MICA: Therapeutic targeting of histone acetylation in scars

Millions of people worldwide suffer trauma or undergo surgery each year, and in turn they inevitably develop scars. In the skin, mild scarring may present only a minor aesthetic problem, but wound healing and scar formation can proceed out of control in a subset of the population, resulting in disfiguring and painful keloid scars. To date, there has been little clinical success in preventing or reducing scars, but this project will evaluate the anti-scarring potential of two novel drug classes, HDAC inhibitors and BET inhibitors.
The two families of cellular proteins that we will investigate in this project (the potential therapeutic targets) are histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) proteins. HDACs, by removing a chemical modification (acetylation), are capable of altering the activity and stability of many proteins within a cell. They are most well known for altering histones, which are proteins involved in the packaging of DNA. Histone acetylation promotes gene expression; therefore HDACs, by removing histone acetylation, contribute to gene silencing. BETs, the other family of proteins under investigation, facilitate gene expression by binding to acetylated histones and thereby mediate recruitment of transcriptional complexes (potentially this could have an opposing effect to HDACs). This project aims to gain insight into their respective roles in the wound healing process in health and disease.
Our interest in HDACs has stemmed from our own observation that one particular family member, HDAC2, is significantly over-expressed in human scars (normal and keloid). Also, the work of others has shown that drugs that inhibit HDACs have anti-fibrotic potential. What is not yet clear is which HDAC family member(s), and which target proteins are responsible for these anti-fibrotic outcomes. Our hypothesis is that HDAC2 is a particularly important family member that promotes scarring, and its roles will be addressed in this work. Studying the role of BETs during scar formation provides us with a powerful alternative strategy to investigate the functional importance of histone acetylation during this process. Moreover, there is preliminary evidence that BET inhibition could also have anti-fibrotic outcomes. To reconcile how inhibition of two potentially opposite-acting proteins could both be anti-fibrotic, we suspect that they have distinct targets whose expression or activity levels they influence.
Having discovered that HDAC2 is over-abundant in normal and keloid scars, our first objective is to investigate the functional role of HDAC2 during scar formation. We will manipulate its expression and activity in cultured cells from normal skin and keloids, and observe the consequences on scar-associated cell behaviours. We also hope to unveil the HDAC2 target proteins that help mediate its effects. Next, the relative abundance of BET proteins will be evaluated in our models of scar formation (cell cultures, an animal model of wound-induced scar formation, and human scar samples). The BET protein most anticipated to have a functional role during scar formation will then be genetically and pharmacologically manipulated in cultured cells (as with HDAC2) and the cellular effects will be observed. Finally, we will begin to test the value and feasibility of inhibiting HDACs or BETs to counter wound-induced scarring by investigating the effects on an in vivo model of scarring.
With this project we hope to bring these novel anti-scarring treatment strategies closer to the clinic, by improving our understanding of how they work, and by testing the feasibility of their use. It is not only skin that is vulnerable to scarring and fibrosis; many organ systems in the human body (for example, lung, kidney, heart), if diseased will develop fibrosis, and the severity of scarring correlates significantly with disease mortality. Ultimately we hope that our findings can benefit a range of fibrotic conditions.

Despite the ubiquitous nature and severe morbidity associated with scarring, there are no effective means to treat or prevent this problem. We recently found that histone deacetylase (HDAC)2 is significantly over-expressed in normal and keloid scars and suggest it may be viable therapeutic target. Evidence is increasing that HDAC inhibitors can indeed decrease fibrosis; however, it is not yet clear whether this is true for skin scars, or which HDAC family members, and which target proteins are responsible. Our first objective is to use genetic (over-expression/knock-down) and pharmacological approaches to manipulate HDAC2 in primary cultures of normal and keloid-derived dermal fibroblasts, and in this way define its contribution to cell proliferation, survival, differentiation and migration. The target proteins mediating the effects of HDAC2 in this context will also be explored. Next, to gain further insight into the importance of histone acetylation in scars, a second family of enzymes involved in its regulation will be studied. BET (bromodomain and extra-terminal domain) proteins (Brd2, 3, 4 and T) facilitate histone acetylation-mediated gene transcription, and we recently discovered that their inhibition in dermal fibroblasts inhibits proliferation and alters extracellular matrix gene expression. This led us to hypothesize that their inhibition may be of value in treating scars. The abundance and localization of Brd2, 3 and 4 will be analysed in: 1) primary human dermal fibroblast cells (normal and keloid, with or without TGFbeta1); 2) an in vivo model of wound-induced scar formation; and 3) normal and keloid human scar samples. Genetic and pharmacological approaches will again be used, this time to manipulate a BET protein in vitro, to define its contribution to scar-associated cellular changes. Finally, we will examine the value and feasibility of using an HDAC or BET inhibitor to counter wound-induced scarring by investigating the effects in an in vivo model.

The proposed research will characterize the functions of HDAC2 and BETs in normal and keloid scar formation, and the therapeutic value and feasibility of their inhibition. It is via the academic beneficiaries that we predict this work will have its most immediate impact. The areas of research that we foresee may benefit from our findings are: tissue repair, dermatology, fibrosis, epigenetics, and pharmacology (as described in the "Academic Beneficiaries" section).

Our industrial partner, GlaxoSmithKline will benefit from the proposed work in a number of ways. First, they will gain a dedicated team of collaborators that genuinely share both their curiosity about the diverse roles of BETs, and their enthusiasm to see the research outcomes benefit the health of patients. In more concrete terms, the proposed project will provide access to established models of tissue repair and valuable human skin and scar tissue, to assess the efficacy of novel BET inhibitors and highlight potential for new therapies. They will have an opportunity to test their emerging compounds in a system (healing wounds) that will give insight into many biological processes (e.g. inflammation, angiogenesis, scar formation, cell migration and proliferation). This project may also pave the way for potential future collaborations with scientists at St. George's with unique and valuable biological assays.

By proposing to evaluate the anti-scarring potential of two novel drug types; it is hoped that the research outcomes will benefit the millions of people worldwide who suffer trauma or undergo surgery each year, and in turn inevitably develop scars.

Throughout the project, we propose to analyse both normal and keloid scars, with the goal that we will reveal characteristics unique to keloids, which may improve our understanding of their etiology, and provide an exploitable feature from a therapeutic stand point. I hope that this work will benefit the 5-15% of people who are susceptible these debilitating scars. Keloids are pathologically overgrown scars that can be disfiguring and also very painful. They have a extremely negative impact on an individual's quality of life, with both physical and psychological effects. Current treatment strategies are rarely successful, with regrowth virtually inevitable. If this project was to reveal a effective, non-surgical treatment strategy, the societal impact would be substantial.

It is not only skin that is vulnerable to scarring and fibrosis; many organ systems in the human body (e.g., lung, kidney, heart), when diseased will develop fibrosis, and the extent of scarring correlates significantly with disease mortality. If inhibition of HDACs and/or BETs shows promising anti-scarring results in skin in this project, it may indicate that the same strategy would also be of therapeutic benefit to patients suffering from other fibrotic conditions. Finally, by testing HDAC and BET inhibitors in healing wounds, we will gain insight into the effects of these drugs on a number of biological processes (e.g. inflammation, angiogenesis, cell migration and proliferation). At this stage it is impossible to predict precisely which patient populations may ultimately benefit, but it would certainly be of great clinical benefit if the compounds tested exhibited anti-inflammatory, or anti-angiogenic properties.

The clinical and industrial collaborations inherent in this project will provide an excellent career-developing experience for the Post-doctoral Research Fellow and myself. This project also provides a fantastic training opportunity since the Research Fellow will gain expertise in a wide-range of techniques, ranging from in vivo experimentation and histology, to molecular biology. Moreover, our lab environment is collaborative and supportive, and St. George's, University offers excellent development opportunities for staff to expand on their research and professional skills (specific and transferrable).