Breast cancer is the most prevalent form of cancer, with more cases worldwide than any other. This article, inspired by the image for February in our 2022 PDBe calendar, focuses on a protein that is one of the key drug targets for breast cancer treatment.
A widespread disease
In 2020, there were 2.3 million women diagnosed with breast cancer, making it the most prevalent cancer worldwide. Thankfully, treatment for breast cancer is generally more successful than many other cancers, however there were still 685,000 deaths around the world in 2020. Therefore, there is a clear need for further development of treatments and diagnostics to combat this disease.
‘Addicted� to HER2
One protein that has been identified as important in the development of many breast cancers is the Human epidermal growth factor receptor-2 (HER2). This protein receptor is found on the surface of nearly every cell in the human body and has been directly linked to the growth of tumours. Proteins such as HER2, which promote the replication of tumour cells are known as oncogenes. HER2 has a role in multiple signalling pathways, including with the related protein HER3, and is important in the regulation of cell survival and proliferation. These signalling pathways appear to be key to the survival of human tumour cells, known as ‘HER2-addicted� cells. Finding treatments that suppress the oncogenic action of HER2 would slow tumour growth and, combined with other drugs, allow the destruction of these tumours.
Targeting HER2 with antibodies
One method that has effectively been used in clinical treatment of breast cancers is the use of specific antibodies to target HER2. The most common of these is trastuzumab, commonly known by the brand name Herceptin, a humanised monoclonal antibody that binds HER2 at its extracellular subdomain IV (see Figure 1). It is thought that binding of this antibody to the HER2 protein interferes with its interactions with other HER2 molecules or with HER3, thereby disrupting its function. Though there is some evidence that this treatment can kill cancer cells, the predominant effect is in prevention of tumour cell growth.
Figure 1: Structure of the complex between HER2 (blue) and trastuzumab/Herceptin (green) in . The interaction of the trastuzumab antibody occurs at the C-terminal region of the HER2 protein. Directly interacting amino acids from both molecules are displayed as spheres and coloured in magenta.
Though treatment of breast cancer with trastuzumab in conjunction with other therapies, such as chemotherapy, has been found to be effective at killing tumour cells, there are some drawbacks of the use of this antibody in treatment. Firstly, the generation of humanised monoclonal antibodies is not cheap - the cost of trastuzumab can be too high for use in many low/middle income countries. Another issue is the potential for resistance to trastuzumab to build up within the tumour, making it ineffective for further treatment. There is therefore a need for additional, more cost-effective therapeutics that target HER2 to combat breast cancer.
Designing alternative therapeutics
One family of molecules which may present an alternative to antibodies as treatment for breast cancers are ankyrin repeat proteins (ARPs). These proteins are involved in mediation of protein-protein interactions in virtually all species, with more than 2000 known ARPs. They are also found in various different regions of the cell, highlighting their ability to adapt to many different environments. These proteins appear to be good alternatives to antibodies for targeting HER2, due to their small size, high stability, efficient folding and easy expression in bacterial cells. Furthermore, these molecules can be fine-tuned for specific interactions with target proteins, with these adapted proteins known as designed ankyrin repeat proteins (DARPins).
The general structure of DARPins involves identical repeat regions, which are stacked into a compact structure. The stability of these molecules increases with the number of repeats, though commonly there are around 4 to 6 six repeats regions of 33 amino acids. Each of these repeats consists of a helix-turn-helix-β-hairpin structural motif, which includes a β-turn followed by two antiparallel helices, joined by a loop to the next β-turn. It is the exposed β-turns, along with the following helices, that form the interaction sites. Usually these interaction regions are formed from multiple adjacent repeats, leading to high affinity protein-protein interactions.
Figure 2: General structure of a DARPin, in this case with five ankyrin repeats, taken from . The structure is shown in cartoon format and coloured by rainbows from blue (N-terminus) to red (C-terminus). Each ankyrin repeat consists of a helix-turn-helix-β-hairpin structural motif, forming a repeated structure of stacked alpha-helices.
Disrupting HER2 function
The structures that inspired the artwork for February in our 2022 PDBe calendar, are complexes of HER2 with DARPin molecules. In , a DARPin is bound to domain I of HER2, whereas in , the DARPin is instead bound to domain IV. These particular DARPins had already been determined to have strong cytotoxic effects on ‘HER2-addicted� tumour cells, particularly when connected together by a short linker region.
By superimposing these regions of HER2 onto the full extracellular domain structure, the authors could show the binding positions of these DARPin relative to each other (Figure 3). They could also indicate that, due to the short length of the linker region, that these DARPin molecules must interact with separate HER2 molecules, orienting these proteins in a way that prevents the formation of homodimers. Subsequently, this leads to a reduction in phosphorylation by both HER2 and HER3, disrupting the proliferation of HER-addicted tumour cells.
Figure 3: Structures of DARPins from PDB entries and , superimposed onto the corresponding interacting domains of HER2, from PDB entry . The structure of HER2 is shown in cartoons and greyed out, with the exception of the labelled domains I and IV, which are coloured yellow. The interacting DARPins are also shown in cartoon representation and coloured based upon the corresponding HER2 domain, with domain I and IV interactions in blue and red, respectively.
A new anti-cancer drug?
This use of DARPins to disrupt the function of HER2 in tumour cells has progressed further, with one (MP0274) now in phase I clinical trials. This DARPin has been designed with multiple domains, with two of these interacting with HER2 domains II and IV, respectively. Like the DARPins in PDB entries and , this drug has been found to interfere with signalling of both HER2 and HER3, inducing apoptosis and significantly inhibiting growth of HER2-addicted breast cancer cells. Though it is still early in the clinical process, hopefully this drug can provide a new and improved alternative for the fight against breast cancer.
David Armstrong
About the artwork
Cancer originates when cells divide uncontrollably and unchecked. In most instances, the loss of control begins with faulty instructions from genes that encode proteins regulating normal cell function. A large number of genes have been identified that can cause cancer and are involved in diverse functions within a cell. Among them are tumour suppressor genes which normally act like ‘brakes� to inhibit cell growth and division whereas oncogenes act like ‘accelerators� to promote cell growth and division. Mutations in these genes can result in defective proteins that often lead to cancers. This artwork from Joshua Wenley of , features one such oncogene, HER2, that is highly expressed in 20% of breast cancers. His artwork draws inspiration from his personal experience and finds beauty in something that can be very frightening.
View the artwork in the .
Structures mentioned in this article
HER2 bound to Herceptin:
HER2 bound to HER3:
General DARPin structure (bound to receptor):
HER2 bound to DARPins:
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