Notch-EGFR/HER2 Bidirectional Crosstalk in Breast Cancer

Abstract

The Notch pathway is a well-established mediator of cell-cell communication that plays a critical role in stem cell survival, self-renewal, cell fate decisions, tumorigenesis, invasion, metastasis, and drug resistance in a variety of cancers. An interesting form of crosstalk exists between the Notch receptor and the Epidermal Growth Factor Receptor Tyrosine Kinase family, which consists of HER-1, -2, -3, and -4. Overexpression of HER and/or Notch occurs in several human cancers including brain, lung, breast, ovary, and skin making them potent oncogenes capable of advancing malignant disease. Continued assessment of interplay between these two critical signaling networks uncovers new insight into mechanisms used by HER-driven cancer cells to exploit Notch as a compensatory pathway. The compensatory Notch pathway maintains HER-induced downstream signals transmitted to pathways such as Mitogen Activated Protein Kinase and Phosphatidylinositol 3-Kinase (PI3K), thereby allowing cancer cells to survive molecular targeted therapies, undergo epithelial to mesenchymal transitioning, and increase cellular invasion. Uncovering the critical crosstalk between the HER and Notch pathways can lead to improved screening for the expression of these oncogenes enabling patients to optimize their personal treatment options and predict potential treatment resistance. This review will focus on the current state of crosstalk between the HER and Notch receptors and the effectiveness of current therapies targeting HER-driven cancers.

Keywords: EGFR; HER2; Notch; breast cancer; crosstalk.

Figure 1

The HER receptor and pathway: (A) there are four members of the HER family of RTKs: EGFR/HER1, HER2, 3, and 4. Each HER receptor is composed of four functional extracellular domains (domain I, II, III, and IV) as well as a tyrosine rich (Tyr) intracellular domain. Epidermal growth factor (EGF), amphiregulin (AREG), heparin binding EGF (HB-EGF), and heregulin 1, 2, 3, 4 growth factor-ligands bind to domains II and III of EGFR/HER1 and HER3, HER4, respectively. Growth factor-ligand binding initiates HER conformational rearrangement causing exposure of the dimerization domain (DD), which is sandwiched between domains II and IV, as well as allowing the closed conformation of the HER receptor (portrayed by EGFR/HER1, HER3, 4) to assume an open conformation (displayed by HER2). It is important to note the truncated intracellular domain of HER3, which lacks kinase activity therefore, the HER3 receptor is the kinase dead member of the HER family of RTKs. (B) Ligand-mediated exposure of the dimerization domain enables the binding of two like HER receptors to form a homo-dimer (left, EGFR–EGFR) or two different HER receptors to form a hetero-dimer (right, HER2–HER3). It is important to note that the HER2 receptor is in a fixed, open conformation and does not require ligand binding to dimerize with a ligand bound HER receptor. (C) Upon HER dimerization, the HER receptors undergo auto-phosphorylation of their intracellular tyrosine residues. Auto-phosphorylation of the HER receptors primes the receptors to transphosphorylate the tyrosine residues of their binding partners (D) in preparation to initiate activation of downstream targets (E) activated HER receptors initiate the AKT (left) and mitogen activated protein kinase (MAPK) (right) pathways through a series of residue targeted phosphorylations, or a phosphorylation cascade. The Src homology domain 2 (SH2) of p85 (regulatory domain of PI3K) docks to the phosphotyrosine residue of the activated HER receptor (EGFR). p85 docking to EGFR enables Src homology domain 3 (SH3) of p110 (catalytic domain of PI3K) to join the proline rich region (Pro) of p85 to initiate PI3K 3′ phosphorylation of phosphatidylinositol (4, 5)-bisphosphate (PIP₂) transforming PIP₂ into phosphatidylinositol (3, 4, 5)-triphosphate (PIP₃). PIP₃ phosphorylation is aided by the Plekstrin homology domain (PHD) within the catalytic domain of phosphoinositide dependent kinase-1 (PDK1). The lipid binding PH domain recruits AKT to the plasma membrane where it docks to PIP₃ as the physical interactions and activations of PI3K, PTEN, PDK1, as well as AKT occurs at the surface of the cell. PDK1 is able to phosphorylate AKT at the Thr 308 residue as well as aid in PKCa activation. AKT phosphorylation disrupts the formation of the tuberous sclerosis 1/2 (TSC 1/2) dimer, inhibits hydrolyzation of the GTP binding protein, Ras homolog enriched in brain (Rheb), to activate the mammalian target of rapamycin complex 1 (mTORC1) complex as well as the mTORC2 complex. The mTORC1 complex is composed of: G protein beta subunit-like (GβL), the metabolically sensitive regulatory-associated protein of mTOR (Rptor), DEP domain-containing mTOR-interacting protein (DEPTOR), and the serine/threonine kinase mammalian target of rapamycin (mTOR). The fully formed mTORC1 complex is able to promote cell growth, proliferation, and autophagy by directing protein translation by mediating the ability of Rptor to recruit the formation of the eukaryotic initiation factor 4F complex [eIF4F (eIF4E, G, B, etc.)] and ribosomal protein S6 kinase beta-1 (S6K1). The mTORC2 complex is also involved in cellular metabolism but mainly facilitates fluctuations in cytoskeletal formation and degradation throughout the cell via serum and glucocorticoid-regulated kinase 1 (SGK1), for instance. In a similar fashion, the SH2 domain of growth factor receptor bound-2 (Grb2) protein docks to the phosphotyrosine residue of the activated HER receptor (HER2). SH2 interaction with the Grb2 scaffold protein initiates the binding of two SH3 domains to the proline rich region of the son of sevenless (SOS) protein. SOS is a guanine-nucleotide exchange factor (GEF) that acts on Ras-GTPases (Ras-GDP) (rat sarcoma). SOS facilitates the unbinding of guanosine diphosphate (GDP) from Ras, there by destabilizing Ras and enabling guanosine triphosphate (GTP) binding to form Ras-GTP, and initiate the MAPK phosphorylation cascade. A GTPase activating protein (GAP) hydrolyzes Ras bound GTP (Ras-GTP), forming GDP, and halting downstream phosphorylation of the MAPK pathway. Ras activation begins the MAPK phosphorylation cascade, which is a series of serine/threoinine specific protein kinases occurring on: rapidly accelerating fibrosarcoma/mitogen activated protein kinase kinase kinase (Raf/MAPKKK), mitogen/extracellular signal-related kinase/mitogen activated protein kinase kinase (MEK/MAPKK), and extracellular signal-related kinase 1/2/mitogen activated protein kinase (Erk1/2/MAPK). Activation of the MAPK pathway initiates the transcription of a host of genes including: c-Myc, c-Fos, and cyclins to orchestrate cell motility, invasiveness, and proliferation.

Figure 2

The Notch receptor and pathway. (A) The Notch receptor is composed of several domains that can be divided between the extracellular domain (NECD) and the transmembrane and intracellular domain (NTMICD). The NECD begins at the N-terminus (NH₃) end of the Notch receptor and contains ligand binding epidermal growth factor-like (EGF) repeats and the negative regulatory region (NRR). Fringe proteins target the EGF repeats for glycosylation, which can augment their ligand binding ability to the Notch receptor. The NRR is composed of Lin12-Notch repeats (L) and the heterodimerization domain (HD), which aid in maintaining a closed conformation of the Notch receptor until ligand binding. The NTMICD contains: the transmembrane domain (T, TMB), the RBP-Jκ associating module (R) or RAM domain, one of two nuclear localization signals (N), the ankyrin domain, a second nuclear localization signal, the cysteine response region (NCR), the transactivating domain (TAD), and a proline/glutamic acid/serine/threonine-rich motif (PEST) at the C-terminal (COOH) end of the Notch receptor. Ligand bound Notch is cleaved at a series of scissile sites (S1, 2, 3, 4), which are represented just outside of (S1) and inside the transmembrane domain (S2, 3, 4) (TMB enlargement). Furin-like convertase cleaves immature Notch at S1 to create the mature, heterodimeric Notch receptor, which is stabilized by a non-covalently bound calcium ion (Ca). The RAM domain facilitates core binding factor-1 (CBF-1) binding, the ankyrin domain binds mammal-like mastermind-1 (MAML-1) and p300 (p300), which are necessary components of the Notch transcriptional activating complex. The PEST domain is responsible for degradation of the Notch intracellular domain (NICD). (B). There are four Notch receptor paralogs, each similar in overall composition but with slight structural differences that facilitate their diverse attributes. From top to bottom are Notch-1, -2, -3, and -4. Notch-1 is the most studied Notch receptor containing all of the above mentioned domains with 36 EGF-like repeats. Notch-2 is similar to Notch-1 except for differences in its EGF-like repeats as well as the ability of Notch-2 to bind to Jagged-1 upon fringe modification (118). Notch-3 has a slightly truncated EGF-like domain with 34 repeats and lacks a transactivating domain. Notch-4 is the smallest of the Notch receptor paralogs with 29 EGF-like repeats, a shorter NICD due to a lack of a transactivating domain and cysteine response region, as well as the addition of a PDZ-Domain [Post-synaptic Density Protein (PSD)-95] (PDZ) that plays a critical role in proper neuronal development (119). (C) The Notch pathway is activated by a Notch-ligand (jagged-1, -2, DLL-1, 3, 4) expressed on a signal sending cell binding to a Notch receptor (Notch 1, 2, 3, 4) on a signal receiving cell. Ligand bound Notch undergoes a series of cleavages, first by a disintigrin and metalloproteinase/tumor necrosis factor alpha converting enzyme (ADAM/TACE) at the S2 cleavage site to form the Notch extracellular truncation (NEXT) fragment. The NEXT fragment is targeted by the γ-Secretase complex for cleavage. The γ-secretase complex is composed of nicastrin, anterior PHarynx-defective 1 (APH-1), presenilin ENhancer 2 (PEN-2), and the active component of the complex, presenilin-1. γ-Secretase is inhibited by γ-secretase inhibitors (GSIs), which prevent Notch cleavage and transcriptional activation. Proper cleavage of the Notch receptor releases NICD, allowing NICD to travel to and enter the nucleus where NICD binds to promoter bound CBF-1, allowing CBF-1 to release negative co-regulators (NCoR) and recruit transcriptional co-activators. NICD-mediated transcription cannot occur without the recruitment of mammalian mastermind-like-1 (MAML-1) and the histone acetyltransferase, p300, to unwind the compressed DNA allowing transcription of the Notch targeted genes. Notch activates the transcription of canonical target genes such as: hairy enhancer of split-1 (HES-1), hairy/enhancer of split with an YRPW motif-1 (HEY-1), and non-canonical Notch target genes such as survivin.

Figure 3

Notch–EGFR crosstalk in TNBC. Dong et al. ascertained a form of crosstalk between EGFR and Notch receptors that enabled resistance to EGFR targeted therapy, gefitinib, in TNBC cells. It is proposed that EGFR targeted resistance occurs by Notch-1-mediated activation of the AKT pathway. Notch–EGFR crosstalk facilitates an increase in serine-473 residue phosphorylation of AKT, which allows TNBC cells to survive EGFR targeted treatments. Dual inhibition of EGFR via the tyrosine kinase inhibitor (TKI) gefitinib and the Notch pathway by GSIs, N-[N- (3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) in vitro, and compound E in vivo, are synergistically lethal to TNBC tumorigenesis.

Figure 4

Notch–HER2 crosstalk in HER2+ BC (A). Ju et al. determined an intricate form of crosstalk between HER2 and Notch-1 in HER2+ breast cancer. HER2+ breast cancer cells exhibit an increase in survivin protein expression concomitantly with a decrease in survivin mRNA expression. HER2 promotes activation of the AKT pathway, which threonine 308 phosphorylated AKT can inhibit p21-mediated inhibition of cyclin dependent kinase-1 (CDK1) dimerization to cyclin B1. Concomitantly, HER2-mediated activation of the MAPK pathway enables Erk-mediated CDK1-Cyclin B1 dimerization as well as inhibition of ubiquitination and proteasomal degradation of survivin by restricting the formation of the X-linked Inhibitor of apoptosis protein-X-linked associating factor-1(XIAP-XAF-1) complex. Together, AKT and Erk promote dimerization of CDK1 to Cyclin B1, which stabilizes survivin by phosphorylating the threonine 34 residue (Thr³⁴) of the survivin protein. Increased survivin stabilization causes an increase in survivin expression. Survivin mRNA is reduced by Erk-mediated inhibition of γ-secretase, which in turn inhibits Notch receptor-mediated transcriptional activation. (B) Pradeep et al. established an interesting form of crosstalk between the HER2 and Notch-3 receptors in HER2+ DCIS. HER2 up-regulates the transcription of the Notch pathway components: jagged-1, -2 (Jag-1/2), DLL-1, ADAM17, presenilin-1 (Pres-1), as well as Notch-3 and Notch-1 (Notch-3/1) through the MAPK pathway. Increased transcription of the Notch pathway components causes Notch-mediated up-regulation of c-Myc, Cyclin D1, phosphorylation of Serine-473 of Atk (+P-Ser 473 AKT), and down-regulation of the tumor suppressor, PTEN. Combined GSI and trastuzumab (Trast) treatment reduce HER2+ DCIS cell survival and invasiveness.

Figure 5

Notch–HER2 crosstalk in HER2+ DCIS. Farnie et al. discerned a form of Notch–EGFR/HER2 bidirectional communication in an in vitro DCIS breast cancer model that promoted acinar size and mammosphere (BCSC) formation. Dual inhibition of the Notch–EGFR/HER2 receptors using DAPT (GSI) and gefitinib/lapatinib, respectfully, caused decreased acinar size and mammosphere formation while either inhibitor alone effected only acinar size or only mammosphere formation.