Endothelial Gluconeogenesis: a novel target for tumor anti-angiogenesis?

Periodic Reporting for period 1 - EC GNG metabolism (Endothelial Gluconeogenesis: a novel target for tumor anti-angiogenesis?)

Okres sprawozdawczy: 2017-01-01 do 2018-12-31

Before the start of the funded Marie Curie project, the host lab had recently reported that endothelial cell (EC) metabolism (glycolysis and fatty acid) is crucial to control vessel sprouting, a process which is deregulated in several pathologies including cancer. Similar to cancer cells, endothelial cells are "glycolysis-addicted" and highly dependent on this pathway to produce the required energy for sprouting into avascular areas, where glucose becomes limiting.

The overall project hypothesis aimed to investigate endothelial metabolism into more detail, and to discover new pathways driving sprouting angiogenesis. Besides, we also evaluated whether ECs could produce glycolytic intermediates de-novo via a process called gluconeogenesis (GNG). Finally, as metabolism of tumor endothelial cells (TECs) is largely unknown, we investigated their properties in order to improve current anti-angiogenic therapies.

Initial preliminary results showed that ECs express key enzymes of the GNG pathway. However, it appeared that the amount of glucose actually produced by ECs in glucose starved condition was minimal (<2% and could be considered as background, confirmed by tracing studies).

Via knockdown and pharmacological inhibitor strategies, we showed that phosphoenolpyruvate carboxykinase (PEPCK) or fructose-1,6-bisphosphatase (F1,6BPase) are dispensable for EC proliferation, migration and sprouting capacities when cells are cultured in low glucose. However, as GNG only minimally contributes to ECs, we attributed the functional effects observed in WP2 to side pathways diverted from GNG, and therefore did not proceed to WP3-4. Currently, the mechanisms (beyond GNG) are still under investigations and a manuscript citing the Marie Curie grant will be submitted soon.

In addition to the originally proposed experiments focusing on the role of GNG in ECs, we have unravelled the role of fatty acid oxidation as an indispensable metabolic pathway to maintain endothelial quiescence and vasculoprotection [1]. These findings might offer therapeutic opportunities by improving anti-oxidant control and fight against inflammatory and thrombogenic diseases.
Cancer is another disease in which blood vessels become abnormal. For decades, anti-angiogenic therapies have been targeting tumor vessels but resistance mechanisms still persist. Alternative strategy would consist in normalizing tumor vessels, rather than pruning and destroying them [2]. Targeting endothelial metabolism could be particularly well adapted to achieve such normalization, as demonstrated in pre-clinical studies [3].

A reason to anti-angiogenic resistance could be due to tumor vessel heterogeneity, with only a small subset of ECs which are sensitive to the treatments. Therefore we have investigated this question by single cell profiling the transcriptome of tumor (TECs) and normal ECs (NECs) in human and mouse models upon anti-VEGF treatments. Our manuscripts are currently in revision and the Marie Curie fellowship properly acknowledged. The hyper-proliferative activity of TECs, as compared to NECs, was confirmed in a meta-analysis using an online, freely available tool (https://vibcancer.be/software-tools/endodb) developed at the lab [4]. Our EndoDB database will be of great help for biologist to facilitate meta-analysis of transcriptomic studies on ECs, in a broad range of diseases. It could help facilitating the development of new alternative strategies and a better understanding of pathological vessels.

Tuning metabolism in ECs has been shown to restore/normalize key aspects of the tumor endothelium. Another interesting aspect of tumor vessel normalization is its association with immunotherapy to improve cancer treatment perspectives [2]. Preliminary results from our lab (manuscripts in revision, citing the Marie Curie grant), and might hold great promise in association with immunotherapy.

In the last years, endothelial metabolism has been under extensive investigation and we have published several review articles highlighting its potential to treat a broad spectrum of diseases [3,5]. Although the initial hypothesis concerning the role of GNG in ECs was not entirely true, our current results clearly show that it is unneglectable and current analysis hold high potential.
Overall, this proposal has been a great success. The recruited researcher has been able to face difficulties, operate a project change and contribute to several ongoing research studies from the host lab. Altogether, these efforts lead to demonstrate the therapeutic efficacy and potential of targeting endothelial metabolism in tumor vessels to achieve tumor vessel normalization.

References
1. Kalucka J, Bierhansl L, Conchinha NV, et al. Quiescent Endothelial Cells Upregulate Fatty Acid beta-Oxidation for Vasculoprotection via Redox Homeostasis. Cell metabolism 2018; 28: 881-894 e813.
2. Treps L. EnLIGHTenment of tumor vessel normalization and immunotherapy in glioblastoma. The Journal of pathology 2018; 246: 3-6.
3. Eelen G, de Zeeuw P, Treps L, et al. Endothelial Cell Metabolism. Physiol Rev 2018; 98: 3-58.
4. Khan S, Taverna F, Rohlenova K, et al. EndoDB: a database of endothelial cell transcriptomics data. Nucleic acids research 2019; 47: D736-D744.
5. Wong BW, Marsch E, Treps L, et al. Endothelial cell metabolism in health and disease: impact of hypoxia. EMBO J 2017; 36: 2187-2203.

Update of the plan for exploitation and dissemination of results:
A publication overview from the Marie Curie fellow can be found above. As mentioned, several research articles are currently under revision, or will be submitted soon. Thanks to this Marie Curie fellowship, I enhanced my capacity to integrate and structure complex questions and data into scientific proposals and presentations by working on multiple research projects simultaneously.
I presented my results during:
* internal meetings at the VIB: Science Lunch (2017 & 2018); Metabolism Focus group (2017) organised by Dr. S. Maria-Fendt.
* international meetings:
I. "Predictive & Prognostic Markers, Mechanisms of Resistance in Angiogenesis".
The 5th MSKCC Immunotherapy & Angiogenesis Meeting. Algeria, 15-16 Feb 2019.
II. "Multi-omics profiling of tumor endothelial cells reveal novel anti-angiogenic target". SOAPosium 2018. Nantes, France, 14 Dec 2018.
III. "Tumor angiogenesis: mechanisms, therapies, challenges and novel avenues".
The 4th course of angiogenesis with recent advances in immunotherapy. Beirut, Lebanon, 17-18 Feb 2017.

Metabolism in quiescent ECs

Periodic Reporting for period 1 - EC GNG metabolism (Endothelial Gluconeogenesis: a novel target for tumor anti-angiogenesis?)

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