Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions. Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions. Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction. These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction. / published_or_final_version / Physiology / Master / Master of Philosophy

As CFTR plays an important role in HCO3- transport, and HCO3- sensitive soluble adenylyl cyclase (sAC) has been shown to be largely responsible for the cAMP production in spermatogenetic cells, we hypothesized that CFTR-mediated HCO3- transport was important to spermatogenesis via sAC pathway in spermatogenetic and Sertoli cells. Using intracellular pH measurement, we demonstrated that CFTR is involved in HCO3- transport in Sertoli cells. RT-PCR results showed that increased HCO3- concentrations in the culture medium resulted in upregulation of CFTR expression. The results also showed that the intracellular cAMP level in Sertoli cells increased as the extracellular HCO3- concentration increased. HCO3- also caused phosphorylation of the cAMP response element binding (pCREB) proteins transcription factor on serine 133, a modification known to be required by Sertoli cells to support spermatogenesis. This phosphorylation could be inhibited by CFTR inhibitor, further lending support to the notion that CFTR is important for HCO3- transport in Sertoli cells, leading to HCO3- dependent events that are important for spermatogenesis. / CFTR is known to be widely expressed in epithelial cells of male reproductive tracts, but its expression in spermatogenic cells is less well known. We first confirmed the expression of CFTR in spermatogenic cells and mature sperm in rodents. Our study thus focused on the important role of CFTR in the processes related to male fertility including spermatogenesis and sperm capacitation. / Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl- and HCO3- transport. While over 95% of CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. / In conclusion, our study has demonstrated the role of CFTR in male reproductive system. We have further elucidated its possible physiological role and the underlying molecular mechanisms. These studies may pave the way for the development of method strategies for diagnosis and treatment of CFTR related infertility in male. / Our study also detected CFTR in both human and mouse sperm. CFTR inhibitor or antibody significantly reduced sperm capacitation, and the associated HCO 3--dependent events including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower as compared to that of the wild type. These results suggest that CFTR in sperm may be involved in the transport of HCO3- important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD. / We further demonstrated the physiological role of CFTR in spermatogenesis using CFTR knockout mice as an in vivo model. Although TUNNEL staining showed normal percentage of apoptotic cells in seminiferous tubules, Cftr -/- mice had spermatogenetic defects in histology section and fewer number of mature sperm compared with wild type (WT) mice. Consistent with the proposed role of CFTR in spermatogenesis, RT-PCR and Western blot results showed reduced expression of spennatids specific gene, Protamine 1, Protamine 2, and CREM, which have been known to be involved in the process of spermatogenesis, in Cftr-/- mice. / Xu, Wenming. / "January 2008." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4506. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 121-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

上皮間充質轉化（EMT），作為重要的生理和病理事件，廣泛的參與胚胎發育、組織纖維化病變及腫瘤轉移的過程。這一顯著的細胞表型變化包括上皮細胞失去緊密連接和極性，上皮細胞呈現纖維細胞形態以及增強的細胞移動性。囊性纖維變性跨膜電導調節器（CFTR）是一種廣泛表達於上皮細胞的氯離子和碳酸根離子通道。研究證實，CFTR 的蛋白轉運與上皮連接的形成和功能有關，同時 CFTR 的表達受到 EMT 誘導因子 HIF-1 和 TGF-β 的反向調節。另外，CFTR 的表達和功能被證實參與 EMT 相關信號分子 Wnt 和 NF-κB活性的調節。基於上述發現，本研究旨在闡述 CFTR 與 EMT 的相關性。 / CFTR 參與的腎上皮 EMT 以及後續的腎纖維化首先被關注。實驗表明，在腎上皮細胞（MDCK）中，小 RNA 介導的 CFTR 基因敲降或抑製劑引起的CFTR 通道功能缺陷均引起間充質細胞特徵的出現，包括纖維狀細胞形態、細胞連接分子 E-cadherin, ZO-1 和 Occludin 表達下調和間充質細胞標誌分子 Vimentin 和 N-cadherin 上調、上皮細胞跨膜電阻減低以及細胞遷徙能力的增強。有趣的是，在單側尿道結紮的腎纖維化模型中，CFTR 表達被顯著下調。同時，動物實驗證實一個最常見的 CFTR 分子突變（deltaF508 -/-）增加了單側尿道結紮導致的腎纖維化的程度。另外，在缺氧引起的 EMT 過程中CFTR 的表達顯著下調；同時，腎纖維化模型中，HIF-1 和 CFTR 的表達呈現負相關。結果提示，生理及病理的條件下，氧氣的調節可能作為 CFTR 下調及其後續事件的誘因。進一步實驗發現，CFTR 功能抑製或基因突變可以引起Wnt 的富集和 β-catenin 的細胞核轉移。基於以上的實驗結果，在腎纖維化的過程中，CFTR 參與了缺氧引起的 EMT 過程，並通過激活 Wnt/β-catenin 信號調節相關的下游因子。 / 第二部分集中探究了 CFTR 在癌細胞EMT 及腫瘤轉移中的作用及機制。實驗證實，在 TGF-β 誘導的腫瘤細胞 EMT 過程中，CFTR 表達被抑制。TGF-β 可能作為病理狀態下的調節因子，引起腫瘤細胞中 CFTR 表達下調及EMT。抑制 CFTR 通道功能或敲降其蛋白表達導致明顯的間充質細胞特徵，這一變化在不同來源的腫瘤細胞系中呈均一性。相對地，過表達 CFTR 引起細胞遷移和侵潤能力地顯著下降。在體實驗顯示，CFTR 表達與腫瘤的轉移能力呈現負相關。進一步機制研究證明，CFTR 通過調節多重的通路參與 EMT的過程。首先，uPA 的表達和活性受到 CFTR 的反向調節，並且這一調節作用是由激活的 NF-κB 介導的。其次，抑制 CFTR 通道功能引起 β-catenin 的細胞核轉移。 / 綜上所述，研究發現 CFTR 通過調節多重信號參與腎上皮及腫瘤細胞的 EMT。同時，研究顯示 CFTR 的表達和功能與腎纖維化及腫瘤轉移有關。此研究對相關疾病的診斷和預後具有潛在的提示作用。 / Epithelial-Mesenchymal Transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. It is essential for numerous physiological and pathological processes, such as embryonic development, tissue fibrosis and cancer metastasis. The dramatic phenotype changes of EMT include loss of tight junctions and polarity, acquisition of a fibroblastic morphology and increased motility. The cystic fibrosis transmembrane regulator (CFTR) is known as an anion channel and extensively expressed in a variety of epithelial cells. Interestingly, the apical membrane expression of CFTR is reported to be required for the normal organization and function of epithelial junctions. Moreover, EMT inducers, such as HIF-1 and TGF-β, are known to suppress the expression of CFTR in epithelial cells. In addition, CFTR has been reported to be associated with expression and/or activity of Wnt and NF- κB, key factors known to be involved in EMT. Thus, we hypothesized that CFTR might play an important role in EMT. / In the first part of the study, the involvement of CFTR in EMT of kidney epithelial cells and renal fibrosis was investigated. Our experiments revealed that suppression of CFTR by either inhibitor or knockdown induced EMT in Madin- Darby canine kidney epithelial cells (MDCK). This was accompanied by the appearance of fibroblastic morphology, with reduced expression of epithelial junction proteins E-cadherin, ZO-1 and occludin and accumulated expression of the mensenchymal markers vimentin and N-cadherin, as well as reduced transepithelial resistance (TER) and enhanced migratory ability. Interestingly, the expression of CFTR was found significantly down-regulated in unilateral urethral obstruction (UUO) kidney. In addition, CFTR mutant (deltaF508 -/-), the most common mutation found in CF patients, increased the risk of renal fibrosis in UUO model. Our results showed that the expression of CFTR down-regulated in hypoxia induced-EMT in MDCK, and the expression of hypoxia-sensitive transcription factor, HIF-1, is inversely correlated with CFTR in UUO kidney. Accumulation of Wnt and translocation of β-catenin were also observed in CFTR inhibitors-treated MDCK and deltaF508 -/- UUO mice. Taken together, these findings suggest that CFTR may be involved in mediating hypoxia-induced EMT by influencing the Wnt/β-catenin signaling contributing to renal fibrosis. / In the second part of the study, the role of CFTR in EMT during cancer metastasis and the underlying mechanisms were investigated. Recent studies have demonstrated that cancer cells may reinstitute properties of developmental EMT including enhanced migration and invasion. On the other hand, the reverse process, known as mesenchymal-to-epithelial transition (MET), has been implicated in forming a secondary metastatic tumor. Using various tissue-derived cancer cell lines including human colorectal cancer cell line LIM1863, human lung carcinoma cell line A549, and human breast cancer cell lines MCF7 and MDA-MB-231, we report that induction of EMT by TGF-β sharply reduces CFTR expression in various tissue derived cancer cell lines, while overexpression of CFTR can reverse the TGF-β- induced EMT phenyotype. Interfering with CFTR function either by its specific inhibitor or lentiviral miRNA-mediated knockdown mimicks TGF-β-induced EMT and enhances cell migration and invasion. Ectopic overexpression of CFTR in a highly metastatic cancer cell lines downregulates EMT markers and suppresses cell invasion and migration in vitro, as well as the ability of the cells to metastasize to the lung in vivo. The EMT-suppressing effect of CFTR is found to be associated with its ability to alter NF-κB targeting urokinase-type plasminogen activator (uPA) and the nuclear translocation of β-catenin. Taken together, the present study has demonstrated a previously undefined role of CFTR as an EMT suppressor in cancer. / In summary, our findings have demonstrated a regulatory role of CFTR in EMT in both normal kidney epithelial cell line and various cancer cell lines. We conclude that CFTR plays important roles in renal fibrosis and cancer progression/metastasis by modulating EMT process through multiple pathways. The insights afforded by these studies will provide critical new information about the function of CFTR as a suppressor of EMT, which may have potential application in diagnosis and prognosis of fibrosis and cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Jieting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 136-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epithelial-Mesenchymal Transition --- p.1 / Chapter 1.1.1 --- Concept and features of EMT --- p.2 / Chapter 1.1.2 --- Roles of EMT in development and diseases --- p.10 / Chapter 1.1.3 --- The Regulators of EMT --- p.13 / Chapter 1.2 --- Structure and function of CFTR --- p.18 / Chapter 1.2.1 --- General structure and channel functions of CFTR --- p.18 / Chapter 1.2.2 --- Gene mutations and CF --- p.18 / Chapter 1.3 --- Potential role of CFTR in EMT --- p.20 / Chapter 1.3.1 --- CFTR in formation of cell-cell junction and membrane polarity --- p.20 / Chapter 1.3.2 --- CFTR and EMT inducers --- p.21 / Chapter 1.3.3 --- CFTR and EMT related pathways and factors --- p.22 / Chapter 1.4 --- Hypothesis and aim of the study --- p.22 / Chapter Chapter 2 --- CFTR involves in hypoxia induced EMT in renal fibrosis --- p.24 / Chapter 2.1 --- Abstract --- p.24 / Chapter 2.2 --- Introduction --- p.25 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Knockdown of CFTR induces EMT in MDCK --- p.30 / Chapter 2.3.2 --- Inhibition of CFTR channel function induces EMT in MDCK --- p.30 / Chapter 2.3.3 --- CFTR is downregulated during the process of renal fibrosis --- p.36 / Chapter 2.3.4 --- CFTR defect increases the risk of renal fibrosis --- p.39 / Chapter 2.3.5 --- Hypoxia/HIF-1α rather than TGF-β as the inducer of CFTR repression during EMT and renal fibrosis --- p.44 / Chapter 2.3.6 --- CFTR as a negative regulator of Wnt/β-catenin signaling in renal epithelium --- p.51 / Chapter 2.4 --- Discussion --- p.57 / Chapter 2.5 --- Conclusion --- p.61 / Chapter 2.6 --- Materials and Methods --- p.61 / Chapter 2.6.1 --- Cell culture and treatments --- p.61 / Chapter 2.6.2 --- Plasmids and transient transfection --- p.62 / Chapter 2.6.3 --- Western blot analysis --- p.62 / Chapter 2.6.4 --- Measurement of trans epithelial electric resistance --- p.64 / Chapter 2.6.5 --- Wound-healing migration assay --- p.64 / Chapter 2.6.6 --- Animals and Obstructive model --- p.64 / Chapter 2.6.7 --- HE and Masson's trichrome stain --- p.65 / Chapter 2.6.8 --- Immunofluorescent and immunohistochemistry staining --- p.65 / Chapter 2.6.9 --- Statistical analysis --- p.66 / Chapter Chapter 3 --- CFTR down-regulation mediates EMT during cancer metastasis --- p.67 / Chapter 3.1 --- Abstract --- p.67 / Chapter 3.2 --- Introduction --- p.67 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Repression of CFTR during TGF-β induced EMT in cancer cells --- p.73 / Chapter 3.3.2 --- Hypoxia does not have significant effect on CFTR expression --- p.78 / Chapter 3.3.3 --- Repression of CFTR channel function induces EMT in cancer cells --- p.81 / Chapter 3.3.4 --- Knockdown/overexpression of CFTR induces/inhibits EMT and malignant phenotypes --- p.84 / Chapter 3.3.5 --- CFTR inhibits lung metastasis in vivo --- p.94 / Chapter 3.3.6 --- Anti-metastatic effect of CFTR involves NF-κB targeting uPA --- p.104 / Chapter 3.3.7 --- Correlation between CFTR and β-catenin --- p.112 / Chapter 3.4 --- Discussion --- p.116 / Chapter 3.5 --- Conclusion --- p.122 / Chapter 3.6 --- Materials and methods --- p.122 / Chapter 3.6.1 --- Cell culture and treatments --- p.122 / Chapter 3.6.2 --- Lentiviral production and transduction --- p.123 / Chapter 3.6.3 --- Plasmids and stable transfection --- p.124 / Chapter 3.6.4 --- RT-PCR analysis --- p.124 / Chapter 3.6.5 --- Western blot analysis --- p.126 / Chapter 3.6.6 --- Immunofluorescence staining --- p.126 / Chapter 3.6.7 --- Cell growth assay --- p.127 / Chapter 3.6.8 --- Migration assay --- p.127 / Chapter 3.6.9 --- Invasion assay --- p.128 / Chapter 3.6.10 --- In vivo tumor growth assay --- p.128 / Chapter 3.6.11 --- In vivo metastasis assay --- p.128 / Chapter 3.6.12 --- Human EMT PCR array --- p.129 / Chapter 3.6.13 --- uPA activity assay --- p.129 / Chapter 3.6.14 --- Statistical analysis --- p.129 / Chapter Chapter 4 --- General discussion --- p.130 / Chapter 4.1 --- Normal function of CFTR in epithelial polarity and barrier function --- p.130 / Chapter 4.2 --- Down-regulation of CFTR is associated with EMT-related diseases --- p.131 / Chapter 4.3 --- CFTR functions as a central mediator of different EMT signals --- p.132 / Chapter 4.4 --- Future directions --- p.134 / Chapter 4.5 --- Conclusion --- p.135 / References --- p.136 / Declaration --- p.151

CFTR基因突變或者功能缺失是否導致包括胃腸道在內的各種組織惡性腫瘤的發生風險增加目前仍然是一個充滿爭議的問題。同時，眾所周知，緊密連接分子在腫瘤發生和轉移的過程發揮了關鍵的作用。本論文首次發現了CFTR基因與一種緊密連接分子AF-6/afadin的在人類結直腸腫瘤中的表達水平呈高度相關，并研究了CFTR和AF-6/afadin之間潛在的相互作用及其在結直腸腫瘤轉移中的功能。 / 論文的第一部份首先用實時定量PCR和免疫組織化學的方法比較了CFTR在結直腸腫瘤和正常組織的表達情況，發現CFTR表達水平在腫瘤組織中有顯著的下降。令人感興趣的是，我們同時發現CFTR和AF-6/afadin在腫瘤組織中的表達呈高度正相關，并由此展開了後續的體外實驗，研究對CFTR與AF-6/afadin之間可能的相互聯繫。利用免疫螢光染色和免疫共沉澱的方法，我們發現了這兩種蛋白分子共表達在結直腸腫瘤細胞的接觸面，并存在相互作用。用CFTR突變蛋白的免疫共沉澱實驗進一步發現，這種相互作用需要CFTR分子在細胞膜表面的正確定位及其PDZ結構域結合位點。實驗還發現與CFTR的相互作用加強了AF-6/afadin與細胞骨架蛋白系統的結合。在結直腸腫瘤細胞中CFTR基因敲减导致了AF-6/afadin蛋白定位混亂，從細胞連接位點轉移到細胞漿內，并因此破壞了上皮細胞的緊密性。極性生長細胞的跨上皮電阻降低而滲透性增強的實驗結果證實了CFTR基因敲減導致的上皮細胞緊密性的破壞。同時，AF-6/afadin蛋白水平也隨著CFTR基因敲減而降低，但mRNA水平未發生明顯的改變。蛋白降解系統的抑製劑逆轉了CFTR基因敲減細胞中AF-6/afadin蛋白的減少，提示CFTR基因敲減增加了AF-6/afadin的蛋白降解。這些實驗結果揭示了通過與細胞連接分子AF-6/afadin的相互作用以及調節，CFTR可能在上皮細胞極性的調節以及腫瘤發展過程中起重要作用。 / 論文的第二部份研究了CFTR和AF-6/afadin在結直腸腫瘤細胞上皮細胞間充質化（EMT）和轉移過程中的功能及機制。我們之前的工作已經揭示抑制CFTR的功能可以誘導結直腸腫瘤LIM1863細胞的EMT過程。本研究在另外三株不同的結直腸腫瘤細胞（SW480，SW1116和HRT-18）中進一步證實了抑制CFTR誘導的EMT過程。細胞形態轉變，上皮細胞標誌物的下調，間充質細胞標誌物的上調以及受損的上皮細胞緊密性均證實了對CFTR的抑制可以在這三種細胞中成功誘導EMT的發生。我們發現在以上所有細胞EMT的過程中，AF-6/afadin的蛋白表達水平都發生了顯著的下調。在HRT-18細胞中過表達AF-6/afadin，可以逆轉由CFTR抑製劑誘導的上皮細胞標誌分子的下調和間充質標誌分子的上調，表明抑制CFTR誘導的EMT過程是由AF-6/afadin參與介導的。此外，CFTR基因敲減導致結直腸腫瘤細胞的惡性表型強化，包括減弱的細胞粘附性，增強的貼壁依賴性生長、侵襲和遷移。另外，CFTR基因敲減激活了ERK的磷酸化，過表達AF-6/afadin可以阻斷ERK途徑的激活。CFTR基因敲減而增強的細胞侵襲性也可以被外源性AF-6/afadin或者ERK途徑的抑製劑U0126完全逆轉，提示作為AF-6/afadin的下游靶信號，ERK介導了CFTR在腫瘤侵襲中的作用。更重要的是，我們分析了CFTR和AF-6/afadin的表達水平與結直腸癌病人腫瘤進展的關係，發現在嚴重TNM腫瘤分期或者有腫瘤遠處轉移的病人中CFTR的表達水平顯著低於輕型分期或未发生转移的病人中的水平，而且CFTR和/或AF-6/afadin低表達的病人的預後更差。這些實驗結果顯示CFTR的缺失可能通過抑制AF-6/afadin和激活ERK通路而與EMT和結直腸癌癥轉移的過程高度相關。 / 綜上所述，本研究揭示了以往未報道過的CFTR在結直腸腫瘤發病機理中的功能，提示CFTR可以用作一種新的腫瘤的潛在預後指標。 / The question whether mutation or dysfunction of CFTR increases the risk of malignancies in various tissues, including the gastrointestinal tract, remains highly controversial. Meanwhile, it is well-known that adherens junctions play critical roles in the process of cancer development and metastasis. In this thesis we found for the first time a highly correlation between expression levels of CFTR and an adherens junction molecule AF-6/afadin in human colorectal tumours, and investigated the potential interaction between CFTR and AF-6/afadin and their functional roles in the metastasis of colorectal cancer. / In the first section of this thesis, we started our studies with comparing the expression of CFTR between human colorectal tumours and normal colorectal tissues. Real time quantitative PCR and immunohistochemistry results revealed a dramatically reduced CFTR level in the cancer tissues. Intriguingly, we noticed a highly positive correlation between CFTR and AF-6/afadin expression in tumours, which prompted the further in vitro investigation of possible interaction between CFTR and AF-6/afadin. Using immunofluoresent staining and co-immunoprecipitation, we found that the two proteins were colocalized at cell-cell junctions and interacted with each other in colorectal cancer cell lines. Further Co-IP experiments performed with CFTR mutations revealed that this protein interaction requires the proper localization of CFTR in cell membrane and its PDZ-interacting domain. Moreover the interaction with CFTR strengthens the binding of AF-6/afadin to the cytoskeleton system. Knockdown of CFTR in colorectal cancer cells resulted in the disorganized localization of AF-6/afadin protein from junctional sites to the cytoplasm and impaired epithelial tightness, which was confirmed by significantly reduced transepithelial resistance and increased permeability of polarized cells. Meanwhile, the protein level of AF-6/afadin was down-regulated in CFTR-knockdown cells, while no significant changes were detected at the mRNA level. Protein degradation inhibitor reversed the repression of AF-6/afadin protein in CFTR knockdown cells, suggesting the protein degradation of AF-6/afadin was increased by CFTR knockdown. These data revealed that CFTR interacts with and regulates the cell adhesion molecular AF-6/afadin in colorectal cells, which may be important in the regulation of epithelial cell polarity and cancer development. / In the second section of this thesis, we studied the functional roles and mechanisms of CFTR and AF-6/afadin in the epithelial-mesenchymal transition (EMT) and metastasis of human colorectal cancer cells. Our previous work has revealed inhibition of CFTR can induce EMT in a colorectal cancer cell line, LIM1863. This study further confirmed the induction of EMT by inhibiting CFTR in several other colorectal cancer cell lines (SW480, SW1116 and HRT-18), which was evaluated by morphological changes, down-regulation of epithelial markers or up-regulation of mesenchymal markers, and impaired epithelial cell tightness. In all these cell lines, we found that the protein levels of AF-6/afadin were significantly reduced. Over-expression of AF-6/afadin in HRT-18 cells reversed the down-regulated epithelial markers and up-regulated mesenchymal markers induced by CFTR inhibition, indicating that the CFTR inhibition-induced EMT is mediated by AF-6/afadin. Moreover, knockdown of CFTR in HRT-18 or RKO cells resulted in enhanced malignant phenotypes, including decreased cell adhesion, increased anchorage-independent cell growth, invasion, and migration. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was activated by CFTR knockdown, which was abolished by over-expression of AF-6/afadin. The enhanced invasiveness of CFTR knockdown cells was also completely inhibited by either exogenous AF-6/afadin or ERK inhibitor, U0126, suggesting that ERK, the downstream target of AF-6/afadin, is involved in mediating the effect of CFTR in cancer invasion. More importantly, we analyzed the association of CFTR and AF-6/afadin expression levels with tumour progression of patients with colorectal cancer, and revealed that CFTR expression was significantly lower in patients with more severe TNM stage or with metastasis to distant organs than those with milder stage or with no metastasis. The prognosis was poorer in patients with lower expression of CFTR and/or AF-6/afadin than those with higher expressions. These data showed that dysfunction of CFTR is highly associated with EMT and colorectal cancer metastasis, probably via repression of AF-6/afadin and activation of ERK pathways. / In summary, the present study has revealed a previously undefined role of CFTR in the pathogenesis of colorectal cancer and indicated its potential as a new prognostic indicator. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Tingting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 113-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Publications --- p.vi / Conference Abstract --- p.vii / Declaration --- p.viii / Acknowledgements --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Colorectal Cancer --- p.1 / Chapter 1.1.1. --- Structure of Human Normal Colon and Rectum Epithelium --- p.1 / Chapter 1.1.2. --- Staging of Colorectal Cancer --- p.3 / Chapter 1.1.3. --- Metastasis of Colorectal Cancer --- p.3 / Chapter 1.1.4. --- K-Ras mutation and It Downstream Pathways in Colorectal Cancer Metastasis --- p.11 / Chapter 1.1.5. --- Prognosis of Colorectal Cancer --- p.14 / Chapter 1.2. --- Epithelial Cell Junctional Complexes --- p.14 / Chapter 1.2.1. --- Junctional Complexes and Epithelial Cell Polarity --- p.15 / Chapter 1.2.2. --- Classic Cadherin-catenin Complex --- p.17 / Chapter 1.2.3. --- Novel Nectin-afadin Complex --- p.19 / Chapter 1.2.4. --- Cell Polarity and Cancer Progression --- p.23 / Chapter 1.3. --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.24 / Chapter 1.3.1. --- Structure of CFTR --- p.24 / Chapter 1.3.2. --- Mutations of CFTR --- p.24 / Chapter 1.3.3. --- Functions of CFTR --- p.26 / Chapter 1.3.4. --- Cancer Risk of CF Patients --- p.33 / Chapter 1.4. --- Hypothesis and Aims --- p.34 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1. --- Materials --- p.35 / Chapter 2.1.1. --- Reagents and Chemicals --- p.35 / Chapter 2.1.2. --- Antibodies --- p.35 / Chapter 2.1.3. --- Primers --- p.35 / Chapter 2.1.4. --- Solutions and Buffers --- p.35 / Chapter 2.1.5. --- Human Specimens --- p.36 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Cell Culture --- p.36 / Chapter 2.2.2. --- Transfection --- p.36 / Chapter 2.2.3. --- Selection of Stable Clones --- p.40 / Chapter 2.2.4. --- RNA Extraction and RT-PCR --- p.40 / Chapter 2.2.5. --- Quantitative Real Time PCR --- p.41 / Chapter 2.2.6. --- Protein Extraction and Western Blotting --- p.42 / Chapter 2.2.7. --- Immunostaining --- p.45 / Chapter 2.2.8. --- In vitro Cell Functional Assays --- p.46 / Chapter 2.2.9. --- Epithelial Tightness Measurement --- p.48 / Chapter 2.2.10. --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Interaction of CFTR with AF-6/afadin and Its Importance in Maintaining Colorectal Epithelial Cell Polarity --- p.50 / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Objectives --- p.53 / Chapter 3.3. --- Experimental plan --- p.54 / Chapter 3.4. --- Results --- p.55 / Chapter 3.4.1. --- The expression of CFTR and AF-6/afadin is decreased and positively correlated in human colorectal cancer --- p.55 / Chapter 3.4.2. --- CFTR colocalizes and interacts with AF-6/afadin in human colorectal cancer cells --- p.58 / Chapter 3.4.3. --- PDZ binding motif and membrane localization of CFTR are necessary for the interaction between CFTR and AF-6/afadin --- p.64 / Chapter 3.4.4. --- Knockdown of CFTR interferes with cell junction formation in colorectal cancer cells --- p.66 / Chapter 3.5. --- Discussion --- p.71 / Chapter Chapter 4 --- CFTR as a Suppressor and Prognosis Indicator of Metastasis in Human Colorectal Cancer --- p.77 / Chapter 4.1. --- Introduction --- p.77 / Chapter 4.2. --- Objectives --- p.80 / Chapter 4.3. --- Experimental plan --- p.81 / Chapter 4.4. --- Results --- p.82 / Chapter 4.4.1. --- CFTR inhibition-induced EMT in colorectal cancer cells involves AF-6/afadin --- p.82 / Chapter 4.4.2. --- Knockdown of CFTR aggravates malignant phenotype of colorectal cancer cells --- p.86 / Chapter 4.4.3. --- AF-6/afadin mediates the effect of CFTR on cell invasion in colon cancer through ERK --- p.91 / Chapter 4.4.4. --- CFTR and AF-6/afadin expression is correlated with the prognosis of colorectal cancer --- p.97 / Chapter 4.5. --- Discussion --- p.100 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.105 / Chapter 5.1. --- The diversified roles of CFTR in epithelial cells --- p.105 / Chapter 5.2. --- The unfolding relationship between CFTR and cancer development --- p.107 / Chapter 5.3. --- Future studies --- p.109 / Chapter 5.4. --- Conclusions --- p.112 / Reference List --- p.113 / Chapter Appendix A --- Reagents and Chemicals --- p.128 / Chapter Appendix B --- Antibody List --- p.131 / Chapter Appendix C --- Primer List --- p.132 / Chapter Appendix D --- Solution Recipe --- p.133

Indiana University-Purdue University Indianapolis (IUPUI) / The patients with Cystic fibrosis (CF), an inherent genetic disorder, suffer from chronic bacterial infection in the lung. In CF, modification of epithelial cells leads to alteration of the lung environment, such as inhibition of ciliary bacterial clearance and accumulation of thickened mucus in the airways. Exploiting these conditions, opportunistic pathogens like Pseudomonas aeruginosa cause lifelong persistent infection in the CF lung by forming into antibiotic-resistant aggregated communities called biofilms. Airway infections as well as inflammation are the two major presentations of CF lung disease. P. aeruginosa strains isolated from CF lungs often contain mutations in the mucA gene, and this mutation results in higher level expression of bacterial polysaccharides and toxic lipoproteins. In a previous work, we have found a putative lipoprotein gene (PA4326) which is overexpressed in antibiotic-induced biofilm formed on cultured CF-derived airway cells. In the current work, we speculated that this particular putative lipoprotein affects cellular cytotoxicity and immune-stimulation in the epithelial cells. We found that mutation of this gene (ΔPA4326) results in reduced airway cell killing without affecting other common virulence factors.Moreover, we observed that this gene was able to stimulate secretion of the proinflammatory cytokine IL-8 from host cells. Interestingly, we also found that ΔPA4326 mutant strains produced less pyocyanin exotoxin compared to the wild type. Furthermore, our results suggest that PA4326 regulates expression of the pyocyanin biosynthesis gene phzM, leading to the reduced pyocyanin phenotype. Overall, these findings implicate PA4326 as a virulence factor in Pseudomonas aeruginosa. In the future, understating the molecular interplay between the epithelial cells and putative lipoproteins like PA4326 may lead to development of novel anti-inflammatory therapies that would lessen the suffering of CF patients.