Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1

As a major growth factor transducer, Ras is an upstream activator of mTORC1, which further integrates nutrient and energy inputs. To ensure a contextual coupling of cell division via Ras/MAPK-signalling and growth via mTORC1-signalling, feedback loops from one pathway back to the other are required. Here we describe a novel feedback from mTORC1, which oppositely affects oncogenic H-ras- and K-ras-signalling output, and as a consequence stemness properties of tumourigenic cells. Amino acid stimulation of mTORC1 increases the processed form of SREBP1, a major lipidome regulator. We show that modulation of the SREBP1 levels downstream of S6K1 has opposite effects on oncogenic H-ras and K-ras nanoscale membrane organisation, ensuing signalling output and promotion of mammospheres expressing these oncogenes. Our data suggest that modulation of phosphatidic acid, a major target of SREBP1 controlled lipid metabolism, is sufficient to affect H-ras and K-ras oppositely in the membrane. Thus mTORC1 activation increases H-ras-, but decreases K-ras-signalling output in cells transformed with the respective oncogene. Given the different impact of these two Ras isoforms on stemness, our results could have implications for stem cell biology and inhibition of cancer stem cells

Cancer stem cell drugs target K-ras signaling in a stemness context

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.Peer reviewe

Altered TUBB3 expression contributes to the epothilone response of mitotic cells

BACKGROUND: Epothilones are a novel group of microtubule (mt) targeting cancer drugs that bind to the β-subunit of the αβ-tubulin dimer. Epothilones inhibit cell proliferation and induce cell death by interfering with the normal mt function. In this study, we examined the consequences of altered expression of human β-tubulin isotypes in terms of the epothilone drug response in human lung and breast cancer cell lines. METHODS: The β-tubulin isotypes TUBB2A–C, TUBB3 and TUBB were silenced or overexpressed in A549, A549EpoB40 and MCF7 cell lines in the presence or absence of epothilones. The drug effects on cell proliferation, mitosis and mt dynamics were determined using live cell microscopy and immunofluorescence assays. RESULTS: Loss of TUBB3 enhanced the action of epothilones. TUBB3 knockdown increased the severity of drug-induced mitotic defects and resulted in stabilisation of the mt dynamics in cells. Moreover, exogenous expression of TUBB3 in the epothilone resistant cell line conferred the response to drug treatments. In contrast, reduced levels of TUBB2A–C or TUBB had not apparent effect on the cells' response to epothilones. CONCLUSION: Our results show that the expression of TUBB3 contributes to the cellular response to epothilones, putatively by having an impact on the mt dynamics

PO-092 Inhibition of the mTORC1-pathway can feedback-activate H-RAS or K-RAS

ABSTRACTIntroductionPI3K/mTORC1- and Ras/MAPK-signalling pathways are aberrantly regulated in most cancers. Specific resistances to drugs targeting these pathways can emerge during tumour evolution. Preexisting, innate resistance mechanisms, such as stemming from feedback-loops, should ideally be known already during drug-target nomination. However, as the example of B-Raf-inhibitors that paradoxically activate MAPK-signalling has shown, feedback mechanisms may only become apparent at very late drug development stages.Material and methodsHEK cells expressing FRET-pairs of Ras proteins were used to study specific effects on Ras isoforms (nanoclustering). Breast cancer cells were grown in 2D for Western blotting of Ras and mTORC1-pathway proteins, or as spheres to analyse stemness traits.Results and discussionsHere, we describe two broad feedback loops from the mTor-pathway back to the nanoscopic membrane signalling complexes (nanocluster) of H-ras and K-ras4B (hereafter K-ras). Increased nanoclustering typically correlates with increased Ras output. The first, upstream loop leads to an inadvertent rapalog induced promotion of stemness traits and tumorigenicity in Ras transformed cells. This is due to an induction of the H-ras nanocluster scaffold galectin-1, when FKBP12 levels are low. Surprisingly we find that rapalogs do not only bind to but induce a loss of FKBP12 protein. Thus, rapalog treatment induces galectin-1, which stimulates H-ras signal output and stemness traits. Secondly, modulation of the activity in the mTORC1 pathway downstream of the major lipidome regulator SREBP1, oppositely regulates H-ras and K-ras nanoclustering. Thus, ablation of SREBP1 increases K-ras, but decreases H-ras nanoclustering and signal output. We show that altered levels of phosphatidic acid downstream of SREBP1 are sufficient for the opposite regulation of the two Ras isoforms.ConclusionThe described feedback loops may only become apparent in certain tumour settings. For example, tumour promotion during rapalog-treatment may only be relevant in H-ras mutant cancers, which make up a small portion of human cancers. In those cases, rapalog efficacy may be improved in combination with novel anti-galectin-1 drugs. Targeting the mTORC1 pathway downstream of SREBP1, may have opposite effects in H-ras and K-ras mutant cancers. Thus, care may have to be taken when targeting the mTORC1-pathway in a mutant Ras setting