Claudin 1 is a driver of EMT in primary liver cancer

Cancer of liver cells (hepatocellular carcinoma) is often diagnosed late as it may grow slowly and for unnoticed for years; consequently most patients present with advanced disease, the outlook for which is currently very poor. The process of epithelial to mesenchymal transition (EMT), which is required for many cancers to undergo metastasis and set up secondary tumours in other organs and tissues, occurs in advanced disease and in the case of liver cancer very little is known about EMT.

A recent study published in the journal Oncogene shows that a molecule that is normally involved in holding epithelial cells together in tissues, Claudin 1, can in fact promote EMT and allow cancer cells to become more invasive. Increased levels of Claudin 1 in liver cancer cells causes promotion of genes involved in EMT through up-regulation of key transcription factors ZEB1 and SLUG. Another important EMT transcription factor, SNAIL, was shown to not be involved, but interestingly a protein called c-Abl was found to be very important for this process.

c-Abl, is best known for being part of a mutation that occurs commonly in a form of leukaemia called CML and a number of drugs targeting c-Abl have been developed to treat CML. For example, Imatinib (Glivec) is an inhibitor that blocks the BCR-Abl fusion oncogene product which has been very useful for doctors  treating CML. In this present study, the group showed that by silencing the protein c-Abl in cells that have Claudin 1 over-expressed, they could reverse the up-regulation of ZEB1 and SLUG and prevent the invasiveness conveyed by EMT. This study demonstrates that in the future when doctors look at hepatocellular carcinoma they could look for increased levels of Claudin 1 as a possible indicator of the invasiveness of the cancer. With more work, this marker could be validated and possibly even exploited as a druggable target for the treatment of liver cancer.

Mentioned Articles

Suh Y, Yoon CH, Kim RK, Lim EJ, Oh YS, Hwang SG, An S, Yoon G, Gye MC, Yi JM, Kim MJ, Lee SJ.

Claudin-1 inducesepithelial-mesenchymal transition through activation of the c-Abl-ERK signalingpathway in human liver cells.

Oncogene. 2013 Oct 10;32(41):4873-4882. doi: 10.1038/onc.2012.505.

Brief: A test for potentially harmful human induced pluripotent stem cells

Yamashita et al. have produced a method for determining if human induced pluripotent stem cells (iPSC) are likely to form tumours and published their results in Scientific Reports.

Taking a patient's cells, engineering them to gain stem cell properties, such as being able to self-renew and develop into multiple different cell types, and re-injecting them into that patient's tissue is an emerging new therapeutic field. However there are concerns that these cells, once transplanted into a patient, may become cancerous. The methods used to make human iPSCs often involves making normal cells express stem cell factors (which in themselves can be oncogenic - cancer forming) and the oncogene c-myc.

By making these iPSCs into cartilage and monitoring for the formation of tumours, the group could see which iPSCs were potentially harmful. This test could prove crucial in validating iPSCs for regenerative medicine.

Mentioned Articles

Yamashita A, Liu S, Woltjen K, Thomas B, Meng G, Hotta A, Takahashi K, Ellis J, Yamanaka S, Rancourt DE.

Cartilage tissue engineering identifies abnormal human induced pluripotent stem cells.

Sci Rep. 2013 Jun 13;3:1978. doi: 10.1038/srep01978.

Micro-RNAs and Retinoic Acid take their Toll on prostate and breast cancer

One of the first lines of defence against infection is the innate immune system. This mounts a non-specific response to infectious agents including bacteria and viruses. Recognition of bacterial and viral components is accomplished by a family of receptors called Toll Like Receptors (TLRs) found on the surface of innate immune cells. TLRs can also be expressed by other cells such as cancer cells.

Previous studies have shown that activation of TLRs can affect the levels of micro RNAs (miRNAs) in cells and therefore regulate which proteins are expressed (I have mentioned the role of miRNAs in cancer in previous posts). In this present study, Galli et al. found that activation of TLR3 in prostate and breast cancer cells caused an increase in four miRNAs. These miRNAs target a class of proteins called DNA methyltransferases, resulting in the loss these proteins from the cancer cells. Without these DNA methyltransferases a protein called retinoic acid receptor beta (RARβ) is produced by the cancer cells.

In a breakthrough, the group showed that drugging tumours first with an activator of TLR3 followed by retinoic acid (an activator of RARβ), caused tumours to be smaller compared to control treated tumours. This study therefore presents promising pre-clinical data for the treatment of breast and prostate cancer with this exciting combination therapy.

Mentioned Articles

Galli R, Paone A, Fabbri M, Zanesi N, Calore F, Cascione L, Acunzo M, Stoppacciaro A, Tubaro A, Lovat F, Gasparini P, Fadda P, Alder H, Volinia S, Filippini A, Ziparo E, Riccioli A, Croce CM.

Toll-like receptor 3(TLR3) activation induces microRNA-dependent reexpression of functional RARβand tumor regression.

Proc Natl Acad Sci U S A. 2013 Jun 11;110(24):9812-7. doi: 10.1073/pnas.1304610110. Epub 2013 May 28.

Chen R, Alvero AB, Silasi DA, Steffensen KD, Mor G.

Cancers take theirToll--the function and regulation of Toll-like receptors in cancer cells.

Oncogene. 2008 Jan 7;27(2):225-33. doi: 10.1038/sj.onc.1210907.

Researchers hot on the TRAIL of new combination therapy for glioblastoma

Some cancers are particularly difficult to treat, particularly brain tumours. Glioblastoma is an aggressive form of cancer in the brain which, although responds initially to treatment, is refractory and often always fatal. Treating cancerous cells with a protein called TRAIL is a specific treatment which causes cancer cell death in around 50% of cancer cell lines. Bagci-Onder et al. employ some interesting molecular techniques to studying the effect of TRAIL on glioblastoma cell viability.

Neuronal stem cells were used to deliver soluble TRAIL protein to glioblastoma cells and the way in which this happens was modelled by artificially colouring both cell types and watching their interactions when grown together. Two out of three glioblastoma cell lines tested were sensitive to TRAIL treatment and underwent a form of highly controlled cell death called apoptosis.

The killing of these cells by TRAIL treatment was dependent on the levels of two proteins on the cell surface called death receptors; death receptor 4 (DR4) and death receptor (DR5). The group observed that the higher the levels of these receptors, the more receptive to TRAIL treatment the cells were. This led the group to postulate that increasing death receptor expression could enhance the effectiveness of TRAIL in treating glioblastoma.

To see how they could increase death receptor expression in cancerous cells, the group used a panel of different drugs known to affect the way cells function. They found that targeting histone deacetylases (HDACs) [see previous post] with a drug called MS-275 caused an increase in death receptor levels at the surface of cells. As expected, this also led to an increased sensitivity to TRAIL treatment. Importantly, the one glioblastoma cell line which was initially resistant to TRAIL treatment became sensitive to the killing power of TRAIL if cells were also treated with MS-275. This study highlights a potential new combination treatment for glioblastoma.

Mentioned Articles

Bagci-Onder T, Agarwal A, Flusberg D, Wanningen S, Sorger P, Shah K.

Real-time imaging of the dynamics of death receptors and therapeutics that overcome TRAIL resistance in tumors.

Oncogene. 2013 Jun 6;32(23):2818-27. doi: 10.1038/onc.2012.304. Epub 2012 Jul 23.

"Types of primary brain tumours : Cancer Research UK : CancerHelp UK." Cancer Research UK: the UK's leading cancer charity : Cancer Research UK . N.p., n.d. Web. 13 June 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.

TGF-β stimulates Pyk2 expression as part of an epithelial-mesenchymal transition program required for metastatic outgrowth of breast cancer.

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.

Proline-rich tyrosine kinase 2 (Pyk2) promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition.

PLoS One. 2011 Apr 20;6(4):e18878. doi: 10.1371/journal.pone.0018878.

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.

mTORC1

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.

mTORC2

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.

mTORC1 signaling requires proteasomal function and the involvement of CUL4-DDB1 ubiquitin E3 ligase.

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.

Ubiquitin hydrolase UCH-L1 destabilizes mTOR complex 1 by antagonizing DDB1-CUL4-mediated ubiquitination of raptor.

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.

Deconvoluting mTOR biology.

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

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)

The Grainyhead transcription factor Grhl3/Get1 suppresses miR-21 expression and tumorigenesis in skin: modulation of the miR-21 target MSH2 by RNA-binding protein DND1.

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.

Inhibition of microRNA function by antimiR oligonucleotides.

Silence. 2012 Jan 9;3(1):1. doi: 10.1186/1758-907X-3-1.

Martin SA, Lord CJ, Ashworth A.

Therapeutic targeting of the DNA mismatch repair pathway.

Clin Cancer Res. 2010 Nov 1;16(21):5107-13. doi: 10.1158/1078-0432.CCR-10-0821. Epub 2010 Sep 7.