Cadherin gene family members are known to be involved in the differentiation of cytotrophoblasts of the human placenta. In particular, the regulation of cadherin expression is coupled with the development of an invasive phenotype and the formation of the multinucleated syncytiotrophoblast. To investigate further the mechanisms underlying the differential regulation of cadherins during these developmental processes, we have examined the role of the transcription factor known as Twist. Twist is a basic HLH (helix-loop-helix) factor which has been shown to regulate cadherin expression in a variety of human tissues under normal and pathological conditions. Using an siRNA strategy, I have determined that Twist regulates both E-cadherin and N-cadherin in distinct subtypes of human trophoblastic cells in vitro. In particular, suppression of Twist gene expression in poorly invasive BeWo choriocarcinoma cells by using Twist-specific siRNA resulted in a concomitant increase in E-cadherin mRNA and protein levels in these cells. In contrast, transfection of highly invasive extravillous cytotrophoblasts with Twist siRNA decreased N-cadherin mRNA levels in a concentration-dependent manner. Taken together, these observations indicate that Twist differentially regulates E-cadherin and N-cadherin in human trophoblastic cells, two cadherin subtypes that govern the differentiation of these cells along the non-invasive and invasive pathways respectively. Although, the results of my studies do not directly demonstrate this biological function of Twist, they support the speculation that alterations in Twist expression levels will result in cadherin-mediated disorders of pregnancy associated with aberrant trophoblast differentiation.

Cadherin gene family members are known to be involved in the differentiation of cytotrophoblasts of the human placenta. In particular, the regulation of cadherin expression is coupled with the development of an invasive phenotype and the formation of the multinucleated syncytiotrophoblast. To investigate further the mechanisms underlying the differential regulation of cadherins during these developmental processes, we have examined the role of the transcription factor known as Twist. Twist is a basic HLH (helix-loop-helix) factor which has been shown to regulate cadherin expression in a variety of human tissues under normal and pathological conditions. Using an siRNA strategy, I have determined that Twist regulates both E-cadherin and N-cadherin in distinct subtypes of human trophoblastic cells in vitro. In particular, suppression of Twist gene expression in poorly invasive BeWo choriocarcinoma cells by using Twist-specific siRNA resulted in a concomitant increase in E-cadherin mRNA and protein levels in these cells. In contrast, transfection of highly invasive extravillous cytotrophoblasts with Twist siRNA decreased N-cadherin mRNA levels in a concentration-dependent manner. Taken together, these observations indicate that Twist differentially regulates E-cadherin and N-cadherin in human trophoblastic cells, two cadherin subtypes that govern the differentiation of these cells along the non-invasive and invasive pathways respectively. Although, the results of my studies do not directly demonstrate this biological function of Twist, they support the speculation that alterations in Twist expression levels will result in cadherin-mediated disorders of pregnancy associated with aberrant trophoblast differentiation.

Benign ovarian tumors and majority of epithelial ovarian cancers possess steroid receptors including estrogen receptors (ERs). However, the estrogen-ER signaling in ovarian carcinomas is not completely understood. Tumorigenesis is a multiple-step process involving dysregulated cell growth and metastasis. Tumor cells acquire the capacity of migration and invasion by temporal phenotypical and genotypical changes termed epithelial-mesenchymal transition (EMT). Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In this study, I have focused on the role of 17β-estradiol (E2) in ovarian tumorigenesis. EMT related genes including E-cadherin, Snail, Slug, and Twist were examined. E2 treatment led to clear morphological changes and an enhanced cell migratory propensity. These morphologic and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin was strikingly suppressed, whereas EMT-associated transcription factors Snail and Slug were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of the endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated control in E-cadherin. In addition, the E2-induced cell migration was neutralized by Snail and Slug siRNAs, implying that both transcription factors are indispensable for the pro-metastatic actions of E2. Importantly, by using selective ER agonists as well as over-expression and siRNA approaches, it was identified that E2 triggered the metastatic behaviors exclusively through an ER⍺-dependent pathway. In contrast, overexpression of ERβ opposed the phenotypic changes and down-regulation of E-cadherin induced by ER⍺. In addition, microarray analysis was performed to characterize more putative downstream mediators of E2. Expression levels of 486 genes were found to be altered by at least 50% upon E2 treatment, and included several genes involved in oncogenesis, cell cycle control, apoptosis, signal transduction and the gene expression machinery. These candidate genes may be valuable for better delineating the ER pathways and functions. In summary, this study provides compelling arguments that estrogen can potentiate tumor progression by EMT induction, and highlight the crucial role of ER⍺ in ovarian tumorigenesis.

Colon cancer is characterized by the progressive loss of E-cadherin, a Ca2+-dependent adherens junction component and epithelial marker. Furthermore, inhibition of the phosphatase SHP2, essential in the cell survival signaling via the epidermal growth factor receptor pathway, has been shown to upregulate E-cadherin in breast cancer cell lines. This suggests that unregulated increases in SHP2 may promote a cancer cell phenotype. The aim of this study was to define molecular mechanisms by which dietary Ca2+ is chemoprotective against colon cancer. The extracellular Ca2+-sensing receptor (CaSR) has been implicated in this process and we speculated that there was a relationship between CaSR activation and E-cadherin and SHP2 expression levels on colonic epithelia. A colonic adenocarcinoma cell line which lacks endogenous E-cadherin expression, SW480, was used as a model cell. CaSR levels were manipulated by transient transfection and E-cadherin and SHP2 expression levels were determined by Western blotting. The E-cadherin expression pattern was also assessed with RT-PCR and immunocytochemistry. We found that after CaSR activation via Ca2+ or other known CaSR agonists (neomycin sulphate, spermine), E-cadherin expression was increased and SHP2 expression was decreased. However, the E-cadherin expression pattern was altered in the presence of a dominant-negative CaSR (R185Q). Furthermore, pharmacological inhibition of p38 MAPK inhibited CaSR-mediated increases of E-cadherin protein in SW480 cells. Inhibition of p38 MAPK had no effect on CaSR-stimulation of E-cadherin transcript or promoter activity. SHP2 decreases usually seen after Ca2+-treatment were reduced after pharmacological inhibition of JNK in the presence of high Ca2+. CaSR activation also increased cell-cell adherence as assessed by electronic cell sizing and this adherence was lost when both CaSR-mediated changes in E-cadherin and SHP2 expression were inhibited. We conclude that CaSR activation in colonic epithelial cancer cells stimulated E-cadherin increases, through a mechanism involving p38 MAPK, as well as inhibited SHP2 expression, through a JNK-mediated mechanism. Further, these protein changes post-CaSR activation cause an increase in cell-cell adherence. Our results suggest that the chemoprotective nature of dietary Ca2+ supplements may involve reciprocal regulation of E-cadherin and SHP2 via the CaSR. / Thesis (Master, Physiology) -- Queen's University, 2009-06-09 17:56:44.843

Benign ovarian tumors and majority of epithelial ovarian cancers possess steroid receptors including estrogen receptors (ERs). However, the estrogen-ER signaling in ovarian carcinomas is not completely understood. Tumorigenesis is a multiple-step process involving dysregulated cell growth and metastasis. Tumor cells acquire the capacity of migration and invasion by temporal phenotypical and genotypical changes termed epithelial-mesenchymal transition (EMT). Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In this study, I have focused on the role of 17β-estradiol (E2) in ovarian tumorigenesis. EMT related genes including E-cadherin, Snail, Slug, and Twist were examined. E2 treatment led to clear morphological changes and an enhanced cell migratory propensity. These morphologic and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin was strikingly suppressed, whereas EMT-associated transcription factors Snail and Slug were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of the endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated control in E-cadherin. In addition, the E2-induced cell migration was neutralized by Snail and Slug siRNAs, implying that both transcription factors are indispensable for the pro-metastatic actions of E2. Importantly, by using selective ER agonists as well as over-expression and siRNA approaches, it was identified that E2 triggered the metastatic behaviors exclusively through an ER⍺-dependent pathway. In contrast, overexpression of ERβ opposed the phenotypic changes and down-regulation of E-cadherin induced by ER⍺. In addition, microarray analysis was performed to characterize more putative downstream mediators of E2. Expression levels of 486 genes were found to be altered by at least 50% upon E2 treatment, and included several genes involved in oncogenesis, cell cycle control, apoptosis, signal transduction and the gene expression machinery. These candidate genes may be valuable for better delineating the ER pathways and functions. In summary, this study provides compelling arguments that estrogen can potentiate tumor progression by EMT induction, and highlight the crucial role of ER⍺ in ovarian tumorigenesis.

Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer that is characterized by a high rate of invasion and destruction to the surrounding tissues, with patients showing poor 5-year survival rate. A pre-malignant stage of cellular atypia and loss of stratification within the epithelium (dysplasia) occurs prior to the establishment of OSCC, which is manifested as white (leukoplakia) or red (erythroplakia) lesions. Treatment of dysplasia/OSCC involves surgical intervention with the removal of an adequate safety margin. However, high grade dysplasia and OSCC exhibit high recurrence rates. To date the only method used to diagnose oral epithelial dysplasia (OED) and OSCC is haematoxylin and eosin staining of biopsies and examination by an experienced pathologist. Furthermore, the mechanisms of dysplasia formation, transition to OSCC, and high recurrence rates are little understood. Recent data from the Ward lab suggests that the cell surface tumour suppressor protein E-cadherin plays an important role in regulating many cellular functions in epithelial cells. Loss of E-cadherin in carcinomas has been well studied and is linked with tumour invasion, metastasis and poorer patient outcomes. In this thesis, I have investigated expression of E-cadherin in low grade (LG) and high grade (HG) dysplasia and T1 and T4 OSCC patient biopsies to determine whether loss of this protein occurs prior to tumour cell invasion. Furthermore, microarray data analysis identified Epithelial Membrane Protein-1 (EMP-1) as a putative early marker of tumorigenesis, with alterations of N-cadherin, CD44 and 5T4 oncofoetal antigen expression during tumorigenesis inferred from our studies in embryonic stem cells. Immunofluorescence microscopy (IFM) analysis revealed that normal oral epithelium exhibits cell surface E-cadherin, EMP-1 and 5T4 expression but generally lacks N-cadherin and CD44 reactivity. The statistically significant loss of both E-cadherin and EMP-1 was observed in low and high grade dysplastic tissue and OSCC biopsies (p < 0.001). 5T4 expression was decreased in HG dysplasia (p < 0.001), suggesting this may be a useful assay for discriminating between LG and HG dysplastic tissues. N-cadherin or CD44 did not show any statistical significance in expression between normal, LG/HG dysplasia and T1/T4 OSCC, although there was a trend for increased N-cadherin expression in T4 OSCC. Significantly, Ecadherin expression was decreased or absent from surgical margins of LG/HG dysplasia and T1 OSCC, and EMP-1 and 5T4 were absent from the margins of LG and HG dysplastic tissue, indicating that these margins are abnormal. Therefore, loss of Ecadherin and EMP-1 are early events associated with dysplastic tissue and may be a useful method to confirm the removal of sufficient surgical safety margin. The OSCC cell line BICR56 was assessed for marker expression and exhibited a phenotype consistent with normal oral epithelium. Inhibition of E-cadherin protein in BICR56 cells using a neutralising antibody led to decreased cell surface localisation of 5T4 antigens, suggesting that E-cadherin expression is critical for maintenance of a normal epithelial phenotype. In conclusion, both E-cadherin and EMP-1 are useful markers for detecting early cancerous changes and 5T4 expression can discriminate between LG and HG dysplasia. This study has also shown significant abnormalities within the surgical safety margins of LG/HG dysplasia and T1 OSCC biopsies, which requires further investigation, and may explain the high recurrence rates seen in such patients.

Cadherin gene family members are known to be involved in the differentiation of cytotrophoblasts of the human placenta. In particular, the regulation of cadherin expression is coupled with the development of an invasive phenotype and the formation of the multinucleated syncytiotrophoblast. To investigate further the mechanisms underlying the differential regulation of cadherins during these developmental processes, we have examined the role of the transcription factor known as Twist. Twist is a basic HLH (helix-loop-helix) factor which has been shown to regulate cadherin expression in a variety of human tissues under normal and pathological conditions. Using an siRNA strategy, I have determined that Twist regulates both E-cadherin and N-cadherin in distinct subtypes of human trophoblastic cells in vitro. In particular, suppression of Twist gene expression in poorly invasive BeWo choriocarcinoma cells by using Twist-specific siRNA resulted in a concomitant increase in E-cadherin mRNA and protein levels in these cells. In contrast, transfection of highly invasive extravillous cytotrophoblasts with Twist siRNA decreased N-cadherin mRNA levels in a concentration-dependent manner. Taken together, these observations indicate that Twist differentially regulates E-cadherin and N-cadherin in human trophoblastic cells, two cadherin subtypes that govern the differentiation of these cells along the non-invasive and invasive pathways respectively. Although, the results of my studies do not directly demonstrate this biological function of Twist, they support the speculation that alterations in Twist expression levels will result in cadherin-mediated disorders of pregnancy associated with aberrant trophoblast differentiation. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Benign ovarian tumors and majority of epithelial ovarian cancers possess steroid receptors including estrogen receptors (ERs). However, the estrogen-ER signaling in ovarian carcinomas is not completely understood. Tumorigenesis is a multiple-step process involving dysregulated cell growth and metastasis. Tumor cells acquire the capacity of migration and invasion by temporal phenotypical and genotypical changes termed epithelial-mesenchymal transition (EMT). Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In this study, I have focused on the role of 17β-estradiol (E2) in ovarian tumorigenesis. EMT related genes including E-cadherin, Snail, Slug, and Twist were examined. E2 treatment led to clear morphological changes and an enhanced cell migratory propensity. These morphologic and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin was strikingly suppressed, whereas EMT-associated transcription factors Snail and Slug were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of the endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated control in E-cadherin. In addition, the E2-induced cell migration was neutralized by Snail and Slug siRNAs, implying that both transcription factors are indispensable for the pro-metastatic actions of E2. Importantly, by using selective ER agonists as well as over-expression and siRNA approaches, it was identified that E2 triggered the metastatic behaviors exclusively through an ER⍺-dependent pathway. In contrast, overexpression of ERβ opposed the phenotypic changes and down-regulation of E-cadherin induced by ER⍺. In addition, microarray analysis was performed to characterize more putative downstream mediators of E2. Expression levels of 486 genes were found to be altered by at least 50% upon E2 treatment, and included several genes involved in oncogenesis, cell cycle control, apoptosis, signal transduction and the gene expression machinery. These candidate genes may be valuable for better delineating the ER pathways and functions. In summary, this study provides compelling arguments that estrogen can potentiate tumor progression by EMT induction, and highlight the crucial role of ER⍺ in ovarian tumorigenesis. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

The complex circuitry of the visual system contains around one hundred functionally distinct neuronal types that become specified and connect with the appropriate synaptic partners during development. Previous studies have indicated that immature retinal ganglion cells already express subset-specific molecules that guide them to make precise synaptic choices. In the mammalian retina, members of the cadherin family of adhesion molecules are attractive candidates for this role. To test this idea I began by investigating the expression of cadherins 1-26 in the mouse retina and superior colliculus using in situ hybridization. I then studied the connectivity of cadherin-expressing neurons by analyzing mouse lines in which a marker was inserted after the start codon of each of six cadherin genes of interest. In this way, I identified functional circuits in the visual system that are marked by cadherins. One such circuit is formed of direction-selective retinal ganglion cells (DSGCs), which fire in response to objects moving in one (preferred) direction, and their synaptic partners, the starburst amacrine cells. There are four DSGC subsets, distinguished by their preference for dorsal, ventral, nasal, or temporal motion on the retina. I determined that cadherin 6 is selectively expressed by the two DSGCs subtypes that respond to dorsal or to ventral movement. In collaboration with other lab members I used in situ hybridization and gene expression profiling to identify other molecular markers that distinguish between the four DSGC subsets and that distinguish DSGCs from other retinal ganglion cells. Finally, I used birthdating and lineage tracing methods to ask when DSGCs become molecularly specified. I determined that at least two subsets of DSGCs are specified at or shortly after their birth. For cadherin 6-positive DSGCs, I went on to show that they are specified even before their birth, and that they arise from committed retinal progenitors. Globally, my experiments aimed not only to examine cadherin expression and function in the visual system, but also to demonstrate a method of using molecular signatures to probe the mechanisms of neural circuit assembly in the central nervous system.

In epithelial cells, formation of stable adherens junction is essential for a number of important cell processes. The central protein responsible for creating cell-cell adhesion is known as E-cadherin. When the lamellipodia of migratory cells make contact, the cell is signaled to send E-cadherin/β-catenin complexes to the point of contact. Upon proper binding of two E-cadherin molecules further E-cadherins are signaled to cluster at the point of contact through cis lateral interactions and a passive diffusion trap mechanism. The actin cytoskeleton is also signaled through Rac1 to interact with the nascent adherens junction. As the adherens junction matures there are further actin cytoskeleton rearrangements and alterations to cell shape due to variable expression of the Rho GTPases. When adhesion in the adherens junction is stable the cell is able to become polarized by the assembly of tight junctions. Interference with any of the steps that lead to the development of a stable, mature, adherens junction results in various disease states such as cancer. Cancer can develop in epithelial cells due to E-cadherin dysfunction, particularly gastric, breast, ovarian, head and neck, and prostate cancer are seen. E-cadherin dysfunction can be caused by interference with proper transcription, N-glycosylation, and recycling. Transcription is most commonly disrupted due to acetylation of the E-cadherin promoter by improperly modulated transcriptional repressor, such as Snail. Aberrant Nglycosylation and/or modification with branching β1, 6 GlcNAc can interfere with the creation of stable adherens junction by interfering with E-cadherin binding. Increased endocytosis of E-cadherin via irregular Rho GTPase activity destabilizes adherens junctions. These interferences effect an epithelial to mesenchymal transition that can act as a metastatic cancer phenotype. E-cadherin serves a crucial function in cell-cell adhesion and preventing cells from exhibiting malignancy. It has been shown that restoration of its function in cancer cell lines reduces the invasiveness of cancer cells and returns to the cell to a normal epithelial phenotype. Knowledge of E-cadherin, its regulators, and association with the actin cytoskeleton will undoubtedly have clinical impacts in cancer treatment. However, understanding of E-cadherin is still incomplete, in particularly more studies need to be done in the area of Rho GTPases and N-glycosylation, There has also been recent controversy in identifying the principal molecule that links the actin cytoskeleton and α- catenin to mediate the binding of the E-cadherin/β-catenin complex to actin.