EGFR and HER2 small molecules inhibitors as potential therapeutics in veterinary oncology

EGFR and HER2 receptors are crucial signaling molecules tyrosine kinase involved in human cancer. Aberrant signaling is associated with a variety of cancers, frequently with poor prognosis. Currently, EGFR and HER2 receptors are being targeted by small molecules, which offer a huge benefit to those patients afflicted by aggressive forms of cancer, improving their prognosis. Both human and canine cancers share molecular, biological, histopathological, and clinical similarities, including EGFR and HER2 expression in some forms of cancer. However, despite the use of one tyrosine kinase inhibitor approved to treat canine mastocytoma, canine cancers overexpressed EGFR and HER2 do not yet have targeted therapy, leading to high morbidity and mortality. Targeting EGFR and HER2 receptors in canine cancers using comparative approaches in human cancer could lead to better outcomes.


Introduction
Cancer is the most important cause of death in dogs worldwide [1][2][3]. As observed in human patients EGFR and HER2 receptors, tyrosine kinase receptors that play a fundamental role in the control of fundamental transduction and signaling cellular pathways involved in cell survival, cell proliferation, angiogenesis, cell adhesion, cell motility, development, and organogenesis, their dysregulation can result in both development and progression of a variety of tumors [4,5]. Their overexpression and amplification are associated with poor prognosis in both species, however by contrast with human medicine, small animal veterinary oncology lacks small molecule inhibitors targeting EGFR and HER2, despite the evidence of in vitro activity in canine cancer cell lines of some approved EGFR and HER2 small molecule inhibitors for use in humans [6,7], in which using inhibitors can provide better prognosis. The objective of this literature research was to review and discuss the role of EGFR and HER2 in the biology of both human and veterinary cancer, and the potential benefit of using EGFR and HER2 small molecule inhibitors in veterinary practice.
The structure presents the typical kinase bi-lobed folding. The N-terminal lobe contains mainly β-strands and a one α-helix, whereas the C-terminal lobe is mostly α-helical [16]. The two lobes are connected through a flexible hinge region and separated by a cleft functioning as docking site for ATP [16].
Globally, EGFR and HER2 consist of an extracellular domain, a single hydrophobic transmembrane αhelix, a juxtamembrane segment, and a catalytic domain with tyrosine kinase activity [12]. ErBb family members exist as monomers, the ligand binding to the extracellular domain results in the formation of either homo or heterodimers, a process that activates the catalytic cytoplasmic domain and the C-terminal phosphorylation initiates further downstream signaling pathways [8].
Although EGFR can HER2 is the preferred dimerization partner to all the other members, the heterodimers forming with HER1 and HER3 manifest a strong signaling activity [8,10,17]. The binding domains of ErbB family are stimulated by eleven polypeptide growth factor ligands distributed in 4 subgroups, which results in the formation of dimers [18]. In contrast to EGFR, HER2 receptors are unable to bind growth factors physiologically. However, HER2 can form functional homodimers under non-physiological expression, an important phenomenon in carcinogenesis [19].

EGFR and HER2 signaling pathways
4 EGFR and HER2 signaling pathways correspond to complex networks, their specificity and potency are determined by several factors, and however, the most important determinant is the variety of binding proteins associated with the carboxy-terminal tail from each member of the family [20]. The autophosphorylated sites are determined both by the ligand identity and by the heterodimer partner [20].
HER2 can binds to a much quantity of phosphotyrosine-binding proteins in contrast to other receptors of the ErbB family [21]. Additionally, EGFR and HER2 heterodimers present higher affinity and broader specificity to various ligands than other heterodimeric receptor complexes in this family, and have slow rates of growth-factor dissociation, slow endocytosis and more recycle to the cell surface [22,23]. This confers potent mutagenic signaling [24] due to the recruitment of multiple signaling pathways [10]. Ras (Ras/Raf/MEK/ERK1/2 pathway), phospholipase C (PLCγ), Shc-activated mitogen-activated protein kinase (MAPK) and STAT (signal transducer and activator of transcription) proteins, especially STAT3 and STAT5 pathway are common targets of all the family ligands and PI3K/AKT are activated by the majority of the active dimers [5,8,25,26]. The activation of simultaneous signaling cascades, including the MAPK pathway, the stress-activated protein cascade, protein kinase C (PKC) and the Akt pathway results in activation of transcriptional activity in the nucleus [8]. This variety of process involves both the proto-oncogenes fos, myc and Sp1 and Egr1 a family of zinc-finger-containing transcription factors and GA-binding protein (GABP) one of the Ets family members [25].
EGFR and HER2 signaling pathways play fundamental roles in cell survival, cell proliferation, angiogenesis, cell adhesion, cell motility, development, and organogenesis [5,25]. However, their aberrant signaling results in key events leading to cancer development and progression [18].

EGFR, HER2, and oncogenesis
EGFR and HER2 dysregulation represent a potent oncogenic trigger [12,27,28]. The underlying mechanisms leading to this dysregulated activity include extracellular and cytoplasmatic domains mutations, which cause increased biosynthesis and levels of ErbB proteins. These mechanisms are tumorspecific [28].
Through receptors deregulation matrix metalloproteinases (MMPs) process its ligands, facilitating autocrine activation and growth signals auto-sufficiency [1,28]. Loss of suppressor genes functions results in EFGR and HER receptors recycling, phenomena that induces the insensibility to growth inhibition [12,28]. PI3K and STAT signaling inhibit apoptosis, a potent mechanism regulator of cellular survival, additionally, STAT and MAPK signaling provide to cells with unlimited replicative potential [12]. Both STAT3 activation and vascular endothelial growth factor (VEGFR) transactivation results in sustained angiogenesis [29]. Finally, PLCγ, MAPK and MMP pathways are associated with invasion and metastasis [12,30].

EGFR and HER2 expression in human cancers
When EGFR and HER2 are deregulated, they can become potent oncogenic triggers [5,31]. Resulting effects from aberrant EGFR and HER2 signaling pathways induce the hallmarks of cancer (selfsufficiency in growth signals, inhibitory signals insensitivity, apoptosis evasion, angiogenesis, unlimited replication, invasive potential, and metastasis) [31,32]. Deregulated receptors induce self-ligands processing, and hence, can establish autocrine-signaling loops, which provides EGFR and HER2 the ability to generate their own growth signals [5]. Frequently, loss of regulation occurs because of degradation evasion, and gain sustained signaling. Evasion results whether from increased reutilization or reduced degradation [33,34]. Additionally, the loss of function of tumor suppressor proteins and constitutively activated receptors [35] increase the effect of inhibitory signals insensitivity [5].

EGFR and HER2 expression in veterinary oncology
Despite the role of EGFR and HER2 is well characterized in human medicine, in veterinary medicine is still in its early stages [53]. In veterinary oncology, several studies have documented the overexpression of EGFR and HER2 and its correlation with prognosis, however, these results have been in certain degree contradictory [54], hence further research is needed [5]. In veterinary cancer, EGFR expression has been demonstrated in a variety of cancer, including head and neck squamous cell carcinoma [55], hepatocellular carcinoma [56,57] , astrocytoma [5,43], and glioblastoma [58]. There is an increasing body of evidence about the function of EGFR either in normal and neoplastic mammary tissue in dogs, and its potential as a pharmacological target [59].
In canine mammary tumors, one of the most common in dogs a recent study reports the positive correlation between EGFR expression by immunohistochemistry with angiogenesis, histological grade of malignancy, mitotic grade, and clinical stage in canine mammary tumors [60]. Another research evaluated EGFR immunohistochemical expression in 138 tumor specimens, and revealed overexpression in 38 tissues (42.2%), this finding was associated with age, and tumoral size, and showed a relation between overexpression and malignant behavior [61]. Based on 5 molecular phenotypes established in breast cancer according to estrogens, progesterone, and HER2 status [62], in canine mammary tumors researchers have attempted to evaluate the potential of this system [62][63][64]. One of these studies have revealed 45 de 241 to triple negative (18.7%), 32 (71.1%) was positive to EGFR, and 13 (28.9%) was negative, and they were associated with pathological parameters (grade III, central necrosis, lymphatic infiltration, and high mitotic index) [62]. Another recent research has quantified EGFR in canine mammary tumors using ELISA in 75 specimens of 45 affected patients and 8 controls and demonstrated a statistically significant difference between both groups and a correlation with relapse, and distant metastasis during follow-up, and both reduced global survival and disease-free time. These findings provide the rationale to the implementation to therapeutic intervention, in particular in cases with aggressive behavior [65].
In HER2, its expression has been documented in diverse types of cancer in dogs, especially in canine mammary tumors, which exists larger emphasis, there exists a correlation between overexpression and malignant behavior [61,66]. Additionally, certain evidence in other types of cancer, such as transitional cell carcinoma in urinary bladder [7,67], and canine osteosarcoma, has also reported [68]. Clonal aberrations have been identified in the HER2 gene in some types of cancer in dogs [69].

First generation of EGFR and HER2 inhibitors Lapatinib
Lapatinib distosylate monohydrate (Tykerb®, GlaxoSmithKline) an oral small molecule reversible inhibitor [73] that targets both EGFR and HER2, was approved by the FDA in March 2007 to be used in combination with capecitabine (Xeloda®; Roche) for patients with metastatic breast cancer overexpressing HER2 and who have received prior therapy including an anthracycline, a taxane and trastuzumab [14]. Lapatinib binds the kinase ATP-binding cleft of EGFR on its inactive conformation in contrast to other HER family inhibitors (erlotinib and gefitinib) that exhibit type I inhibition mechanism [70], additionally uses an allosteric pocket formed by the conformation change of the DFG motif [74].
Lapatinib binds with the inactive conformation of HER4 and HER2 due shares the same contacting residues between both [75], its EGFR, HER2, and HER4 inhibition leads to inhibition of substrate phosphorylation and blocks MAPK and PI3K/Akt and treated cells depending on tumor type can undergo either apoptosis or growth arrest [73].

Second generation of EGFR and HER2 inhibitors Neratinib
Neratinib maleate (Nerlynx®, Puma Biotechnology, Inc), is an irreversible small molecule inhibitor initially designed to target specifically HER receptor using a model of homology for the catalytic domain [76]. Neratinib binds covalently with a cysteine residue in the adenosine triphosphate (ATP)-binding pocket of HER receptor kinases (Cys773 in EGFR and Cys805 in HER2), which is tough as a property to compete with the high concentration of ATP and provide prolongs inhibition of kinase catalytic activity [77]. It is considered as a pan-HER inhibitor because the cysteine residue required for binding is conserved in these three HER receptors [78]. Neratinib inhibits phosphorylation of MAPK [77], retinoblastoma gene product, blocks cell cycle progression, cyclin D1 expression, increase p27 levels (an inhibitor of cell cycle progression), which result in G1-S arrest and an increase in cells with sub-G1 DNA content, associated with apoptosis [77]. Nerlynx® is indicated as adjuvant treatment of patients with early stage HER2overexpressed following adjuvant trastuzumab-based therapy [79].
Quinazoline ring binds to catalytic domain as observed with inhibitors EGFR and HER2 [80] [81] According to efficacy on in vitro and in vivo assays in lung cancer [82,83] afatinib was approved in 2013 as Gilotrif ®, Boehringer Ingelheim Pharmaceuticals, Inc, to the therapy of non-small cell lung cancer. Its potency in vitro is better than gefitinib and lapatinib due to covalent nature [82]. Afatinib has been effective to a variety of cell lines of breast cancer (BT-474, SUM190-PT, SUM 149-PT y T47D). In fact, this finding was crucial to elucidate its action against Her3, associated with the ability to confer cellular survival to cancer cells throughout the PI3K/Akt pathway [84]. Afatinib has demonstrated synergistic activity to mTOR inhibitor rapamycin and trastuzumab [83], and longer duration of action in vitro in contrast with reversible inhibitors erlotinib, gefitinib y lapatinib [82,85]. Nowadays the potential role of afatinib as adjuvant therapy with paclitaxel in breast cancer is investigated, however, the results have not yet released [86]. In breast cancer, afatinib has been administered because of HER2 inhibition with promising results in HER overexpressing patients with combination to first-line therapies [87]. Adverse effects documented are very similar to those observed with lapatinib (diarrhea and skin reactions).

Dacomitinib
Dacomitinib inhibits irreversibly EGFR, HER2, and HER4. Its preclinical efficacy was demonstrated only on EGFR T790M, and some HER2 cell lines [88][89][90]. In both phases, I and II, dacomitinib was safe and efficient in patients with non-small cell lung cancer, even compared with patients treated with erlotinib [91]. Adverse effects observed in different trials are diarrhea, dermatitis acneiform, and other types of skin toxicities, and stomatitis [91]. In 2018 was approved the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 19 deletion or exon 21 L858R substitution mutations.

Comparative oncology: opportunities for both species
The insights in the molecular and genomic research of cancer have improved the understanding about the similarities between humans and dogs and have led to the concept of comparative oncology, which has the objective to accelerate simultaneously the cancer drug development in both species [92]. Whole canine genome sequence and assembly released in 2005 and improved in 2014 has revealed strong similarities in genes implied in cancer development and progression [93,94]. Additionally, it is widely accepted that both humans and dogs share certain features in cancer, including determined risk factors, such as age, hormonal, environmental influence, histological types, overexpression of a variety of biomarkers such as cellular proliferation (Ki67, AgNor), p53 mutations, EGFR, MMPs, cyclooxygenase 2 (COX2), molecular, clinical behaviors and outcomes, however also exist significant differences [95][96][97][98].

Tyrosine kinase inhibitors in veterinary oncology
Actually, in veterinary oncology only exists one small molecule inhibitor of RTKs approved to clinical use in canine patients by FDA, toceranib phosphate (Palladia ®, Pfizer Animal Health), an oral small molecule inhibitor targeting VEGFR2, platelet-derived growth factor receptor (PDGFRα) and c-kit [99].
Due Palladia® has structural similarity with Sunitinib® an oral small molecule inhibitor targeting VEGFR2, VEGFR3, PDGFRα/β, KIT, CSF1R, FLT-3, and RET, probably has an additional activity to these molecules [100]. Toceranib phosphate was initially developed as an antiangiogenic drug, however, due to its wider pharmacological profile that includes KIT and FLT-3 causes antitumor activity [101].
Its first clinical efficacy in canine cancer patients was a phase 1 clinical trial, which included 57 dogs with diverse temporal types including sarcomas, carcinomas, melanomas, myelomas, and mastocytomas. The response in 16 patients was observed: 6 with complete response and 10 with partial response and stable disease. This study documented a biological activity of 54% [99].
After its FDA approved licensed in June 2009, has been utilized as an extra label in other temporal types that failed to standard treatments, including metastatic osteosarcoma, anal sacs adenocarcinoma, thyroid carcinoma, head, and neck carcinoma, and nasal carcinoma documented clinical benefit in 63 of 85 dogs [102]. Additionally, have been documented responses in a case of [103], synergistic activity with piroxicam in transitional and squamous cell carcinoma and with vinblastin in mastocytoma [104,105].
In combination with radiotherapy its efficacy was evaluated, was observed the objective response in 76.4%, 58.8% of canine patients achieved a complete response and 17.6% partial response, which suggest clinical benefit [106].
An interesting in vitro study with Gefitinib (Iressa1®, AstraZeneca), an oral small molecule inhibitor targeting EGFR, conducted in a cellular line of CMT (REM134) demonstrated a favorable effect in cellular proliferation, migration. Additionally, other small molecule inhibitors such as AG825, which targeting HER2 and GW583340, which targeting EGFR and ERBB2, have documented activity, with the potential use in veterinary oncology clinical trials [6].

Conclusions
As documented in human cancer, in canine patients the aberrant expression of EGFR and HER2 receptors constitute a potent oncogenic trigger, however in canine patients still clearly unresolved the expression patterns and the precise molecular structure, which limit its inclusion in validated clinical trials. Despite the increasing evidence of the biological activity of EGFR and HER2 small molecule inhibitors using in human medicine in canine cancer cell lines, their clinical use not have been yet received special attention, in contrast to toceranib phosphate and masitinib mesylate in veterinary oncology. Actually, there is an increasing interest in developing targeted therapies in veterinary oncology. The in vitro efficacy documented regarding some small molecule tyrosine kinase inhibitors, can constitute an interesting starting point to initiate the investigation of therapeutic potential to target EGFR and HER2 in veterinary cancer and then improve the clinical outcomes, survival rates, and optimize health quality of life of these patients. Veterinary cancer high prevalence requires targeted therapies, and the routinely uses of EGFR and HER2 small molecules inhibitors represent an excellent opportunity to address this worldwide problem that affects dogs.

Disclosure statement
No potential conflict of interest was reported by the authors.