Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

Basal breast cancer is a heterogeneous disease whose unfavourable outcome is determined by a high risk of tumour relapse and metastasis formation. The potential of a cancer cell to adapt to foreign environments is favoured by oncogenic cell plasticity, which is supported by epigenetic reprogramming. It was previously demonstrated that MYC acts as an oncogenic reprogramming factor by inducing epigenetic rewiring at enhancers (Poli et al., 2018). This causes the activation of oncogenic pathways and pro-metastatic transcription factors such as SOX9, but scant pieces of evidence support a causal link between epigenetic alteration of oncogenic enhancers and cell plasticity. In the present work, we investigated the establishment of an alternative epigenetic program during tumorigenesis in a basal breast cancer xenograft derived model. We found that tumorigenic cells, primary tumour derived cells and metastasis derived cells showed intrinsically different phenotypic and epigenetic signatures, and that metastatic derived cells were characterized by the acquisition of pro-metastatic features, such as migration and invasion, that may increase their metastatic potential. Specifically, we provided data supporting the notion that changes of the chromatin landscape during tumour progression increased the responsiveness of cancer cells to environmental cues that they may encounter during dissemination and colonization of distant organs. We focused on investigating the role played by putative regulatory elements localized around the SOX9 locus, whose chromatin accessibility and interaction with the SOX9 promoter were increased in metastatic cells. We observed that SOX9 expression was responsive to the activation of the retinoic acid (ATRA) pathway, and our data suggests that this response may be strengthened by transcriptional memory priming SOX9 regulatory elements after a first exposure, so that the response is faster and more robust after the second one. SOX9 transcription modulation and ATRA response were also shown to be linked to the activation of a quiescence program specific of metastatic cells, which we hypothesise may favour cells during the dissemination steps of the metastatic cascade.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13566号 / 論医博第2293号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 柊, 卓志, 教授 篠原, 隆司, 教授 後藤, 慎平 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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