Acute promyelocytic leukemia (APL) is one of the subtypes of acute myeloid leukemia carrying t(15;17), and constitutes 10 to 15% of adult AMLs. One of the mechanisms of gene inactivation is hypermethylation of promoter-associated CpG islands. Cancers are characterized by global hypomethylation with locus-specific hypermethylation and hence silencing of tumor suppressor genes. Apart from tumor suppressor genes, microRNA, a class of non-coding RNA measuring 19-25 nucleotides, with tumor suppressive function is also found to be inactivated by DNA methylation in hematological malignancies. microRNAs repress target gene translation and hence expression by binding to 3'-untranslated region of corresponding mRNA. Because TP53 mutation is frequently involved in solid cancer carcinogenesis but is rarely found in APL, TP53 network may be dysregulated through epigenetic inactivation of tumor suppressor gene/miRNAs engaged in TP53 tumor suppressor network. This thesis aimed to study DNA methylation of tumor suppressor genes and miRNAs engaged in TP53 tumor suppressor network in APL. Overall survival (OS) and event free survival (EFS) of patients with or without candidate gene/miRNA hypermethylation were compared to examine their prognostic significances. Promoter methylation of DAPK1, p14ARF, miR-34a, miR-34b/c and miR-605 were studied in 10 normal bone marrow samples, NB4 cell line and 60 APL primary samples at diagnosis by methylation-specific PCR (MSP). DAPK1, miR-34a, miR-34b/c and miR-605 were completely unmethylated in normal bone marrow samples but completely methylated in NB4. Treatment of NB4 by 5'-Aza-2'-deoxyctidine (5-azadC) resulted in promoter demethylation together with re-expression of DAPK1, miR-34a, miR-34b/c and miR-605. Promoter methylation of DAPK1, p14ARF, miR-34a were absent while miR-34b/c and miR-605 methylation were detected in 43% and 10% APL samples respectively. However, methylation of miR-34b/c and miR-605 bore no prognostic significance. Overexpression of miR-34b in NB4 resulted in inhibition of proliferation. In short, methylation of DAPK1, miR-34a, miR-34b/c and miR-605 is associated with gene/miRNAs silencing. miR-34b/c is frequently methylated whereas miR-605 is methylated in small number of APL patients. miR-34b/c is a tumor suppressive miRNA in APL. Methylation of miR-34b/c may contribute to APL leukemogenesis. / published_or_final_version / Medicine / Master / Master of Philosophy

Mixed lineage leukemia (MLL) gene undergoes chromosomal translocation with over 60 different fusion partner genes in human leukemias. The resultant MLL-fusion oncoproteins are profoundly implicated in leukemias with poor prognosis. Epigenetic dysregulations have been frequently reported in MLL-rearranged leukemogenesis. Our study aims to investigate the correlations between epigenetic alterations, including both histone modification and DNA methylation, and gene dysregulation in MLL-rearranged leukemia. My study focused on MLL-EEN fusion protein, which causes an onset of acute myeloid leukemia (AML). A novel Mll-Een expressing cell line, VLA33, was derived from the bone marrow of Mll-Een knockin mouse with AML phenotype. The cells were mainly myeloblast cells, possessing clonogenic ability and showed upregulation of Hoxa cluster genes. Previous study demonstrated that the protein arginine methyltransferase 1 (PRMT1) plays a significant role in MLL-EEN leukemogenesis through conferring H4R3 asymmetric dimethylation (H4R3me2a) mark on HoxA9 locus. Consistently, our ChIP analysis demonstrated enrichment of H4R3me2a at the Hoxa promoters while knockdown of Prmt1 attenuated the expression of Hoxa genes and reduced in vitro clonogenic potential of VLA33 cells. CD41, Runx1 and Tgm2 genes, which showed elevated expression in VLA33 cells, were identified as potential target genes of Mll-Een/Prmt1 complex. However, enrichment of active H4R3me2a was only observed at Runx1 promoters, but not at the regulatory regions of CD41 and Tgm2. Inhibition of Prmt1 by inhibitor AMI-1 reduced Runx1 and CD41 expression. Although Prmt1 knockdown reduced the enrichment of H4R3me2a at Runx1 promoter, it did not suppress the expression of Runx1. These data suggest the involvement of other regulatory mechanism and Prmt1 is not the sole factor causing gene dysregulation. CD41 is a marker of murine definitive hematopoietic progenitors. Interestingly, the CD41+ VLA33 cells demonstrated a trend of enhanced self-renewal ability in colony-forming assay as compared with CD41-/low cells. The CD41 expression was positively correlated with Mll-Een and Prmt1 expression. In addition, CD41+ cells expressed higher level of Hoxa9, Bmi-1, Runx1, Tal-1 and Lmo2 genes that are associated with HSC activities, suggesting reactivation of stem-cell regulatory program in CD41+ leukemia cells, which confer as leukemia stem cell population. The association between DNA methylation and MLL-EEN leukemogenesis was also investigated. The results demonstrated the establishment of stem cell Hox code, which was correlated with DNA hypomethylation status at Hox gene promoters in Mll-Een leukemia cells. Besides, Hox activation through DNA hypomethylation was independent of Prmt1-mediated histone modification, but was found associated with reduction of Bmi-1 binding at Hox loci. In conclusion, my study identified novel dysregulated genes in Mll-Een leukemogenesis. My findings provide insight into the reactivation of stem-cell program in leukemia cells through epigenetic dysregulation, which furthers our understanding of MLL-rearranged leukemogenesis. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies worldwide with very poor prognosis. It is generally believed that accumulation of irreversible alterations in critical oncogenes and tumor suppressor genes during the long-term inflammation finally leads to the hepatocellular pathogenesis. Although under intensive investigation, the molecular pathogenesis of HCC still remains to be further elucidated. In this study, we aimed to identify novel genetic alterations critical to the pathogenesis of HCC, especially in hot regions with recurrent chromosomal instability. Amplification of broad regions of 8q is one of the most frequent genetic alterations in HCC, suggesting the existence of oncogenes in addition to MYC at 8q24. By screening the publicly available microarray database and clinical samples, we found frequent amplification and overexpression of Serum and Glucocorticoid Kinase 3 (SGK3) in clinical HCC specimens, and SGK3 genomic activation was significantly associated with poor outcome of HCC patients. Functional assays revealed that SGK3 could increase G1/S cell cycle progression, cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. We provided evidences that SGK3 could promote HCC growth and survival through inactivating GSK3-β and BAD respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1, has more significance than overexpression of AKT in predicting poor outcome of HCC patients. Our findings suggested the existence of an AKT-independent SGK3 pathway, which may function in parallel with AKT pathway in the pathogenesis of HCC. In addition to large chromosomal alterations, small changes in nucleotides may also make substantial contributions to carcinogenesis. Recent advances in high-throughput deep sequencing technology have provided a powerful tool to understand the whole cancer transcriptome and identify novel genetic alterations related to cancer progression. In this study, we identified a high proportion of allele imbalance in genes related to cellular stress response by sequencing the whole transcriptome of 3 paired HCC tissues. A novel nucleotide variation which resulted in a R438H amino acid change was identified in the coding region of the gene Oxidative Stress Induced Growth Inhibitor 1 (OSGIN1), and the variant 438H form of OSGIN1 was found to be specifically retained in the tumor tissues in a cohort of HCC patients. OSGIN1 was found to be closely associated with chemotherapeutic reagents and exhibited strong tumor suppressive function in HCC by directly inducing cell apoptosis. The wild type OSGIN1 was found to have stronger tumor suppressive function than the variant allele, and this might be due to their different ability to localize to mitochondria. The significantly decreased basal apoptotic index in HCC patients carrying OSGIN1 variant allele and their poor prognosis further suggested that the specific retention of 438H OSGIN1 might be important in HCC progression. In summary, we found a frequently amplified oncogenic SGK3 signaling pathway, as well as the allele-specific imbalance of tumor suppressive OSGIN1 in the pathogenesis of HCC. Further characterization of their mechanisms in hepatocarcinogenesis may help provide novel prognostic biomarkers and therapeutic targets in HCC treatment. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and particularly common in China and Southeast Asia. It ranks the 2nd and 4th most common fatal cancer in males and females, respectively, in Hong Kong. Current treatments are not always effective, as recurrence and metastasis in HCC are difficult to tackle and the underlying mechanisms not fully understood. Aberration of Wnt signaling has been implicated in HCC; in this study, we investigated the underlying mechanisms of how aberrant Wnt signaling promoted HCC development. With Taqman Low Density Array (LDA) analysis on 38 pairs of HCC and the corresponding non-tumorous livers for 59 Wnt signaling related-genes, we found significant overexpression of the Wnt signaling intermediate, Dishevelled (Dvl)-3, in HCC (p = 0.014). This observation in LDA was confirmed in 36 additional HCC cases. Among a total of 74 cases studied, 28.38% showed more than 3-fold overexpression in the tumors as compared with the corresponding non-tumorous livers. Dvl3 overexpression positively correlated with the presence of venous invasion. We also observed significant correlation of Dvl3 expression with accumulation of β-catenin, a downstream effecter of Wnt/β-catenin signaling (p=0.028). We further characterized the functional roles of Dvl3 in contributing to the stem cell-like and metastatic properties of HCC. We found that Dvl3 knockdown in HCC cells suppressed cell proliferation, sphere formation, tumorigenicity in immunodeficient mice, chemo-resistance, and expression of stemness genes. We then examined whether Wnt/β-catenin was effectively modulated by Dvl3 and found that Dvl3 overexpression and knockdown, respectively, promoted and reduced the TOP/FOP luciferase reporter activity in HCC cells. This was accompanied by the expression of β-catenin target genes, EpCAM and LGR5, both of which are associated with HCC stemness. Furthermore, rescue with wild-type or constitutively active β-catenin partially restored the in vivo tumorigenicity suppressed by Dvl3 knockdown, indicating a partial role of β-catenin in mediating the effects of Dvl3 on HCC stemness. In addition, since cell migration is a critical determinant in metastasis, we assessed the HCC cell migratory ability in vitro using transwell migration assays and observed suppression of the cell migration ability upon Dvl3 knockdown. Also, the in vivo orthotopic model confirmed a role of Dvl3 in promoting metastasis, as stable Dvl3 knockdown in HCC cells resulted in a reduction in lung metastasis. Interestingly, the effect of Dvl3 on cell migration was independent of β-catenin, as knockdown of β-catenin had no effect on HCC cell migration in vitro. It was also not related to the phosphorylation of MYPT in Rho-ROCK signaling, which itself was previously implicated in HCC cells metastasis and reported as a downstream signaling of Dvl in development. In summary, our study has identified roles of Dvl3 in HCC stemness properties and cell migration and this may provide functional implication of Dvl3 overexpression, which significantly correlated with venous invasion in human HCCs. Also, β-catenin is partly responsible for the role of Dvl3 in HCC stemness but independent of that in cell migration. Functional characterization of Dvl3 in HCC may help future development of therapy targeting Dvl3 of Wnt signaling pathways. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Liver cancer is the fifth most commonly diagnosed and the second most lethal malignancies worldwide, in which hepatocellular carcinoma (HCC) represents the majority subtype. High mortality rate of HCC is due to lack of effective treatments and early detection methods. Activation of cadherin-17 (CDH17)/β-catenin axis is found by our team in HCC and targeting components of this axis associated with anti-tumorigenesis. With limited knowledge on this axis in HCC, I plan to study molecules related to this axis as a way to uncover the cellular mechanism of this axis in liver tumorigenesis. Gene profiling data was re-analyzed to search for CDH17-associated genes in HCC clinical samples. The patient cohort was segregated into CDH17-high and CDH17-low group according to tumor/adjacent non-tumor expression ratio of CDH17. Serine peptidase inhibitor, Kazal type 1 (SPINK1) was found highly expressed in CDH17-high cases and its over-expression accounted for 73 % of total studied cases. Gene manipulation and inhibitor study in HCC cell lines suggested SPINK1 as a downstream molecule of CDH17/β-catenin axis in HCC. Further in silico analysis predicted potential binding sites of two transcriptional factors downstream of CDH17/β-catenin axis, lymphoid enhancer-binding factor 1 (LEF1) and T-cell factor 7 (TCF7), on SPINK1 promoter. Deletion or mutation of their binding sites on SPINK1 promoter suppressed the transcription of SPINK1 gene, while transient suppression of these two transcriptional factors resulted in reduction of SPINK1 level. As the direct link between SPINK1 and CDH17/β-catenin axis was confirmed, SPINK1 was hypothesized to possess tumorigenic properties like its upstream molecule CDH17. Suppression of SPINK1 using RNA interference in PLC and MHCC97-H HCC cells hampered growth, migration and colony formation abilities of suppressed cells. These phenotypic alterations accompanied with an inactivation of tumorigenic c-Raf/MEK/ERK pathway. These findings demonstrate the tumorigenic properties of SPINK1 in HCC. Next, the therapeutic potential of targeting SPINK1 in HCC was tested by using purified monoclonal antibody raised against recombinant SPINK1 protein (C4). C4 was capable in suppressing SPINK1 level based on results of immunocytochemisty, enzyme-linked immunosorbent assay and immunoneutralization. Treatment of HCC cells using C4 suppressed growth, migration and colony formation ability of cells by inactivating MAPK pathway. In subcutaneous tumor xenograft study, treating tumor-bearing mice with C4 at 8 mg/kg three times weekly inhibited tumor growth by around 65 %. These findings demonstrate C4 is a potential therapeutic for counteracting liver tumorigenesis. In conclusion, I have demonstrated for the first time SPINK1 as a novel downstream molecule of CDH17/β-catenin axis involved in HCC progression via activating MAPK pathway. Targeting this molecule with its specific monoclonal antibody is a potential approach for cancer therapy. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is the fifth most prevalent human malignancy and the third leading cause of cancer deaths in the world. MicroRNAs (miRNA) are conserved, small noncoding RNA molecules that regulate gene expression of protein-coding gene posttranscriptionally. Dysregulation of miRNA is implicated in many human malignancies including HCC, yet little is known regarding the regulatory mechanisms of these small noncoding RNAs. Hypoxia is a prevalent! tumor microenvironment in HCC because of its rapid growth often to large size and plays a key role in modulating tumor aggressiveness. In the present study, we investigated the effects of hypoxia on microRNA expression in human HCCs, identified and characterized hypoxia-inducible microRNAs that are important for the development of aggressive phenotypes. To initialize the study, we examined the miRNA expression profiles with TaqMan human microRNA Low-Density Array and identified a panel of microRNAs differentially expressed in HCC cells under hypoxic conditions. We observed that miR-210 was consistently upregulated by hypoxia in a total of 7 different HCC cell lines, via a HIF1α-dependent mechanism. In human HCCs, miRL210 overexpression significantly correlated with poorer overall and disease-free survival of patients, as well as aggressive pathologic features, including advanced tumor stages of HCC and the presence of venous invasion. These findings established miRL210 as a surrogate marker of aggressive HCC with high metastatic potential. In most human malignancies, cancer metastasis contributes to about 90% of cancer-related mortality. Given the correlation of miR-210 levels with poorer patient survival and aggressive clinical features of HCC, we then characterized the metastatic role of miRL210 by functional assays in the second part of the study. The findings from in vitro and in vivo experiments using both gain- and loss-of-function approaches led us to conclude that the hypoxic induction of miRL210 enhanced metastatic potential of HCC cells. The pro-metastatic effect of miRL210 was attributed, at least partly, to the downregulation of TIMP2 by hypoxia, through a feedback loop circuit consisting of HIF1α, miRL210, and HIF3α. The impact of miR-210 on HCC metastasis was not the only scope of this study since hypoxia has long been recognized as a major obstacle in chemotherapy. Given that activation of the HIF1α-miR-210 axis was frequently observed in hypoxic HCC cells, in the last part of the study we also investigated whether hypoxic induction of miRL210 promoted cell survival against cytotoxic treatments, including cisplatin and 5-flurouracil. Here, we demonstrated that induction of HIF1α-miR-210 axis conferred chemoresistance to HCC cells under hypoxic conditions, and inhibition of miR-210 re-sensitized HCC cells to these cytotoxic drugs. Mechanistically, we also revealed that RAD52 was a direct functional target of miRL210 that linked hypoxia to chemoresistance in HCC cells. The overall findings of this study have enriched our understanding of miR-210 as a mediator of hypoxic responses in HCC, in particular metastasis and chemoresistance. We have highlighted the clinical significance of this microRNA by showing that miR-210 can serve not only as a prognostic marker for HCC progression, but also as a mediator for the hypoxic tumor microenvironment to modulate tumor aggressiveness. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Ovarian cancer is the most lethal gynecological cancer. This is mainly due to widespread peritoneal dissemination and malignant ascites, in which angiogenesis, the formation of new blood vessels, is critical to both ascites development and its metastasis. Loss of E-cadherin is a well-established marker that characterizes the progression of metastatic tumors, including ovarian cancer. The release of a soluble form of E-cadherin (sE-cad) has been frequently associated with a rapid reduction of functional E-cadherin at the cell surface. Importantly, sE-cad is significantly present in ascites from women with stage III/IV ovarian cancer when compared to women with benign ovarian cysts. However, despite the clinical significance, most studies have focused on its role in weakening cell-cell adhesion, whether sE-cad itself has any biological function is not fully understood. Here it is shown for the first time a potent angiogenic role for sE-cad released from ovarian carcinoma. Soluble form of E-cadherin promoted the migration, permeability, and tubulogenesis of endothelial cells. These activities were also observed with a sE-cad/Fc chimera, and targeted inhibition using E-cadherin blocking antibodies completely prevented the sE-cad mediated effects. In addition, it was further revealed that sE-cad could be released from ovarian cancer cells in form of exosomes, a form of extracellular vesicles that play an important role in distant intercellular communication. sE-cad-positive exosomes were able to stimulate the angiogenic phenotype in vitro and functional neovascularization in a Matrigel implant model in vivo. The use of E-cadherin blocking antibodies resulted in diminished angiogenesis, confirming that the effect was sE-cad-positive exosomes specific. In search of the underlying mechanism by which sE-cad-positive exosomes promoted angiogenesis in endothelial cells which lacked E-cadherin, sE-cad was found to heterodimerize with VE-cadherin. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector β-catenin, but not p120 catenin. Similarly, the angiogenic phenotype could be reversed by inhibition of VE-cadherin, PI3K/Akt and β-catenin. A mass spectrometric proteomic analysis of the isolated exosomes revealed distinct membrane-bound proteases, especially disintegrin and metalloproteinase 10 (ADAM10) and matrix metalloproteinase 25 (MMP25) commonly associated with ovarian cancer progression, are implicated in sE-cad production. Small interfering RNA-mediated down-regulation of ADAM10 and MMP25 significantly inhibited sE-cad production. Moreover, hepatocyte growth factor, a multifaceted cytokine which is frequently elevated in ovarian cancer ascites, was shown to increase the expression of ADAM10 and MMP25 concomitant with an elevated level of sE-cad. Together, these results uncover a novel angiogenic role of sE-cad and a new mechanism of the action of sE-cad in tumor progression. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Although influenza is usually acute self-limiting respiratory infection, influenza viruses are among the most common pathogens that threaten the health of humans and animals worldwide. Various anti-viral therapeutic agents are currently used for treatment and prophylaxis of influenza virus, but the problem is that the targets of these drugs are easily mutated and result in resistance. Therefore, medications that have broad spectrum coverage are urgently needed to combat with the disease. Since nucleoprotein (NP), which is encoded by influenza virus genome, is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle, numerous studies are focused on this protein in attempts to develop broad-spectrum anti-influenza therapeutics. Recently, Kao et al. found that the addition of a novel small molecule nucleozin could lead to large aggregates of NP, which in turn caused cessation of virus replication. Give that the interaction between NP and nucleozin is still not unveiled, it is crucial to identify the binding sites using X-ray crystallography. The full length influenza A/WSN/33 (H1N1) NP gene was cloned into pET28 vector, with His-tag in its C-terminus and overexpressed in E.coli strain Rosetta 2. Cell culture was purified by HisTrap HP and Superdex-200 16/60 gel filtration columns. Crystals were grown using the vapour diffusion method and the NP-nucleozin complex was prepared by soaking native crystal in solution containing 0.25 mM nucleozin for 2h. Crystals of the complex can diffract to 3.0 Å at the Shanghai Synchrotron Radiation Facility. The structure of NP was determined by molecular replacement and it belongs to space group C121 with two NP trimers per asymmetric unit. After further refinement, two nucleozin molecules were found in each asymmetric unit, and each of them could bind with two NP molecules at the same time. The ligand binding pockets were formed by the combination of Y289/N309 pocket from one NP molecule, and R382 pocket from another NP molecule. Therefore, the function of nucleozin is to bridge two NP molecules and lead to NP aggregation, which are in agreement with functional studies on nucleozin. Furthermore, computational models of the NP-nucleozin binding are provided to reveal the mechanism of nucleozin induced aggregation. In addition, recent work on interaction between NP and another novel molecule named compound A has also been briefly described and compared with NP-nucleozin complex at the end of this thesis. Collectively, this study presents a new paradigm for better understanding of how NP and nucleozin interact with each other and hence result in NP aggregates, which is envisaged to accelerate the development of anti-influenza therapeutic agents. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Oncogenic mutations in lung cancer further our knowledge about cancer initiation and progression, and may guide personalized treatment. The fact that targeted therapy is most effective in subsets of patients with defined molecular targets indicates the need for classification of clinically-related molecular tumor phenotypes based on the presence of oncogenic mutations, including EGFR mutations and EML4-ALK rearrangements. The identification of EGFR mutations, in up to half of lung adenocarcinomas in Asians, could predict clinical sensitivity to tyrosine kinase inhibitor (TKI). However, testing for mutations is not always possible due to tumor tissue availability. The therapeutic decision sometimes remains a clinical one especially for elderly lung cancer patients but no known mutation status. We studied the survival outcomes of targeted therapy versus conventional chemotherapy in elderly patients with lung cancer when we did not yet have routine EGFR mutation testing and demonstrated comparable survival outcomes in targeted therapy compared to chemotherapy, implying that survival with targeted therapy could be better if the treatment population could be selected with EGFR mutations. Though testing for EGFR mutation in tumor biopsy have later become routine practice and remains the accepted reference for therapeutic decision, the detection of EGFR mutations in plasma DNA with high diagnostic performance will be useful adjunct for diagnostic and therapeutic monitoring. Among patients with EGFR mutations in tumor biopsy, the concurrent detection of EGFR mutation in plasma DNA was found to confer a less favorable prognosis in terms of overall survival than those patients with EGFR mutations in tumor biopsy but the corresponding mutation was not detected in plasma. Other oncogenic mutations with therapeutic implications in lung tumors are yet to be fully explored, like ALK, KRAS, ROS1 or NTRK1 mutations. It is not exactly the tumor – but the mutations in the tumor that need to be explored with reference to clinical behavior. Even with EGFR mutation with well-established clinical implications, further exploration into its mechanistic functions will help in understanding of drug resistance. Lung cancer cell lines established from patients with known mutation profiles could be useful tools for studying the biology of known molecular targets as well as for therapeutic testing. Four new lung adenocarcinoma and one mesothelioma cell lines were established from patients with different clinical characteristics and oncogenic mutation profiles. These cell lines with defined mutation profiles will provide tools for exploration of lung cancer and mesothelioma biology with respect to molecular therapeutic targets. The Large Tumor Suppressor 2 (LATS2) gene was a differentially expressed gene between EGFR mutant and wildtype lung adenocarcinomas. The differential LATS2 expression levels were predictive of survival in patients with resected lung AD and may modulate tumor growth via different signaling pathways in EGFR mutant and wild-type tumors. The identification of oncogenic mutations has led to a new paradigm of targeted therapy in lung cancer. Further improvements in outcome of lung cancer management will stem from research into the biology of oncogenic mutations and their clinico-pathological correlations, which would fuel parallel development of clinically efficacious targeted therapies. / published_or_final_version / Medicine / Master / Doctor of Medicine

Spermatogenesis is regulated by steroid hormones which induce expression of various genes responsible for the growth, proliferation and differentiation of spermatogonia to form mature haploid spermatozoa. The surrounding somatic cells including Leydig and Sertoli cells support the whole process in vivo. Previously, we used the post-vitamin A treated vitamin-A-deficient (PVA-VAD) rat model to study spermatogenesis, and identified 24 genes that are differentially up-regulated after retinol treatment. Vad1.2 is one of the up-regulated transcripts expressed in the rat testis from postnatal day 25. Vad1.2 transcript is localized to the round and elongating spermatids in the adult mouse testis. In silico analyses showed that Vad1.2 transcript is down-regulated in patients with teratozoospermia and non-obstructive azoospermia, suggesting that Vad1.2 may have important roles in spermatogenesis. However, how Vad1.2 affects spermatogenesis remains unclear. Therefore, the present study was designed to study the functional roles of Vad1.2 protein in mice using gene targeting approach and investigate the molecular changes in mice with Vad1.2 deficiency. Vad1.2 polyclonal antibody was raised against the full-length mouse Vad1.2 recombination protein and affinity purified. Vad1.2 protein was localized to the cytoplasm and flagellum of condensing spermatids, specifically to the fibrous sheath (FS) in cauda epididymal spermatozoa. Vad1.2 conditional knockout vector was constructed and used to generate Vad1.2 null mice. Vad1.2-/- male mice developed normally but were subfertile with reduced sperm count and motility. Vad1.2-/- male mice had smaller testis and higher incident of sloughing of immature germ cells into the seminiferous lumens when compared to the wild-type. Yet, the rates of germ cell proliferation and apoptosis were similar between the wild-type and the mutant testis. Interestingly approximately 50% of the mutant cauda epididymal spermatozoa showed deformed flagella and demonstrated structural defects typically associated with bending of flagellum at the principal piece or at the midpiece/principal piece junctions. The acrosome, nucleus and mitochondrial sheath of these spermatozoa appeared normal, while the flagellum displayed structural abnormalities including deformation of the two longitudinal columns of the FS and disruption of a portion of FS, suggesting that Vad1.2 might be involved in the biogenesis of FS in spermatogenesis. Furthermore, Vad1.2 interacted with Akap4 in vivo, and the two proteins were co-immunoprecipitated from the testis or cauda epididymal spermatozoa lysates. Akap4 and Vad1.2 were localized to the tail region of the testicular spermatids and cauda epididymal spermatozoa. The expression levels of pro- and mature Akap4 in Vad1.2-/- testes were markedly increased when compared with the wild-type mice. However, a significant decrease of Akap4 was found in the mutant cauda epididymal spermatozoa, suggesting that most of the mature Akap4 failed to incorporate into the FS. Taken together, Vad1.2 plays an important role in spermatogenesis and Vad1.2 deficiency leads to subfertility in mice with the deformed flagella in mature spermatozoa. Further studies on the regulation of FS formation may uncover the underlying molecular changes associated with Vad1.2 deficiency, and may provide fundamental information for treatment of infertile patients with FS defect in the spermatozoa. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy