Hitting the supply lines – targeting macropinocytosis as source for nutrients in pancreatic cancer
Prosjekt
- Prosjektnummer
- 2020038
- Ansvarlig person
- Kay Oliver Schink
- Institusjon
- Oslo universitetssykehus HF
- Prosjektkategori
- Karrierestipend
- Helsekategori
- Cancer
- Forskningsaktivitet
- 1. Underpinning
Rapporter
Macropinocytosis is an important route for cancer cells to acquire amino acids, allowing them to take up extracellular proteins and use them as a metabolic fuel source. In this project, we aim to identify new regulators of macropinocytosis and test whether their inhibition can be used to starve tumors that depend on this process.Macropinocytosis, or cellular drinking, is a process where extracellular fluid is internalized into large vesicles called macropinosomes. Certain cancer cells, such as pancreatic cancer cells expressing oncogenic KRAS, use macropinocytosis to scavenge nutrients and sustain their growth. Many tumors are in nutrient-poor environments where amino acid availability limits proliferation. Macropinocytosis allows cells to take up extracellular proteins into macropinosomes, degrade them, and obtain amino acids to support growth. Despite its relevance, little is known about how macropinocytosis is regulated and which proteins are involved. This study investigates the regulation of macropinocytosis in pancreatic cancer cells, focusing on the role of nutrient availability and nutrient sensors such as the mTORC complex. In addition, we assess whether inhibiting macropinocytosis can affect the growth of pancreatic cancer cells.
As part of this project, we identified and characterized proteins involved in macropinocytosis regulation, including Phafin2 and its interacting partner Jip4 (Schink et al., 2021; Tan et al., 2021). Building on this work, we have now identified additional regulators. Using a proteomic screen, we found that the kinase MAP4K3 plays a role in macropinocytosis regulation. MAP4K3 localizes to newly formed macropinosomes and determines whether these vesicles successfully mature and remain in the cell. Overexpression of MAP4K3 suppresses macropinocytosis, while its deletion enhances it. At the same time, we observed that MAP4K3 deletion inhibits cell migration and invasion in 3D matrices, while its overexpression increases these processes. A related kinase, MAP4K5, shows a similar effect. MAP4K5 also localizes to macropinosomes, and its deletion enhances macropinocytosis. Further characterization showed that MAP4K3 and MAP4K5 regulate macropinocytosis through small Rab GTPases. We are currently investigating the molecular mechanisms behind this regulation in more detail.
NEI
Macropinocytosis is an amino acid supply route for cancer cells, which can take up extracellular proteins and use these to fuel tumor metabolism. In this project, we aim to identify novel regulators of macropinocytosis and test if inhibition of them can be used to starve macropinocytosis-dependent tumors.Macropinocytosis - cellular drinking - is a cellular process in which extracellular fluid is taken up into large vesicles, called macropinosomes. Cancer cells - for example pancreatic cancer expressing oncogenic KRAS – use macropinocytosis as a nutrient scavenging mechanism, which enables them to grow faster. Many tumors are poor in nutrients, and their grow is limited by the available amino acids. Macropinocytosis enables these cells to take up extracellular proteins into macropinosomes and digest the proteins to gain additional amino acids to fuel their growth. Despite its importance, relatively little is known how macropinocytosis is regulated and which proteins are involved. This study aims to examine the regulation of macropinocytosis in pancreatic cancer cells and to determine the effect of nutrient availability and known nutrient sensors, such as the mTORC complex, on its regulation. Additionally, the potential impact of inhibiting macropinocytosis on the growth of pancreatic cancer cells will also be investigated. In the course of this project, we were able to identify and characterize several proteins involved in the regulation of macropinocytosis, specifically the protein Phafin2 and its interacting protein Jip4 (Schink et al, 2021, Tan et al, 2021). Building on these works, we have now identified additional proteins which regulate macropinocytosis. By using a proteomic screen, we have identified the protein MAP4K3 as a novel player in macropinocytosis.
Currently, our main aim is to understand the role of MAP4K3 in macropinocytosis. We have generated stable cell lines expressing fluorescently labelled MAP4K3 and we find that the protein transiently localizes to forming macropinosomes. Moreover, we find that MAP4K3 overexpression reduces macropinocytosis. Conversely, deletion of MAP4K3 using CRISPR/Cas9 enhances macropinocytosis, suggesting that MAP4K3 could act as a negative regulator of macropinocytosis. We have further evidence that MAP4K3 potentially regulates a switch between cell migration and macropinocytosis. Cells overexpressing MAP4K3 show enhanced cell migration and 3D invasion, whereas knockout of MAP4K3 reduces migration and invasion. Several lines of evidence in the literature suggest that macropinocytosis and cell migration are mutually exclusive. Our novel data suggests that the kinase MAP4K3 could regulate this process.
As MAP4K3 is a kinase, we have now started to search for targets that get regulated by this kinase. One of the potential downstream effectors is the actin binding protein DBNL. DBNL localizes to forming macropinosomes at the same time as MAP4K3, suggesting that it could regulate the actin cytoskeleton and be itself regulated by MAP4K3. We further find that DBNL depletion shows – similar to MAP4K3 – enhanced macropinocytosis. This indicates that MAP4K3 and DBNL could act in the same pathway.
Beyond the study of the molecular regulators of macropinocytosis, we are currently working on translational projects to target macropinocytosis in pancreatic cancer. To this end, we have now established a PDAC-derived organoid system which will allow us to test the role of macropinocytosis as a nutrient supply route for pancreatic cancer cells. We recently also made an importantfinding in anothernutrient supply route, autophagy.We found that the protein DFCP1 is a dynamin-related ATPase which is involved in the initial steps of autophagosome formation (Nähse et al, 2023).
NO
Macropinocytosis is an amino acid supply route for cancer cells, which can take up extracellular proteins and use these to fuel tumor metabolism. In this project, we aim to identify novel regulators of macropinocytosis and test if inhibition of them can be used to starve macropinocytosis-dependent
tumors.Macropinocytosis - cellular drinking - is a cellular process in which extracellular fluid is taken up into large vesicles, called macropinosomes.
Cancer cells - for example pancreatic cancer expressing oncogenic KRAS – use macropinocytosis as a nutrient scavenging mechanism, which enables them to grow faster. Many tumors are poor in nutrients, and their grow is limited by the available amino acids. Macropinocytosis enables these cells to take up extracellular proteins into macropinosomes and digest the proteins to gain additional amino acids to fuel their growth.
Despite its importance, relatively little is known how macropinocytosis is regulated and which proteins are involved. This study aims to examine the regulation of macropinocytosis in pancreatic cancer cells and to determine the effect of nutrient availability and known nutrient sensors, such as the mTORC complex, on its regulation. Additionally, the potential impact of inhibiting macropinocytosis on the growth of pancreatic cancer cells will also be investigated.
In the course of this project, we were able to identify and characterize several proteins involved in the regulation of macropinocytosis, specifically the protein Phafin2 and its interacting protein Jip4 (Schink et al, 2021, Tan et al, 2021). Building on these works, we have now identified additional proteins which regulate macropinocytosis. By using a proteomic screen, we have identified the protein MAP4K3 as a novel player in macropinocytosis. Currently, our main aim is to understand the role of MAP4K3 in macropinocytosis.
We have generated stable cell lines expressing fluorescently labelled MAP4K3 and we find that the protein transiently localizes to forming macropinosomes. Moreover, we find that MAP4K3 overexpression reduces macropinocytosis. Conversely, deletion of MAP4K3 using CRISPR/Cas9 enhances macropinocytosis, suggesting that MAP4K3 could act as a negative regulator of macropinocytosis.
We have further evidence that MAP4K3 potentially regulates a switch between cell migration and macropinocytosis. Cells overexpressing MAP4K3 show enhanced cell migration and 3D invasion, whereas knockout of MAP4K3 reduces migration and invasion. Several lines of evidence in the literature suggest that macropinocytosis and cell migration are mutually exclusive. Our novel data suggests that the kinase MAP4K3 could regulate this process.
Beyond the study of the molecular regulators of macropinocytosis, we are currently working on translational projects to target macropinocytosis in pancreatic cancer. First, we have established a collaboration which will allow us to test if inhibition of macropinocytosis can block pancreatic cancer growth. Moreover, we are currently establishing an organoid system using pancreatic cancer organoids to study the role of macropinocytosis in a physiological model. While these experiments are in early stages, we have obtained patient-derived cancer cell organoids, in which we will study the effect of macropinocytosis inhibition. In addition, we have obtained mouse primary pancreatic organoids, which will allow us to model KRAS*-driven early steps of tumor formation and will allow us to study macropinocytosis in these early phases.
NO
Macropinocytosis is an amino acid supply route for cancer cells, which can take up extracellular proteins and use these to fuel tumor metabolism. In this project, we aim to identify novel regulators of macropinocytosis and test if inhibition of them can be used to starve macropinocytosis-dependent tumors.Macropinocytosis - cellular drinking - is a process by which cells can take up large ammounts of extracellular fluid into large vesicles, so-called macropinosomes. Cancer cells - for example pancreatic cancer expressing oncogenic KRAS - exploit this to take up additional nutrients, which enables them to grow faster. So far, relatively little is known how macropinocytosis is regulated and which proteins are involved.
In this project, we aim to investigate how cancer cells, especially pancreatic cancer cells - regulate macropinocytosis. We will also test if cells regulate macropinocytosis in response to available nutrients and test if known nutrient sensors - such as the mTORC complex - are involved in this regulation. Finally, we will test if inhibition of macropinocytosis can inhibit growth of pancreatic cancer cells.
We have identified several proteins involved in the regulation of macropinocytosis. We have found that the protein Phafin2 is a critical regulator of early macropinosome formation and that it locally regulates the actin cytoskeleton at forming macropinosomes. Deletion of Phafin2 reduces macropinocytosis. In line with this finding, we found that Phafin2 is required for effective nutrient acquisition by pancreatic cancer cells. We have further found that Phafin2 is highly expressed in many cancers, suggesting that it could help these cancers with nutrient acquisition. These results were published in the high-quality open access journal Nature Communications, with project leader Kay Oliver Schink as first author.
By using a proteomics screen, we have further identified an interactor of Phafin2, the protein Jip4. Jip4 localizes to macropinosomes, where it interacts with Phafin2. We find that Jip4 it is involved in the formation of recycling tubules at macropinosomes, and likely helps to transport cargo back to the cell surface. This process is important to ensure the homeostasis of the plasma membrane, and we have previously found that mis-regulation of this process can lead to enhanced cancer cell migration.This manuscript was published in Journal of Cell Science, with PhD student Kia Wee Tan as the first author.
We are currently investigating the role of a potential new regulator of macropinocytosis, the protein kinase MAP4K3 (GLK). This subproject is investigated by a postdoc hired on this project. We find that MAP4K3 localizes to forming macropinosomes at the same time as Phafin2, and overexpression of MAP4K3 causes forming macropinosomes to collapse. This suggests that MAP4K3 could be a negative regulator of macropinosome formation. We are currently generating stable cell lines and CRISPR knockout cells to further investigte the role of MAP4K3.
NO
Macropinocytosis is an amino acid supply route for cancer cells, which can take up extracellular proteins and use these to fuel tumor metabolism. In this project, we aim to identify novel regulators of macropinocytosis and test if inhibition of them can be used to starve macropinocytosis-dependent tumors.Macropinocytosis - cellular drinking - is a process by which cells can take up large ammounts of extracellular fluid into large vesicles, so-called macropinosomes. Cancer cells - for example pancreatic cancer expressing oncogenic KRAS - exploit this to take up additional nutrients, which enables them to grow faster. So far, relatively little is known how macropinocytosis is regulated and which proteins are involved.
In this project, we aim to investigate how cancer cells, especially pancreatic cancer cells - regulate macropinocytosis. We will also test if cells regulate macropinocytosis in response to available nutrients and test if known nutrient sensors - such as the mTORC complex - are involved in this regulation. Finally, we will test if inhibition of macropinocytosis can inhibit growth of pancreatic cancer cells.
To identify the cellular proteins which regulate macropinocytosis, we have performed a proteomics screen. From this screen, we gained a list of several candidates that we are currently analyzing. We found that one of the candidate proteins - the protein Jip4 - localizes to macropinosomes, where it interacts with the macropinocytosis regulator Phafin2. Our data shows that it is involved in the formation of recycling tubules at macropinosomes, and likely helps to transport cargo back to the cell surface. This process is important to ensure the homeostasis of the plasma membrane, and we have previously found that mis-regulation of this process can lead to enhanced cancer cell migration. A manuscript based on this data has been submitted and is also available as a preprint on BioRxiv (Tan et al,https://doi.org/10.1101/2020.09.29.319111).
In addition, we are currently investigating a second candidate, the kinase MAP4K3. This kinase has been proposed to regulate autophagy in response to amino acid starvation. We therefore speculate that this kinase might be a good candidate which might regulate macropinocytosis in response to amino acid starvation. This protein will be investigated by a postdoctoral researcher hired on the project, who has started in November 2020.
NEI
Vitenskapelige artikler
Mierke CT, Hu X, Yao M, Schink KO, Sheetz M
Editorial: Tumor cell mechanosensitivity: molecular basis.
Front Cell Dev Biol 2024;12():1484725. Epub 2024 sep 6
PMID: 39310224
Nähse V, Schink KO, Stenmark H
ATPase-regulated autophagosome biogenesis.
Autophagy 2024 Jan;20(1):218. Epub 2023 des 27
PMID: 37722386
Nähse V, Stenmark H, Schink KO
Omegasomes control formation, expansion, and closure of autophagosomes.
Bioessays 2024 Jun;46(6):e2400038. Epub 2024 mai 9
PMID: 38724256
Nähse V, Raiborg C, Tan KW, Mørk S, Torgersen ML, Wenzel EM, Nager M, Salo VT, Johansen T, Ikonen E, Schink KO, Stenmark H
ATPase activity of DFCP1 controls selective autophagy.
Nat Commun 2023 Jul 08;14(1):4051. Epub 2023 jul 8
PMID: 37422481
Frikstad KM, Schink KO, Gilani S, Pedersen LB, Patzke S
3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines.
Bio Protoc 2022 Mar 20;12(6):e4360. Epub 2022 mar 20
PMID: 35434191
Schink KO, Tan KW, Spangenberg H, Martorana D, Sneeggen M, Stévenin V, Enninga J, Campsteijn C, Raiborg C, Stenmark H
The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes.
Nat Commun 2021 11 12;12(1):6577. Epub 2021 nov 12
PMID: 34772942
Zdzalik-Bielecka D, Poswiata A, Kozik K, Jastrzebski K, Schink KO, Brewinska-Olchowik M, Piwocka K, Stenmark H, Miaczynska M
The GAS6-AXL signaling pathway triggers actin remodeling that drives membrane ruffling, macropinocytosis, and cancer-cell invasion.
Proc Natl Acad Sci U S A 2021 07 13;118(28).
PMID: 34244439
Tan KW, Nähse V, Campsteijn C, Brech A, Schink KO, Stenmark H
JIP4 is recruited by the phosphoinositide-binding protein Phafin2 to promote recycling tubules on macropinosomes.
J Cell Sci 2021 07 15;134(14). Epub 2021 jul 20
PMID: 34109410
Doktorgrader
Helene Spangenberg
Cellular mechanisms of vesicle generation and closure
- Disputert:
- mai 2022
- Hovedveileder:
- Camilla Raiborg
Kia Wee Tan
Membrane Remodeling in Macropinocytosis
- Disputert:
- oktober 2021
- Hovedveileder:
- Kay Oliver Schink
Deltagere
- Kay Oliver Schink Prosjektleder
- Harald Alfred Stenmark Prosjektdeltaker
- Camilla Raiborg Prosjektdeltaker
- Anette Weyergang Prosjektdeltaker
- Helene Spangenberg Doktorgradsstipendiat (annen finansiering)
- Trude Wetas Brukerrepresentant
- Lisette Pamela Sandoval Galarce Postdoktorstipendiat (finansiert av denne bevilgning)
- Kia Wee Tan Doktorgradsstipendiat (annen finansiering)
- Øyvind Ødegård Fougner Postdoktorstipendiat (finansiert av denne bevilgning)
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