Saturday, 9 February 2013

A new selective inhibitor of Histone Deacetylase 6

Chemotherapy, treatment of cancer with drugs, requires the development of compounds which have the effect of slowing tumour growth, stopping growth all together or even causing death to cancer cells within the tumour. Generally, compounds can either act to positively stimulate a response within cells or to inhibit one or more processes. These compounds will normally be designed to target a single protein or multiple proteins in the same family. There are advantages and disadvantages to targeting more than one protein: on the plus side you may help kill cancer cells by stopping several proteins in the same signalling pathway or prevent more than one pathway, on the downside you can increase the chance that the compound will also target non-cancerous cells and increase side effects for patients. One way to lessen this effect is to try and specifically target the compound to only the tumour thus limiting its effects on surrounding healthy cells, an alternative is to make the compound more specific for its intended target and reduce off-target effects. Compounds that not only show these properties, but also enter the body easily, are absorbed at a desirable rate and are successfully cleared from the body without creating dangerous metabolites, are likely to make suitable drugs. Here is where the fields of drug design, pharmacology and cancer research combine in an attempt make more effective therapeutic agents for the treatment of cancer.

Drug development can take several approaches, firstly there are a number of compounds that exist in nature such as the anti-cancer drug Taxol which was found in the Pacific Yew tree Taxus brevifolia in the late 1960s. Often, but not always, chemists can find ways to make these natural products from reactions between other chemicals, increasing the amount of drug that can be produced from these sometimes rare natural products. Additionally, once the chemical structure of these natural products is known, medicinal chemists can make small changes to a compound's structure and look to see what effect this has on the anti-cancer activity of the resulting compounds, this is called structure activity relationship (SAR). These methods are the usual route of drug discovery by academic groups, however in the pharmaceutical industry vast libraries of compounds which have drug-like properties are screened for interactions with desired target proteins, or their ability to stop cancer cell growth. This effort is called high throughput screening (HTS), it is expensive but often finds compounds which can undergo SAR to finally produce potential drugs.

Kaliszcak and colleagues recently developed an anti-cancer drug based on two natural inhibitors of a class of proteins called histone deacetylases (HDACs). I mentioned in a previous post that DNA is tightly held together in a structure called chromatin, to build chromatin DNA is wound around proteins called histones. These histones can be modified by adding acetyl groups which loosens the chromatin structure making DNA more accessible, HDACs can remove these acetyl groups causing DNA to become more tightly wound and inaccessible. HDACs also target proteins other than histones for deacetylation such as alpha-tubulin, a protein involved in the cell cycle that can be acetylated. The HDAC6 inhibitor prevented the deacetylation of alpha-tubulin by HDAC6 and caused an increase in the levels of the acetylated form. This effect on alpha-tubulin makes inhibiting HDACs, including HDAC6, a potential treatment for cancer. However, many current HDAC inhibitors are very unspecific and have serious side effects. The inhibitor developed by Kaloszcak and colleagues targets HDAC6 but at much lower concentrations of drug than required to target other HDACs such as HDAC1, suggesting it is more selective for HDAC6. This inhibitor had potent anti-cancer effects, causing a reduction in the growth of a wide variety of cancer cell types including: breast, colon, lung, ovarian and prostate cancer. 

The group also showed that their HDAC6 inhibitor caused cancer cells to undergo cell death. They did this by measuring the DNA content of cells after treatment with their HDAC6 inhibitor; DNA content decreased, a sign that cells are undergoing cell death. They noticed that proteins called caspases, important in a specific type of controlled cell death called apoptosis, were increased. Finally, when used in a mouse model of cancer, these inhibitors worked to reduce tumour growth with no general toxicity suggesting this HDAC6 inhibitor may do well if transferred into human clinical trials. 

Mentioned Articles

Kaliszczak M, Trousil S, Aberg O, Perumal M, Nguyen QD, Aboagye EO. (2013)
Br J Cancer. 2013 Feb 5;108(2):342-50. doi: 10.1038/bjc.2012.576. Epub 2013 Jan 15. (2009) Paclitaxel (Taxol) : Cancer Research UK : CancerHelp UK. [online] Available at: [Accessed: 9 Feb 2013].

Aldana-Masangkay GI, Sakamoto KM.(2011)
J Biomed Biotechnol. 2011;2011:875824. doi: 10.1155/2011/875824. Epub 2010 Nov 7.

Gryder BE, Sodji QH, Oyelere AK (2012)
Future Med Chem. 2012 Mar;4(4):505-24. doi: 10.4155/fmc.12.3.

Annunziato, A. (2008) 
Nature Education 1(1)

Wednesday, 6 February 2013

Let-7 prevents an agressive shift towards EMT in breast cancer cells

I have written previously about examples of transcription factors (Twist) which can bind DNA to help the initiation of transcription, and chromatin re-remodelling factors (FOXA1) which can constrict or release tightly wound DNA. Transcription makes a copy of DNA called messenger RNA, this contains the gene tic information coding for the protein to be made, as well as regions before and after that gene DNA. When the human genome was sequenced a large proportion of our DNA was discovered to not code for specific genes and was termed “junk DNA”. The recent ENCODE project was a massive effort to study the human genome in more detail, interestingly they found that most of this “junk DNA” actually has a function. So what does this DNA do? Some of the DNA codes for microRNAs, these are small pieces of RNA which can negatively regulate the second major step of making protein, translation. These microRNAs contain a “seed” sequence which can share similarity with one or more messenger RNA sequences, microRNAs help guide cellular machinery to messenger RNA that prevents it from being translated into protein. The numbers of different microRNAs present in cells can therefore have a dramatic effect on the levels of many proteins in the cell, including those for processes such as cell growth and cell survival. Cancer cells exhibit changes in microRNA levels which allows them to alter protein expression to increase growth and avoid cell death.

Let-7 was discovered in a species of roundworm called Caenorhabditis elegans and is one of the first observed microRNAs; it is also expressed in mammalian cells such as humans and has been investigated in the context of cancer. Let-7, along with another microRNA miR-200, has been shown to control the epithelial to mesenchymal transition (EMT) in breast cancer cells, a process mentioned here in a previous blog post. A recent study by Guo and colleagues looked at a protein called oncostatin M, which is released by breast cancer cells and can cause them to undergo metastasis through EMT. They first show that oncostatin M is indeed present in breast cancer cells. Next, they take breast cancer cells that show low levels of oncostatin M that are less aggressive and treat them with oncostatin M, this makes them undergo EMT and become more motile and better at invading into a substance called Matrigel which mimics human tissue. When cells were treated with oncostatin M levels of the microRNAs Let-7 and miR-200 were reduced suggesting that they may negatively regulate oncostatin M induced EMT. To test this, the group artificially raised levels of both these microRNAs which as expected reduced the effect of oncostatin M to cause EMT. Additionally, preventing the action of Let-7, but interestingly not miRNA200, resulted in an increase in EMT.

Finally, the group probed the potential pathway of proteins involved in the Let-7  and miR200 dependent repression of oncostatin M induced EMT. They showed that a protein called HMGA2 is the “master regulator” of oncostatin M induced EMT, and is targeted by Let-7 microRNA. To increase HMGA2 protein levels in cancer cells and induce EMT, Let-7 levels are reduced by a protein called Lin28. Oncostatin M increases Lin28 levels through the transcription factor Stat3.This pathway is important for the initiation of EMT in breast cancer cells, whereas miR200 is directly inhibited by Stat3 allowing an increase in the levels of a miR200 target protein ZEB1. ZEB1 is a transcription factor important in initiation and maintenance of EMT. This study has therefore revealed that Let-7 and miR200 microRNAs act as brakes to prevent  cells undergoing EMT. By utilising a Stat3 signalling pathway, cancer cells can overcome this brake and become more aggressive by entering into EMT.

Mentioned Articles

Guo L, Chen C, Shi M, Wang F, Chen X, Diao D, Hu M, Yu M, Qian L, Guo N. (2013)
Stat3-coordinated Lin-28-let-7-HMGA2 and miR-200-ZEB1 circuits initiate and maintain oncostatin M-driven epithelial-mesenchymal transition.
Oncogene. 2013 Jan 14. doi: 10.1038/onc.2012.573. [Epub ahead of print]

Iorio MV, Croce CM.(2012)
Causes and consequences of microRNA dysregulation.
Cancer J. 2012 May-Jun;18(3):215-22. doi: 10.1097/PPO.0b013e318250c001.

Peter ME. (2009)
Let-7 and miR-200 microRNAs: guardians against pluripotency and cancer progression.
Cell Cycle. 2009 Mar 15;8(6):843-52. Epub 2009 Mar 22.

Sunday, 3 February 2013

FOXA1 is a gatekeeper, as well as a marker, for less aggressive breast cancer

Breast cancer is a complex disease, comprising of several recognised subtypes which can be distinguished by factors including the signature of the proteins within the tumour cells. Two such subtypes include luminal and basal breast cancer, the latter is more agressive and has a poorer prognosis for patients. Recent work has suggested that basal breast cancer cells may arise from luminal cells, although the mechanism for this is unclear.

A protein called FOXA1 may be the key to this transformation and Bernardo and colleges explore this in a recent paper in the journal Oncogene. The double helix is the basic structure of DNA (although exciting research from a group in Cambridge has identified four stranded DNA helices in cancer cells). Proteins and modifications to the double helix DNA compact it within the nucleus of the cell into a tightly wound structure known as chromatin. Like transcription factors such as twist, chromatin re-modellers including FOXA1, are capable of altering which genes are turned on and subsequently made into proteins. But chromatin re-modellers do this by altering the structure of chromatin, making the genes in DNA less or more accessible and in turn changing which proteins are made. Changing levels of FOXA1 in cells can therefore have dramatic results on the pattern of proteins, this can lead to changes in the function of those cells.

The group first took breast cancer cells which represent either luminal or basal subtypes and measured how much FOXA1 was present. In agreement with previous studies, luminal breast cancer cells had high levels of FOXA1, whereas this protein was almost undetectable in basal breast cancer cells. The researchers then took luminal breast cancer cells, artificially reduced the levels of FOXA1 and measured which genes were being transcribed (the first stage in making proteins of genes). This gives a fingerprint of all the genes turned on or off in a cell, called the "transcriptome". Interestingly, the loss of FOXA1 in luminal cells led to a decrease in luminal genes and an increase in basal genes switched on. This change from a luminal to basal gene signature with loss of FOXA1 expression was accompanied by increased motility and invasiveness of breast cancer cells.

FOXA1, therefore acts to prevent the conversion from a luminal to basal type breast cancer, and therefore limits the agressiveness of the tumour. This study supports the evidence that FOXA1 expression marks a less aggressive form of breast cancer with better prognosis, however it also suggests that efforts to target FOXA1 as a treatment for luminal breast cancers may have serious unwanted consequences such as increasing aggressiveness.

Mentioned Articles
Bernardo GM, Bebek G, Ginther CL, Sizemore ST, Lozada KL, Miedler JD, Anderson LA, Godwin AK, Abdul-Karim FW, Slamon DJ, Keri RA. (2013)
FOXA1 represses the molecular phenotype of basal breast cancer cells.
Oncogene. 2013 Jan 31;32(5):554-63. doi: 10.1038/onc.2012.62. Epub 2012 Mar 5.

Yamaguchi N, Ito E, Azuma S, Honma R, Yanagisawa Y, Nishikawa A, Kawamura M, Imai J, Tatsuta K, Inoue J, Semba K, Watanabe S. (2008)
FoxA1 as a lineage-specific oncogene in luminal type breast cancer.
Biochem Biophys Res Commun. 2008 Jan 25;365(4):711-7. Epub 2007 Nov 26. [Abstract and figures free]

Thorat MA, Marchio C, Morimiya A, Savage K, Nakshatri H, Reis-Filho JS, Badve S. (2008)
Forkhead box A1 expression in breast cancer is associated with luminal subtype and good prognosis.
J Clin Pathol. 2008 Mar;61(3):327-32. Epub 2007 Nov 23. [Abstract only free] (2013) Four-stranded ‘quadruple helix’ DNA structure proven to exist in human cells - Research - University of Cambridge. [online] Available at: [Accessed: 3 Feb 2013].

Phillips, T. & Shaw, K. (2008) Chromatin remodeling in eukaryotes. Nature Education 1(1)