As of March 31st 2015 I am the proud co-author of “Myc drives Pten/p53-deficient proliferation and metastasis due to Il6-secretion and Akt-suppression via Phlpp2” – snappy title, right? – which was published “online first” in the journal Cancer Discovery. The abstract and author manuscript can be found here. The polished article will appear in the May print issue, I believe.
The first author, Dawid G. Nowak, was my supervisor at Cold Spring Harbor Laboratory this summer, where I participated in a ten-week undergraduate research programme. The main aim of the paper was to characterise molecular differences between prostate cancer (PC) cells of the primary tumour (i.e. at the prostate) and metastasised prostate cancer cells. A well-known change that occurs during the progression of PC is the loss of so-called tumour suppressor genes, in particular they are the proteins called PTEN and p53. Tumour suppressor genes are responsible for preventing healthy cells from overproliferating, so when they are lost there are fewer “molecular brakes” in the cell to stop aggressive growth. In addition to losing tumour suppressors, cancer cells also activate so-called oncogenes, and these can be viewed as drivers of growth. So, broadly speaking, when tumour suppressors are lost and oncogenes constitutively switched on a cancer cell is created.
Dawid’s work shows for the first time that as PC cells become metastatic they switch from using one main “driver” oncogene known as Akt to using a different oncogene known as Myc. Firstly, they just made this observation in various tissue samples and biopsies, but then the lab, led by Lloyd Trotman, tried to elucidate the mechanism by which this “oncogene switch” occurs. What Dawid found was that an extracellular signalling factor called IL-6, normally involved in inflammatory processes, activates Myc, which in turn activates a phosphatase – an enzyme that dephosphorylates its targets – called Phlpp2. One of Phlpp2’s targets is Akt, and Akt is inactive when dephosphorylated. Thus, once this process is set into motion Myc rapidly overtakes Akt as the main driving oncogene.
Needless to say, one of the methods employed in the study was the CRISPR knockout approach. The idea of the CRISPR experiment was to show that in a PC cell line derived from a patient’s bone metastasis (PC3), which has already lost the tumour suppressors PTEN and p53, we could knock out Myc and at the same time decrease the levels of Phlpp2. And indeed this is what the following Western blot shows (copied directly from Figure 6 of the paper) – on the left is the control and on the right the CRISPR approach has completely knocked out Myc; PCNA is a marker for cell proliferation and beta-actin is the loading control, which shows that equivalent amounts of total protein are present in both lanes:
And then in the obverse experiment Myc was overexpressed in these PC3 cells (again the Western blot is copied from Figure 6 and the left is the control, whereas the right is the experiment). When this was achieved the levels of Phlpp2 also increased and furthermore, the levels of phosphorylated/active Akt decreased:
One could now argue, however, that this was merely a correlation but not a causative effect. So to prove that the proposed signalling – Myc activates Phlpp2, which inactivates Akt – was actually occurring, the last experiment was performed in PC3 cells in which, in addition to PTEN and p53, Phlpp2 had also been deleted. Here the levels of phosphorylated Akt no longer decreased because the responsible phosphatase was not present (again from Figure 6; left: control, right: Myc overexpression):
All in all quite an elegant story I think. Hopefully the scientific community studying PC will be able to use these results in further experiments. For my part I certainly learnt a great deal from Dawid last summer and enjoyed helping to prepare the manuscript for publication.
Nowak DG, Cho H, Herzka T, Watrud K, DeMarco DV, Wang VM, Senturk S, Fellmann C, Ding D, Beinortas T, Kleinman D, Chen M, Sordella R, Wilkinson JE, Castillo-Martin M, Cordon-Cardo C, Robinson BD, Trotman LC (2015) MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2. Cancer Discovery 5: 636-651