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Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

Charlotte M. de Winde, Samantha L. George, Abbey B. Arp, Agnesska C. Benjamin, Eva Crosas-Molist, Yukti Hari-Gupta, Alexander Carver, Valerio Imperatore, Victor G. Martinez, Victoria Sanz-Moreno, Sophie E. Acton

Preprint posted on 24 July 2020 https://www.biorxiv.org/content/10.1101/2020.07.23.218578v1

Article now published in iScience at http://dx.doi.org/10.1016/j.isci.2021.102976

Podo’s (Podoplanin) path to cancer!

Selected by Ankita Jha

Context:

Cancer metastasis is a multi-step process and is often characterized by cancer cells switching between different modes of migration. This plasticity allows cancer cells to effectively invade and migrate through a complex extracellular matrix (ECM). The complexity of ECM like availability of ligands, stiffness of the matrix and confinement also regulates this plasticity. Cells usually attain mesenchymal migratory mode in the presence of a high adhesive environment but highly contractile cells can switch to amoeboid migration mode which is characterized by the formation of blebs allowing cells to migrate through complex ECM. Amoeboid migration is characterized by high acto-myosin contractility driven by RhoA activity that can be regulated by cell surface receptors or mechanical signaling. Recent work suggests that membrane glycoproteins can also drive acto-myosin contractility in the cells; the authors of this work wanted to test if membrane glycoprotein Podoplanin also regulates contractility and drives amoeboid morphology during migration.

What is  Podoplanin:

Podoplanin is a small glycoprotein with an extracellular domain rich in O-glycoside type carbohydrate chains, a transmembrane domain, and an intracellular domain. The intracellular domain binds to the ERM (Ezrin-Rdixin-Moesin). RhoA activity by Podoplanin has been suggested to be regulated by its interaction with the receptor CLEC-2 or its binding to ERM proteins that lead to the release of RhoA-GDP which increases its activation (Ugorski et al., 2016). Podoplanin is also known to interact with other transmembrane proteins like CD44 which can also drive RhoA activation, though the mechanism behind it remains elusive (Martín-Villar E et al., 2010).  Here, the authors set out to test whether Podoplanin plays a role in regulating contractility and driving amoeboid morphology during migration.

Major findings:

Authors show that PDPN (the gene encoding Podoplanin) expression is higher in melanoma cells and has round morphology which suggests higher contractility and amoeboid mode of invasion and migration. This increase in contractility is independent of Braf mutation (increase in MAPK, ERK signaling) which causes hyperactivation of proliferation and increases metastasis. Higher Podoplanin in melanoma cells is correlated with higher contractility, rounded morphology, and an increase in migratory and invasive behavior in the melanoma cells in-vivo. Invasiveness in melanoma cells can often be driven by an increase in the proteolytic activity of these cells but the authors show that increase in the invasive behavior of melanoma cells with high Podoplanin is independent of proteolytic activity. Podoplanin also causes de-differentiation in the tumor cells that are linked with loss of melanocyte functions such as pigmentation.

Illustration showing high levels of Podoplanin leads to more amoeboid like morphology with an increase in the motility and invasion in melanoma cells. The illustration was done by Ankita Jha.

What I like about this preprint:

Cancer cells are highly plastic. This is maintained by acto-myosin contractility though we have only started to understand the cell surface signaling that is linked to the contractility pathway during cancer progression. Changes in glycosylation patterns have been characteristic to many cancer types and glycoproteins are an ideal source for cancer detection and disease progression (Zhao et al., 2008). This paper links one of glycoproteins, Podoplanin, that is detected to be high in many different cancer types with the contractility pathway driving plasticity in the cancer cells. This work also generates many open questions in understanding the alternate pathways regulating contractility and hence the plasticity in cancer cells. Some of those questions are as follows.

My questions to the authors:

  1. Podoplanin and other glycoproteins are reported to be upregulated in different cancer types, I was wondering if authors and the field think that these glycoproteins have some redundancy?
  2. It is interesting to note that down-regulation or low levels of Podoplanin are corroborated with less contractility in melanoma cells, previous work shows that contractility in melanoma cells is transcriptionally regulated by TGF-β signaling (Cantelli et al., 2015). Do authors think Podoplanin is also involved in transcriptional regulation of contractility? How does it work in coherence with TGF-β signaling in melanoma cells?
  3. High levels of Podoplanin enhances bleb like morphology. Where is podoplanin localized in the cells undergoing bleb based motility? Since the intracellular domain of Podoplanin can bind to Ezrin that can interact with the cortical actin, would authors suggest that Podoplanin has a role in maintain bleb dynamics during amoeboid migration?
  4. Different mechanisms of activation of Podoplanin have been suggested in the literature and one prominent mechanism that comes forward is its interaction with other cell surface proteins like CD44. CD44 can also occur as an adhesion molecule promoting cell-cell and cell-ECM interactions. Can Podoplanin promote similar adhesion to the ECM? By what mechanism can CD44 interact with Podoplanin?

References:

Martín-Villar E, Fernández-Muñoz B, Parsons M, et al. Podoplanin associates with CD44 to promote directional cell migration. Mol Biol Cell. 2010;21(24):4387-4399. doi:10.1091/mbc.E10-06-0489

Cantelli, G. et al. (2015) ‘TGF-β-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination’, Current Biology, 25(22), pp. 2899–2914. doi: 10.1016/j.cub.2015.09.054.

Ugorski, M., Dziegiel, P. and Suchanski, J. (2016) ‘Podoplanin – a small glycoprotein with many faces’, American Journal of Cancer Research, 6(2), pp. 370–386.

Zhao, J. et al. (2008) ‘Protein biomarkers in cancer: Natural glycoprotein microarray approaches’, Current Opinion in Molecular Therapeutics, 10(6), pp. 602–610.

Tags: #amoeboid, #cellmigration, melanoma

Posted on: 19 August 2020

doi: https://doi.org/10.1242/prelights.24107

Read preprint (No Ratings Yet)

Author's response

Charlotte M. de Winde shared

  1. Podoplanin and other glycoproteins are reported to be upregulated in different cancer types, I was wondering if authors and the field think that these glycoproteins have some redundancy?

 

Potentially, for some aspects of their function. Podoplanin however seems to have many different functions which depend on the cell type, the expression of binding partners (e.g. CD44), and also the availability of CLEC-2 from other cell types in the tissue microenvironment. We have been surprised at just how many pathways podoplanin seems to be involved in. Our current line of investigation is linking podoplanin to transcriptional regulation, watch this space.

 

  1. It is interesting to note that down-regulation or low levels of Podoplanin are corroborated with less contractility in melanoma cells, previous work shows that contractility in melanoma cells is transcriptionally regulated by TGF-β signaling (Cantelli et al., 2015). Do authors think Podoplanin is also involved in transcriptional regulation of contractility? How does it work in coherence with TGF-β signaling in melanoma cells?

 

TGF-b was an obvious target to look at, TGF-b can drive dedifferentiation of melanoma, and drive contractility, but although podoplanin signaling can stimulate production of TGF-b in lymphoid stroma, we found no change in these melanoma cell lines. We can only conclude that that are both important pathways, but perhaps independent from one another.

 

  1. High levels of Podoplanin enhances bleb like morphology. Where is podoplanin localized in the cells undergoing bleb based motility? Since the intracellular domain of Podoplanin can bind to Ezrin that can interact with the cortical actin, would authors suggest that Podoplanin has a role in maintain bleb dynamics during amoeboid migration?

 

Yes indeed, podoplanin is a membrane protein, and linked directly to the cortex through ERM proteins. We have some preliminary data in melanoma cell lines showing membrane localization of podoplanin during bleb formation, and data from fibroblasts has shown us that when we inhibit podoplanin-driven contractility through binding CLEC-2, then blebbing is inhibited, but further, that ERM proteins are relocated to the cytoplasm.

 

  1. Different mechanisms of activation of Podoplanin have been suggested in the literature and one prominent mechanism that comes forward is its interaction with other cell surface proteins like CD44. CD44 can also occur as an adhesion molecule promoting cell-cell and cell-ECM interactions. Can Podoplanin promote similar adhesion to the ECM? By what mechanism can CD44 interact with Podoplanin?

 

CD44 is a direct binding partner for podoplanin, binding via their extracellular domains, and certainly changes how podoplanin signals. We have another paper in preparation looking at podoplanin and its binding partners, and we find that podoplanin and CD44 colocalise more in the presence of CLEC-2 – so when contractility is inhibited. It’s safe to say that there is a lot about the downstream signaling that we still don’t understand. As for adhesion, we don’t think that podoplanin is directly involved in binding ECM, but indirectly, through controlling cytoskeletal contractility, podoplanin activity does regulate cell adhesion at least in vitro.

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