Notch in cancer and cancer metabolism : six degrees of intracellular turbulence

Notch signaling is an evolutionarily conserved cell-to-cell contact-dependent signaling mechanism in multicellular organisms directing cellular fates both in early development and adult tissues. In metazoans the Notch pathway consists of multiple paralogs of receptors and ligands constituting a complex juxtacrine communications network orchestrating organismal homeostasis. Binding of receptors on signal-receiving cells to the ligands on signal-sending cells leads to proteolytic cleavage and release of the intracellular domain of Notch (NICD). NICD subsequently translocates to the cell nucleus to activate Notch downstream gene expression machinery by binding to the Notch-dependent transcriptional regulator CSL. Notch is highly context-dependent, and the nature of Notch-mediated outcomes is governed by multiple factors such as crosstalk with other signaling pathways, post-translational modifications, and CSL- binding type preference. Notch is ultimately a cell fate decider with a temporal specificity, where context and time can determine whether Notch inhibits or promotes a cellular outcome. The importance of the Notch pathway is further emphasized by the dramatic effects of dysregulated Notch signaling, which often leads to life-threatening diseases and cancer, such as CADASIL and T-ALL. In this thesis I have glimpsed behind the veil into the unknowns of Notch signaling and investigated several novel aspects and peculiarities relating to Notch deregulation in cancer, and to Notch regulation via post-translational modifications. “When Notch and Pim Unite”, Notch1 ICD undergoes post-translational phosphorylation by Pim kinases occurring at the nuclear localization signal within the PPD-domain, thus modulating the nuclear transport and transactivation of N1ICD. This impacts tumor growth and metabolism in breast cancer, and migration in prostate cancer. In “A Metabolic Turn of Events” we discover that Notch signaling is able to reprogram the metabolism in breast cancer where high Notch levels induce the PI3K/Akt pathway leading to a shift towards aerobic glycolysis, while low Notch leads to a forced switch to glycolysis following mitochondrial oxidative phosphorylation defects. The Notch deficiency subsequently sensitizes the cancer cells for low glucose conditions. Next we unleash “Systematic KOs”, when we knockout CSL in MDA-MB-231 breast cancer cells which leads to increased tumor growth and an activated hypoxic response. Furthermore, comparison of the Notch wild-type and CSL knock-out transcriptomic signatures reveals an upregulation of over 1700 genes not part of the Notch gene signature, suggesting that CSL transcriptionally controls a number of genes not part of the canonical Notch signature. Lastly, we are “Falling Into Hypoxity” as canonical Notch1 is shown to induce HIF2α and trigger a HIF1α-to-HIF2α switch in medulloblastoma. However, Notch1 remains tumor suppressive in CAM-xenographs and the genetic removal of HIF2α increases tumor growth. Taken together, this thesis contributes new puzzle pieces to building a complete picture of the Notch signaling pathway, its role in cancer, and provides new vistas for future anti-Notch therapies

Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8(-/-)) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo, K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8(-/-) colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium

Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells

Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy.</p

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.</p

Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells

Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms