déjà dans l’épithélium morphologiquement normal du fumeur. L'altération de l'expression de p53 est donc un événement très précoce dans la genèse du CEB et la positivité de p53 augmente ensuite avec la sévérité des lésions. Dans le but de caractériser les voies de contrôle de p53 au cours de la carcinogenèse épidermoïde bronchique, nous avons analysé l'expression de 3 autres protéines, MDM2, p14arf et la nucléophosmine (NPM), dans une série de 200 biopsies. L’expression de MDM2, NPM et p14arf est respectivement altérée aux stades de dysplasie légère, modérée et sévère. Au stade de dysplasie sévère, l’expression de p14arf, inhibiteur de MDM2, peut soit disparaître, soit se concentrer dans les nucléoles. Tant la perte de p14arf que sa concentration dans les nucléoles sont associées à une augmentation de l’expression de MDM2. Nous avons également observé que la délocalisation de NPM depuis le nucléoplasme vers le nucléole est hautement corrélée à celle de p14arf. En IF, NPM et p14arf colocalisent dans le nucléoplasme dans les échantillons de bas grade ou dans les nucléoles dans les lésions de haut grade. Par contre, la protéine MDM2 n’est détectée dans les nucléoles quelles que soient les localisations de p14arf ou de NPM et quel que soit le stade. Ces données sont en faveur de l’hypothèse selon laquelle la localisation de p14arf dans les nucléoles empêcherait la formation des complexes p14arf–MDM2 et pourrait ainsi faciliter la carcinogenèse pulmonaire. Nos résultats suggèrent également que la présence de NPM dans le nucléoplasme pourrait jouer un rôle protecteur contre le dommage cellulaire et la transformation maligne tandis que sa délocalisation vers les nucléoles et ce, peut-être par la délocalisation de p14arf, favoriserait la carcinogenèse bronchique. Enfin, suite à la réalisation d’une méta-analyse montrant le rôle de la cyclooxygénase 2 (COX-2) en tant que facteur pronostique dans les CBNPC de stade précoce, l’expression de la protéine COX-2 a été analysée par IHC dans 106 biopsies. Cette étude montre que cette protéine s’accumule exclusivement à partir du stade de dysplasie sévère, permettant ainsi de séparer les lésions bronchiques de bas et de haut grade. Avec une haute valeur prédictive positive (100 %) et une bonne valeur prédictive négative (82,35 %), COX-2 apparaît donc comme un marqueur précoce potentiel à tester pour le dépistage du cancer bronchique. En parallèle à ces travaux, nous avons collecté des biopsies fraîchement congelées aux différents stades de la carcinogenèse épidermoïde pour une analyse du transcriptome. La première étape a consisté à définir le profil d’expression génique par la technique des microdamiers. Après son extraction et sa rétrotranscription, l’ARN a été marqué et hybridé sur des lames commercialisées par Agilent Technologies. Au total, 122 échantillons, dont minimum 12 de chaque catégorie, ont été hybridés avec un contrôle commun (issu d’un mélange de biopsies bronchiques normales de non-fumeurs). Une analyse en composante principale a montré que la hiérarchie de la classification histologique était bien représentée par les profils d’expression génique des biospies aux différents stades. Quelle que soit la liste de gènes de départ sélectionnée pour réaliser le regroupement hiérarchisé, nous avons observé, de manière constante et robuste, une différence majeure de profil d’expression génique entre, d’une part, les tissus bronchiques les plus « normaux » (normal normofluorescent, histologiquement normal mais hypofluorescent et hyperplasie) et, d’autre part, toutes les autres catégories histologiques dites « anormales ou modifiées » à partir de la métaplasie jusqu’au carcinome invasif. Parmi les épithéliums bronchiques « modifiés », deux grands groupes se distinguent : d’un côté, les métaplasies et dysplasies légères, qui sont très souvent bénignes et réversibles pour la plupart, et de l’autre côté, les dysplasies sévères, les CIS et les carcinomes invasifs, lésions à risque beaucoup plus élevé d’évoluer vers un cancer ou déjà malignes. Les échantillons au stade de dysplasie modérée se classent tantôt dans le deuxième groupe avec les dysplasies légères tantôt dans le troisième avec les dysplasies sévères. Il semble donc que le stade histologique de la dysplasie modérée soit un groupe hétérogène n’ayant pas de réalité biologique et qu’il serait plus adéquat de les reclasser dans un des 2 autres groupes sur base des arguments moléculaires. Nous avons obtenu les listes de gènes dont l’expression varie de manière statistiquement significative entre les différentes étapes de la carcinogenèse épidermoïde bronchique. Par ailleurs, nous avons pu mettre en évidence un profil d’expression génique permettant de discriminer les lésions bronchiques de mauvais pronostic (dysplasies sévères ou plus) de celles de meilleur pronostic (tissu normal et anomalies morphologiques de bas grade). Les gènes qui constituent ce profil permettant d’identifier les lésions de mauvais pronostic sont des candidats pour la détection précoce du cancer bronchique. De plus, nos données de génomique confirment la surexpression de certains ARNs messagers correspondant à des protéines dont la surexpression a été rapportée dans les études d’IHC comme COX-2, MDM2, Ki-67, les cytokératines, les métallopeptidases, les cyclines. Par ailleurs, certaines protéines dont le rôle dans les cancers pulmonaires invasifs a déjà été décrit mais pas aux stades pré-invasifs, parmi lesquelles la protéine PTH-like, des protéines liées à TNF ou d’autres liées à IGF, E2F ou à Wnt, semblent impliquées aux stades précoces de la carcinogenèse épidermoïde bronchique. Enfin, de nouveaux acteurs possibles de ce processus ont été mis en évidence. Nous avons également étudié l’évolution de l’expression des microARNs au cours de la carcinogenèse bronchique par des RT-PCR quantitatives (LDA) sur 60 des biopsies dont nous avons étudié le transcriptome (6 par catégorie) et en utilisant la classification en 3 groupes issue des analyses des microdamiers d’expression génique. Cette étude montre que plusieurs miARNs sont différentiellement exprimés durant la carcinogenèse bronchique. De plus, deux étapes successives et distinctes ont été mises en évidence pour l’évolution de l’expression des miARNs au cours de la carcinogenèse bronchique. Aux stades les plus précoces, correspondant à la progression de l’épithélium normal du non-fumeur vers l’épithélium histologiquement normal du fumeur et l’hyperplasie jusqu’au groupe des anomalies morphologiques relativement bénignes (métaplasie, dysplasies légère et modérée), on observe une réduction significative de l’expression de la grande majorité des miARNs. Aux stades plus tardifs de la carcinogenèse (dysplasie sévère, CIS et CEB), même si 74 % des miARNs altérés restent encore régulés négativement par rapport à leur niveau d’expression dans l’épithélium normal du non-fumeur, la proportion de miARNs dont l’expression est augmentée (43 %) s’accroît par rapport à leur niveau d’expression dans les stades qui les précèdent (métaplasie, dysplasies légère et modérée). En outre, lorsque l’on compare ce dernier groupe à celui des lésions plus sévères (dysplasie sévère et CIS), qui progressent fréquemment vers des carcinomes invasifs, 80 % des miARNs augmentent leur niveau d’expression. Par ailleurs, l’expression de certains microARNs évolue de manière linéaire. En particulier, l’expression de miR 34c, cible transcriptionnelle de p53, et celle de miR 15a, inhibiteur de Bcl2, diminuent progressivement entre les différents stades à partir du tissu bronchique normal du non-fumeur jusqu’au CEB. Enfin, les profils d’expression des miARNs permettent de prédire avec précision la classification histologique non seulement entre les lésions de bas grade (métaplasie, dysplasies légère et modérée) et de haut grade (dysplasie sévère et CIS) mais également entre les CIS et les carcinomes invasifs. Les miARNs qui constituent ces signatures sont donc des candidats potentiels pour la détection précoce du cancer bronchique. Conclusions Nos travaux montrent que les anomalies moléculaires apparaissent aux stades les plus précoces de la transformation maligne de l’épithélium bronchique. L’expression protéique des marqueurs étudiés -p53, MDM2, p14arf, NPM, Ki-67, COX-2, c-erbB-2, et EGFR- permet d’affiner la classification des lésions bronchiques précancéreuses et de mieux comprendre les voies de la carcinogenèse précoce. Les profils d'expression des gènes et des microARNs apportent une approche originale des étapes successives du processus de carcinogenèse épidermoïde bronchique et ont mis en évidence des signatures permettant de discriminer les lésions qui sont à très haut risque de progression vers un cancer invasif ou qui sont déjà néoplasiques de celles de meilleur pronostic. Les marqueurs qui constituent ces profils sont des candidats potentiels pour la détection précoce du cancer bronchique.

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

The esophageal squamous cell carcinoma (ESCC) is known to be one of the most difficult malignancies to treat among digestive carcinomas. The esophageal squamous cell carcinoma and adenocarcinoma are the two most common cell types of esophageal cancer in the world. Even though the incidence of esophageal adenocarcinoma has been dramatically increasing in Western populations during the past two decades, esophageal squamous cell carcinoma remains the predominant type of esophageal malignancy in the remainder of the world. In Taiwan, ESCC is the ninth leading cause of cancer deaths, the 6th among male. In spite of advances in surgical techniques and perioperative management in recent decades, current modalities of therapy for this disease still offer poor survival and cure rates. Even in resectable diseases, the 5-year survival rates were only less than 20%. Recently, esophageal squamous cell carcinoma and adenocarcinoma were increasingly recognized as two entities with different biologic behavior and outcome. Consequently, the surgical risks and oncologic benefits of esophagectomies for esophageal squamous cell carcinoma patients are controversial and not confessed. From 1991 to 2003, 216 esophageal squamous cell carcinoma patients underwent esophagectomy were enrolled and analyzed retrospectively. Among these patients, 166 patients underwent transthoracic esophagectomy and 50 patients underwent transhiatal esophagectomy. The overall hospital mortality and postoperative complication rates were 9.7% and 49%, respectively. The overall 5-year survival rate was 16.8%. The hospital mortality rate, postoperative complication rate, length of hospital stay and amount of intra-operative blood loss or transfusion were not significantly different between both groups. But, shorter operative time was noticed in transhiatal group (p<0.001). Patients underwent either transthoracic or transhiatal esophagectomy had comparable long-term survival. The pTNM stage was independent prognostic factors for patients underwent transthoracic esophagectomy. However, location of tumors (p=0.0085) and pathologic tumor length (p=0.0118) were significant predictors for patients underwent transhiatal esophagectomy. In this part of study, we found that both transthoracic and transhiatal esophagectomies could provide comparable survival benefits for esophageal squamous cell carcinoma patients. However, the traditional pTNM staging system might underestimate the severities of ESCC patients who underwent transhiatal esophagectomy. Due to the dismal results of ESCC patients after surgery and the findings of multi-environmental and/or genetic factors involved in the carcriogenesis of ESCC, further realizing the molecular mechanisms of carcinogens in the development of esophageal squamous cell carcinoma is crucial for prevention and treatment for this disease. Epidemiological analysis showed that the prevalence of esophageal squamous cell carcinoma varied in different geographic areas. The various prevalence of disease in different parts of the world reflects different forms and extents of exposure to these etiological agents involving the development of this disease. In stead of tobacco and alcohol, recent reports indicated that betel quid (BQ) chewing also significantly correlated with the occurrence of esophageal squamous cell carcinoma in Taiwanese. The mechanisms behind the BQ-related esophageal squamous cell carcinoma in Taiwan are worthy for further investigation. Previous studies have shown that certain carcinogens may induce a ¡§fingerprint-like¡¨ ¡V like pattern of mutations at the p53 gene, both in terms of mutation type and codon specificity. However, the role of p53 mutation in the etiology of esophageal squamous cell carcinoma has not been rigorously studied in Taiwan. The incidence of p53 mutations in ESCC associated with risk factors has not been explored in Taiwanese. Accordingly, 75 primary esophageal squamous cell carcinoma specimens were collected for examining the incidence of mutations in the conserved regions of p53 gene by using polymerase chain amplification and direct sequencing of amplified products. There were 37 mutations of p53 gene detected in 45.5% (34/75) of tumor specimens. These mutations significantly clustered in exon 5 (21/37) of p53 gene. The incidence of p53 mutations didn¡¦t associate with clinicopathological characteristics and habits of cigarette smoking or alcohol drinking. However, BQ chewer exhibited significantly higher incidence of p53 gene mutations than non-chewer (67.6% vs. 32.4%, p=0.007). After controlling confounding factors of cigarette smoking and alcohol drinking, BQ chewing still showed significant impact on the incidence of p53 mutation in esophageal squamous cell carcinomas (RR=4.233; 95% CI, 1.317-13.603). The A:T to G:C transition (8/37, 21.6%) and G:C to T:A transversion (5/23, 13.5%) were the prevalent spectrum of p53 gene mutations. All A:T to G:C transitional mutations occurred in patients with habits of betel quid chewing and cigarette smoking. Noticeably, alcohol drinking could enhance this peculiar spectrum of p53 mutation in esophageal squamous cell carcinoma. Therefore, p53 gene might be one of the molecular targets of betel quid carcinogens in the development of esophageal squamous cell carcinoma in Taiwanese. Determination of the importance of p53 gene mutation in ESCC requires further study. To elucidate the role of cell cycle associated genes in ESCC, 40 primary esophageal squamous cell carcinoma patients were included in this part of study. Tissue samples were analyzed for cell proliferation, DNA content, mutation of p53 gene, and expression of p16, p21waf1/cip1, pRb and p53 proteins. In this part of study, 75% of tumors exhibited aneuploid DNA content. Significantly higher S-phase fractions were detected in tumor samples (p<0.001). The p53 immunostaining was detected in 62.5% (25/40) of tumor tissues and 50% of tumors were p21waf1/cip1 overexpression. The p16 protein was only detected only in 8 of the 40 ESCC (20.0%) tissue samples by immunohistochemistry. The nucleus stained Rb protein was detected in 38 ESCC tissue samples and all of them were phosporylated status. The phosphorylation of pRb at Ser-795, Ser-789 and Ser-807/811 was detected in 87.5% (35/40), 72.5 (29/40) and 42.5% (17/40) of ESCC tissue samples, respectively. Expression of p16 protein, total pRb or phospho-Rb expression status did not correlate with the clinicopathological parameters of patients. The overexpression of p21waf1/cip1 protein didn¡¦t correlate with p53 gene status, but significantly correlated with the existence of abnormal DNA content (P=0.028). Advanced pTNM stage, lymph node metastasis and p21waf1/cip1 overexpression conferred survival disadvantages in univariate analysis (P=0.013, 0.045 and 0.017, respectively). A Cox multivariable analysis revealed pTNM stage (IIB/III/IV vs. I/IIA; p=0.024) associated with p21waf1/cip1 overexpression (positive vs. negative; p=0.035) as independent prognostic factors in esophageal squamous cell carcinomas. Surprisingly, p21waf1/cip1 overexpression significantly compromised the survival of patients with mutated p53 gene (p=0.035). However, no significant dismal effect of p21waf1/cip1 overexpression can be seen in patients with wild-type p53 gene (P=0.175). Consequently, overexpression of p21waf1/cip1 is correlated with chromosomal instability and serves as an adverse prognostic predictor for esophageal squamous cell carcinoma patient. Its dismal effect is more prominent when p53 gene is mutated. The ribonucleotide reductase (RNR) is an S phase-specific dimeric enzyme and is the rate-limiting enzyme of DNA synthesis pathway responsible for the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides (dNTPs), which are the building blocks for DNA replication and repair in all living cells. The RNR enzyme was formed by the association of RRM1 and RRM2 subunits. Normally, the levels of RRM2 expression modulate the RNR enzymatic activity. However, RRM2 also plays an important role in other aspects of the malignant phenotype such as tumor development and drug resistance. Recently, the p53-inducible ribonucleotide reductase small subunit homologue, p53R2, has been isolated and shown to play a crucial role in DNA repair after DNA damage. However, the function of p53R2 is still unclear especially in tumor cells. By immunohistochemistry, the expression of RRM2 and p53R2 proteins were detected in 94.1% (80/85) and 55.3% (47/85) of ESCCs tissue samples, respectively. No significant correlation could be found between RRM2 protein expression and gender, depth of tumor invasion, lymph-node involvement and pTNM stage. The p53R2 expression status also did not correlate with the gender of patients and the depth of tumor invasion. However, the presence of p53R2 protein expression significantly correlated with pTNM stages of tumors (p=0.027) and lymph node metastasis (p=0.009). In Cox multivariable regression analysis, p53R2 expression (positive vs. negative; p=0.011, HR: 3.096, 95% CI: 1.294-7.407) together with pTNM stage (IIB/III/IV vs. I/IIA; p=0.005, HR: 2.496, 95% CI: 1.320-4.719) was shown to have independent prognostic impact on survival of ESCC patients. Accordingly, in the analysis of cell cycle associate genes, the p53 mutations associated with loss of Rb pathway function would be the critical event in the development of human esophageal squamous cell carcinoma. In ESCC, loss of p53 pathway function is attributable to p53 mutations. However, the Rb pathway might be perturbed by inactivation of p16 and over-expressed p21waf1/cip1 protein in ESCC tissues. Consequently, the perturbed Rb function results in up-regulation ribonucleotide reductase activity, causing DNA precursors overproduction and cell proliferation. Using immunohistochemistry, our findings provide the first evidence of RRM2 and p53R2 expression in human ESCCs. We identified the different prognostic effects of RRM2 and p53R2 expression in human ESCCs. The identification of different roles of p53R2 and RRM2 involved in the carcinogenesis of esophageal squamous cell carcinomas might be useful for designing more effective RRM2 or p53R2 specific target therapy for esophageal squamous cell carcinoma to improve the clinical outcome of patients with esophageal carcinoma.

DNA repair mechanisms counteract the formation of deleterious DNA lesions and maintain genomic integrity. Nucleotide excision repair (NER) is an important DNA repair pathway because of its extraordinarily large substrate specificity. P53 protein regulates NER pathway in a transcription-dependent or transcription-independent manner. Inherited polymorphisms of NER pathway genes (XPC, HR23B, XPA, DDB2, XPB, XPD, ERCC1, XPF, and XPG) and TP53 gene may contribute to individual variations in genetic susceptibility to OSCC and correlate with the prognosis of 204 OSCC patients. We carried out a hospital-based case-control study to investigate the association of 25 various polymorphisms of nine NER pathway genes and TP53 gene with the risk for OSCC. There were 34 newly diagnosed OSCC patients and 135 frequency-matched controls without BQ chewing and smoking habit as well as 313 newly diagnosed OSCC patients with BQ chewing or smoking habit and 312 frequency-matched controls being recruited between November 2003 and July 2007 at Kaoshiung Veterans General Hospital. Genotyping was performed using the PCR-RFLP techniques or TaqMan real-time PCR method. The significant association between polymorphisms of NER pathway genes and OSCC risk was mainly found among subjects with BQ chewing or smoking habit. In the single locus analysis, GA and AA genotypes of ERCC1 G-641A (AOR, 0.64; 95% CI, 0.45-0.93 and AOR, 0.48; 95% CI, 0.29-0.79, respectively; p for trend, 0.002), CT genotype of XPF C-850T (AOR, 1.53; 95% CI, 1.08-2.18; p for trend, 0.014), as well as GG genotype of XPB A-1039G (AOR, 0.51; 95% CI, 0.26-0.98; p for trend 0.034) were significantly associated with the risk of OSCC. Furthermore, -641G/ -425T or -641G/ -425C haplotype of ERCC1 (AORs, 1.34; 95% CI, 1.02-1.77 and AOR, 1.56; 95% CI, 1.18-2.07, respectively; p for trend 0.002) as well as -850T/ -247T and -850T/ -247C haplotype of XPF (AOR, 1.45; 95% CI, 1.09-1.94 for; p for trend 0.016) were strongly associated with the risk of OSCC. A trend toward increased risk of OSCC was observed when people with the increasing number of at risk genotypes in the combined analyses of nine NER pathway genes with (p for trend, <0.001) or without (p for trend 0.001) TP53 gene. Finally, in the stratification analysis, the combined effects of nine NER pathway genes had a significantly increased risk of OSCC among younger group (¡Ø50 years old), Fukienece population, BQ chewers, light smokers, or light drinkers. Besides, in the prognosis analysis of 204 OSCC patients, HR23B A-823C, polymorphisms of XPA gene, XPD C-643G, XPG C787G, and the number of at risk genotypes of NER pathway genes were associated with pathologic stage, T classification, or N classification. The association between NER genetic polymorphisms and survival of patients was only found in XPA C-1778T polymorphism. These results suggested that the single polymorphism of XPB A-1039G, ERCC1 G-641A and XPF C-850T, the joint effect of genetic polymorphisms of NER pathway genes, and gene-environment combined effect were associated with the risk of OSCC. Furthermore, in the analysis of NER genetic polymorphisms and prognosis of OSCC, we found polymorphisms of XPA gene might be a prognostic factor for OSCC.