NRG4 suppresses breast cancer metastasis via ERBB4-YAP1-mediated down-regulation of MMPs.

BackgroundCancer metastasis is a multi-step event including epithelial-to-mesenchymal transition (EMT). Breast cancer metastasis suppressor 1 (BRMS1) is a novel metastasis suppressor protein without anti-proliferating activity. However, a detailed underlying mechanism by which BRMS1 attenuates cancer cell EMT and invasion remained to be answered. In the present study, we report an additional mechanism by which BRMS1 attenuates Transforming growth factor-beta1 (TGF-β1)-induced breast cancer cell EMT and invasion.MethodsExperimental analysis involving chromosome immunoprecipitation (ChIP) and luciferase reporter assays were used to validate hypoxia inducible factor-1alpha (HIF-1α) as a transcriptional regulator of TWIST1 and Snail. Quantitative RT-PCR was used to analyze transcript expression. Immunoblotting and immunofluorescence were used to analyze protein expression. Matrigel-coated in vitro invasion insert was used to analyze cancer cell invasion.ResultsBRMS1 strongly inhibited TGF-β1-induced breast cancer cell EMT and invasion. Unexpectedly, we observed that BRMS1 downregulates not only TWIST1 but also Snail expression, thereby inhibiting breast cancer cell invasion. In addition, we provide evidence that HIF-1α is required for Snail and TWIST1 expression. Further, BRMS1 reduced TGF-β1-induced HIF-1α transcript expression through inactivation of nuclear factor kappaB (NF-κB).ConclusionCollectively, the present study demonstrates a mechanical cascade of BRMS1 suppressing cancer cell invasion through downregulating HIF-1α transcript and consequently reducing Snail and TWIST1 expression.

Lipodystrophies (LDs) are rare disorders characterized by the partial or complete loss of subcutaneous adipose tissue, leading to severe metabolic complications. Although metreleptin therapy has shown beneficial effects, its therapeutic efficacy is limited, particularly in patients with partial LD. Neuregulin 4 (NRG4), a batokine secreted by brown adipose tissue, regulates lipid metabolism and hepatic function, but its relevance in LD has not been investigated. In this study, we observed significantly reduced serum NRG4 levels in patients with LD compared to matched healthy controls. NRG4 levels declined further during metreleptin therapy, potentially reflecting fat mass reduction or limited treatment response. To explore functional relevance, we treated a transgenic LD mouse model with recombinant NRG4. While NRG4 enhanced thermogenic gene expression in brown and inguinal white adipose tissue, it did not improve systemic metabolic parameters or hepatic steatosis. In vitro, NRG4 failed to rescue impaired adipogenesis and thermogenesis in brown adipocytes from LD mice but increased insulin-stimulated fatty acid uptake in white adipocytes, indicating a preserved functional response despite differentiation defects. NRG4 also activated hepatic AMPK signaling without improving lipid accumulation. These findings suggest that NRG4 promotes adipose tissue remodeling but is insufficient to restore systemic metabolic homeostasis in LD. Together, our data indicate that NRG4's beneficial effects may depend on the presence of functional adipose tissue, which is profoundly impaired in LD. Consequently, while NRG4 may support local plasticity in adipose tissue, it is insufficient as a therapy for metabolic restoration in LD.

BackgroundCREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer.MethodsQuantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis.ResultsThis study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression.ConclusionsOur data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0672-x) contains supplementary material, which is available to authorized users.

BackgroundEarly-branching metazoans, such as sponges (Porifera), can provide valuable insight into the emergence of complex gene regulatory systems and cancer-related mechanisms in early metazoan evolution. BRMS1 (breast cancer metastasis suppressor 1) is a component of the Sin3-HDAC complex involved in chromatin remodeling and transcriptional regulation. In humans, BRMS1 inhibits cancer metastasis by modulating signaling pathways that control cell migration, adhesion, and proliferation. Despite its biomedical importance, the evolutionary origin and basic functions of BRMS1 remain largely unexplored.ResultsWe identified and characterized a BRMS1 homolog from the cave sponge Eunapius subterraneus and compared it with human BRMS1 and BRMS1-like paralogs. Phylogenetic analyses revealed that BRMS1 and BRMS1-like arose from a duplication of an ancestral BRMS1 gene during early vertebrate evolution. Structural modeling showed that sponge BRMS1 shares high similarity with human BRMS1. Co-immunoprecipitation assays demonstrated that sponge BRMS1 physically associates with human BRMS1 in mammalian cells. In both sponge and human cells, sponge BRMS1 localized predominantly to the nucleus, similar to human BRMS1 and BRMS1-like. Functional assays in human breast cancer cells revealed that sponge BRMS1 suppresses proliferation, colony formation, and migration to a degree comparable to its human homologs.ConclusionsOur findings demonstrate that the key structural features, subcellular localization, and biological functions of BRMS1 are conserved between sponges and humans. The ability of a sponge BRMS1 homolog to integrate into human protein complexes and suppress cancer cell migration and proliferation suggests that fundamental BRMS1 activities arose early in metazoan evolution, independent of anatomical and functional complexity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-026-02616-5.

The interaction between circulating tumor cells and blood components, mainly platelets, plays an important role during metastasis. In this study, we prepared liposomes containing the platelet aggregation inhibitor Cilostazol (Cil-L). The objective of this study was to investigate the effect of this Cil-L on platelet aggregation and complex formation with murine 4T1 breast cancer cells in vitro and to determine their anti-metastatic potency in a spontaneous metastasis model of 4T1 breast cancer. Cil-L significantly inhibited the aggregation of platelets by up to 78% and completely abolished the complex formation of 4T1 tumor cells in the presence of activated platelets in vitro. Intravenous (i.v.) injection of Cil-L into mice significantly reduced the aggregability of mouse platelets by 60% measured ex vivo. To gain deeper insight into the mode of metastasis formation in a spontaneous metastasis model, 4T1 breast cancer cells were transplanted into the mammary fad pad of mice and metastasis to the mouse lungs was investigated with regard to tumor cell settlement and metastatic growth. We could demonstrate that the formation of pulmonary metastases was significantly reduced by 55% when mice were treated intravenously with 100 nmol Cil-L 6 h before tumor cell inoculation and then daily for 2 weeks. We conclude that Cil-L reduced metastasis by restricting the aggregability of mouse platelets, which probably prevents the interaction between circulating 4T1 tumor cells and platelets, making the Cil-L a useful tool for the inhibition of breast cancer metastasis in mice.

Growth factor receptors such as the ErbB receptor tyrosine kinases can preserve intestinal barrier integrity in the face of inflammation by activating anti‐apoptotic pathways and stabilizing epithelial tight junction components. Activation of ErbB4 with pharmacological administration of its specific ligand neuregulin‐4 (NRG4) promotes epithelial cell survival by anti‐apoptotic pathways (Src and PI3K/Akt) and reduces intestinal inflammation. However, it is unclear whether ErbB4 is an essential regulator of intestinal permeability and if endogenous NRG4 is required for the response to intestinal inflammation. In this study, we investigated the role of endogenous NRG4 in intestinal barrier function in vivo.METHODSBaseline intestinal permeability in 11–17 week old wild type and NRG4 knockout mice was determined by oral gavage with FITC‐dextran (4kDa) followed by analysis of blood collected by cardiac puncture 4 h later. Barrier recovery from bacterial antigen challenge was determined 24 h after FITC‐dextran gavage and LPS‐induced injury. Ileal epithelial apoptosis was assessed by TUNEL and cleaved caspase‐3 staining of formalin‐fixed paraffin embedded tissue.RESULTSNRG4−/−mice had compromised barrier function compared to wild type littermates at baseline, as shown by an increase in FITC‐dextran leakage to the blood (1.4‐fold increase). Furthermore, NRG4 deletion reduced the ability to recover barrier 24 hours after LPS‐induced injury (2.4‐fold increase, p<0.05). NRG4−/− mice had exaggerated ileal epithelial cell apoptosis 24 h after LPS as well, measured by TUNEL (32 positive cells per 100 crypts (KO) vs 14.58 positive cells per 100 crypts (WT), p<0.05) and cleaved caspase‐3 staining.CONCLUSIONEndogenous NRG4 promotes integrity of the intestinal epithelial barrier. Further investigation of the role of NRG4 within the intestinal mucosa may lead to more effective strategies for targeting barrier dysfunction seen in diseases such as inflammatory bowel disease or necrotizing enterocolitis.Support or Funding InformationSupported by NIH award DK095004 and a Senior Research Award from The Crohn's and Colitis Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Over one-third of human breast tumors that become resistant to endocrine therapies have acquired mutations in the estrogen receptor (ER alpha) that result in constitutive receptor activity in the absence of estrogen, and these tumors are much less sensitive to suppression by current standard-of-care antiestrogens such as tamoxifen and fulvestrant. We reported recently that the novel adamantyl antiestrogen, K-07, was effective in inhibiting the proliferation of breast tumor xenografts containing the two most frequent constitutively active mutant forms of the ER, Y537S ER and D538G ER, and that this antiestrogen reduced ER levels and the expression of ER-regulated genes (Zhao et al., Cancer Research 77(20):5602-5613, 2017). Because mutant ERs are usually enriched in recurrent, metastatic breast cancers compared to the primary breast cancer, we have now examined the effectiveness of compound K-07 in preventing the growth of breast cancer metastases and in extending survival in a metastatic tumor model. MCF-7 breast cancer cells expressing luciferase and Y537S ER or D538G ER (ca. 50% mutant ER and 50% wild type ER) were injected i.v. by the tail vein into NOD-SCID-gamma (NSG) female mice. The constitutively active ER-containing breast cancer cells established metastases in liver, bone and brain that increased in number and size over time (day zero – 80 days) as monitored by IVIS imaging and immunohistochemical (IHC) analysis. Daily oral treatment with K-07 (80 mg/kg orally for 30 days and then 40 mg/kg) versus oral vehicle greatly reduced the metastasis of mutant ER-containing breast cancer cells. Notably, mice with mutant ER-containing metastases treated with K-07 survived much longer than mice given daily control vehicle. In fact, by day 60, only 25% of vehicle treated mice with mutant ER breast cancers were alive whereas all K-07 treated D538G mice and 80% of Y537S mice were still alive. Hence, the findings indicate that this antiestrogen can reduce the in vivo metastasis of breast cancers driven by constitutively active mutant ERs and extend host survival. The findings suggest that the antiestrogen K-07 may be suitable for further translational and clinical examination of its efficacy in suppression of metastasis in patients with breast cancers containing constitutively active mutant ERs. Citation Format: Mary J. Laws, Sung Hoon Kim, Jian Min, Yuechao Zhao, Yvonne Ziegler, David Chu, Ben H. Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen. Suppression of breast cancer metastasis and extension of host animal survival by a new adamantyl antiestrogen, K-07, in a preclinical breast cancer metastasis model driven by constitutively active mutant estrogen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 946.

Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that suppresses metastasis in multiple human and murine carcinoma cell lines. BRMS1 interacts with several nuclear proteins including SIN3:HDAC chromatin remodeling complexes that are involved in repressing transcription. However, recent reports suggest BRMS1 may function in the cytoplasm. BRMS1 has two predicted nuclear localization sequences (NLS) that are located near the C-terminus (amino acids 198–205 and 238–244, NLS1 and NLS2 respectively). We hypothesized that nuclear localization sequences of BRMS1 were essential for BRMS1 mediated metastasis suppression. Replacement of NLS2 with NLS1 (BRMS1NLS1,1), truncation at 238 (BRMS1ΔNLS2), or switching the location of NLS1 and NLS2 (BRMS1NLS2,1) did not affect nuclear localization; but, replacement of NLS1 with NLS2 (BRMS1NLS2,2) or truncation at 197 (BRMS1ΔNLS which removes both NLS) promoted cytoplasmic localization. MDA-MB-231 human metastatic breast cancer cells transduced with BRMS1NLS1,1, BRMS1NLS2,2 or BRMS1NLS2,1 were evaluated for metastasis suppression in an experimental xenograft mouse model. Interestingly, while NLS2 was not necessary for nuclear localization, it was found to be important for metastasis suppression since BRMS1NLS2,2 suppressed metastasis by 85%. In contrast, BRMS1NLS2,1 and BRMS1NLS1,1 did not significantly suppress metastasis. Both BRMS1 and BRMS1NLS2,2 co-immunoprecipitated with SIN3A in the nucleus and cytoplasm; however, BRMS1NLS1,1 and BRMS1NLS2,1 were associated with SIN3A in the nucleus only. Moreover, BRMS1 and BRMS1NLS2,2, but not BRMS1NLS1,1 and BRMS1NLS2,1, down-regulated the pro-metastatic microRNA, miR-10b. Together, these data demonstrate an important role for NLS2 in the cytoplasm that is critical for metastasis suppression and is distinct from nuclear localization.

BackgroundEthanol abuse promotes breast cancer development, metastasis and recurrence stimulating mammary tumorigenesis by mechanisms that remain unclear. Normally, 35% of breast cancer is Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2)-positive that predisposes to poor prognosis and relapse, while ethanol drinking leads to invasion of their ERBB2 positive cells triggering the phosphorylation status of mitogen-activated protein kinase. StAR-related lipid transfer protein 10 (STARD10) is a lipid transporter of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); changes on membrane composition of PC and PE occur before the morphological tumorigenic events. Interestingly, STARD10 has been described to be highly expressed in 35–40% of ERBB2-positive breast cancers. In this study, we demonstrate that ethanol administration promotes STARD10 and ERBB2 expression that is significantly associated with increased cell malignancy and aggressiveness.Material and methodsWe investigated the effect of ethanol on STARD10-ERBB2 cross-talk in breast cancer cells, MMTV-neu transgenic mice and in clinical ERBB2-positive breast cancer specimens with Western Blotting and Real-time PCR. We also examined the effects of their knockdown and overexpression on transient transfected breast cancer cells using promoter activity, MTT, cell migration, calcium and membrane fluidity assays in vitro.ResultsEthanol administration induces STARD10 and ERBB2 expression in vitro and in vivo. ERBB2 overexpression causes an increase in STARD10 expression, while overexpression of ERBB2’s downstream targets, p65, c-MYC, c-FOS or c-JUN induces STARD10 promoter activity, correlative of enhanced ERBB2 function. Ethanol and STARD10-mediated cellular membrane fluidity and intracellular calcium concentration impact ERBB2 signaling pathway as evaluated by enhanced p65 nuclear translocation and binding to both ERBB2 and STARD10 promoters.ConclusionOur finding proved that STARD10 and ERBB2 positively regulate each other’s expression and function. Taken together, our data demonstrate that ethanol can modulate ERBB2’s function in breast cancer via a novel interplay with STARD10.

Significance of BRCA1 expression in breast and ovarian cancer patients with brain metastasis – A multicentre study

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-β, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-β1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-β1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice's lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-β1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-β1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-β1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-β1 silencing and their effect on EMT.

Background:Metastatic breast cancer (MBC) presents a significant challenge in the field of oncology, with limited treatment options and generally poor survival outcomes. Therefore, the discovery of new biomarkers and therapeutic targets is crucial for improving the management of MBC. Our previous studies have shown that JAM2, as a tumor suppressor, can reduce the invasiveness and migration of breast cancer cells by inhibiting the epithelial-mesenchymal transition (EMT) pathway, thus playing a key role in tumor biology. These findings suggest the importance of further investigating the specific mechanisms of JAM2. Methods:This study employed various techniques to comprehensively evaluate the expression and function of JAM2. First, we analyzed clinical samples from breast cancer patients using immunohistochemistry and quantitative PCR to determine JAM2 expression levels and its correlation with disease prognosis and metastasis. Additionally, we established a nude mouse model of breast cancer metastasis by injecting MDA-MB-231 cells with altered JAM2 expression to observe its effects on tumor growth and metastasis. We also used HA-tagged immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to screen for proteins interacting with JAM2 and further analyzed these interactions using Western blot, quantitative PCR, and Transwell invasion assays. Results:The results showed that JAM2 expression was generally low in breast cancer tissues, and its low expression was closely associated with poor prognosis and high metastasis rates. In the nude mouse model, JAM2 overexpression significantly inhibited tumor growth and metastasis. In vitro studies further revealed a direct interaction between JAM2 and IGF2BP2, where JAM2 overexpression significantly reduced IGF2BP2 levels, decreasing cell invasiveness and migration. Conversely, JAM2 knockdown increased IGF2BP2 expression, enhancing the invasiveness of breast cancer cells. Moreover, JAM2 inhibits the expression of the key EMT transcription factor Snail2 by affecting IGF2BP2, further emphasizing its role in inhibiting EMT and metastasis. Conclusion:The findings of this study highlight the important biological function of JAM2 as a metastasis suppressor in breast cancer, providing new insights into the key proteins and pathways it regulates during tumor progression. JAM2 may inhibit the EMT and the invasiveness and migration of breast cancer cells by downregulating the m6A reader protein IGF2BP2, thus reducing the stability of SNAI2 mRNA. This suggests that enhancing JAM2 expression could be a viable therapeutic strategy to combat breast cancer metastasis. Future studies are needed to fully elucidate the mechanisms by which JAM2 exerts its effects and to explore its potential clinical applications. Citation Format: Yang Peng, Han Li, Ailin Lan, Yang Liu, Zehao Cai, Shengchun Liu. Exploring JAM2's Potential in Reducing Breast Cancer Invasiveness through IGF2BP2 Regulation [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-10-25.

<div>Abstract<p><b>Purpose:</b> The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the <i>ERBB2</i> mutations in a high-throughput manner.</p><p><b>Experimental Design:</b> We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous <i>ERBB2</i> mutations using the mixed-all-nominated-in-one (MANO) method.</p><p><b>Results:</b> G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the <i>ERBB2</i> mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent <i>ERBB2</i> mutation in lung cancer. We surveyed the prevalence of concurrent <i>ERBB2</i> mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried <i>ERBB2</i> mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within <i>ERBB2</i> reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib.</p><p><b>Conclusions:</b> Several <i>ERBB2</i> mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of <i>ERBB2</i> mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.</p><p>We identified several <i>ERBB2</i> mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several <i>ERBB2</i> mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. <i>Clin Cancer Res; 24(20); 5112–22. ©2018 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the <i>ERBB2</i> mutations in a high-throughput manner.</p><p><b>Experimental Design:</b> We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous <i>ERBB2</i> mutations using the mixed-all-nominated-in-one (MANO) method.</p><p><b>Results:</b> G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the <i>ERBB2</i> mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent <i>ERBB2</i> mutation in lung cancer. We surveyed the prevalence of concurrent <i>ERBB2</i> mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried <i>ERBB2</i> mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within <i>ERBB2</i> reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib.</p><p><b>Conclusions:</b> Several <i>ERBB2</i> mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of <i>ERBB2</i> mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.</p><p>We identified several <i>ERBB2</i> mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several <i>ERBB2</i> mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. <i>Clin Cancer Res; 24(20); 5112–22. ©2018 AACR</i>.</p></div>

ERBB4 is a tyrosine kinase receptor reported to exert both oncogenic and tumor suppressor activities. These paradoxical effects were suggested to stem from different ERBB4 homo-/hetero-dimers and/or isoforms. By stratifying breast cancer patients for clinical and molecular subtypes and ERBB4 mRNA abundance, we here report that higher ERBB4 levels correlate with longer relapse-free survival in breast cancer patients of HER2-enriched and luminal A molecular subtypes, proposing a cancer-protecting role for this receptor in these specific subgroups. We also observed that HER2-enriched breast cancers express intermediate ERBB4 mRNA levels compared to luminal and triple-negative/basal-like subgroups, which displayed the highest and the lowest levels, respectively. Inspired by these clinical data, we tested the activation of ERBB4 by Neuregulins as a potential anticancer strategy for HER2+ breast cancers. To this end, we employed two HER2+ breast cancer cellular models (BT474 and SKBR3), which express intermediate/high and low ERBB4 levels, respectively. Cell proliferation and motility were evaluated on these cellular models following treatments with Neuregulin 1 (NRG1), which activates both ERBB3 and ERBB4, or Neuregulin 4 (NRG4), which specifically activates ERBB4. Both NRG1 and NRG4 were used alone or in combination with anti-ERBB2 neutralizing antibodies, namely trastuzumab and pertuzumab. In vitro treatment with NRG1 on BT474 cells restrained cell growth and reduced the anti-proliferative efficacy of trastuzumab. In contrast, treatment with NRG1 on SKBR3 cells increased cell proliferation and migration, and partially or completely impaired the anti-proliferative/anti-migratory action of trastuzumab and/or pertuzumab. Importantly, in both the cell lines, treatment with NRG4 robustly potentiated the anti-proliferative action of trastuzumab and pertuzumab. Collectively, our data in HER2+ breast cancer cells highlight that NRG1 may exert both pro- and anti-proliferative effects, and may reduce the efficacy of anti-HER2 agents, whereas NRG4 may boost the anti-proliferative effects of anti-ERBB2 agents. We propose a provocative paradigm shift in the field of growth factors in cancer progression, suggesting the administration of ERBB4 ligands, such as Neuregulin 4, as a strategy to improve the efficacy of anti-ERBB2 agents.