While Breast Cancer Awareness Month has ended, the fight against this pervasive disease continues. Approximately one in eight women will develop breast cancer, and by 2050, over 3 million new cases are anticipated annually[1]. One therapeutic approach focuses on receptor tyrosine kinases (RTKs), a protein family whose presence — or excessive presence — is a major risk factor of breast cancer[2].
Among the 90 known human tyrosine kinase genes, the ErbB (also called HER) family stands out for its association with several cancers, including breast cancer. The first FDA-approved targeted breast cancer therapy, Trastuzumab, emerged from studying these HER proteins, and ongoing advancements in biologics similarly depend on understanding ErbB’s role[3,4].
What is ErbB?
The ErbB family comprises four main receptor proteins: HER1, HER2, HER3, and HER4 (Figure 1). Each receptor features an extracellular ectodomain, a transmembrane region, and an intracellular tyrosine kinase domain, with each protein encoded by a distinct gene[5]:
Figure 1. The structure of the four ErbB proteins, side by side.
- HER1 (EGFR)
HER1 forms homodimers or heterodimers with other ErbB proteins and triggers multiple signaling pathways[6]. EGFR is activated by epidermal growth factor (EGF), transforming growth factor-a, and five other ligands. The protein can form homodimers or heterodimers with the other ErbB proteins upon ligand binding[7]. - HER2
HER2 is the first of the HER proteins to be characterized. The transmembrane receptor protein complex can be found in various cell types, including breast and ovarian cells. However, normal tissues have low HER2 membrane protein expression levels[8]. No HER2-specific ligand has been identified, either. Rather, HER2 can form heterodimers with other HER proteins that activate cell signaling[9]. - HER3
HER3 overexpression has also been observed in breast cancers[10]. Also known as c-erbB-3, HER3 is activated by interactions with polypeptides called neuregulins[11]. When activated, HER3 signals processes that affect cell proliferation and migratory behaviors[10]. HER3 can also form a heterodimer with HER2, a preferred heterodimer capable of stimulating cell cycle progression[12]. - HER4
This receptor tyrosine kinase is essential for normal heart, nervous system, and mammary gland development[13]. The protein binds to several ligands, including heregulins and EGF family ligands that also bind to HER1[14].
How ErbB overexpression affects breast cancer risk
The ErbB family of proteins plays a pivotal role in cell signaling, but when these proteins are overexpressed, they can turn from essential communicators to dangerous drivers of cancer growth. In breast cancer, excessive levels of ErbB receptors—especially HER2—are a significant marker of disease severity and poor prognosis[15-17].
Figure 2.
So, what exactly is happening? Overexpression of ErbB receptors often arises from either activating mutations or gene amplification (Figure 2). Activating mutations — small changes in the DNA sequence — can turn these proteins into perpetual “on” switches for cell growth and division. In contrast, gene amplification means there are simply more copies of the gene, leading to an overwhelming abundance of ErbB proteins on cell surfaces. Both of these mechanisms escalate cancer risk in unique ways:
Mutant signals
Activating mutations, also known as driver mutations, confer cells with phenotypes that drive cancer onset[18]. Many kinds of driver mutations can exist. An accurate assessment of these mutations would require a comprehensive survey of known and novel ErbB mutations, along with their links with kinase activity and drug responses[19].
Amplified abundance
Amplification occurs when more copies of a specific gene are present within a genome. HER2, a typically lowly expressed protein, is amplified in 15-20% of breast cancer cases[20]. HER2-negative breast cancer patients can also acquire HER2 gene amplification during cancer progression[21].
Together, these changes disrupt cell behavior, creating a perfect storm for cancer progression. As ErbB proteins ramp up in activity, they fuel breast cancer’s aggressiveness, resistance to treatment, and spread, highlighting the need for precise therapies targeting these powerful proteins.
At the protein level, these mechanisms yield:
- Increased kinase activity: Activating mutations alter the phenotypes of the individual ErbB protein and any resulting dimers. For example, a HER2 containing an insertion mutation increased EGFR phosphorylation even in the presence of EGFR tyrosine kinase inhibitors[22]. Although those mutants were insensitive to EGFR TKIs, they remained sensitive to HER2-targeted therapies.
- Degradation resistance: Although HER2 does not have a known ligand, HER uses a dimerization arm to form a dimer complex with other ErbB proteins such as EGFR, mediating its activity[23]. The dimerized complex reduces EGFR endocytosis, enhancing its signaling in affected cells[24]. The increased activity stems in part from gene amplification[25].
- Reduced inhibitor sensitivity: Gene amplification reduces the effectiveness of HER2-targeting antibodies[26]. Treating HER2-amplified cancers remains a challenge as gene amplification enhances signal transduction through the other ErbB receptor tyrosine kinases to increase cancer cell metastasis, confer resistance to apoptosis, and resist HER2 inhibitors[27].
Challenges in studying ErbB proteins
To innovate breast cancer treatments, scientists need more insight into ErbB proteins individually and in complexes. Purifying these receptors allows for the development of small-molecule inhibitors and biologics aimed at dimerization:
Identifying small-molecule ErbB inhibitors
An efficient protein purification method would also generate multiple protein variants arising from activating mutations. In turn, a eukaryotic cell-based protocol that synthesized the ectodomains of ErbB proteins was developed[28]. A similar pipeline was designed to identify sites within the HER2-HER3 complex that could be targeted by allosteric inhibitors[29].
Developing biologics that inhibit ErbB dimerization
Dimerization is one mechanism by which HER2 overexpression drives breast cancer. Purifying the dimerized complex would elucidate binding mechanisms and reveal new methods to prevent it from happening. By assessing the binding partners of ErbB proteins, tyrosine kinase inhibitors such as Iressa were able to prevent HER2-overexpressing breast cancer cells[30]. Such an effort is especially useful since existing biologics fail to inhibit heterodimer signaling (e.g. HER2-HER3) or inhibit the growth of HER2-expressing tumors[31].
Isolating ErbB proteins is no easy task, largely due to the inherent challenges of working with these delicate molecules:
- Firstly, ErbB proteins are found at relatively low levels within cells, even when their expression is amplified in cancerous states. This scarcity makes it difficult to gather enough protein for analysis, often requiring enrichment techniques to boost yields[32].
- Once isolated, the proteins themselves pose further issues. Maintaining the structure of ErbB proteins outside their natural cellular environment is notoriously difficult. These proteins typically reside in the cell membrane, where their structure is stabilized by lipid layers. Removing them from this environment can cause them to lose their functional shape, despite careful handling with detergents and lipids[33,34].
- Another hurdle comes from their amphipathic nature. ErbB proteins have both hydrophobic and hydrophilic regions, which means they interact differently with water and other molecules. To stabilize these regions, scientists must use detergents, but prolonged exposure to these detergents can lead to protein degradation, making it challenging to keep samples intact long enough for thorough study[35].
Despite these challenges, purifying ErbB proteins holds enormous promise for advancing breast cancer research. Each breakthrough in these techniques brings us closer to understanding how these proteins drive cancer progression, opening the door to therapies that could target them with precision. As researchers refine these methods, the hope is that tailored treatments will emerge, leading to more effective options for patients facing breast cancer.
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