No description available.

No description available.

Renal cell carcinoma (RCC) is a cancer that originates from renal tubular epithelial cells in the kidneys. Studies focusing on RCC are vital as it accounts for around 3% of all human cancers as of 2014 and accounts for over 90% of cancers affecting the kidneys. Being a heterogenous disease, RCC consists of multiple subtypes including papillary RCC (pRCC) with a 10% incidence rate. The heterogenous nature of RCC makes it difficult to determine biomarkers and therapeutic treatments that can provide adequate treatment targeting RCC in different patients, which stresses the need to discover as many therapeutic targets as possible. OIP5, also known as Mis18β, is a 25-kDa protein that is accumulated in the telophase-G1 centromere during mitosis and plays a role in centromere/kinetochore structure formation and functionality. As Mis18β, it interacts with Mis18α in a heterotetramer complex to bind and localize centromeric protein A (CENP-A) to the proper centromere region to allow for formation for centromere/kinetochore formation and function. OIP5 is normally highly expressed in the testes but recent studies have suggested that OIP5 overexpression is linked to the progression of some types of human cancers, including promoting pRCC cell proliferation and tumorigenesis. Furthermore, past studies have shown that other genes may impact RCC progression through regulation of cell growth and substrate activation extending past just the cell cycle. PCSK5 is another target of interest of focus in this study. Known also as PC6, it is a proprotein convertase which acts on precursor proteins to turn them into their active forms through post-translational modification and plays a role in cell growth and bone remodeling. This study aims to investigate the role of OIP5 during RCC progression both in vitro and in vivo with a focus on pRCC as well as investigate the expression of PCSK5 in conjunction with OIP5 and pRCC. In vitro studies confirmed previous results that OIP5 promotes colony formation in ACHN pRCC cells. Using a bioluminescent reporter alongside ACHN OIP5 and ACHN empty vector (EV) cell lines, mice were injected through renal subcapsule transplantation. It was seen that, while OIP5 produced larger tumors over a wider area after 12 weeks, metastasis did not appear to occur as largely as hoped though this may be due to limitations of the model. Analysis of the ACHN xenografts showed increased expression of PCSK5 and PLK1 in the OIP5 tumors. For PLK1, this is likely due to the close nature of its function with OIP5 during mitosis while PCSK5 may be regulated by OIP5 through an unknown mechanism or through microRNA miR-101-5p. Despite this, mRNA levels of PCSK5 were insignificant between OIP5 and EV groups. pRCC patient data showing PCSK5 expression levels showed a minor correlation with OIP5 expression, as well as higher expression at later pRCC stages and leading to reduced survival probability. Overall, this thesis analyzes the relationship of OIP5 in RCC, specifically pRCC, and introduces PCSK5 as another intriguing target for further study in treating pRCC. / Thesis / Master of Science (MSc) / This project focuses on renal cell carcinoma (RCC) which is a cancer that originates from renal tubular epithelial cells in the kidneys. The elusive and vast nature of RCC leads to many subtypes that differ histologically and biologically and thus require different diagnoses and treatments. By investigating the papillary RCC (pRCC) subtype, the goal is to identify potential biomarkers for pRCC that may aid in future clinical diagnosis and treatment. OIP5 is a protein scarcely investigated and past studies have shown its overexpression linked to pRCC progression and tumorigenesis, but further metastatic investigation is warranted. This study aimed to confirm OIP5 overexpression plays a role in cancer as well as use a luciferase firefly reporter gene to monitor OIP5’s role in tumorigenesis and metastatic potential in vivo using a live mouse model. Results suggest that OIP5 overexpression plays a role in enhanced tumor growth in vivo. PCSK5 is another fairly novel gene of interest in this study, and while research involving PCSK5 and RCC is severely limited, recent studies imply it may be affected by different oncogenes such as OIP5 and specific microRNA during ccRCC and pRCC progression. Therefore, this study investigated the presence of PCSK5 in OIP5 pRCC tissue and tumors. The results suggest that PCSK5 has a higher presence in OIP5 overexpressed cells and tissue as well as a higher mRNA presence. Figures created using public genomic datasets show a correlation between PCSK5 and OIP5 expression during the later stages of pRCC progression and with lower survival probabilities. This indicates the possibility of interaction between OIP5 and PCSK5 and if true, provides a potential link for finding more biomarkers for potential future therapeutic targeting.

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. In the first part of the thesis, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by crossing the conditional Mdm4 and Mdm2 alleles to an erythroid-specific-cre (EpoRGFP-Cre ) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, interestingly, Mdm4 only contributes to p53 regulation at a specific phase of the differientation program. Moreover, a large body of evidence indicates that aberrant expression of either MDM2 or MDM4 impairs p53 tumor suppression function and consequently favors tumor formation. Overexpression of MDM2 was observed in 10% of 8000 human cancers from various sites, including lung or stomach, and MDM4 was found amplified and/or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach and breast cancers. Remarkably, selective MDM4 amplification occurs in about 65% of human retinoblastomas. In contrast, MDM2 amplifications are relatively rare (about 5%) in retinoblastomas, indicating that depending on the tumor context (cell type, initiating oncogene, …), MDM4, rather than MDM2, overexpression might be selected for as a more efficient mean of suppression of p53 function. As part of a large effort to better understand why different cell types require distinct combinations of mutations to form tumours, we will examine the molecular basis for selective up-regulation of Mdm4 in retinoblastomas. In this context, we have successfully generated 2 conditional transgenic mouse lines expressing either mycMdm2 or mycMdm4 driven by the PCAGGs promoters in the ROSA26 locus. Since a cassette containing a floxed transcriptional stop element is inserted upstream of the transgenes, we can achieve tissue-specific expression and spatio-temporal regulation of the transgenes by using different Cre and CreER. By the use of N-terminal myc-tag fused with the transgenes, we are able to compare the expression levels of the transgenes. Finally, due to C-terminal IRES-GFP element, we can easily identify transgene expressing cells. One of our aims is to use this Mdm4 conditional transgenic mouse line as the first, non-chimeric, mouse model of retinoblastoma that can be used as an appropriate preclinical model to improve treatment of this disease.

No description available.

No description available.

<p>The genes encoding the Ras family of small GTPases are mutated to yield constitutively active GTP-bound oncoproteins in one-third of all human cancers. In many other cancers lacking Ras mutations, Ras is activated by other means. One common example of such activation is found in breast cancer, in which epidermal growth factor receptor (EGFR) family receptor tyrosine kinases, including EGFR and HER2 (ErbB-2/Neu), are frequently amplified and overexpressed, which in turn activates Ras. In human cells, activation of the Ral guanine nucleotide exchange factor, or RalGEF, effector pathway is necessary for Ras-mediated tumorigenesis and metastasis. RalGEFs activate the two highly similar Ral GTPases, RalA and RalB. While RalA has been shown to be required for Ras-mediated tumorigenesis, RalB is important for tumor metastasis. Activated Ral GTPases bind to and activate a limited number of effector proteins, including RalBP1, Sec5, and Exo84, to affect numerous diverse activities of the cell. This dissertation research sought to determine which of these well-characterized Ral effector proteins were required for oncogenic mutant Ras-induceded tumorigenesis and metastasis of human cells, as well as to examine the role of RalA in breast cancer cells that can activate Ras through EGFR and HER2 overexpression. </p><p> RNA interference-mediated loss-of-function analysis demonstrated that Sec5 and Exo84 are required for oncogenic Ras-mediated tumorigenesis, and, at least in part, metastasis. Additionally, both gain-of-function and inhibition studies showed that RalA activation is induced by EGFR and HER2 in breast cancer cell lines stimulated with EGF. Furthermore, stable suppression of RalA expression inhibited tumorigenic growth of breast cancer cells, and RalA activation was shown to be higher in a majority of mammary adenocarcinomas versus matched patient normal mammary tissue. These studies provide new insights into the importance of RalA activation in breast cancer, as well as the molecules downstream of RalA and RalB that may be responsible for mediating their effects on tumorigenesis and metastasis.</p> / Dissertation

The Hippo signaling pathway has been implicated in both mammalian organ size regulation, as well as tumor suppression. Specifically, the Hippo pathway plays a critical role regulating the activity of transcriptional co-activator, and downstream effector, Yes-associated protein (YAP), which modulates pro-proliferative transcriptional elements. Recent investigations have demonstrated that this pathway is activated in non-regenerating livers and its inhibition leads to liver overgrowth and tumorigenesis. The majority of the existing evidence regarding the role of the Hippo pathway in hepatocyte proliferation is based on in vitro studies and knock-out animal models. However, the role of the Hippo pathway during the natural process of liver regeneration, remains unknown. Here alterations in the Hippo signaling pathway were investigated, namely its interaction with angiomotin-like 2 (AmotL2) and Set7, during liver regeneration using a 70% rat partial hepatectomy (PH) model. Overall, results indicated no significant difference between AmotL2 levels in control and regenerating tissue at various time points during liver regeneration. No significant alterations in YAP methylation during liver regeneration were found compared to control tissue. In the end, results regarding the role of both AmotL2 and Set7 provided inconclusive evidence about their roles during the regenerative process. Given the role of the Hippo pathway in hepatocyte proliferation, a hypothesis was made that this pathway may play a role in pediatric liver tumors. YAP localization was evaluated using immunohistochemical analysis in tumor sections from patients with hepatoblastoma or hepatocellular carcinoma. Once again, the results were inconclusive at the time of the preparation of this manuscript due to technical difficulties in achieving satisfactory staining of the specimens. Further studies will be directed at elucidating the role of the Hippo pathway during liver regeneration as well as developing better conditions for the immunohistochemical staining of human liver specimens.

TBX2, a member of the T-box family of transcription factors, plays important roles in embryonic development. Aberrant expression of TBX2 is observed in many cancers, and serves as an oncogene to maintain tumor cell proliferative and malignant properties. We found that TBX2 was expressed in both embryonic myoblasts and adult proliferative satellite cells, but was quickly down regulated during muscle differentiation in mouse models, which suggests an important function of TBX2 in the early myogenesis. Using molecular and cellular biology approaches we showed that TBX2 forms complex with myogenin and MyoD, and then recruits HDAC1 to muscle-specific promoters to repress the myogenin and MyoD dependent differentiation of myoblasts. In rhabdomyosarcoma (RMS), which is typically referred to as a muscle derived cancer, we found TBX2 was over expressed in both major subtypes of RMS. The deregulated TBX2 repressed the expression of cell cycle regulators, such as p21 and p14/p19, and the tumor suppressor PTEN in RMS tumor cells. Knock down of TBX2 significantly decreased the proliferation rate of RMS cells. We also found that loss of TBX2 significantly inhibited tumorigenesis of RMS cells by decreasing cell proliferation, mobility, migration, anchorage-independent growth and xenograft formation. To determine why TBX2 was deregulated in RMS cells, we performed cellular biological experiments to understand how TBX2 is regulated by cell signaling pathways and growth factors in both normal muscle myoblasts and RMS tumor cells. In normal murine myoblasts and primary murine ARMS tumor cells TBX2 was up regulated by FGF-2 treatment, but in primary murine ERMS cells TBX2 expression showed no response to FGF-2 stimulation. In human RMS cell lines a modest up regulation of TBX2 was detected by treatment of FGF-2. RMS cells constitutively express PAX3 and PAX7 which are expressed and function in myogenic precursors, but are quickly degraded in myoblasts and during myogenesis. We found that TBX2 was a downstream target of PAX3 in RMS cells, as well as the ARMS specific fusion proteins PAX3/7-FOXO1. Our novel findings on TBX2 highlight the significant roles of TBX2 in muscle development and adult muscle regeneration, where TBX2 represses MRF activities to inhibit myogenic differentiation and promote proliferation of myoblasts. Also, our work establishes essential oncogene effects of TBX2 in driving and maintaining RMS proliferation and tumorigenesis by repressing cell cycle regulatory factors, p21 and p19/p14, and tumor suppressor of PTEN. Therefore, this work provides an exciting opportunity for development of new therapeutic treatments for TBX2 driven RMS cancer.

TBX2 and TBX3, which function as repressors, are members of the T-Box transcription factor family which are conserved throughout the metazoan lineage. TBX2 is highly expressed in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, and many other cancers. Previously, our lab dissected the oncogenic properties of TBX2 and its regulation of p14, p21 and PTEN. TBX3 is also expressed in some cancer types, however, its expression profile in RMS is severely down-regulated. TBX3 is shown to repress TBX2 in chondrocytes, but the characterization and regulation of TBX3 is poorly understood in the muscle lineage. Polycomb Repressive Complex 2 (PRC2), a gene silencing complex, acts to methylate histone H3 lysine 27 (H3K27me) of target gene promoters. The catalytic subunit of PRC2, EZH2, is up-regulated in RMS and data from our lab has shown that depletion of EZH2 up-regulated TBX3 and down-regulated TBX2 in C2C12 cells, an immortalized murine cell line. The hypothesis of this project was that there would be a PRC2-TBX3-TBX2 axis in RMS cells. To examine if TBX3 represses TBX2, TBX3 was transiently expressed in RMS cells representing both subtypes of RMS and we found that TBX2 was downregulated in each cell line. In a stable RH30 cell line with ectopic TBX3, TBX2 was down-regulated and PTEN expression was up-regulated. To determine if TBX2 repression by TBX3 was direct, a TBX3 ChIP assay was performed on the TBX2 promoter as well as the PTEN promoter. We found a strong enrichment of TBX3 on the TBX2 promoter but not on the PTEN promoter. Accordingly, we also observed that TBX3 over-expression impaired tumorigenesis through reduced cell proliferation, migration, and anchorage dependent growth. Also, we found that a stable RD cell line with ectopic TBX3 could promote differentiation, strongly suggesting that these results could have therapeutic value. Next, a shEZH2 plasmid was transfected into RMS cell lines ask if TBX3 was regulated by the PRC2 complex as we had observed in C2C12 cells. Just as we hypothesized, TBX3 was up-regulated and TBX2 was down-regulated. Similar to the previous TBX3 overexpression experiments, the EZH2 depleted RMS cell lines also showed decreased cell proliferation and migration rate. Also, an EZH2 knock down treatment induced differentiation in RMS cell lines. Therefore, understanding this potent regulation axis could provide an excellent opportunity for treatment of RMS cancer in the future.