The Utility of ERBB4 and RB1 Immunohistochemistry in Distinguishing Chromophobe Renal Cell Carcinoma From Renal Oncocytoma.

Objectives. Differentiating renal oncocytoma (RO) from chromophobe renal cell carcinoma (ChRCC) can occasionally be challenging. We evaluated the expression of RB1 and ERBB4 in RO and ChRCC, and compared the immunohistochemistry (IHC) results to RB1 and ERBB4 gene abnormalities detected by fluorescence in situ hybridization (FISH). Materials and Methods. Fifty-three kidney resections (ChRCC, n=28; RO, n=25) were stained for RB1 and ERBB4 IHC and FISH was performed to evaluate gene copy number analysis. Results. A loss of RB1 staining was identified in 64% (18/28) of ChRCCs, which was not found in any ROs (0/25; P <.001). FISH analysis revealed 36% (10/28) of ChRCCs contained a RB1 hemizygous deletion with a concordance of 56% (10/18) between the IHC and FISH findings. No RB1 gene copy number variations were detected in any of the ROs (0/25; P <.001) and retained expression of RB1 by IHC. ERBB4 showed cytoplasmic/membranous staining in all ROs and ChRCCs. However, 75% (21/28) of ChRCCs also contained nuclear positivity for ERBB4, which was uncommonly seen in ROs (3/25, 12%; P < .001). A hemizygous ERBB4 gene deletion was detected in 46% of ChRCCs (13/28), but none of the ROs (0/25; 0%). Loss of labeling by RB1 or nuclear staining for ERBB4 IHC identified 25 of 28 (89%) of ChRCCs. Conclusion. In summary, the loss of RB1 expression is a highly specific diagnostic biomarker in distinguishing ChRCC from RO. Nuclear ERBB4 expression also appears to be a sensitive diagnostic biomarker for ChRCC, albeit the mechanism is unknown.

Neuregulin and ErbB expression is regulated by development and sensory experience in mouse visual cortex.

Neuregulins (NRGs) are protein ligands that impact neural development and circuit function. NRGs signal through the ErbB receptor tyrosine kinase family. NRG1/ErbB4 signaling in parvalbumin-expressing (PV) inhibitory interneurons is critical for visual cortical plasticity. There are multiple types of NRGs and ErbBs that can potentially contribute to visual cortical plasticity at different developmental stages. Thus, it is important to understand the normal developmental expression profiles of NRGs and ErbBs in specific neuron types in the visual cortex, and to study whether and how their expression changes in PV inhibitory neurons and excitatory neurons track with sensory perturbation. Cell type-specific translating ribosome affinity purification and qPCR was used to compare mRNA expression of nrg1,2,3,4 and erbB1,2,3,4 in PV and excitatory neurons in mouse visual cortex. We show that the expression of nrg1 and nrg3 decreases in PV neurons at the critical period peak, postnatal day 28 (P28) after monocular deprivation and dark rearing, and in the adult cortex (at P104) after 2-week long dark exposure. In contrast, nrg1 expression by excitatory neurons is unchanged at P28 and P104 following sensory deprivation, whereas nrg3 expression by excitatory neurons shows changes depending on the age and the mode of sensory deprivation. ErbB4 expression in PV neurons remains consistently high and does not appear to change in response to sensory deprivation. These data provide new important details of cell type-specific NRG/ErbB expression in the visual cortex and support that NRG1/ErbB4 signaling is implicated in both critical period and adult visual cortical plasticity.

Altered ErbB4 splicing and cortical parvalbumin interneuron dysfunction in schizophrenia and mood disorders.

Working memory requires the activity of parvalbumin (PV) interneurons in the dorsolateral prefrontal cortex (DLPFC). Impaired working memory and lower PV expression in the DLPFC are reported in schizophrenia and to a lesser degree in mood disorders. We previously proposed that activity-dependent PV expression is lower in schizophrenia due to a shift in the splicing of erb-b2 receptor tyrosine kinase 4 (ErbB4) transcripts from major to inactive minor variants that reduces excitatory drive to PV interneurons. Here, we tested the hypothesis that the degree of major-to-minor shift in ErbB4 splicing predicts the level of PV expression across schizophrenia and mood disorders. Levels of ErbB4 splice variants and PV mRNA were quantified by PCR in the DLPFC from 40 matched tetrads (N = 160 subjects) of schizophrenia, bipolar disorder (BD), major depressive disorder (MDD), and unaffected comparison subjects. Relative to unaffected comparison subjects, the magnitude of increases in minor variant levels and decreases in major variant levels was greatest in schizophrenia, intermediate in BD, and least in MDD. The same rank order was present for the magnitude of increases in the composite splicing score, which reflects the degree of major-to-minor shift across all ErbB4 splice loci, and for the magnitude of deficient PV expression. Finally, the composite splicing score negatively predicted PV expression across all subject groups. Together, these findings demonstrate a shared relationship between ErbB4 splicing and PV expression and suggest that scaling of the major-to-minor shift in ErbB4 splicing may influence the severity of deficient PV interneuron activity across diagnoses.

Expressions and clinical significance of HER4 and CD44 in sinonasal mucosal malignant melanoma.

Sinonasal mucosal malignant melanoma (SNMMM) is a rare disease. The aim of this study was to investigate the expressions of HER4 and CD44 in human SNMMM tissues and their relationship with the clinicopathological features and prognosis of patients. In total, 64 paraffin-embedded samples of SNMMM treated in our hospital from 29 December 1999 to 24 June 2011 were collected. HER4 and CD44 were detected in the tissues of SNMMM by immunohistochemistry. The differences in the HER4 and CD44 expressions in the tissues were evaluated and matched with clinicopathological parameters and the survival rate, respectively. The positive rates of the HER4 and CD44 expressions were 70.3 and 65.6%, respectively; the positive expression of HER4 was correlated with a positive expression of CD44 (P<0.05). The positive expression of HER4 was correlated with the prognosis of SNMMM patients (P<0.05). There was no significant correlation between a positive expression of CD44 and the prognosis of patients (P>0.05). The expressions of HER4 and CD44 were not significantly correlated with sex, age, pigment, tumor site, etc. (P>0.05). Our results further emphasize a correlation between HER4 and CD44 expressions in SNMMM tissues and point out that a positive HER4 expression might be an important factor in valuing the prognosis of patients with SNMMM.

Clinical significance of overexpression of NRG1 and its receptors, HER3 and HER4, in gastric cancer patients.

BACKGROUND: Neuregulin 1 (NRG1), a ligand for human epidermal growth factor (HER) 3 and HER4, can activates cell signaling pathways to promote carcinogenesis and metastasis. METHODS: To investigate the clinicopathologic significance of NRG1 and its receptors, immunohistochemistry was performed for NRG1, HER3, and HER4 in 502 consecutive gastric cancers (GCs). Furthermore, HER2, microsatellite instability (MSI), and Epstein-Barr virus (EBV) status were investigated. NRG1 gene copy number (GCN) was determined by dual-color fluorescence in situ hybridization (FISH) in 388 available GCs. RESULTS: NRG1 overexpression was observed in 141 (28.1%) GCs and closely correlated with HER3 (P = 0.034) and HER4 (P < 0.001) expression. NRG1 overexpression was significantly associated with aggressive features, including infiltrative tumor growth, lymphovascular, and neural invasion, high pathologic stage, and poor prognosis (all P < 0.05), but not associated with EBV, MSI, or HER2 status. Multivariate analysis identified NRG1 overexpression as an independent prognostic factor for survival (P = 0.040). HER3 and HER4 expressions were observed in 157 (31.3%) and 277 (55.2%), respectively. In contrast to NRG1, expression of these proteins was not associated with survival. NRG1 GCN gain (GCN ≥ 2.5) was detected in 14.7% patients, including two cases of amplification, and was moderately correlated with NRG1 overexpression (κ, 0.459; P < 0.001). CONCLUSIONS: Although our results indicate a lack of prognostic significance of HER3 and HER4 overexpression in GC, overexpression of their ligand, NRG1, was associated with aggressive clinical features and represented an independent unfavorable prognostic factor. Therefore, NRG1 is a potential prognostic and therapeutic biomarker in GC patients.

ErbB4 protects against neuronal apoptosis via activation of YAP/PIK3CB signaling pathway in a rat model of subarachnoid hemorrhage.

Neuronal apoptosis is a central pathological process in subarachnoid hemorrhage (SAH)-induced early brain injury. Previous studies indicated that ErbB4 (EGFR family member v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) is essential for normal development and maintenance of the nervous system. In this study, we explored the neuroprotective effects of ErbB4 and its downstream YAP (yes-associated protein)/PIK3CB signaling pathway in early brain injury after SAH in a rat model using the endovascular perforation method. Rats were neurologically evaluated with the Modified Garcia Scale and beam balance test at 24h and 72h after SAH. An ErbB4 activator Neuregulin 1ß1 (Nrg 1ß1), ErbB4 siRNA and YAP siRNA were used to explore this pathway. The expression of p-ErbB4 and YAP was significantly increased after SAH. Multiple immunofluorescence labeling experiments demonstrated that ErbB4 is mainly expressed in neurons. Activation of ErbB4 and its downstream signals improved the neurological deficits after SAH and significantly reduced neuronal cell death. Inhibition of ErbB4 reduced YAP and PIK3CB expression, and aggravated cell apoptosis. YAP knockdown reduced the PIK3CB level and eliminated the anti-apoptotic effects of ErbB4 activation. These findings indicated that ErbB4 plays a neuroprotective role in early brain injury after SAH, possibly via the YAP/PIK3CB signaling pathway.

Abnormal wiring of CCK+ basket cells disrupts spatial information coding.

The function of cortical GABAergic interneurons is largely determined by their integration into specific neural circuits, but the mechanisms controlling the wiring of these cells remain largely unknown. This is particularly true for a major population of basket cells that express the neuropeptide cholecystokinin (CCK). Here we found that the tyrosine kinase receptor ErbB4 was required for the normal integration into cortical circuits of basket cells expressing CCK and vesicular glutamate transporter 3 (VGlut3). The number of inhibitory synapses made by CCK+VGlut3+ basket cells and the inhibitory drive they exerted on pyramidal cells were reduced in conditional mice lacking ErbB4. Developmental disruption of the connectivity of these cells diminished the power of theta oscillations during exploratory behavior, disrupted spatial coding by place cells, and caused selective alterations in spatial learning and memory in adult mice. These results suggest that normal integration of CCK+ basket cells in cortical networks is key to support spatial coding in the hippocampus.

Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1.

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFß signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-ß1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-ß1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

MicroRNA-146b, a Sensitive Indicator of Mesenchymal Stem Cell Repair of Acute Renal Injury.

: The role of mesenchymal stem cells (MSCs) in kidney injury repair has been studied widely. However, the underlying molecular mechanism remains unclear. We profiled the altered microRNAs in renal tissues from cisplatin-induced acute kidney injury (AKI) rats treated with or without rat bone marrow MSCs (rMSCs). We observed that microRNA-146b (miR-146b) expression was considerably upregulated in renal tissues from AKI rats compared with that in healthy rats, and the expression decreased following MSC treatment after cisplatin administration. At the early stage of AKI, serum miR-146b levels exhibited a rapid increase that was even faster than that of two conventional renal function indexes: serum creatinine and blood urea nitrogen levels. Furthermore, the serum miR-146b levels in AKI patients were higher than those in healthy people. In vitro exposure to cisplatin also increased miR-146b expression in renal tubular epithelial cells (TECs). miR-146b knockdown protected renal TECs from cisplatin-induced apoptosis and promoted their proliferation. Moreover, ErbB4 was identified as a direct target of miR-146b, and miR-146b inhibition induced ErbB4 expression, resulting in enhanced proliferation of injured renal TECs. In addition, restoration by rMSCs could be controlled through ErbB4 downregulation. In conclusion, elevated miR-146b expression contributes to cisplatin-induced AKI, partly through ErbB4 downregulation. miR-146b might be an early biomarker for AKI, and miR-146b inhibition could be a novel strategy for AKI treatment. SIGNIFICANCE: The present study found that microRNA-146b (miR-146b) might be a novel biomarker for acute kidney injury and an indicator for its recovery after treatment with mesenchymal stem cells (MSCs). The results showed that in acute kidney injury induced by cisplatin, miR-146b in serum increased more quickly than did the usual indexes of kidney injury and decreased with restoration of MSCs. In addition, inhibition of miR-146b could ameliorate the apoptosis induced by cisplatin and potentially improve the proliferation by freeing ErbB4 and its downstream proteins.

Chronic administration of calcitriol enhanced neuregulin-1/ErbB signaling in rat myocardium.

Although emerging evidence suggests that vitamin D has beneficial effects in the cardiovascular health, the underlying mechanisms are far from fully elucidated. Given the indispensable role of neuregulin-1 (NRG1)/ErbB signaling in the cardiovascular system, the present study investigated the influences of prolonged administration of calcitriol, the active form of vitamin D, on the NRG1/ErbB system. We examined the protein expression of NRG1, ErbB receptors (ErbB2 and ErbB4) and their phosphorylated forms in the myocardium of rats following 6-week administration of calcitriol (50 ng/kg/day or 100 ng/kg/day). We further assessed the myocardial vitamin D receptor (VDR) to confirm the effect of calcitriol treatment. Additionally, serum neuregulin-1 level was also analyzed. Generally, calcitriol enhanced myocardial VDR expression and NRG1/ErbB signaling. Calcitriol increased NRG1 protein level at the higher dose, while both doses promoted ErbB2 and phosphorylated ErbB2 expression. Although calcitriol has no significant influence on ErbB4 expression, phosphorylated ErbB4 receptors were enhanced at the higher dose. Furthermore, the serum neuregulin-1 concentration was increased at both doses. Overall, our data firstly showed that chronic calcitriol administration enhanced NRG1/ErbB signaling in the heart, indicating a novel mechanism underlying the cardiac effects of vitamin D.

Sub-chronic Antipsychotic Drug Administration Reverses the Expression of Neuregulin 1 and ErbB4 in a Cultured MK801-Induced Mouse Primary Hippocampal Neuron or a Neurodevelopmental Schizophrenia Model.

It has been reported that specific environmental influences during the postpartum period might contribute to the development of schizophrenia (SZ). Administration of MK801 during early development led to persistent brain pathology. Glutamate decarboxylase 1 (GAD67) and parvalbumin (PV), and neuregulin 1 (NRG1)/ErbB4 signaling were closely associated with SZ pathology. We postulated therefore that NMDA receptor antagonists exposure during the postpartum period may be associated with expression dysregulation of some of the SZ candidate proteins. To test this, we used mouse primary hippocampal neurons and neonatal male mice treated with the NMDA receptor antagonist, MK801 at postnatal day 4 (P4) or P7, followed by the treatments of antipsychotic drugs (i.e., olanzapine, risperidone, and haloperidol). The expressions of GAD67, PV, NRG1, and ErbB4 in in vitro and in vivo SZ models were detected with Western blot analysis and immunohistochemistry, respectively. Behavioral tests (locomotion activity, social interaction, novel object recognition and prepulse inhibition) were measured. We found MK801 decreased the expression of GAD67, PV, NRG1 and ErbB4, and induced obvious behavioral alterations, while antipsychotics reversed these alterations. These results suggest that exposure to the NMDA receptor antagonist in early development may lead to long-lasting influence on the expression of specific proteins, such as GAD67, PV, NRG1, and ErbB4. Moreover, our results suggest that rescue of the activation of the NRG1/ErbB4 signaling pathway may be one of the mechanisms by which antipsychotic drugs have an antipsychotic effect.

Tumor-promoting function of single nucleotide polymorphism rs1836724 (C3388T) alters multiple potential legitimate microRNA binding sites at the 3'-untranslated region of ErbB4 in breast cancer.

ErbB4 can act as either a tumor-suppressor gene or an oncogene in breast cancer. Multiple genetic factors including single nucleotide polymorphisms (SNPs) affect gene expression patterns. Multiple 3'-untranslated region (3'-UTR) SNPs reside within the target binding site of microRNAs, which can strengthen or weaken binding to target genes. The present study aimed to predict potential 3'­UTR variants of ErbB4 that alter the target binding site of microRNAs (miRNAs) and to clarify the association of the potential variant with the risk of developing breast cancer. In silico prediction was performed to identify potential functional SNPs within miRNA target binding sites in the 3'­UTR of ErbB4. Thus, 146 patients and controls were genotyped using restriction fragment length polymorphism-polymerase chain reaction. In addition to the Cochran-Armitage test for trend, allele and genotype frequency differences were determined to investigate the association between rs1836724 and the susceptibility to breast cancer. Bioinformatics analysis identified rs1836724 to be a polymorphism in the seed region of four miRNA binding sites (hsa-miR335-5p, hsa-miR-28-5p, has­miR­708­5p and has­miR­665), which may participate in the development of breast cancer. Logistic regression data indicated that the T allele of the polymorphism [OR (95% CI)=1.72 (1.056­2.808), P=0.029] is associated with the risk of breast cancer. Using bioinformatics tools, a correlation was indicated between the presence of the T allele and a reduction in ErbB4 RNA silencing based on miRNA interaction. Furthermore, case subgroup data analysis revealed an association between the C/T genotype and an ER positive phenotype [OR (95% CI)=6.00 (1.082­33.274), P=0.028] compared with the T/T genotype. ErbB4 and estrogen receptor 1 (ESR1) are regulated by identical miRNAs thus there may be a competition for binding sites. Due to this pattern, if the interaction between miRNAs with one gene is reduced, it may be consistent with the increase in interaction with another one. Therefore, more interaction with rs1836724 C variant within ErbB4 may be associated with higher expression of ESR1 (ER­positive phenotype). miRNAs interact with ErbB4 mRNA more frequently when it carries C allele at the rs1836724 position compared with the T carriers. Therefore, the identical miRNA interacts with ESR1 less frequently when ErbB4 mRNA has a C allele. Therefore, ESR1 expression may be higher when ErbB4 mRNA has a C allele.

Elevated Coexpression of KITENIN and the ErbB4 CYT-2 Isoform Promotes the Transition from Colon Adenoma to Carcinoma Following APC loss.

PURPOSE AND EXPERIMENTAL DESIGN: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer cells, and we identified a novel EGFR-independent oncogenic signal of EGF that works under coexpressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contribute to further progression of intestinal adenoma following APC loss. RESULTS: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven KITENIN expression induces increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes compared with those of nontransgenic mice. Among the four ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells coexpressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of the ErB4-CYT-2 mRNA as well as increased EGFR expression were observed in intestinal adenoma of APC(min/+) mice, which makes the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APC(min/+) mice, intestinal tumor tissues in the crossed mice showed the characteristics of early-stage invading adenocarcinoma. In patients with colorectal cancer, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no significant differences in tumor tissue expression were found between different colorectal cancer stages. Furthermore, the mRNA expression of KITENIN and that of ErbB4-CYT-2 were positively correlated in human colorectal cancer tissue. CONCLUSIONS: Elevated coexpression of KITENIN and ErbB4-CYT-2 promotes the transition of colon adenoma to adenocarcinoma within an APC loss-associated tumor microenvironment.

HER4 tumor expression in breast cancer patients randomized to treatment with or without tamoxifen.

The human epidermal growth factor receptor (HER) 4 is a relative of HER2 and has been associated to endocrine breast cancer and prediction of tamoxifen response. In addition to PI3K/Akt and MAPK pathway activation, ligand binding to HER4 triggers proteolytic cleavage and release of an intracellular receptor domain (4ICD) with signaling properties. The aim of the present study was to analyze HER4 protein expression and intracellular localization in breast cancer tissue from patients randomized to treatment with or without adjuvant tamoxifen. To investigate HER4 expression and localization in response to estradiol (E2) and 4-hydroxytamoxifen (4-OHT) exposure, we also performed in vitro studies. Cytoplasmic, nuclear and membrane expression of HER4 protein was evaluated by immunohistochemical staining in tumor tissue from 912 breast cancer patients. Three different breast epithelia cancer cell lines were exposed to E2 and 4-OHT and mRNA expression was analyzed using qPCR. Further, nuclear and cytoplasmic proteins were separated and analyzed with western blotting. We found an association between nuclear HER4 protein expression and ER-positivity (P=0.004). Furthermore, significant association was found between cytoplasmic HER4 and ER-negativity (P<0.0005), PgR-negativity (P<0.0005), tumor size >20 mm (P=0.001) and HER2-negativity (P=0.008). However, no overall significance of HER4 on recurrence-free survival was found. After E2 exposure, HER4 mRNA and protein expression had decreased in two cell lines in vitro yet no changes in nuclear or cytoplasmic protein fractions were seen. In conclusion, nuclear HER4 seem to be co-located with ER, however, we did not find support for overall HER4 expression in independently predicting response of tamoxifen treatment. The possible influence of separate isoforms was not tested and future studies may further evaluate HER4 significance.

Local delivery of the Neuregulin1 receptor ecto-domain (ecto-ErbB4) has a positive effect on regenerated nerve fiber maturation.

The Neuregulin/ErbB system plays an important role in the peripheral nervous system, under both normal and pathological conditions. We previously demonstrated that expression of soluble ecto-ErbB4, the released extracellular fragment of the ErbB4 receptor, stimulated glial cell migration in vitro. In this study we examined the possibility of manipulating this system in vivo in order to improve injured peripheral nerve regeneration. Transected rat median nerves of adult female Wistar rats were repaired with a 10-mm-long graft made by muscle-in-vein combined nerve guide previously transduced with either the adeno-associated viral (AAV) vector AAV2-LacZ or AAV2-ecto-ErbB4. Autologous nerve grafts were used as control. Both stereological and functional analyses were performed to assess nerve regeneration. Data show that delivery of soluble ecto-ErbB4 by gene transfer in the muscle-in-vein combined nerve guide has a positive effect on fiber maturation, suggesting that it could represent a potential tool for improving peripheral nerve regeneration.

The Significance of Neuregulin-1/ErbB Expression in Autogenous Vein Grafts in a Diabetic Rat Model.

Diabetes mellitus (DM) is an important risk factor for increased vein graft failure after bypass surgery. The neuregulin-1 (NRG-1)/ErbB signaling system plays a critical role in neointimal formation after vascular injury as well as the proliferation and migration of mitogen-induced vascular smooth muscle cells; however, changes in NRG-1/ErbB signaling leading to vein grafts attrition in DM remain largely unexplored. Therefore, the aim of this study was to investigate changes in NRG-1/ErbB signaling in vein grafts in diabetic rats. To do this, a rat model of DM was established by streptozotocin injection followed by engraftment of autologous jugular veins to carotid arteries to induce intimal hyperplasia. After vein graft harvest, a pathohistological examination was performed; changes in NRG-1 and ErbB expression were also assessed. NRG-1 and ErbB expression localized to endothelial cells and vascular smooth muscle cells, which is consistent with the arterialization of vein grafts. NRG-1, ErbB2, and ErbB4 expression significantly decreased in vein grafts over time. Our findings show that NRG-1/ErbB signaling is impaired in vein grafts of diabetic rats, suggesting an important role for this pathway in the pathogenesis of intimal hyperplastic lesions in vein grafts of patients with DM.

miR-193a-3p functions as a tumor suppressor in lung cancer by down-regulating ERBB4.

ERBB4, one of four ErbB receptor tyrosine kinase family members, plays an important role in the etiology and progression of lung cancer. In this study, we found that the ERBB4 protein levels were consistently up-regulated in lung cancer tissues, whereas the mRNA levels varied randomly, suggesting that a post-transcriptional mechanism was involved in regulating ERBB4 expression. Because microRNAs are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for microRNAs that can potentially target ERBB4. We identified specific targeting sites for miR-193a-3p in the 3'-UTR of ERBB4. We further identified an inverse correlation between miR-193a-3p levels and ERBB4 protein levels, but not mRNA levels, in lung cancer tissue samples. By overexpressing or knocking down miR-193a-3p in lung cancer cells, we experimentally confirmed that miR-193a-3p directly recognizes the 3'-UTR of the ERBB4 transcript and regulates ERBB4 expression. Furthermore, the biological consequences of the targeting of ERBB4 by miR-193a-3p were examined in vitro via cell proliferation, invasion, and apoptosis assays and in vivo using a mouse xenograft tumor model. We demonstrated that the repression of ERBB4 by miR-193a-3p suppressed proliferation and invasion and promoted apoptosis in lung cancer cells and that miR-193a-3p exerted an anti-tumor effect by negatively regulating ERBB4 in xenograft mice. Taken together, our findings provide the first clues regarding the role of miR-193a-3p as a tumor suppressor in lung cancer through the inhibition of ERBB4 translation.

ErbB4 as a potential molecular target in the treatment of esophageal squamous cell cancers.

ErbB4 is an important member of ErbB subfamily of tyrosine kinases receptor with overexpression in several tumors; however its biological role in esophageal cancer is poorly understood till date. The main objective of this study was to examine whether miRNA-140-5p could target and control ErbB4 expression at transcriptional level. The ErbB4 expressions in different cell lines were evaluated by western blotting and luciferase assay. Moreover, cell proliferation, apoptosis, and cell invasion studies were investigated using MTT, flow cytometry, and transwell assays. miRNA-140-5p remarkably downregulated the ErbB4 expression in EC9706 and TE-1A cell lines. Furthermore, miRNA-140-5p transfected cell significantly controlled the cell proliferation and enhanced the apoptosis of multiple cells. Additionally, miRNA-140-5p had marked effect on the DNA synthesis and caspase 3/7 activity in comparison to control cells. Specifically, miRNA-140-5p inhibited/repressed the cancer cell invasion and migration in a sign to have important biological role in esophageal carcinomas. Taken together, miRNA-140-5p could act as a potential molecular target in ErbB4 overexpressing ESCC cell lines paving the way for effective esophageal cancer treatment.

The localization of HER4 intracellular domain and expression of its alternately-spliced isoforms have prognostic significance in ER+ HER2- breast cancer.

Human epidermal growth factor receptors (HERs) are known to play a pivotal role in breast cancer, both as prognostic markers and as therapeutic targets. The importance of Her4 expression is, however, still controversially discussed; there are few reports on the clinical significance of HER4, its splice variants, and cleaved HER4 intracellular domains (4ICD) which function differently depending on their localization in breast cancer. In 238 primary invasive breast cancer patients, we analyzed the expression levels of HER4 extracellular (JM-a and JM-b) and intracellular (CYT-1 and CYT-2) domains as well as 4ICD localization, and tested the relationship with clinicopathological characteristics and prognosis. The predominantly-expressed extracellular domain was JM-a, and lower CYT-2 dominance was a factor related to better relapse-free survival. CYT-2-dominance with higher nuclear 4ICD expression was a favorable prognostic marker especially in patients with the ER+ HER2- subtype treated with endocrine therapy. The absence of cytoplasmic 4ICD staining was related to better prognosis in CYT-1-dominant patients. In conclusion, analysis of splicing variants and 4ICD localization should be considered when targeting HER4 as a novel ER+/HER2- breast cancer treatment.

Activated PI3K/AKT and MAPK pathways are potential good prognostic markers in node-positive, triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) patients are a poor prognostic subgroup, and currently, there is no biomarker for targeted therapy. PATIENTS AND METHODS: Tissue samples were obtained from 75 TNBC patients with lymph-node metastases who had received adjuvant chemotherapy. We examined 11 biomarkers, including PIK3CA and AKT1mutation, with regard to event-free survival (EFS) and overall survival (OS) of patients. RESULTS: In the tumor tissues, phospho-AKT (pAKT) expression was significantly related to HER4 expression. Expression of each of these biomarkers was significantly related to longer EFS (P = 0.024 and 0.03, respectively). pERK expression was also a good prognostic factor regarding EFS and OS in TNBC (P = 0.002 and 0.006, respectively). We also identified a correlation between epidermal growth factor receptor positivity and insulin-like growth factor receptor type 1 positivity (P = 0.001). pERK and T-stage (1-3 versus >3) were independent good prognostic factors by multivariate analysis. CONCLUSIONS: We determined that tumors expressing pAKT or pERK are a good prognostic subtype in node-positive TNBC. Different targeted therapies may be necessary for TNBC that involves activation of PI3K/AKT or MAPK pathways.