The AC133 epitope of the pentaspan transmembrane glycoprotein CD133 has been used as a cell-surface marker for normal and cancer stem cells from a broad range of tissue types. Despite the utility of CD133 as a marker, little is known regarding its regulation and biological function. To study these poorly understood aspects of CD133, I took two main experimental approaches: RNA interference (RNAi) screening and affinity purification coupled with mass spectrometry (AP-MS) to identify CD133 regulatory genes and CD133 protein-protein interactions (PPIs), respectively. Both of these experimental approaches relied on a human embryonic kidney (HEK) 293 cell line that exogenously expresses affinity tagged CD133 (HEK293/AC133). This cell line allowed me to perform a large-scale RNAi screen to interrogate 11,248 genes for their involvement in cell-surface AC133 recognition. This resulted in the identification of the N-glycosylation pathway as a direct contributor to CD133 plasma membrane localization and cell-surface AC133 detection. I used the same RNAi screening approach on the colon adenocarcinoma cell line Caco-2, which express CD133 from its native promoter, to identify factors that regulate endogenous CD133 transcription. I was able to demonstrate that AF4 promotes CD133 transcription in a number of cancer cell lines. Furthermore, I showed that CD133 expression in an acute lymphoblastic leukemia (ALL) cell line SEM, which is dependent on the mixed-lineage leukemia (MLL)-AF4 gene fusion, is critical for the viability of these cells. To gain further insight into the function of CD133, I performed AP-MS using HEK293/AC133 cells to identify CD133 PPIs. I identified histone deacetylase 6 (HDAC6) as a CD133 protein interaction partner. I found that HDAC6 negatively regulates CD133 trafficking into the endosomal-lysosomal degradation pathway. CD133 binds HDAC6 to prevent inhibition of HDAC6 deacetylase activity by phosphorylation. Protection of HDAC6 from phosphorylation promotes HDAC6 deacetylation of β-catenin, which results in β-catenin dependent signalling and the suppression of cancer cell differentiation. My thesis provide functional roles for CD133 as a pro-proliferative protein and as a key signalling protein in certain cancer cell lines.

Note: Portions of this abstract have been previously published in the journal Blood, Basiorka et al. Blood. 2016 Oct 13, and has been reproduced in this manuscript with permission from the publisher. Myelodysplastic syndromes (MDS) are genetically diverse hematopoietic stem cell malignancies that share a common phenotype of cytological dysplasia, ineffective hematopoiesis and aberrant myeloid lineage maturation. Apoptotic cell death potentiated by inflammatory cytokines has been considered a fundamental feature of MDS for over two decades. However, this non-inflammatory form of cell death cannot account for the inflammatory nature of these disorders. We report that a hallmark of lower-risk (LR) MDS is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptosis, a caspase-1-dependent programmed cell death induced by danger-associated molecular pattern (DAMP) signals. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress pyroptosis-related transcripts, inflammasome proteins and manifest activated NLRP3 inflammasome complexes that direct caspase-1 activation, IL-1β and IL-18 maturation and pyroptotic cell death. Using the S100A9 transgenic (S100A9Tg) mouse model that phenocopies human MDS, we demonstrated that forced expression of S100A9 was sufficient to drive pyroptosis in vivo, implicating pyroptosis as the principal mechanism of HSPC cell death in S100A9Tg mice. The lytic cell death releases intraceullar contents that include alarmins and catalytically active ASC specks, which can propagate bystander inflammation. Notably, MDS mesenchymal stromal cells (MSC) and stromal-derived linages were found to predominantly undergo pyroptosis, with marked activation of caspase-1 and NLRP3 inflammasome complexes. These findings may account for the clusters of both HSPC and stromal cell death previously described in the bone marrows of patients with MDS. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPC and bone marrow (BM) plasma. Further, both somatic gene mutations and S100A9-induced signaling activate NADPH oxidase (NOX), generating reactive oxygen species (ROS) that initiate cation influx, cell swelling and β-catenin activation. Accordingly, ROS and active β-catenin were significantly increased in MDS BM mononuclear cells (BM-MNC) and S100A9Tg mice compared to normal controls, as well as in human cell lines harboring gene mutations and in murine models of gene mutation knock-in or gene loss. ROS and β-catenin nuclear translocation were significantly reduced by NLRP3 or NOX inhibition, indicating that S100A9 and somatic gene mutations prime cells to undergo NOX1/4-dependent NLRP3 inflammasome assembly, pyroptosis and β-catenin activation. Together, these data explain the concurrent proliferation and inflammatory cell death characteristic of LR-MDS. Given that loss of a gene-rich area in del(5q) disease results in derepression of innate immune signaling, we hypothesized that this genetic deficit would trigger assembly of the NLRP3 inflammasome complex, akin to the pathobiological mechanism characteristic of non-del(5q) MDS. To this end, we utilized two distinct murine models of del(5q) disease, namely in the context of Rps14 haploinsufficiency and concurrent loss of mDia1 and microRNA (miR)-146a. In both models, pyroptosis was not evident in the HSPC compartment; however, early erythroid progenitors displayed high fractions of pyroptotic cells. This was associated with significant increases in caspase-1 and NLRP3 inflammasome activation, ROS and nuclear localization of β-catenin, which was extinguished by inflammasome or NOX complex inhibition. These data suggest that early activation of the inflammasome drives cell death and prevents terminal maturation of erythroid precursors, accounting for the progressive anemia characteristic of del(5q) disease, whereby hematopoietic defects are primarily restricted to the erythroid compartment. Importantly, these data implicate a similar pathobiological mechanism in del(5q) MDS as is observed in non-del(5q) patients. The identification of the NLRP3 inflammasome as a pathobiological driver of the LR non-del(5q) and del(5q) MDS phenotype allows for novel therapeutic agent development. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS, and are sufficient to restore effective hematopoiesis. In del(5q) murine models, inhibition of the NLRP3 inflammasome significantly improved erythroid colony forming capacity by a mechanism distinct from that of lenalidomide, highlighting the translational potential for targeting this innate immune complex in this subset of MDS. Thus, alarmins and founder gene mutations in MDS license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. Furthermore, aggregated clusters of the NLRP3 adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] are referred to as ASC specks. During pyroptosis, ASC specks are released from dying cells and function as DAMP signals that propagate inflammation. In this way, specks are a surrogate marker for NLRP3 inflammasome activation and pyroptotic cell death. Given that pyroptosis is the predominant mechanism of cell death in MDS and ASC specks are readily quantified by flow cytometry, we hypothesized that BM or peripheral blood (PB) plasma-derived ASC specks may be a biologically rational biomarker for the diagnosis of MDS. The percentage of ASC specks were significantly increased in MDS BM plasma compared to normal, healthy donors, which was validated by confocal microscopy. PB plasma-derived ASC specks were significantly greater in LR- versus HR-MDS, consistent with the greater extent of cell death and myeloid-derived suppressor cell (MDSC) expansion in LR disease. As hyperglycemia induces NLRP3 inflammasome activation, plasma glucose levels were measured to adjust for this confounding variable. Subsequently, the percentage of glucose-adjusted, PB plasma-derived ASC specks was measured in a panel of specimens of varied hematologic malignancies. The corrected percentage of ASC specks was significantly increased in MDS compared to normal donors and to each other malignancy investigated, including other myeloid and lymphoid leukemias, myeloproliferative neoplasms and overlap syndromes, like chronic myelomonocytic leukemia (CMML). These data indicate that the glucose-adjusted ASC speck percentage is MDS-specific and may be a valuable diagnostic biomarker. At a cutoff of 0.039, the biomarker minimizes misclassification error and achieves 95% sensitivity and 82% specificity in classifying MDS from normal donors, other hematologic malignancies and T2D. Lastly, the biomarker declined with treatment response to lenalidomide in LR-MDS patients, but not to erythropoietin stimulating agent (ESA) or hypomethylating agent (HMA) therapy. As such, the percentage of ASC specks represents the first biologically rational, diagnostic biomarker for MDS that can be implemented with current diagnostic practices to reduce diagnostic error.

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K⁺ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Indiana University-Purdue University Indianapolis (IUPUI) / Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/β-Catenin signaling pathway, may impact the IVD. Stabilization of β-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of β-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) β-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β-catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF-activated PTEN-deficient mouse melanoma was significantly suppressed in vivo after blocking β-catenin signaling in CAFs. Further analysis revealed that expression of phospho-Erk1/2 and phospho-Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial-mesenchymal transition (EMT)-like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF-MEK-ERK signaling cascade in BRAF-activated melanoma and may offer a new approach to abrogate host-dependent drug resistance in targeted therapy.

No / Wnt signaling normally functions in cell determination and proliferation and is essential for embryonic development. It does this by regulating target genes through a tightly regulated but complex signaling cascade. Overexpression of these genes due to aberrant Wnt activity can lead to uncontrolled cell growth and survival, and ultimately oncogenesis. Wnt signaling is also involved in epithelial–mesenchymal transition that contributes to tumor progression and metastasis evidence that tumor growth can be suppressed irrespective of other neoplastic promoters when the Wnt pathway is blocked and this has led to interest in its use as a therapeutic target. Recent developments in our understanding of the Wnt signaling cascade have led to research into drugs that specifically target different levels in this pathway, and the identification of β-catenin as the primary cause of dysregulated Wnt signaling has led to a number of protein knockdown strategies. Moreover, increased knowledge of the 300–400 Wnt inducible genes has provided a large untapped source of new potential therapeutic targets. Existing drugs such as nonsteroidal anti-inflammatory drugs and vitamin A and D derivatives have also shown efficacy in disrupting the Wnt signaling pathway and, together with a new generation of derivatives, they may soon be in clinical trials. This chapter details the Wnt signaling pathway, its role in different cancers, and some potential therapeutic targets that may show promise as effective cancer treatments.

Das Ansprechen primärer Rektumkarzinome auf eine präoperative Radiochemotherapie ist äußerst heterogen. Resistente Karzinome stellen aufgrund der schlechteren Prognose ein großes klinisches Problem dar. Ziel ist daher eine Individualisierung der Therapie und die Identifikation neuer therapeutischer Angriffspunkte, um resistente Tumoren für die Therapie zu sensibilisieren. In vorangegangenen Studien konnte gezeigt werden, dass zwischen Respondern und Nonrespondern TCF4, ein Transkriptionsfaktor des Wnt/β-Catenin-Signalwegs, differentiell exprimiert ist. Zudem führt die Herunterregulation von TCF4 in kolorektalen Karzinomzelllinien zu einer Radiotherapiesensibilisierung. Um zu untersuchen, ob es sich um eine TCF4-spezifische Beobachtung handelt oder ob der Wnt/β-Catenin-Signalweg Radiotherapieresistenz vermittelt, wurde dieser Signalweg in den Zelllinien SW480, SW837, LS1034 und RPE inhibiert. Anschließend wurde die Radiotherapieresistenz der Zellen untersucht. Die Inhibition des Wnt/β-Catenin-Signalwegs mittels siRNA gegen β-Catenin führte zu einer signifikanten Radiotherapiesensibilisierung von SW480, SW837 und LS1034. Die Behandlung von kolorektalen Karzinomzelllinien und der Normalzelllinie RPE mit dem kleinmolekularen Wnt/β-Catenin-Signalweg-Inhibitor XAV939 führte zum Teil zu einer Radiotherapiesensibilisierung. Insgesamt scheint der Wnt/β-Catenin-Signalweg eine entscheidende Rolle in der Entstehung der Radio(chemo)resistenz zu spielen und stellt somit ein potentielles therapeutisches Ziel für zukünftige Therapieansätze des Rektumkarzinoms dar. Zudem wurde dem Wnt/β-Catenin-Signalweg eine weitere biologische Bedeutung zugeordnet. Ziel zukünftiger Bemühungen ist die weitere Validierung dieser Ergebnisse, die Aufdeckung der zugrunde liegenden Mechanismen und die Übertragung in ein in-vivo-Modell.

Les Wnts représentent une famille de glycoprotéines de signalisation qui sont connues pour les nombreux rôles qu'ils jouent durant le développement embryonnaire et dans la cancerogénèse. Plusieurs Wnts, leurs récepteurs (Fzd) et d'autres composants des voies de signalisation des Wnt sont exprimés dans l’ovaire postnatal, et il a été démontré que l’expression de certains de ces gènes est régulée pendant le développement et l'ovulation/luteinization folliculaires. Toutefois, leurs rôles physiologiques dans l’ovaire demeurent mal définis. Pour étudier le rôle de WNT4 dans le développement folliculaire, nous avons entrepris d’identifier ses cibles transcriptionnels dans les cellules de la granulosa. Pour ce faire, nous avons employé la souris Catnbflox(ex3)/flox(ex3), chez laquelle une activation constitutive de la voie de Wnt/β-catenin a lieu suite à l’action de la recombinare Cre. Des cellules de la granulosa de ces souris ont été mises en culture et infectées avec un adenovirus pour causer la surexpression de WNT4 ou l’expression de Cre. L’ARN a alors été extrait de ces cellules et analysé par micro-puce. Les résultats ont démontré qu’une forte proportion des gènes induits par WNT4 étaient des gènes impliqués dans la réponse cellulaire au stress. Presque tous gènes induits par WNT4 ont également été induits par Cre, indiquant que WNT4 signale via la voie Wnt/β-catenin dans ces cellules. Nos résultats suggèrent donc que WNT4 favorise la survie des follicules par l’induction de gènes de réponse au stress dans les cellules de la granulosa, augmentant ainsi la résistance cellulaire à l'apoptose. / The Wnts comprise a large family of local-acting, secreted glycoprotein signaling molecules that are known mostly for the numerous roles they play in embryonic development and cancer. Several Wnts, their cognate receptors of the Frizzled (Fzd) family and other components of the Wnt signaling pathways are expressed in the postnatal ovary, and several have been shown to exhibit specific patterns of regulation in response to gonadotropin stimulation. Nonetheless, their role(s) in ovarian physiology remain poorly defined. To study the role of WNT4 in follicle development, we endeavoured to identify its transcriptional targets in granulosa cells. To this end, we used the Catnbflox(ex3)/flox(ex3) mouse model, in which constitutive activation of the Wnt/β-catenin pathway is obtained following Cre-mediated genetic recombination. Cultured granulosa cells from these mice were infected with adenoviruses to either overexpress WNT4 or to express Cre. RNA from these cells was then extracted and subjected to microarray analysis. Results revealed that a large proportion of the genes induced by WNT4 were genes previously shown to mediate cellular stress responses. Nearly all genes that were up-regulated by WNT4 were also induced by the Cre, indicating that WNT4 signals via the Wnt/β-catenin pathway in these cells. Our findings suggest that WNT4 mediates ovarian follicle survival by inducing a stress response in granulosa cells, thereby increasing their resistance to apoptosis.

Les Wnts représentent une famille de glycoprotéines de signalisation qui sont connues pour les nombreux rôles qu'ils jouent durant le développement embryonnaire et dans la cancerogénèse. Plusieurs Wnts, leurs récepteurs (Fzd) et d'autres composants des voies de signalisation des Wnt sont exprimés dans l’ovaire postnatal, et il a été démontré que l’expression de certains de ces gènes est régulée pendant le développement et l'ovulation/luteinization folliculaires. Toutefois, leurs rôles physiologiques dans l’ovaire demeurent mal définis. Pour étudier le rôle de WNT4 dans le développement folliculaire, nous avons entrepris d’identifier ses cibles transcriptionnels dans les cellules de la granulosa. Pour ce faire, nous avons employé la souris Catnbflox(ex3)/flox(ex3), chez laquelle une activation constitutive de la voie de Wnt/β-catenin a lieu suite à l’action de la recombinare Cre. Des cellules de la granulosa de ces souris ont été mises en culture et infectées avec un adenovirus pour causer la surexpression de WNT4 ou l’expression de Cre. L’ARN a alors été extrait de ces cellules et analysé par micro-puce. Les résultats ont démontré qu’une forte proportion des gènes induits par WNT4 étaient des gènes impliqués dans la réponse cellulaire au stress. Presque tous gènes induits par WNT4 ont également été induits par Cre, indiquant que WNT4 signale via la voie Wnt/β-catenin dans ces cellules. Nos résultats suggèrent donc que WNT4 favorise la survie des follicules par l’induction de gènes de réponse au stress dans les cellules de la granulosa, augmentant ainsi la résistance cellulaire à l'apoptose. / The Wnts comprise a large family of local-acting, secreted glycoprotein signaling molecules that are known mostly for the numerous roles they play in embryonic development and cancer. Several Wnts, their cognate receptors of the Frizzled (Fzd) family and other components of the Wnt signaling pathways are expressed in the postnatal ovary, and several have been shown to exhibit specific patterns of regulation in response to gonadotropin stimulation. Nonetheless, their role(s) in ovarian physiology remain poorly defined. To study the role of WNT4 in follicle development, we endeavoured to identify its transcriptional targets in granulosa cells. To this end, we used the Catnbflox(ex3)/flox(ex3) mouse model, in which constitutive activation of the Wnt/β-catenin pathway is obtained following Cre-mediated genetic recombination. Cultured granulosa cells from these mice were infected with adenoviruses to either overexpress WNT4 or to express Cre. RNA from these cells was then extracted and subjected to microarray analysis. Results revealed that a large proportion of the genes induced by WNT4 were genes previously shown to mediate cellular stress responses. Nearly all genes that were up-regulated by WNT4 were also induced by the Cre, indicating that WNT4 signals via the Wnt/β-catenin pathway in these cells. Our findings suggest that WNT4 mediates ovarian follicle survival by inducing a stress response in granulosa cells, thereby increasing their resistance to apoptosis.

Bei verschiedenen Krebs-Typen kann die molekulare Behandlung der Krebs-Stammzellen ein effektives Ziel sein, die Therapie-Resistenz und die Metastasierung der Tumore zu hemmen. Basaler Brustkrebs enthält Zellen mit Stammzell-Eigenschaften; hingegen sind rationale Therapien gegen diese Zellen nur wenige etabliert. Ich zeige in meiner Doktorarbeit, dass Rezeptor-Tyrosinkinase-Met-Signalvermittlung die Aktivität der Hippo-Komponente YAP im basalem Brustkrebs verstärkt. Weitere Analysen zeigten erhöhte YAP-Aktivität in den Krebsstammzell-Populationen. Durch Verwendung genetischer und pharmakologischer Methoden konnte ich zeigen, dass die Interferenz mit YAP die Tumor-Bildung verzögert und die luminal-basale Transdifferenzierung verhindert sowie das Krebsstammzell-Überleben reduziert. Gen-Expressions-Analysen von YAP-Knockout-Brustdrüsen zeigten eine starke Reduzierung der Expression der β-Catenin-Zielgene, was indiziert, dass YAP für nukleare β-Catenin-Aktivität essentiell ist. Ich habe weiter gefunden, dass nukleares YAP mit β-Catenin und TEAD4 interagiert und an gemeinsamen regulatorischen Gene-Regionen überlappt. Die Analyse von proteomischen Daten von primären Brustkarzinomen des Menschen zeigte eine signifikante Hoch-Regulation der YAP-Signatur in basalem im Vergleich zu andern Brustkarzinom-Typen, was suggeriert, dass YAP-Aktivität auf den basalen Brustkrebs limitiert ist. Unsere Daten beleuchten die Wichtigkeit von YAP als essentiellen Tumor-Initiator und Krebs-Stammzell-Regulator beim basalen Brustkrebs und zeigt, dass dessen Aktivität als Prognose-Faktor wichtig ist, wenn man basalen mit luminalem Brustkrebs vergleicht. Dies suggeriert, dass Behandlung des YAP/TEAD4/β-Catenin-Komplexes mit neuen, spezifischen Pharmaka, die die Wissenschaftler in Akademie- und Industrie-Instituten im Moment entwickeln, eine potentielle therapeutische Richtung darstellen, um basalen Brustkrebs in der Zukunft zu behandeln. Dies ist der wichtigste Aspekt meiner Doktorarbeit. / In various cancer types, targeting cancer stem cells (CSCs) can serve as an effective approach for limiting therapy resistance and metastasis. While basal breast cancers encompass cells with CSC features, rational therapies remain poorly established. Here, I show that receptor tyrosine kinase (RTK) Met signalling promotes the activity of the Hippo component YAP in basal breast cancer. Further analysis revealed enhanced YAP activity within the CSC population. Utilising both genetic and pharmaceutical approaches, I show that interference of YAP activity delays tumour formation, prevents luminal to basal trans-differentiation and reduces CSC survival. Gene expression analysis of YAP knock-out mammary glands revealed a strong decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, I find that nuclear YAP interacts and overlaps with β-catenin and TEAD4 at common gene regulatory regions. Analysis of proteomic data from primary breast cancer patients identified significant upregulation of the YAP signature in basal compared to other breast cancers, suggesting that YAP activity is limited to basal breast cancers. Our data highlight the importance of YAP as a crucial tumour initiator and cancer stem cell regulator in basal breast cancer and demonstrates that its activity has prognostic value, when comparing basal with luminal breast cancers. This suggests that targeting the YAP/TEAD4/β-catenin complex through specific new drugs, which researchers in academia and industry are presently developing, is a potential therapeutic avenue for treating basal breast cancers in the future. This is the most important aspect of my thesis.