Of Platelets, cancer cells, EMT and Metastasis.

Hello again!

This time I’ll be writing about a paper that recently appeared in the journal Cancer Cell. The paper is something of a landmark because it has showed how the mere interaction of platelets with cancer cells is sufficient to induce activation of EMT (Epithelial Mesenchymal Transition). It details how the presence of a protein called TGFß derived from platelets, in combination with direct contact between cancer cells and platelets, can activate the NF-kB and Smad signalling pathways that confer invasiveness to cancer cells, thus making metastasis possible.

TGF-beta signalling pathway,courtesy Cell Signaling Technologies.

NF-kB Signalling, courtesy Cell Signaling Technologies.

The paper (see journal reference at the bottom for a link) is elegant because of the way critical questions were asked and addressed… the main questions were if platelets could stimulate metastasis and if so, by what means and methods.

The first question was quite easy to address. They took colon carcinoma cells from a cell line called MC38GFP and breast cancer cells from a cell line called Ep5, they grouped cells from each of those lines into two groups each. One of each group was treated with platelets while the other wasn’t, and the cells were then injected into mice to look for the frequency of metastasis. and bingo, the platelet treated cells showed a higher frequency of metastasis.


Number of metastatic foci.

Treatment with Platelets tends to vastly increase the number of metastatic foci compared to untreated cells.

There you go, first question answered. The researchers then examined whether platelet treatment induced an EMT-like phenotype, and they did this by looking at the mRNA and protein concentrations of various genes and their products that are markers of EMT, such as MMP-9 (a matrix metalloproteinase) and found that the expression of such marker genes and proteins is highly upregulated. Some of the markers used were Snail, Vimentin, Fibronectin and PAI-1 for a mesenchymal phenotype, and E-cadherin expression as a marker of epithelial phenotypes (or the loss thereof).

(D) Relative fold change in mRNA expression in MC38GFP or Ep5 cells treated with buffer or platelets for 40 hr (n = 3). Values are normalized to Gapdh expression. (E) Detection of E-cadherin protein levels by immunoblotting of lysates ofMC38GFPorEp5cells treated as in (D). Amounts of platelets equal to those used to treat cells were also loaded as control (no cells). b-tubulin was used as loading control. (F) Zymography for MMP-9 in the conditioned medium of MC38GFP or Ep5 cells treated as in (D). Amounts of platelets equal to those used to treat cells were also loaded as control (no cells). (G) MC38GFP and Ep5 cells were added at the top of transwells coated with Matrigel and treated with buffer or platelets. The total number of cells that invaded to the bottom of the transwell was counted after 48 hr (n = 3). For (A), (B), (D), and (G) bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 were determined by Student’s t test.

The question then would be how platelets could induce EMT, could TGFß be to blame? To find out they carried out bioinformatics analysis of data from microarray experiments looking for differentially expressed genes when cells were treated with platelets. They found that expression of EMT genes was upregulated and there was increased expression of genes in the TGFß pathway.

Gene upregulation data from microarray analysis when platelet stimulation is induced. Gene enrichment analysis using Gene Ontology functional categorization is presented in the table. You can see how the upregulated genes are all associated with EMT and with TGF-beta dependent activation processes.

This indicated to them that platelets induced the EMT phenotype through the TGFß pathway. They then wanted to find out if TGFß alone was responsible or if direct contact with platelets also had something to do with said upregulation.

To do this, they set up a rather cute experiment where they used a PAI-1 reporter gene to assay TGFß activation (this is possible because PAI-1 is downstream in that pathway and can be used as a surrogate marker)

They assayed levels of activation when platelets AND TGFß were added as opposed to TGFß alone. They observed more PAI-1 activation in the former than the latter, thus indicating that the presence of platelets and TGFß together act synergistically in activating the pathways involved. The central role of TGFß in the process was confirmed by blocking TGFß using an inhibitory antibody and a small molecule, which led to loss of PAI-1 expression.

The question then, of course, was why the presence of both platelets and TGFß would have more effect in EMT activation compared to TGFß alone. What other pathways would be activated such that the added effect could be accounted for?

To test this, they used reporter based assays for multiple pathways involved in cancer, and found that the releasate from platelets (platelet free) activated the JNK pathway, while the presence of cells and the releasate activated the JNK and NF-kB pathways.

Now this is where things got really interesting. Firstly, they confirmed whether NF-kB was actually able to account for the results of cell-inclusive treatment experiments by using Ep5 cells with mutant NF-kB and a reporter with reference to a control with a normal NF-kB and the corresponding reporter.

They found that mutant cells didn’t show NF-kB activation when treated with platelets, and that these cells had the same metastatic potential as cells that were treated with TGFß alone, thus fully implicating this pathway in platelet contact induced EMT.

These results were further validated when treatment with an NF-kB inhibitor also downregulated the expression of several key markers of EMT, such as MMP-9, in cells with intact NF-kB signalling.

They found that inhibiting cells with the NF-kB inhibitor switched off a reporter in that pathway, but not one in the other pathway, indicating that TGFß activation was independent of NF-kB activation, while the effects were synergistic.

And finally, here’s the piece de resistance of the paper; they showed that blocking TGFß secretion in megakaryocytes and platelets or wrecking the TGFß pathway was enough to prevent metastasis, they did this using mice in which TGFß expression was knocked out where seeding with cancer cells prevented metastasis to the lungs. They also showed that even cells pre-treated with wild type platelets for a while before introduction into TGFß fl/fl mice wasn’t enough to trigger metastasis.

This basically confirms that platelet derived TGFß signalling is absolutely necessary, while NF-kB signalling in synergy with it renders tumour cells potentially more metastatic, but isn’t per se sufficient for metastasis.

They note that this could have therapeutic implications, since TGFß inhibition in platelets doesn’t have physiological effects on normal cells, and if this could be replicated in humans it might serve as a brilliant therapeutic strategy.

However, as always, blocking metastasis, it would appear, would only really be useful in tumours where metastasis hasn’t occurred, how established metastatic disease should be dealt with is still a very open, and problematic question.

Journal Reference: Labelle et al, Direct Signaling between Platelets and Cancer Cells Induces an Epithelial-Mesenchymal-Like Transition and Promotes Metastasis, Cancer Cell. http://www.sciencedirect.com/science/article/pii/S1535610811003564

That is all from me this time round.





3 responses to “Of Platelets, cancer cells, EMT and Metastasis.

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  2. Pingback: Of Platelets, cancer cells, EMT and Metastasis. | Exploreable | from Flow Cytometry to Cytomics | Scoop.it

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