Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Modulation of TGF-β signaling by Hypoxia

A tumor is a fast-growing malignant tissue. This creates areas inside the tumor that are distant from local blood vessels to be able to get enough oxygen. This hypoxic condition activates a transcription factor called hypoxia inducible factor (HIF). HIF responses help a cell to adapt to decreased oxygen by activating glycolytic and angiogenesis pathways and by regulating apoptotic responses. Hypoxia drives the upregulation of a growth factor called transforming growth factor beta (TGF-beta). Similar to a hypoxia response, TGF is an important regulator of cell fate. TGF-β and HIF pathways regulate partially overlapping target genes. This regulation can also be cooperative. The TGF-beta signal is initiated by activation of plasma membrane receptors that then activate effector proteins called small mothers against decapentaplegic (Smad) homologs. In healthy tissue, TGF-β keeps cell proliferation and growth under control. During cancer progression, TGF-beta has shown a dual role, whereby it inhibits initial tumor formation but, conversely, in an existent tumor, TGF-beta drives malignant progression. Along with HIF and TGF-beta also protein dephosphorylation is an important regulatory mechanism of cell fate. Protein dephosphorylation is catalyzed by protein phosphatases such as Protein phosphatase 2A (PP2A). PP2A is a ubiquitous phosphatase that can exist in various active forms. PP2A can specifically regulate TGF-beta signaling either by enhancing or inhibiting the receptor activity. This work demonstrates that during hypoxia, PP2A is able to fine-tune TGF-beta signal by specifically targeting Smad3 effector in a Smad7-dependent manner. Inactivation of Smad3 in hypoxia leads to malignant conversion of TGF-beta signaling.

Life without Phd? – Phd Oxygen Sensor Proteins in Hypoxic Carcinoma Cell Survival

The microenvironment within the tumor plays a central role in cellular signaling. Rapidly proliferating cancer cells need building blocks for structures as well as nutrients and oxygen for energy production. In normal tissue, the vasculature effectively transports oxygen, nutrient and waste products, and maintains physiological pH. Within a tumor however, the vasculature is rarely sufficient for the needs of tumor cells. This causes the tumor to suffer from lack of oxygen (hypoxia) and nutrients as well as acidification, as the glycolytic end product lactate is accumulated. Cancer cells harbor mutations enabling survival in the rough microenvironment. One of the best characterized mutations is the inactivation of the von Hippel-Lindau protein (pVHL) in clear cell renal cell carcinoma (ccRCC). Inactivation causes constitutive activation of hypoxia-inducible factor HIF which is an important survival factor regulating glycolysis, neovascularization and apoptosis. HIFs are normally regulated by HIF prolyl hydroxylases (PHDs), which in the presence of oxygen target HIF α-subunit to ubiquitination by pVHL and degradation by proteasomes. In my thesis work, I studied the role of PHDs in the survival of carcinoma cells in hypoxia. My work revealed an essential role of PHD1 and PHD3 in cell cycle regulation through two cyclin-dependent kinase inhibitors (CKIs) p21 and p27. Depletion of PHD1 or PHD3 caused a cell cycle arrest and subjected the carcinoma cells to stress and impaired the survival.