Maria Sundvall

Signaling via ErbB2 and ErbB3 Associates with Resistance and Epidermal Growth Factor Receptor (EGFR) Amplification with Sensitivity to EGFR Inhibitor Gefitinib in Head and Neck Squamous Cell Carcinoma Cells

Purpose: The epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) has shown antitumor activity in clinical trials against cancers, such as non–small cell lung cancer and head and neck squamous cell carcinoma (HNSCC). Research on non–small cell lung cancer has elucidated factors that may predict response to gefitinib. Less is known about molecular markers that may predict response to gefitinib in HNSCC patients. Experimental Design: We analyzed possible associations of responsiveness to gefitinib with molecular markers of the EGFR/ErbB receptor family signaling pathway using 10 established HNSCC lines in vitro. IC50 of gefitinib sensitivity was determined using clonogenic survival assays. ErbB signaling was assessed by Western and real-time reverse transcription-PCR analyses of EGFR, ErbB2, ErbB3, and ErbB4 expression levels as well as by phosphorylation analysis of pEGFR, pErbB2, pErbB3, pAkt, and pErk. EGFR sequences encoding kinase domain and EGFR gene copy numbers were determined by cDNA sequencing and real-time PCR, respectively. Finally, responsiveness to gefitinib was compared with responsiveness to the anti-EGFR antibody cetuximab (Erbitux). Results: Expression levels of pErbB2 (P = 0.02) and total ErbB3 protein (P = 0.02) associated with resistance to gefitinib. Combining gefitinib with pertuzumab (Omnitarg), an antibody targeting ErbB2 heterodimerization, provided additional growth-inhibitory effect over gefitinib alone on relatively gefitinib-resistant HNSCC cell lines. The same markers did not predict resistance to cetuximab. In contrast, a similar trend suggesting association between EGFR gene copy number and drug sensitivity was observed for both gefitinib (P = 0.0498) and cetuximab (P = 0.053). No activating EGFR mutations were identified. Conclusions:EGFR amplification may predict sensitivity to gefitinib in HNSCC. However, other EGFR/ErbB receptor family members than EGFR may contribute to resistance to gefitinib. ErbB2 and ErbB3 may have potential as predictive markers and as therapeutic targets for combination therapy in treatment of HNSCC with gefitinib.

Cleavable ErbB4 Isoform in Estrogen Receptor–Regulated Growth of Breast Cancer Cells

ErbB1 and ErbB2 receptors are well-characterized targets for anticancer drugs, but the clinical relevance of the related ErbB4 receptor is unknown. Here, we have assessed the clinical significance of the proteolytically cleavable ErbB4 isoforms in breast cancer patients and investigated their functions in vitro. The expression of transcripts encoding the cleavable ErbB4 isoforms associated with estrogen receptor-alpha (ER) expression (P < 0.001) and a high histologic grade of differentiation (P </= 0.002) in real-time reverse transcription-PCR analysis of 62 breast cancer samples. Despite high ErbB4 mRNA expression levels in a subset of samples, ErbB4 gene amplification was not observed. High ErbB4 protein expression levels, as assessed by immunohistochemistry, associated with a favorable outcome in ER-positive cases from a series of 458 breast cancer patients (P = 0.01), whereas no association between ErbB4 expression and survival was found among women with ER-negative cancer (P = 0.86). However, nuclear ErbB4 immunoreactivity was associated with poor survival as compared with women whose cancer had membranous ErbB4 staining (P = 0.04). In vitro, overexpression of a cleavable ErbB4 isoform in ER-positive breast cancer cells resulted in translocation of a proteolytically released intracellular ErbB4 receptor fragment into the nucleus, as well as, enhanced proliferation, anchorage-independent growth, and estrogen response element-mediated transcriptional activity. These results suggest that the association of ErbB4 expression with clinical outcome is dependent on the subcellular localization of ErbB4 and that a proteinase-cleavable ErbB4 isoform promotes growth of ER-positive breast cancer and enhances ER-mediated gene transcription.

ErbB4 and Its Isoforms: Selective Regulation of Growth Factor Responses by Naturally Occurring Receptor Variants

ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family that mediates responses to neuregulins and other EGF-like growth factors. ErbB4 is a central regulator of cardiovascular and neural development as well as differentiation of the mammary gland. A role for ErbB4 has also been implicated in malignancies and heart diseases. Four structurally and functionally distinct ErbB4 isoforms have recently been identified. One pair of isoforms differs within their extracellular juxtamembrane domains. These juxtamembrane ErbB4 isoforms are either susceptible or resistant to proteolytic processing that release a soluble receptor ectodomain. Another pair of ErbB4 isoforms differs within their cytoplasmic tails. Analysis of the intracellular signal transduction pathways indicates that both cytoplasmic ErbB4 isoforms can couple to the Shc-MAPK signaling pathway, while the other one is incapable of coupling to the phosphoinositide 3-kinase (PI3-K)-Akt pathway. The differences in the activation of signaling cascades are reflected in the cellular responses stimulated via the cytoplasmic isoforms. Both cytoplasmic ErbB4 isoforms can stimulate proliferation, but the isoform that cannot activate PI3-K is defective in stimulating cellular survival and chemotaxis. Together these four naturally occurring receptor variants provide a new level of diversity to the control of growth factor-stimulated cellular responses. Thus, the ErbB4 isoforms may have distinct and specific roles in the regulation of various developmental and pathological processes.

A metabolic prosurvival role for PML in breast cancer

Cancer cells exhibit an aberrant metabolism that facilitates more efficient production of biomass and hence tumor growth and progression. However, the genetic cues modulating this metabolic switch remain largely undetermined. We identified a metabolic function for the promyelocytic leukemia (PML) gene, uncovering an unexpected role for this bona fide tumor suppressor in breast cancer cell survival. We found that PML acted as both a negative regulator of PPARγ coactivator 1A (PGC1A) acetylation and a potent activator of PPAR signaling and fatty acid oxidation. We further showed that PML promoted ATP production and inhibited anoikis. Importantly, PML expression allowed luminal filling in 3D basement membrane breast culture models, an effect that was reverted by the pharmacological inhibition of fatty acid oxidation. Additionally, immunohistochemical analysis of breast cancer biopsies revealed that PML was overexpressed in a subset of breast cancers and enriched in triple-negative cases. Indeed, PML expression in breast cancer correlated strikingly with reduced time to recurrence, a gene signature of poor prognosis, and activated PPAR signaling. These findings have important therapeutic implications, as PML and its key role in fatty acid oxidation metabolism are amenable to pharmacological suppression, a potential future mode of cancer prevention and treatment.

Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor

Recruitment of vascular smooth muscle cells (SMC) by endothelial cells (EC) is essential for angiogenesis. Endothelial‐derived heparin binding EGF‐like growth factor (HB‐EGF) was shown to mediate this process by signaling via ErbB1 and ErbB2 receptors in SMCs. 1) Analysis of ErbB‐ligands demonstrated that primary ECs expressed only HB‐EGF and neuregulin‐1. 2) Primary SMCs expressed ErbB1 and ErbB2, but not ErbB3 or ErbB4. 3) Consistent with their known receptor specificities, recombinant HB‐EGF, but not neuregulin‐1, stimulated tyrosine phosphorylation of ErbB1 and ErbB2 and migration in SMCs. 4) Neutralization of HB‐EGF or inhibition of ErbB1 or ErbB2 blocked 70–90% of the potential of ECs to stimulate SMC migration. Moreover, 5) angiopoietin‐1, an EC effector with a role in recruitment of SMC‐like cells to vascular structures in vivo, enhanced EC‐stimulated SMC migration by a mechanism involving up‐regulation of endothelial HB‐EGF. Finally, 6) immunohistochemical analysis of developing human tissues demonstrated that HB‐EGF was expressed in vivo in ECs associated with SMCs or pericytes but not in ECs of the hyaloid vessels not associated with SMCs. These results suggest an important role for HB‐EGF and ErbB receptors in the recruitment of SMCs by ECs and elaborate on the mechanism by which angiopoietins exert their vascular effects.—Iivanainen E., Neli‐markka L., Elenius V., Heikkinen S.‐M., Junttila T. T., Sihombing L., Sundvall M., Määttä J. A., Laine V. J. O., Ylä‐Herttuala S., Higashiyama S., Alitalo K., Elenius K. Angiopoietin‐regulated recruitment of vascular smooth muscle cells by endothelial‐derived heparin binding EGF‐like growth factor. FASEB J. 17, 1609–1621 (2003)

Role of ErbB4 in Breast Cancer

Members of the ErbB subfamily of receptor tyrosine kinases are important regulators of normal mammary gland physiology, and aberrations in their signaling have been associated with breast tumorigenesis. Therapeutics targeting epidermal growth factor receptor (EGFR = ErbB1) or ErbB2 in breast cancer have been approved for clinical use. In contrast, relatively little is known about the biological significance of ErbB4 signaling in breast cancer. This review focuses on recent advances in our understanding about the role of ErbB4 in breast carcinogenesis, as well as in the potential clinical relevance of ErbB4 in breast cancer prognostics and therapy.

Association of Wwox with ErbB4 in Breast Cancer

WWOX, WW domain-containing oxidoreductase, is a tumor suppressor that is altered in many human cancers, including breast cancer. Wwox interacts with the ErbB4 receptor, reduces nuclear translocation of the cleaved intracellular domain of ErbB4, and inhibits its transactivation function mediated through Yes-associated protein. Here, we assessed the clinical significance of the Wwox-ErbB4 association. We determined Wwox protein expression by immunohistochemistry in a series of 556 breast cancers. Wwox expression was absent in 36% of the cancers, and loss of Wwox expression was associated with unfavorable outcome (P = 0.02). Membranous location of ErbB4 was associated with favorable survival compared with women whose cancer lacked such ErbB4 expression (P = 0.02). Wwox expression was strongly associated with membranous ErbB4 localization (P = 0.0003) and with overall ErbB4 expression (P = 0.0002). Coexpression of membranous ErbB4 and Wwox was associated with favorable outcome compared with cases with membranous ErbB4 and no Wwox immunoreactivity (P = 0.002). In vitro, Wwox associated with the two ErbB4 isoforms, JM-a CYT-1 and JM-a CYT-2, expressed in breast cancer. Moreover, expression of Wwox both in vitro and in vivo led to accumulation of total full-length membrane-associated ErbB4. These results suggest that expression of Wwox is associated with ErbB4 expression and that their coexpression has prognostic significance in breast cancer.

Isoform-specific monoubiquitination, endocytosis, and degradation of alternatively spliced ErbB4 isoforms

Endocytosis and subsequent lysosomal degradation serve as a well characterized mechanism to fine-tune and down-regulate EGFR signaling. However, other members of the EGFR/ErbB receptor family have been reported to be endocytosis-impaired. Here we demonstrate that endocytosis of ErbB4 is regulated in an isoform-specific manner: CYT-1 isoforms were efficiently endocytosed whereas CYT-2 isoforms were endocytosis-impaired. CYT-1 isoforms in endocytic vesicles colocalized with Rab5 and Rab7 indicating trafficking via early endosomes to late endosomal/lysosomal structures. A PPXY motif within the CYT-1-specific sequence that lacks from CYT-2 was necessary both for ubiquitination and endocytosis of CYT-1 isoforms and provided a binding site for a WW domain-containing ubiquitin ligase Itch. Itch catalyzed ubiquitination of ErbB4 CYT-1, promoted its localization into intracellular vesicles, and stimulated degradation of ErbB4 CYT-1. Dominant negative Itch suppressed ErbB4 CYT-1 endocytosis and degradation. These data indicate that ErbB4 isoforms differ in endocytosis and degradation by a mechanism mediated by CYT-1-specific PPXY motif interacting with a WW domain-containing E3 ubiquitin ligase.

Function of ERBB4 is determined by alternative splicing.

Alternative splicing is a central tool of evolution that significantly increases the size of transcriptomes and generates functional specification. Within the human ERBB receptor gene family, only ERBB4 is known to produce functionally distinct isoforms as a result of alternative splicing. While ErbB4 signaling has been demonstrated to regulate cellular processes involved in embryogenesis, carcinogenesis and cardiovascular and psychiatric diseases, relatively little is known about the contribution of the individual isoforms in the different biological contexts. Here, we review recent findings as well as provide novel data about the distribution and functions of the ERBB4 splice variants. These observations represent an example of how minor alterations in the transcripts of a single gene can result in even antagonistic cellular responses. The observations also underline the significance of understanding the unique functions of isoforms of a potential drug target gene.

Differential nuclear localization and kinase activity of alternative ErbB4 intracellular domains.

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene β-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.