Seasonal variation and correlation between corticosterone and the antioxidant uric acid during stress in the Northern Cardinal (Cardinalis cardinalis).

Glucocorticoids modify metabolism and other physiological processes in order to mount an appropriate response to stress. This stress response is affected by a combination of seasonal changes, life-history events, and environmental factors. Determining seasonal variability and the potential connection between stress hormones and metabolism is fundamental in understanding seasonal physiological changes in animals. Here, we compared an indicator of stress (corticosterone) with an indicator of metabolic activity (uric acid-a non-enzymatic antioxidant and end product of protein metabolism) during capture and restraint in Northern Cardinals (Cardinalis cardinalis) during fall, winter, spring, and summer. Baseline corticosterone, the acute change in corticosterone (Δ10-corticosterone), and integrated corticosterone during capture stress were significantly different among seasons. For all corticosterone measurements birds captured in summer consistently had the lowest values while spring birds were highest. The lower corticosterone stress response in summer may decrease the likelihood of abandonment and thus protect investment in eggs/chicks. Higher glucocorticoid secretion in spring may benefit birds competing for nesting sites. No differences in uric acid levels (baseline, 60 min, and acute change over 60 min- Δ60-uric acid) were found among seasons. While plasma uric acid significantly decreased over an hour in all seasons examined, there were no significant correlations between baseline corticosterone and uric acid, time-60 corticosterone and uric acid, and Δ10-corticosterone and Δ60-uric acid. We conclude that the relationship between corticosterone and metabolism, as measured by uric acid, is indirect, and seasonal variation occurs with corticosterone secretion but not with uric acid, as measured here.

Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake.

Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2-regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2-regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.

Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis.

INTRODUCTION: Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer. METHODS: We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed. RESULTS: Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform. CONCLUSIONS: Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.

Neuregulin 1-activated ERBB4 interacts with YAP to induce Hippo pathway target genes and promote cell migration.

The receptor tyrosine kinase ERBB4, a member of the epidermal growth factor receptor (EGFR) family, is unusual in that ERBB4 can undergo intramembrane proteolysis, releasing a soluble intracellular domain (ICD) that modulates transcription in the nucleus. We found that ERBB4 activated the transcriptional coactivator YAP, which promotes organ and tissue growth and is inhibited by the Hippo tumor-suppressor pathway. Overexpressing ERBB4 in cultured mammary epithelial cells or adding the ERBB4 ligand neuregulin 1 (NRG1) to breast cancer cell cultures promoted the expression of genes regulated by YAP, such as CTGF. Knocking down YAP or ERBB4 prevented the induction of CTGF expression by NRG1, as did treating cells with the ERBB inhibitors lapatinib or erlotinib, which reduced ERBB4 cleavage. NRG1 stimulated YAP activity to an extent comparable to that of EGF (epidermal growth factor) or LPA (lysophosphatidic acid), known activators of YAP. NRG1 stimulated YAP-dependent cell migration in breast cancer cell lines. These observations connect the unusual nuclear function of a growth factor receptor with a mechanosensory pathway and suggest that NRG1-ERBB4-YAP signaling contributes to the aggressive behavior of tumor cells.

Convergent and divergent cellular responses by ErbB4 isoforms in mammary epithelial cells.

UNLABELLED: Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions. IMPLICATIONS: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer.

EGF receptor activates MET through MAPK to enhance non-small cell lung carcinoma invasion and brain metastasis.

MET amplification as a mechanism of acquired resistance to EGF receptor (EGFR)-targeted therapies in non-small cell lung carcinoma (NSCLC) led to investigation of novel combinations of EGFR and MET kinase inhibitors. However, promiscuous interactions between MET and ERBB family members have made it difficult to evaluate the effects of MET on EGFR signaling, both independent of drug treatment and in the context of drug resistance. We addressed this issue by establishing a 32D model cell system wherein ERBBs or MET are expressed alone and in combination. Using this model, we determined that EGFR signaling is sufficient to induce MET phosphorylation, although MET activation is enhanced by coexpression of ERBB3. EGFR-MET cross-talk was not direct, but occurred by a combined regulation of MET levels and intermediary signaling through mitogen-activated protein kinases (MAPK). In NSCLCs harboring either wild-type or mutant EGFR, inhibiting EGFR or MAPK reduced MET activation and protein levels. Furthermore, MET signaling promoted EGFR-driven migration and invasion. Finally, EGFR-MET signaling was enhanced in a highly metastatic EGFR-mutant cell subpopulation, compared with the indolent parental line, and MET attenuation decreased the incidence of brain metastasis. Overall, our results establish that EGFR-MET signaling is critical for aggressive behavior of NSCLCs and rationalize its continued investigation as a therapeutic target for tumors harboring both wild-type and mutant EGFR at early stages of progression.

Genotype-selective combination therapies for melanoma identified by high-throughput drug screening.

UNLABELLED: Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGF receptor and AKT sensitized treatment-resistant BRAF mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results show the use of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF- and RAS-driven melanomas. SIGNIFICANCE: We have used drug combinatorial screening to identify effective combinations for mutant BRAF melanomas, including those resistant to vemurafenib, and mutant RAS melanomas that are resistant to many therapies. Mechanisms governing the interactions of the drug combinations are proposed, and in vivo xenografts show the enhanced benefit and tolerability of a mutant RAS -selective combination, which is currently lacking in the clinic.