The role of Notch signaling in cancer and metastasis

The Notch signaling pathway is an evolutionary conserved cell-to-cell communication pathway that regulates many important aspects in the development and tissue homeostasis of multicellular animals. It operates in a wide range of cellular contexts and dysregulated Notch signaling has been linked to a number of diseases, including cancer. The Notch signaling pathway consists of receptors and ligands that are expressed on juxtaposed cells, giving rise to short distance signaling. Upon ligand binding to a receptor, a series of proteolytic cleavages releases an intracellular part of the receptor, the Notch intracellular domain (NICD). NICD translocates to the nucleus where it interacts with a DNA-binding protein, CSL, to induce downstream target genes. Hyperactivated Notch signaling has been found to operate in various forms of cancers and an accumulating body of preclinical and clinical evidence supports that this contributes to cancer malignancy. However, the cellular events downstream of activated Notch signaling that facilitates tumor progression are still poorly understood. In this thesis, I have investigated several aspects of how dysregulated Notch signaling contributes to tumor growth and progression, with an emphasis on breast cancer. The data presented here demonstrate several novel interactions between Notch signaling and other oncogenic signaling mechanisms. Firstly, we report that Notch signaling can modulate hypoxia signaling, by specific activation of the hypoxia-inducible factor HIF2alpha. Notch regulates HIF2alpha at the transcriptional level, which triggers a HIF1alpha-toHIF2alpha hypoxic switch. Moreover, a substantial part of the Notch-induced transcriptome in medulloblastoma cells is dependent on HIF2alpha in normoxia. These data demonstrate, for the first time, that the Notch signaling status influences hypoxia signaling, which adds to our understanding of how Notch and hypoxia interplay in a tumor context (paper I). Secondly, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a downstream target of Notch signaling in breast cancer. Enhanced levels of Notch signaling give rise to increased IL-6 mRNA expression and protein secretion, leading to autocrine and paracrine activation of Janus kinase/signal transducers and activators of transcription signaling (JAK/STAT). We show that Notch signaling induces IL-6 in a non-canonical way, as the activation is independent of CSL, but dependent on the inhibitors of nuclear factor kappa-B kinase alpha and beta (IKK and IKK), as well as p53. These data suggest that hyperactivated Notch signaling promotes tumor inflammation in breast cancer through activation of IL-6 (paper II). Thirdly, we show that elevated levels of Notch signaling in breast cancers leads to activation of the phosphatidylinositol 3-kinase/AKT (PI3K/AKT) signaling pathway in breast cancer, which in turn leads to reprogramming of tumor metabolism and increased rates of aerobic glycolysis. In this work, we also provide evidence that low levels of Notch signaling give rise to an attenuation of mitochondrial activity, as well as lower protein levels of p53, which likewise triggers a metabolic shift with enhanced glycolysis. The increase in aerobic glycolysis upon hyperand hypoactivated Notch signaling was observed both in vitro and in an in vivo xenograft breast cancer model. Importantly, however, hyperactivated Notch signaling gave rise to a dramatically increased tumor potential in vivo. In sum, these data show that both hyperand hypoactivated Notch signaling breast cancer cells increase rates of aerobic glycolysis, although having dramatically different tumor potential in vivo (paper III). Lastly, to investigate more basic aspects regarding the regulation of Notch signaling, we have in paper IV and V studied how Notch receptors and ligands are trafficked in the cell, and how this influences Notch signaling output. In paper III, we characterize a mutant version of Jagged1, Nodder (Jagged1), and show that this ligand neither binds to, nor activates, Notch receptors in trans. In the absence of Notch signaling, Jagged1 and Jagged1 ligands does not differ significantly in regards to cellular localization and the ability to interact with the E3 ubiquitin ligase Mind bomb. However, under conditions of active Notch signaling, Jagged1 ligands are ubiquitnated and internalized, in contrast to Jagged1 ligands, which accumulate at the cell surface. In paper IV, in a similar manner, we show that activated atypical protein kinase C  (PKC) signaling influences Notch signaling differently depending on the Notch signaling activation status. When Notch signaling is activated, PKC promotes relocalisation of membrane bound Notch receptors to the nucleus, with enhanced production of NICD accompanied with increased downstream signaling. However, when Notch signaling is inactive, PKC instead further represses downstream Notch signaling, by relocating Notch receptors at the membrane to intracellular endosomal compartments. In conclusion, the work presented in this thesis contributes to a better understanding of how dysregulated Notch signaling contributes to cancer malignancy; knowledge that can be utilized in the development of anti-Notch therapies. List of publications I. Mutvei AP, Jin S, Lendahl U. Notch activates HIF2alpha and triggers a HIF1alpha-to-HIF2alpha switch in tumor cells Manuscript II. Jin S*, Mutvei AP*, Chivukula IV, Andersson ER, Ramskold D, Sandberg R, Lee KL, Kronqvist P, Mamaeva V, Ostling P, Mpindi JP, Kallioniemi O, Screpanti I, Poellinger L, Sahlgren C, Lendahl U. Non-canonical Notch signaling activates IL-6/JAK/STAT signaling in breast tumor cells and is controlled by p53 and IKKα/IKKβ. Oncogene. 2013 Oct 10;32(41):4892-902. III. Landor SK, Mutvei AP, Mamaeva V, Jin S, Busk M, Borra R, Gronroos TJ, Kronqvist P, Lendahl U, Sahlgren CM. Hypoand hyperactivated Notch signaling induce a glycolytic switch through distinct mechanisms. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18814-9. IV. Hansson EM, Lanner F, Das D, Mutvei A, Marklund U, Ericson J, Farnebo F, Stumm G, Stenmark H, Andersson ER, Lendahl U. Control of Notch-ligand endocytosis by ligand-receptor interaction. J Cell Sci. 2010 Sep 1;123(Pt 17):2931-42. V. Sjoqvist M*, Antfolk D*, Ferraris S, Rraklli V, Granqvist C, Mutvei AP, Imanishi SY, Holmberg J, Jin S, Eriksson JE, Lendahl U, Sahlgren CM. PKC regulates Notch receptor routing and activity in a Notch signaling-dependent manner. Accepted, Cell Research List of abbreviations APC Adenomatous polyposis coli ATP Adenosine triphosphate bHLH Basic helix-loop-helix CSL CBF1/Suppressor of Hairless/LAG-1 DAPT 5-difluorophenylacetyl-L-alanyl-2-phenylglycine-1,1-di-methylethyl ester EGF Epidermal growth factor FIH Factor-inhibiting HIF1 GSI Gamma-secretase inhibitor HIF Hypoxia-inducible factor