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The glycoprotein stanniocalcin-1 (STC1) is a paracrine factor in mammals which plays roles in various (patho)physiological functions, such as inflammation and carcinogenesis. Considerable numbers of studies showed dysregulation of STC1 expression in different types of human cancers. A previous study from our group, using clinicopathological data of 216 hepatocellular carcinoma (HCC) patients revealed greater STC1 gene expression in tumors than the paired normal samples. However, patient samples with greater STC1 level exhibited smaller tumor size. In fact, multiple cell types, growth factors and matrix components in tumor microenvironment (TME) control cancer progression. Emerging evidence support the important role of infiltrating immune cells on tumor progression. Among those, tumor associated macrophages (TAM) in TME is known to be an essential driver of tumor inflammation and progression, exerting a yin-yang influence to determine if the tumor is suppressed or paving the way to metastasize. Hepatocellular carcinoma (HCC) is mainly caused by chronic inflammation. With hindsight, the roles of STC1 in inflammation and carcinogenesis were documented. However, the observation on the negative correlation of STC1 expression with tumor size in HCC patients and the roles of STC1 on the interactions between tumor cells and macrophages are not clear. In Chapter 2, the inverse correlation of STC1 expression with tumor size was addressed. Human metastatic HCC cell line, MHCC97L which was stably transfected with empty vector (P) and STC1 (S1) were used. Nude mice xenograft model showed that tumor size and volume formed from S1 cells were significantly smaller than that from P cells. The observation agreed with the clinical data aforementioned. In vitro studies demonstrated S1 cells had lower plating efficiency, migratory and proliferative potential, illustrating a lower tumorigenicity. Biochemical analyses on the rate of glycolysis, extracellular O2 consumption, ATP production and Western blot studies on mTOR/p70S6K/rpS6 pathway showed the S1 cells adopted a lower energy metabolism. The data may explain the negative correlation between STC expression level and tumor size. In cancer microenvironment, infiltration of host immune cells, especially macrophages, contributes to inflammation and tumor progression. In Chapter 3, it was hypothesized that cancer cell-derived STC1 alter macrophage functions. Therefore, the effects of STC1-overexpressing MHCC97L on macrophages were studied. To mimic their interactions, Boyden chamber insert model was adopted to co-culture MHCC97L (97L/P and 97L/S1) and THP-1. Our data illustrated 97L/S1 suppressed migratory response of THP-1, with or without the addition of monocyte chemoattractant protein 1 (MCP-1) as the chemoattractant. Quantitative PCR showed downregulation of cytokine/chemokine receptors (CCR2, CCR4, CSF-1R) in THP-1 when co-cultured with 97L/S1. This prompted us to study the alterations of pathways related to cell motility in THP-1 by 97L/S1. Transcriptomic analysis detected 1784 differentially expressed genes (DEGs) between THP-1 cells co-cultured with 97L/P and 97L/S1. Ingenuity Pathway Analysis (IPA) prioritized an inhibition of RhoA signaling, which is known to stimulate cell motility. Western blotting analysis supported the IPA prediction and the cell migration data to show a significant reduction of MLC2 phosphorylation, leading to impaired formation of stress fibers, cell contraction and cell motility. The preceding chapters focused on cancer cell-derived STC1 on HCC cells or THP-1 derived macrophages. In Chapter 4, it was hypothesized that macrophage-derived STC1 may also play a role in macrophage differentiation and inflammation, which modulate tumorigenicity of HCC during macrophage-cancer cell interactions. Thus, the roles of endogenous STC1 in macrophage differentiation and functions were investigated. Using human leukemia monocytic cell line THP-1, a pilot study showed a treatment with phorbol 12-myristate 13-acetate (PMA) significantly upregulated STC1 expression and pro-inflammatory cytokines. In follow-up studies, THP-1 was pharmacologically stimulated to differentiate into (i) classically activated macrophages (CAM)/ M1 state, and (ii) alternatively activated macrophages (AAM)/ M2 state. Greater STC1 expression was found to be associated with CAM. To examine the role of STC1 in CAM, siRNASTC1 was used for gene knockdown. Conditioned medium collected from siRNASTC1-treated CAM inhibited migration of HCC cell line Hep3B. Transcriptomic analysis of siRNASTC1-treated CAM revealed an upregulation on TBC1D3G gene, which is involved in the release of extracellular vesicles (EVs) in macrophage to mediate inflammation. This study demonstrated the association between STC1 and macrophage-mediated inflammation. Collectively, the above studies elucidated the influence of STC1 on cancer cell metabolism, macrophage differentiation and function. It warrants further investigations to unravel the therapeutic potential of STC1 in inflammation and carcinogenesis.

Mosaicism occurs due to postzygotic genetic alterations during early embryonic development. The phenomenon is common, present in all humans, animals, and plants, and is associated with phenotypic variability and heterogeneity. Mosaic pathogenic gene variants result in a mosaic disease state, in which the individual can present with mild, generalized disease, a localized disease phenotype in specific organs and tissue regions, or full-blown clinical features which are indistinguishable from the heterozygous disease state. Multiple studies have described the prevalence and clinical correlations associated with low-level mosaicism for various genetic disorders, including several tumor suppressor gene (TSG) syndromes, which are well-known to display mosaicism. However, the extent of mosaicism research varies widely between TSG syndromes. Currently there is no comprehensive, up to date review covering multiple TSGs and focusing on mosaicism prevalence, diagnostic strategies and transmission risk. Here, in this literature review, I focus on 8 common tumor suppressor genes NF1, NF2, TSC1, TSC2, RB1, PTEN, VHL, and TP53; reporting the following disease aspects: • Role and function of each tumor suppressor gene, disease prevalence, inheritance pattern, penetrance/expressivity pattern, age of onset clinical features, organs affected, and benign or malignant tumors seen • Different types of mosaicism, including critical review of recent, representative publications for each tumor suppressor gene syndrome • Established criteria for clinical diagnosis of inherited versus mosaic disease, molecular diagnosis, and current methods of genetic analysis Then more extensively, this thesis discusses the most informative, representative original studies for each TSG and provides a summary which covers: • The number of mosaic patients analyzed and the spectrum of clinical features of the cohort they were sampled from • The spectrum of variant allele frequency (VAF), tissue types analyzed, and different analysis methods performed • Whether or not the mosaic patients met clinical criteria for diagnosis of inherited disease • The number of patients who were persistently classified as no mutation identified (NMI) after genetic analysis • Spectrum and type of mosaic mutational event(s) identified • Age of onset and age range of mosaic patients • Patient ascertainment and family history (sporadic or familial cases) and • Type of mosaicism seen Furthermore, it compares and discusses disease severity, possibility of malignancy, and genotype-phenotype correlations for each TSG. Ultimately, by juxtaposing these TSGs, this review aims to centralize existing knowledge about mosaicism and provide insight into how molecular techniques can be broadly applied for better diagnosis of mosaic disease. / 2022-11-10T00:00:00Z

abstract: Cancer is a disease which can affect all animals across the tree of life. Certain species have undergone natural selection to reduce or prevent cancer. Mechanisms to block cancer may include, among others, a species possessing additional paralogues of tumor suppressor genes, or decreasing the number of oncogenes within their genome. To understand cancer prevention patterns across species, I developed a bioinformatic pipeline to identify copies of 545 known tumor suppressor genes and oncogenes across 63 species of mammals. I used phylogenetic regressions to test for associations between cancer gene copy numbers and a species’ life history. I found a significant association between cancer gene copies and species’ longevity quotient. Additional paralogues of tumor suppressor genes and oncogenes is not solely dependent on body size, but rather the balance between body size and longevity. Additionally, there is a significance association between life history traits and genes that are both germline and somatic tumor suppressor genes. The bioinformatic pipeline identified large tumor suppressor gene and oncogene copy numbers in the naked mole rat (Heterocephalus glaber), armadillo (Dasypus novemcinctus), and the two-fingered sloth (Choloepus hoffmanni). These results suggest that increased paralogues of tumor suppressor genes and oncogenes are these species’ modes of cancer resistance. / Dissertation/Thesis / Pipeline results for cancer genes / Phylogenetic regressions with correction tests / Pipeline results for housekeeping genes / Masters Thesis Biology 2019

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Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. HPVs are divided into high-risk (HR) or low-risk (LR) types based on their oncogenic potential. HPVs 16 and 18 are considered HR types and can cause cervical cancer. HPVs 6 and 11 are classified as LR and are associated with condyloma acuminata (genital warts). Viral proteins of both HR and LR HPVs must be able to facilitate a replication competent environment. The E7 proteins of LR and HR HPVs are responsible for maintenance of S-phase activity in infected cells. HR E7 proteins target all pRb family members (pRb, p107 and p130) for degradation. LR E7 does not target pRb or p107 for degradation, but does target p130 for degradation. Immunohistochemistry experiments on HPV 6 infected patient biopsies of condyloma acuminata showed that detection of p130 was decreased in the presence of the whole HPV 6 genome. Further, the effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Experiments were performed using human foreskin keratinocytes transduced with HPV 6 E7, HPV 16 E7 or parental vector. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the viii presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. Experiments were also conducted to detect E7-binding partners. Cyclin C and cullin 5 were identified as proteins capable of binding to both HPV 6 E7 and HPV 16 E7. Preliminary experiments showed that decreasing protein levels of p600, a binding partner of both HPV 6 E7 and HPV 16 E7, by RNA interference might affect p130 stability. Elucidating the mechanisms of p130 degradation may identify potential targets for preventing degradation of p130 and allowing restoration of cell cycle control.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA is subject to various types of damage that can impair cellular function or cause cell death. DNA damage blocks normal cellular processes such as replication and transcription and can have catastrophic consequences for the cell and for the organism. It has long been thought that the G1/S cell cycle checkpoint allows time for DNA repair by delaying S-phase entry. The p53 tumor suppressor pathway regulates the G1/S checkpoint by regulating the cyclin-dependent kinase inhibitor p21Waf1/Cip1, but p53 also regulates the nucleotide excision DNA repair protein XPC. Here, using p53-null cell lines we show that additional mechanisms stabilize XPC protein and promote NER in concert with the G1/S checkpoint. At least one mechanism to stabilize and destabilize XPC involves ubiquitin-mediated degradation of XPC, as the ubiquitin ligase inhibitor MG-132 blocked XPC degradation. The retinoblastoma protein, RB, in its unphosphorylated form actually stabilized XPC and promoted NER as measured by host-cell reactivation experiments. The data suggest that XPC protein and XPC-mediated NER is tightly linked to the G1/S checkpoint even in cells lacking functional p53.

<p>miRNAs therapeutics have emerged as a potential cancer therapeutics due to their unique ability to target multiple genes, allowing a single miRNA to act as a multi-drug cocktail. However, toxicity associated with current delivery vehicles as well as the sensitivity of the miRNA to serum nucleases are critical hurdles that stand against their clinical utility. Ligand-targeted delivery approaches such as small molecules, aptamers, antibodies or glycoconjugates provide a promising strategy to achieve specific delivery of the therapeutic cargo to the intended targeted cells without any toxicity. We hypothesized that combining both ligand targeted approach along with modifying the miRNA would provide a perfect delivery system which does not only achieve specific delivery but also reduce the therapeutic dose. The data presented in this dissertation shows (i) that miR-34a delivered using a combination of targeting ligand (DUPA) and an endosomal escape agent (nigericin) enables selective delivery of miR-34a to prostate cancer cells, (ii) that the use of full chemical modification approach enhances miR-34a stability and activity both in vitro and in vivo. Overall, these results provide an advancement in the miRNA delivery field and will help the development of miRNA based therapeutics against cancer.</p>

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