THE ROLE OF IFITM3 IN MAMMARY GLAND DEVELOPMENT AND MAMMARY STEM CELLS

Le cellule staminali della ghiandola mammaria sono un modello per lo studio dei processi molecolari che regolano lo sviluppo fisiologico dell\u2019organo e lo sviluppo tumorale. Diversamente da molti organi, la ghiandola mammaria incorre in cicli di rigenerazione e involuzione associati alla gravidanza. Per questo la ghiandola mammaria \ue8 uno dei primi organi nel quale si \ue8 ipotizzata la residenza di cellule staminali. Dal concetto che le cellule staminali regolano la crescita e la differenziazione della ghiandola con l\u2019avvento della gravidanza \ue8 stato ipotizzato che cellule tumorali con propriet\ue0 staminali possano contribuire all\u2019eterogeneit\ue0 del tumore. Al fine di scoprire come il normale sviluppo dell\u2019architettura della ghiandola mammaria sia originata dalle cellule staminali e il ruolo delle cellule staminali tumorali nella progressione tumorale, in questo lavoro di tesi \ue8 stata indagata la funzione di IFITM3. IFITM3 \ue8 stato inizialmente identificato dal laboratorio della dott.ssa Zucchi nelle cellule LA7, cellule staminali della ghiandola mammaria di ratto capaci di differenziare in dome, strutture 2D simili agli alveoli che si formano nella ghiandola mammaria durante la gravidanza. \uc8 stato dimostrato dal gruppo Zucchi che la funzione di IFITM3 dipende dal cambio di localizzazione della proteina dal citoplasma alla membrana plasmatica, dove risiede nei lipid raft. Questo \ue8 stato il punto di partenza di questa tesi di ricerca con lo scopo di identificare se IFITM3 partecipi anche alla formazione di strutture tridimensionali complesse della ghiandola mammaria (strutture tubulo-alveolari) e se svolga una funzione nel mantenimento delle cellule staminali della ghiandola mammaria. Ho utilizzato sistemi transienti e stabili per la modulazione dei livelli di espressione di IFITM3 in cellule LA7 e in cellule MCF7, una linea cellulare umana di ghiandola mammaria. Mentre le cellule LA7 possiedono sia la capacit\ue0 di generare sfere sia di differenziare morfologicamente e funzionalmente in tutti i tipi cellulari e strutture 3D della ghiandola mammaria, si ritiene che cloni MCF7 utilizzati in questo studio non possiedano propriet\ue0 di cellule staminali, non formano sfere e possono differenziare unicamente in un tipo di struttura 3D, le cisti simili agli alveoli della ghiandola mammaria. Abbattendo i livelli di espressione di IFITM3 con siRNA/oligo e tecnologia lenti virale ad RNA a forcina corta (shRNA) in cellule impiegate in saggi funzionali in condizioni di coltura 3D, ho scoperto che IFITM3 \ue8 necessario per la formazione di strutture tubulo-alveolari complesse in cellule MCF7 e per il mantenimento delle propriet\ue0 di auto-rinnovamento delle cellule staminali LA7. Dato che le mammosfere e le cisti mammarie rappresentano strutture 3D associate rispettivamente a cellule staminali o differenziate, e dato che la sotto-regolazione di IFITM3 inibisce la formazione di entrambe, possiamo ipotizzare che IFITM3 abbia una funzione diversa in base al tipo cellulare. La sotto-regolazione di IFITM3 in cellule staminali LA7 in condizioni di coltura aderenti e non aderenti ha portato ad una graduale perdita delle cellule, suggerendo che IFITM3 svolga una funzione necessaria al mantenimento dell\u2019auto-rinnovamento delle cellule staminali LA7. In cellule MCF7 la sotto-regolazione di IFITM3 ha portato all\u2019incapacit\ue0 delle cellule di formare alveoli senza effetti sulla proliferazione cellulare. La sovra-regolazione di IFITM3 sia in cellule LA7 sia MCF7 ha portato alla rapida morte cellulare per un meccanismo che \ue8 ancora sotto indagine. Complessivamente ho dimostrato che la sotto-regolazione di IFITM3 porta alla perdita delle propriet\ue0 di auto-rinnovamento II e della capacit\ue0 di essere propagate come cellule staminali delle cellule LA7. La sotto-regolazione di IFITM3 in cellule che non hanno propriet\ue0 staminali di auto-rinnovamento porta all\u2019incapacit\ue0 delle cellule di formare strutture 3D. Lo studio dei profili di espressione genica con tecnologia micorarray ottenuti da cellule LA7 trattate per la sotto-regolazione di IFITM3 supporta il ruolo di IFITM3 nella regolazione del ciclo cellulare, nel trasporto vescicolare e nella modificazione dello stato cromatinico. Geni coinvolti nella proliferazione cellulare sono stati trovati sotto-regolati in seguito alla perdita di IFITM3, insieme a geni del trasporto vescicolare (che coinvolgono proteine che mediano la fusione vescicolare, come le SNARE) che possono essere collegati a IFITM3 in quanto il ruolo di IFITM3 nel prevenire il rilascio delle particelle virali dai compartimenti endosomiali \ue8 associato alla formazione di un poro di fusione e alla fusione delle membrane. I geni associati con la regolazione epigenomica e la proliferazione cellulare suggeriscono che IFITM3 possa avere differenti ruoli in cellule staminali e cellule della ghiandola mammaria differenziate, dato che questi geni sono coinvolti nel cancro, nella crescita cellulare o apoptosi e nel differenziamento cellulare.Mammary stem cells (MaSCs) are a model to understand molecular processes that regulate both normal and cancer development. In contrast to many organs, the mammary gland undergoes cycles of re-generation and involution associated with pregnancy. For this reason, the mammary gland was one of the first organs in which stem cells (SCs) were hypothesized to reside. From the concept that SCs regulate normal mammary gland growth and differentiation with onset of pregnancy, arose the hypothesis that cancer cells with SC properties may contribute to tumor heterogeneity. To understand how normal development of the mammary gland architecture arises from SCs and the role of CSCs in tumor progression, the function of the IFITM3 gene was investigated in this thesis research. IFITM3 was initially identified by Zucchi\u2019s lab in LA7 cells, rat mammary SCs that differentiate into domes, 2D structures similar to alveoli that form in mammary gland at pregnancy. It was demonstrated by the Zucchi group that the function of IFITM3 was dependent on its shuttling from the cytoplasm to the plasma membrane and being part of lipid rafts. This was the starting point of the thesis research with the aims to identify whether IFITM3 also plays a role in the formation of more complex three dimensional (3D) mammary structures (tubule-alveolar structures) and has a role in MaSCs. I used transient and stable IFITM3 expression modulating systems in the LA7 and the human MCF7 mammary cell line. In contrast to the rat LA7 SCs that have both the capacity to generate spheres and differentiate morphologically and functionally into all the mammary cell types and generate tubule-alveolar structures iin 3D culture conditions, the MCF7 clone used in this study are not considered to have SC properties, do not form spheres and can only form one type of 3D differentiated structure called cysts. Targeting IFITM3 in these cells, with siRNA/oligos and short hairpin RNA lentiviral technology in order to down-regulate the expression of the protein, and by performing functional assays in 3D culture conditions, I found that IFITM3 is necessary for the formation of complex alveolar structures in MCF7 cells and for self-renewal of LA7 SCs. As mammary spheres and mammary cysts represent different 3D structures associated with stem and differentiated cells respectively, and since IFITM3 down-regulation inhibits the formation of both, this suggests that IFITM3 may have different functions depending on the cell type. Down-regulation of IFITM3 in LA7 SCs in both adherent or in non-adherent cultures resulted in a gradual loss in the number of cells, suggesting that IFITM3 function is necessary for self-renewal of LA7 SCs. In contrast down regulation of IFITM3 in MCF7 cells resulted in the inability of the cells to form alveoli with no effect in cell proliferation. IFITM3 up-regulation in both LA7 and MCF7 cells resulted in rapid cell death by a mechanism that is still under investigation. Collectively, I demonstrate, that IFITM3 down-regulation results in loss of self-renewal and loss of the ability of LA7 to be propagated as SCs. While IFITM3 down-regulation in mammary cells that do not IV have SC self-renewal capacity results in the inability of the cells to generate 3D structures. Microarray expression experiments, obtained with down-regulation of IFITM3 in LA7 cells, support the IFITM3 function in cell cycle regulation, vesicular transport and in the modification of the chromatin state. In addition to genes involved in cell proliferation found down-regulated with loss of IFITM3, a link among vesicle-transport genes (involving proteins mediating vesicle fusion, such as SNARE), and IFITM3 was also determined, supporting a role of IFITM3 in preventing the release of viral particles from endosomal compartments, associated with membrane fusion and the formation of a fusion pore. Genes associated with epigenomic regulation and cell proliferation suggests that IFITM3 may have multiple and different roles in SCs and in differentiated mammary cells since these genes are involved in cancer, cell growth or apoptosis and in cell differentiation

Therapeutic Targeting of Leukemia Stem Cells to Prevent T-Cell Acute Lymphoblastic Leukemia Relapse

The survival rate of T-cell Acute Lymphoblastic Leukemia (T-ALL) relapse is a dismal 10% of affected adults and 30% of children, largely due to the relapsed disease being more aggressive and treatment resistant than the initial disease. Relapse is thought to occur because conventional chemotherapies are unable to reliably eliminate a unique cell type known as leukemia stem (or propagating) cells (LSCs). LSCs are the only cells within the leukemia with the ability to self-renew and remake or replenish the ALL from a single cell. Currently, the pathways governing self-renewal in LSCs are largely unknown, precluding our ability to successfully and selectively target this important cell type with anti-cancer drugs. More research is needed to identify targetable pathways and develop new technologies for studying LSCs. Here, I determined that the oncogenic phosphatase of regenerating liver 3 (PRL-3) plays a role in leukemia progression, migration, and self-renewal of LSCs in T-ALL in vivo in a zebrafish Myc-induced T-ALL model, while inhibition of PRL-3 reduced LSC numbers in vivo and in vitro. RNA sequencing and GSEA of patient T-ALL samples revealed that PRL-3’s role in self-renewal is at least partly due to activation of Wnt pathway signaling, a known driver of LSC function in T-ALL. While the Wnt pathway seems an ideal target for LSCs, Wnt signaling is critical for many normal and developmental processes. Clinical trials for Wnt inhibitors have shown undesirable toxicity and these drugs are not practical for use in children with T-ALL due to developmental concerns. Thus, a major gap in knowledge concerning leukemia stem cells in T-ALL is the identification of regulators of Wnt signaling, like PRL-3, that are uniquely expressed by leukemia cells and easily targeted with small molecules. To expand my research beyond PRL-3, I have developed a novel zebrafish T-ALL model where Wnt expressing cells fluorescently labeled. These animals can be used as a model for studying LSC function and identifying novel drugs that can target Wnt-expressing T-ALL cells in vivo. I have also developed novel translational technologies that may be used to predict LSC driven relapse in T-ALL. I have optimized a zebrafish larval xenograft model for transplant and rapid drug screening of human T-ALL cell lines and patient samples to gain insight into tumor progression and resistance to chemotherapy. I have also developed a novel pipeline for using cell-free circulating tumor DNA (ctDNA) as a biomarker of disease relapse in patients with ALL, enabling tracking of disease course, assessment of minimal residual disease, and as a potential predictor of patient relapse. Taken together, my research has established PRL-3 as a potential therapeutic target in T-ALL, and provided new insight into the role of a PRL-3/Wnt signaling axis in regulating LSC self-renewal. Additionally, the new models and techniques that I have developed are useful tools in analyzing LSC function, targeting self-renewal, and predicting ALL relapse

Engrailed genes in mammary development and tumorigenesis

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Unveiling the molecular determinants of mammalian skin ageing: A proteomic and transcriptomic approach

As the limits of human lifespan continue to expand, ageing biologists must address the decline in the integrity of bodily tissues with time. Our skin is drastically impacted by both intrinsic and extrinsic ageing processes, driven by gradual accumulation of cellular damage and environmental insults like ultra-violet irradiation. Together intrinsic and extrinsic skin ageing give rise to clinical issues such as xerosis, pruritus and neoplasms. Cosmetic issues, such as unsightly wrinkling, thinning and sagging of the skin also impact human psychological and social wellbeing. Given these issues, studying the molecular mechanisms of intrinsic and extrinsic skin ageing processes is an important element of biological research, as a better understanding of how these processes contribute to reduced tissue integrity will allow us to develop therapies to attenuate the ageing process. Using tissues taken from C57BL/6 male mice and female humans as our models, we analysed skin at early, middle and late stages within both the murine and human lifespans to assess the impact of ageing on changes in the epidermis, dermis and at the basement membrane. By considering tissue taken from photo-protected and photo-exposed sites of humans, we additionally studied the differential changes occurring during intrinsic and extrinsic skin ageing (photo-ageing). Our studies showed that several morphometric changes occur to the epidermis with age in mouse skin, where we observed thinning and cellular loss. Cell proliferation and lamin B1 levels declined, which was coupled with decreased expression of dermal and basement membrane collagens. Many of these observations were ubiquitous in intrinsically aged human skin, where we additionally show unique transcriptional changes at the basement membrane. One little studied pathway in skin ageing is the Hippo pathway, which has crucial roles in epidermal development through its control of epidermal cell proliferation. We identify a novel modulation of the Hippo pathway effector YAP1 in aged mouse and human skin, where we show that nuclear localisation of YAP1 increases during epidermal ageing. Together this body of work demonstrates that C57BL/6 mouse skin ageing shares common mechanisms of intrinsic human skin ageing. Additionally, we show that YAP1 localisation is altered during epidermal ageing, which suggests that the Hippo pathway is sensitive to both changes in the extra-cellular matrix content, and cell-proliferation properties of skin over time

Cancer Biomarkers and Targets in Digestive Organs

Identification and development of cancer biomarkers and targets have greatly accelerated progress towards precision medicine in oncology. Studies of tumor biology have not only provided insights into the mechanisms underlying carcinogenesis, but also led to discovery of molecules that have been developed into cancer biomarkers and targets. Multi-platforms for molecular characterization of tumors using next-generation genomic sequencing, immunohistochemistry, in situ hybridization, and blood-based biopsies have greatly expanded the portfolio of potential biomarkers and targets. These cancer biomarkers have been developed for diagnosis, early detection, prognosis, and prediction of treatment response. The molecular targets have been exploited for anti-cancer therapy and delivery of therapeutic agents. This Special Issue of Biomedicines focuses on recent advances in the discovery, characterization, translation, and clinical application of cancer biomarkers and targets in malignant diseases of the digestive system. The goal is to stimulate basic and translational research and clinical collaboration in this exciting field with the hope of developing strategies for prevention and early detection/diagnosis of cancer in digestive organs, and improving therapeutic and psychosocial outcomes in patients with these malignant diseases