Friday, 19 April 2013

Integral role for Pyk2 in breast tumour outgrowth and metastasis to the lung

There exist a number of well-defined markers of the epithelial to mesenchymal transition (EMT), a process crucial for the outgrowth of tumours to new areas of the body (metastasis), and now a signalling protein Pyk2 has been welcomed into the fold.

Focal Adhesion Kinase (FAK) is an established regulator of EMT in response to the pro-EMT signalling protein Tumour Growth Factor Beta (TGFβ), a recent study has revealed that some of FAK’s functions also require Pyk2 and suggests important roles for Pyk2 in tumour metastasis. A previous study by Sun et al. from The University of Hong Kong has also described a role for Pyk2 in driving EMT in liver cancer (hepatocellular carcinoma).

Normal human breast cells transformed to be cancer-like and cells from patients with aggressive (invasive ductal cell carcinoma, with reaccurance after 5 years) show high levels of Pyk2 protein. One breast cancer cell line is a mixture of less aggressive and more aggressive cancer cells, within these different cell populations the group observed vastly different levels of Pyk2 protein.

This increased Pyk2 expression was associated with increased growth of cells in a three dimensional tumour-like environment. In fact, growth of cells in this 3D model can enhance Pyk2 expression in breast cancer cell lines in a manner that is independent of the pro-EMT factor TGFβ. When growing two dimensionally (i.e. on a plastic culture surface) breast cancer cells show low levels of Pyk2, however treatment TGFβ could increase Pyk2 levels, but not to the same extent as observed in the 3D model. The group suggest that the 3D model exhibits autocrine TGFβ signalling (where TGFβ released by one cell acts directly upon the same cell). Importantly, not only do the cells make more Pyk2, but there are higher levels of the active form of Pyk2 (phosphorylated Pyk2). The signalling mechanism which links TGFβ to the increased Py2k levels observed was shown to involve the actions of a transcription factor called Smad4, this pathway was observed clearest in aggressive breast cancer cells.

Metastasis requires a dramatic change in the shape of cells (a novel mechanism of this was previously described in a past post); the classical EMT shape change is induced by TGFβ, however this was independent of Pyk2. It appears that FAK is the key protein involved here.

The group went on to show that whilst up-regulation of Pyk2 in breast cancer cells is required along with FAK for acquisition of an EMT signature, Pyk2 alone is indispensable for their ability to outgrow their initial “primary” tumour site and metastasise to the lung. Pyk2 could therefore present a therapeutic target for the treatment of invasive, pre-metastatic breast cancer.

Mentioned Articles

Wendt MK, Schiemann BJ, Parvani JG, Lee YH, Kang Y, Schiemann WP.
Oncogene. 2012 Jun 18. doi: 10.1038/onc.2012.230. [Epub ahead of print]

Sun CK, Ng KT, Lim ZX, Cheng Q, Lo CM, Poon RT, Man K, Wong N, Fan ST.
PLoS One. 2011 Apr 20;6(4):e18878. doi: 10.1371/journal.pone.0018878.

Wednesday, 17 April 2013

A novel protein, UCH-L1, regulates the balance of two mTOR protein complexes; potential implications for neurological diseases and cancer

The signaling protein mTOR was first identified as the target of a natural chemical called Rapamycin and so named mammalian Target Of Rapamycin. mTOR can form two distinct complexes called mTORC1 and mTORC2; the two main mTOR binding proteins in these complexes are Raptor (mTORC1) and Rictor (mTORC2). In addition, several other proteins have been shown to associate with these complexes, for a review see this paper.


The mTORC1 complex activates S6K-1 and elF4E, two proteins required for the process of translating the messenger RNA of a gene into the protein via the assembly of amino acids specifically recruited by transfer RNA (tRNA). Increased mTORC1 activity can therefore lead to increased protein synthesis, important for bringing about dramatic cellular changes such as transformation from normal to cancerous states.


The mTORC2 complex, on the other hand, is responsible for activating proteins including: Akt, SGK-1 and PKCalpha which are involved in overall activation and proliferation of cells including those of the immune system. Cancerous cells often show increased Akt activation and this can be either through enhancing mutations in Akt itself or increased activity of Akt activating signaling pathways such as the mTOR pathway.
Since the discovery of Rapamycin, there have been a plethora of drugs developed which target either both mTOCR1, mTORC2 or both complexes. Some of these drugs have even been approved for the treatment of cancer, or are currently progressing through the later stages of clinical trials. However, the processes regulating the shift between mTORC1 and mTORC2 signaling are relatively poorly understood. A recent study by a group from Minnesota in the USA reveals a mechanism by which cells can move mTOR signaling from protein synthesis (mTORC1) to proliferation (mTORC2).

The ubiquitin hydrolase, UCH-L1, was the focus of a recent study as it was found that this protein interrupts mTORC1 signaling, high levels of UCH-L1 were associated with low levels of mTORC1 activity. UCH-L1 does this by displacing a novel mTORC1 interaction partner, namely the DDB1-CUL4 complex, identified in a previous study. This finding is very important because mutations of UCH-L1 have been seen in Parkinson’s disease, and UCH-L1 may also be involved in other brain disorders in which therapeutic inhibition of mTOR is already being investigated. Because of the well known role of mTOR in cancer, UCH-L1 also presents an exciting mechanism for cancer researchers.

Mentioned Articles

Ghosh P, Wu M, Zhang H, Sun H.
Cell Cycle. 2008 Feb 1;7(3):373-81. Epub 2007 Nov 2

Hussain S, Feldman AL, Das C, Ziesmer SC, Ansell SM, Galardy PJ.
Mol Cell Biol. 2013 Mar;33(6):1188-97. doi: 10.1128/MCB.01389-12. Epub 2013 Jan 7.

Wander SA, Hennessy BT, Slingerland JM.
Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy.
J Clin Invest. 2011 Apr;121(4):1231-41. doi: 10.1172/JCI44145. Epub 2011 Apr 1.

Weber JD, Gutmann DH.
Cell Cycle. 2012 Jan 15;11(2):236-48. doi: 10.4161/cc.11.2.19022. Epub 2012 Jan 15.

Monday, 1 April 2013

miR-21 is an "oncomir" important for skin cancer progression

An exciting new study shows that a microRNA called miR-21 acts as an oncogene to promote squamous cell carcinoma of the skin, and its up-regulation is dependent on reduced expression of the transcription factor Grhl3.

MicroRNAs were previously introduced here using the Let7 family as an example. Many different microRNAs are down- or up-regulated in tumour cells and can act as tumour suppressors or oncogenes  Oncogenic miRNAs (oncomirs) are attractive targets for therapeutic intervention as therapeutics can be developed to inhibit their function. A group from the University of California in Irvine CA, USA looked at the epidermis of mice which have the transcription factor Grhl3 genetically deleted. They observed a significant change in a number of microRNAs (Table 1), interestingly three of these were up-regulated 2 fold or more. miR-21, up-regulated to the greatest extent (2.5 +/- 0.6 fold increase), was one of the first oncomirs to be discovered and many of the proteins that it targets for degradation are tumour suppressors. Enforced loss of Grhl3 clearly led to a significant increase in miR-21 expression in the mouse model and importantly in isolated normal human epidermal keratinocytes, the main cell types in the human epidermis.

The group then showed that Grhl3 directly binds to the region of DNA that precedes miR-21, this region normally acts to promote the expression of for miR-21, however Grhl3 binding prevents the function of the miR-21 promoter. Therefore, loss of Grhl3 would remove this promoter repression and allow for the increased miR-21 expression as observed in Grhl3 deficient mice.

Short RNA sequences called antagomirs can be used to inhibit miRNAs including miR-21. When mice were treated with an miR-21 antagomir, reduced miR-21 expression was observed. The group next investigated which genes are targeted by miR-21 a cell line representative of human keratinocytes and karatinocyte tumour cells. These included tumour-suppressor genes involved in the cell cycle such as Cdk6 and Cdc25, and a protein responsible for repairing damaged DNA called Msh2 that is frequently mutated in cancer. 

The effect of miR-21 on these genes was more significant when kerantinocytes were transformed into tumourous cells. The group observed that transformation of kerantinocytes also caused down-regulation of a protein called DND1, DND1 it turns out is a negative regulator of the function of miR-21. Artificial over-expression of DND1 in transformed keratinocytes prevents miR-21 induced down-regulation of Msh2. Conversely, an artificial decrease in DND1 expression caused an enhancement of miR-21 induced Msh2 down-regulation.

The importance of Grhl3 and Msh2 in the progression of skin cancer was highlighted when the group investigated the ability of keratinocytes lacking Grhl3 to form tumours in mice. Loss of Grhl3 caused an increase in tumour volume an importantly was associated with loss of Msh2, showing the importance of this new epidermal regulatory pathway in skin cancer.

Mentioned Articles

Bhandari A, Gordon W, Dizon D, Hopkin AS, Gordon E, Yu Z, Andersen B. (2013)
Oncogene. 2013 Mar 21;32(12):1497-507. doi: 10.1038/onc.2012.168. Epub 2012 May 21.

Stenvang J, Petri A, Lindow M, Obad S, Kauppinen S.
Silence. 2012 Jan 9;3(1):1. doi: 10.1186/1758-907X-3-1.

Martin SA, Lord CJ, Ashworth A.
Clin Cancer Res. 2010 Nov 1;16(21):5107-13. doi: 10.1158/1078-0432.CCR-10-0821. Epub 2010 Sep 7.