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KDM4 – an efficient target for the therapy of triple-negative breast cancer

While breast cancer survival has clearly improved in recent years, there are still tumour types that are resistant to treatment. Women with triple-negative breast cancer have benefitted very little from progress in cancer medicine. Targeted therapies aimed at inhibiting epigenetic regulators might offer a potential new option for the treatment of breast cancer. Prof. Dr. Roland Schüle and Dr. Jochen Maurer have discovered an epigenetic enzyme called KDM4 and come up with a new cell model that significantly facilitates the development of new cancer drugs.

Breast cancer is the most common cancer in women. Strictly speaking, breast cancer refers to a group of disorders. Standard forms of therapy such as chemo-, radiation and antihormone therapy achieve good to very good results overall. If the lymph nodes are not affected, breast cancer patients have a 5-year survival rate of 95 percent. The genetic analysis of cancer has led to a significant change in cancer treatment. It is now possible to treat tumours that were previously largely resistant to treatment. Around 20 percent of breast cancers are extremely aggressive triple-negative breast cancers. Treatment with many approved therapies fails as these types of breast cancer seem to be resistant to many approved therapies. In addition, the probability of recurrence and the risk of metastasis are much higher. Triple-negative breast cancers are particularly dangerous as chemo- and radiation therapies eliminate all but a few cancer cells. And this small population of cells in tumours is sufficient for the tumour to continue to grow.

Many tumours have hormone receptors

How do epigenetic mechanisms enable the differentiation of stem cells? Prof. Dr. Roland Schüle from the Center for Clinical Research (ZKF) at the Freiburg University Medical Centre, is specifically focusing on this topic. © Jürgen Gocke (private)

Diagnosis of triple-negative breast cancers is based on the receptor state: tumours that develop in hormone-sensitive tissues can be recognised by their typical hormone receptors. Around four out of five breast cancer patients test positive for oestrogen and progesterone receptors. They share these characteristics with normal breast cells. This becomes an important issue when the hormones also help the cells to continue to grow. Hormone-sensitive tumours that lack normal growth control, continue to grow when they receive signals from oestrogen and progesterone. If breast cancer cells have hormone receptors, antihormone therapies could help to eliminate growth stimuli and slow down or even stop the growth of metastases.

Triple-negative breast cancer cells express neither oestrogen and progesterone receptors nor HER2/neu receptors (IGF family growth factor) on their surface. This is what makes treatment rather difficult. Cells without oestrogen, progesterone and HER2/neu receptors do not respond to targeted hormonal and antibody therapies. There are different reasons why the cells lack these receptors: genetic deletions or mutations, epigenetic inactivation or even induced resistance to therapy. “A common theory is that the tumour uses this mechanism to evade treatment,” says Prof. Dr. Roland Schüle, scientific director of the Center for Clinical Research (ZKF) at the Freiburg University Medical Centre.

Cancer stem cells with epigenetic abnormalities

Schüle believes that stem cells inside the tumour are the driving force behind this particularly malignant cancer. “The tumour is very heterogeneous. It does not just contain one type of tumour cells, but a whole set of different ones,” Schüle explains. From an epigenetic point of view, the epigenetic modification pattern of tumour stem cells is different from that of other tumour, somatic and germ cells.

Changes at the epigenetic level, such as DNA methylation or histone modification, control gene activity and lead to certain phenotypes. It is now known that the magnitude of epigenetic modifications people experience during their lifetime is several times higher than that of genetic mutations. Epigenetic mechanisms apparently also play a significant role in cancer development. There is well-founded evidence that in certain tumours over 50 percent of all mutations occur in epigenetic proteins.

Scientists in Schüle’s group were able to successfully inhibit the enzyme KDM4, which is responsible for the characteristics of stem cells in the tumour and thus triple-negative breast cancer’s resistance to therapy. (KDM4 catalyses the removal of methyl groups from certain histone lysine residues, and thus plays a key role in regulating gene expression.) Like somatic stem cells, tumour stem cells possess the capacity to self-renew and consistently produce new cancer cells. The differentiated tumour cells, i.e. the tumour mass, can be eliminated with standard therapy; the tumour shrinks, which is taken as a sign of success. “The problem is that the stem cell sits somewhere in the tumour and does not respond to standard cancer therapies. It only divides rarely, and does not express the three receptors,” says Schüle. “It can sit there for months or years before it suddenly starts dividing again.” Can the tumour stem cell have “learned” from the mass of other cells that a chemical attack has taken place and is therefore now trying to protect itself? This is where epigenetics comes into play.

Spheroid cell model and drug design

Dr. Jochen Maurer has developed the new spheroid cell model. It provides new insights into the characteristics of cancer stem cells. © private

The KDM4 demethylase is a promising druggable target. Jochen Maurer from the Centre for Translational Cell Research at Freiburg University Medical Centre, and Schüle’s team showed in a newly developed cell model how KDM4 is inhibited in cancer stem cells and that these cells lose their malignant stem cell properties. Maurer performed pioneering work in that he was able to culture cancer stem cells from patients with triple-negative breast cancer in vitro and keep them alive for up to one year. Normally, the conditions of in vitro experiments cause cells to lose their stem cell characteristics and immediately differentiate. With a particular mixture, a hypoxic atmosphere of only three percent oxygen and a Rho kinase inhibitor, Maurer created the special microenvironment that allows stem cells to remain as stem cells.

He designed a 3D model, a spheroid, in which stem cells and differentiated tumour cells were at different stages. This model simulated the entire tumour outside of the body and allowed the researchers to examine a number of KDM4 inhibitors without having to use animal models. At the same time, Schüle’s team joined forces with a company called Quanticel Pharmaceuticals (ed. note: since October 2015 Celgene Quanticel Research, San Diego, CA, USA) to develop a sophisticated drug design that was able to identify enzyme inhibitors with an antiproliferative effect. “We started off with half a million different molecules that we tested in biophysical approaches for their binding and blockage capacity,” says Schüle. In a lengthy process, the researchers optimised a substance (QC6532), which fits nicely into the binding pocket of the active centre of KDM4, thus paralysing it. Stem cells that are treated with QC6532 lose the ability to self-renew, and they differentiate into tumour cells that respond to standard therapies.

Shrinkage of the tumour at the first attempt

In the new spheroid model, keratin 5 (green) and keratin 8 (red) mark breast cancer stem cells. The cell nucleus is blue. © Dr. Jochen Maurer, Uniklinik Freiburg

The researchers removed all individual cells from a spheroid and treated them with the inhibitor substance for one week to find out whether this is a specific effect or only a toxic effect. They wanted to know whether the cells were subsequently able to form new spheroids. Around a hundred new spheroids formed in the absence of an inhibitor, and only two in the presence of an inhibitor. Cells of the two new spheroids were isolated, but not treated again. The researchers found that “new spheroids did not form even in absence of the inhibitor”. Schüle comments: “We can assume that the effect is specific, but also that it is enough to treat the tumour cell once in a time window in which it is sensitive to the drug.”

Epigenetic modifications usually occur rather slowly, but manifest themselves in the second generation. A substance with low toxicity and only a few side effects prevented KDM4 in vitro from removing methyl groups and activating or inactivating genes involved in proliferation and metabolism. The tumour also shrank in the animal model. Many experiments still need to be carried out. “If we can substantiate our current findings in additional models, we will at least have a general idea of how to eliminate a tumour,” says Schüle.

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