I am very excited to announce our new review paper which is out now in Front Cell Dev Biol [Link]. This is also the first paper from Dr Kamila Marzec who recently joined the lab. Great work!
MASTL kinase is a master regulator of mitosis, essential for ensuring that mitotic substrate phosphorylation is correctly maintained. It achieves this through the phosphorylation of alpha-endosulfine and subsequent inhibition of the tumour suppressor PP2A-B55 phosphatase. In recent years MASTL has also emerged as a novel oncogenic kinase that is upregulated in a number of cancer types, correlating with chromosome instability and poor patient survival. While the chromosome instability is likely directly linked to MASTL’s control of mitotic phosphorylation, several new studies indicated that MASTL has additional effects outside of mitosis and beyond regulation of PP2A-B55. These include control of normal DNA replication timing, and regulation of AKT/mTOR and Wnt/β-catenin oncogenic kinase signalling. In this review, we will examine the phenotypes and mechanisms for how MASTL, ENSA and PP2A-B55 deregulation drives tumour progression and metastasis. Finally, we will explore the rationale for the future development of MASTL inhibitors as new cancer therapeutics.
Great news we have a new co-author publication in Oncogene!
This work was in collaboration with Prof. Neil Wakins here at the Garvan Institute and focuses on the role of Hedgehog (Hh) signaling in small cell lung cancer (SCLC). Small cell lung cancer is a common, aggressive malignancy with universally poor prognosis.
Full details can be found here [link]
TITLE: “The role of canonical and non-canonical Hedgehog signaling in tumor progression in a mouse model of small cell lung cancer”
Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.
A defining feature in over 2/3rds of all solid tumours is the continual loss and gain of whole are small parts of chromosomes. This instability, or CIN for short, strongly implicated in tumour initiation, progression, chemoresistance and poor prognosis. CIN is created through failures during mitosis, whereby whole or parts of a chromosome are segregated incorrectly, thereby created daughter cells with unequal chromosome numbers. Consequently, understanding how mitosis is regulated is essential for uncovering the mechanisms allowing CIN to arise and drive cancer. In our recent publication, we discovered the mechanisms controlling the key regulatory pathway critical to ensuring cells exit mitosis correctly. At the centre of this pathway is a gene call MASTL (short for ‘Microtubule Associated Serine/Threonine Kinase-Like’). The primary function of MASTL is to ensure that the cellular breaks (the phosphatase PP2A), is turned off during mitosis so that the accelerator (Cdk1 kinase) can drive the cell into mitosis. Much like a car, having the accelerator and breaks on at the same time is a bad idea, unless you like the smell of burning rubber. To successfully exit mitosis, and to perfectly segregate chromosomes, the cell must take the foot off the accelerator and turn on the breaks. Because MASTL is the central regulator ensuring the breaks are coordinated with the accelerator, it is essential to understand how MASTL is controlled. To this end, we uncovered that MASTL must be rapidly turned off to allow cells to exit mitosis, and this inactivation is carried out by another cellular brake call PP1 phosphatase (Rogers et al, JCS 2016). Now that we have identified and mapped this novel mitotic exit switch, we hope to be able to shed new light on how CIN drives the initiation and evolution cancer. We believe that with further study we will be able to better predict patient response to chemotherapy, and also identify new ways to ‘switch off’ highly unstable tumours, thereby improving treatment for patients that currently have a very poor prognosis.
Image of Interphase HeLa cell stained for Actin (red), DNA (blue) and the co-localisation of MASTL and PP1 by Proximity Ligation Assay (PLA; green).
Credit: Sam Rogers and Cell Division Lab
Great News, we have a new review article that has just been published online today in Inside the Cell!
Its Open Access, so that means its free for everyone to read!
During mitotic exit, phosphatases reverse thousands of phosphorylation events in a specific temporal order to ensure that cell division occurs correctly. This review explores how the physicochemical properties of the phosphosite and surrounding amino acids affect interactions with phosphatase/s and help determine the dephosphorylation of individual phosphorylation sites during mitotic exit.
The Full Reference and link for the Article can be found below:
Samuel Rogers, Rachael McCloy, D Neil Watkins and Andrew Burgess Mechanisms regulating phosphatase specificity and the removal of individual phosphorylation sites during mitotic exit Inside the Cell [Link]
Here is a very simple guide for determining the level of fluorescence in a given region (e.g nucleus)
- Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
- From the Analyze menu select “set measurements”. Make sure you have AREA, INTEGRATED DENSITY and MEAN GRAY VALUE selected (the rest can be ignored).
- Now select “Measure” from the analyze menu or hit cmd+m (apple). You should now see a popup box with a stack of values for that first cell.
- Now go and select a region next to your cell that has no fluroence, this will be your background.
NB: the size is not important. If you want to be super accurate here take 3+ selections from around the cell.
- Repeat this step for the other cells in the field of view that you want to measure.
- Once you have finished, select all the data in the Results window, and copy (cmd+c) and paste (cmd+v) into a new excel worksheet (or similar program)
- Use this formula to calculate the corrected total cell fluorescence (CTCF).
NB: You can use excel to perform this calculation for you.
CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings)
- Make a graph and your done. Notice that in this example that the rounded up mitotic cell appears to have a much higher level of staining, but this is actually due to its smaller size, which concentrates the staining in a smaller space. So if you just used the raw integrated density you would have data suggesting that the flattened cell has less staining then the rounded up one, when in reality they have a similar level of fluorescence.
How to Cite this if you wold like to:
We have used this method in these papers:
McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., and Burgess, A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 [Link]
Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T & Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA 107: 12564–12569
But you can also find a similar method published here:
Gavet O & Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18: 533-543
Potapova TA, Sivakumar S, Flynn JN, Li R & Gorbsky GJ (2011) Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited. Mol Biol Cell 22: 1191–1206
And my apologies to any others that I have not mentioned.
Big congratulations to our PhD student Sam Rogers, who took out best poster at last weeks inaugural EMBL Australian PhD Symposium, held at UNSW. An amazing achievement considering that he is still only in his first year.
Well done Sam !