Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

Nanoparticle Anisotropy Increases Targeting Interactions on Live-Cell Membranes.

This paper describes how branch lengths of anisotropic nanoparticles can affect interactions between grafted ligands and cell-membrane receptors. Using live-cell, single-particle tracking, we found that DNA aptamer-gold nanostar nanoconstructs with longer branches showed improved binding efficacy to human epidermal growth factor receptor 2 (HER2) on cancer cell membranes. Inhibiting nanoconstruct-HER2 binding promoted nonspecific interactions, which increased the rotational speed of long-branched nanoconstructs but did not affect that of short-branched constructs. Bivariate analysis of the rotational and translational dynamics showed that longer branch lengths increased the ratio of targeting to nontargeting interactions. We also found that longer branches increased the nanoconstruct-cell interaction times before internalization and decreased intracellular trafficking velocities. Differences in binding efficacy revealed by single-particle dynamics can be attributed to the distinct protein corona distributions on short- and long-branched nanoconstructs, as validated by transmission electron microscopy. Minimal protein adsorption at the high positive curvature tips of long-branched nanoconstructs facilitated binding of DNA aptamer ligands to HER2. Our study reveals the significance of nanoparticle branch length in regulating local chemical environment and interactions with live cells at the single-particle level.

Locally misfolded HER2 expressed on cancer cells is a promising target for development of cancer-specific antibodies.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is associated with a poor prognosis, making it an important therapeutic target. Here, we establish a novel cancer-specific anti-HER2 antibody, H2Mab-214. H2Mab-214 reacts with HER2 on cancer cells, but unlike the therapeutic antibody trastuzumab, it does not react with HER2 on normal cells in flow cytometry measurements. A crystal structure suggests that H2Mab-214 recognizes a structurally disrupted region in the HER2 domain IV, which normally forms a ß-sheet. We show that this misfolding is inducible by site-directed mutagenesis mimicking the disulfide bond defects that also may occur in cancer cells, indicating that the local misfolding in the Cys-rich domain IV governs the cancer-specificity of H2Mab-214. Furthermore, we show that H2Mab-214 effectively suppresses tumor growth in xenograft mouse models. Our findings offer a potential strategy for developing cancer-specific therapeutic antibodies that target partially misfolded cell surface receptors.

HER2 phosphorylation induced by TGF-ß promotes mammary morphogenesis and breast cancer progression.

Transforming growth factor ß (TGF-ß) and HER2 signaling collaborate to promote breast cancer progression. However, their molecular interplay is largely unclear. TGF-ß can activate mitogen-activated protein kinase (MAPK) and AKT, but the underlying mechanism is not fully understood. In this study, we report that TGF-ß enhances HER2 activation, leading to the activation of MAPK and AKT. This process depends on the TGF-ß type I receptor TßRI kinase activity. TßRI phosphorylates HER2 at Ser779, promoting Y1248 phosphorylation and HER2 activation. Mice with HER2 S779A mutation display impaired mammary morphogenesis, reduced ductal elongation, and branching. Furthermore, wild-type HER2, but not S779A mutant, promotes TGF-ß-induced epithelial-mesenchymal transition, cell migration, and lung metastasis of breast cells. Increased HER2 S779 phosphorylation is observed in human breast cancers and positively correlated with the activation of HER2, MAPK, and AKT. Our findings demonstrate the crucial role of TGF-ß-induced S779 phosphorylation in HER2 activation, mammary gland development, and the pro-oncogenic function of TGF-ß in breast cancer progression.

Clinical Features and Prognoses of Patients With Breast Cancer Who Underwent Surgery.

Importance: Breast cancer (BC) remains a pervasive malignant neoplasm worldwide, with increasing incidence. However, there are a scarcity of studies examining the clinical characteristics and prognosis of Chinese patients with BC who have undergone surgery. Objective: To evaluate overall survival (OS) and disease-free survival (DFS) in patients with surgically treated BC in China, focusing on histopathology and surgical approach. Design, Setting, and Participants: This cohort study included a retrospective review of the medical records of patients with unilateral BC who underwent surgery between January 2009 and September 2017, with a median follow-up time of 7.69 years. Clinical features were extracted from these records, and survival analysis was performed. Data analysis was conducted in March 2023. Main Outcomes and Measures: Patients' OS and DFS. Results: The study included 14 782 patients (14 724 [99.6%] female patients; mean [SD] age, 51.6 [10.9] years). Invasive ductal carcinoma (IDC) was the most prevalent type, observed in 12 671 patients (85.6%). Stages 0, I, II, III, and IV accounted for 6.4% (919 patients), 32.0% (4579 patients), 40.5% (5791 patients), 20.2% (2896 patients), and 0.9% (126 patients) of cases, respectively. Hormone receptor (HR) positivity was observed in 10 241 patients (75.1%), and 3665 (29.1%) tested positive for ERBB2 (formerly HER2/neu). The HR-negative-ERBB2-negative, HR-negative-ERBB2-positive, HR-positive-ERBB2-negative, and HR-positive-ERBB2-positive subtypes constituted 13.3% (1666 patients), 12.7% (1595 patients), 57.8% (7251 patients), and 16.2% (2034 patients) of cases, respectively. Breast-conserving surgery (BCS) was performed in 2884 patients (19.5%). The 5-year and 10-year OS rates were 92.9% (13 689 of 14 732) and 87.4% (3287 of 3760), while the 5-year and 10-year DFS rates were 89.0% (12 916 of 14 512) and 82.9% (3078 of 3713), respectively. Multivariate analysis found that for patients with IDC, age, BCS, invasive tumor size, tumor grade, lymphovascular invasion (LVI), the number of lymph node metastases (LNMs), distant metastasis, Ki67, and HR status were associated with OS, whereas invasive tumor size, tumor grade, LVI, the number of LNMs, HR status, and ERBB2 status were associated with DFS. After propensity score matching, BCS was equivalent to mastectomy with respect to survival in patients with IDC. Conclusions and Relevance: This cohort study of patients with BC who underwent surgery in China provides valuable insights into the histopathological characteristics and survival outcomes of this population. The diverse histopathological features emphasize the necessity for customized treatment strategies. The relatively low BCS rate in the study population suggests the need for heightened awareness and adoption of this approach, considering its potential advantages for survival.

In silico and experimental methods for designing a potent anticancer arazyme-herceptin fusion protein in HER2-positive breast cancer.

CONTEXT: Breast cancer is the most prevalent type of malignancies among women worldwide and is associated with serious physical and mental consequences. Current chemotherapies may lack successful outcomes; thus, the development of targeted recombinant immunotoxins is plausible. The predicted B cell and T cell epitopes of arazyme of the fusion protein are able to elicit immune response. The results of codon adaptation tool of herceptin-arazyme have improved from 0.4 to 1. The in silico immune simulation results showed significant response for immune cells. In conclusion, our findings show that the known multi-epitope fusion protein may activate humoral and cellular immune responses and maybe a possible candidate for breast cancer treatment. METHODS: In this study, the selected monoclonal antibody constituting herceptin and the bacterial metalloprotease, arazyme, was used with different peptide linkers to design a novel fusion protein to predict different B cell and T cell epitopes by the means of the relevant databases. Modeler 10.1 and I-TASSER online server were used to predict and validate the 3D structure and then docked to HER2-receptor using HADDOCK2.4 web server. The molecular dynamics (MD) simulations of the arazyme-linker-herceptin-HER2 complex were performed by GROMACS 2019.6 software. The sequence of arazyme-herceptin was optimized for the expression in prokaryotic host using online servers and cloned into pET-28a plasmid. The recombinant pET28a was transferred into the Escherichia coli BL21DE3. Expression and binding affinity of arazyme-herceptin and arazyme to human breast cancer cell lines (SK-BR-3/HER2 + and MDA-MB-468/HER2 -) were validated by the SDS-PAGE and cell­ELISA, respectively.

Targeting the HER3 pseudokinase domain with small molecule inhibitors.

HER3 is a potent oncogenic growth factor receptor belonging to the human epidermal growth factor (HER/EGFR) family of receptor tyrosine kinases. In contrast to other EGFR family members, HER3 is a pseudokinase, lacking functional kinase activity. As such, efforts to develop small molecule tyrosine kinase inhibitors against this family member have been limited. In response to HER3-specific growth factors such as neuregulin (NRG, also known as heregulin or HRG), HER3 must couple with catalytically active family members, including its preferred partner HER2. Dimerization of the intracellular HER2:HER3 kinase domains is a critical part of the activation mechanism and HER3 plays a specialized role as an allosteric activator of the active HER2 kinase partner. Intriguingly, many pseudokinases retain functionally important nucleotide binding capacity, despite loss of kinase activity. We demonstrated that occupation of the nucleotide pocket of the pseudokinase HER3 retains functional importance for growth factor signaling through oncogenic HER2:HER3 heterodimers. Mutation of the HER3 nucleotide pocket both disrupts signaling and disrupts HER2:HER3 dimerization. Conversely, ATP competitive drugs which bind to HER3, but not HER2, can stabilize HER2:HER3 dimers, induce signaling and promote cell growth in breast cancer models. This indicates a nucleotide-dependent conformational role for the HER3 kinase domain. Critically, our recent proof-of-concept work demonstrated that HER3-directed small molecule inhibitors can also disrupt HER2:HER3 dimerization and signaling, supporting the prospect that HER3 can be a direct drug target despite its lack of intrinsic activity. In this chapter we will describe methods for identifying and validating small molecule inhibitors against the HER3 pseudokinase.

An effective strategy for ligand-mediated pulldown of the HER2/HER3/NRG1ß heterocomplex and cryo-EM structure determination at low sample concentrations.

Obtaining high-resolution structures of Receptor Tyrosine Kinases that visualize extracellular, transmembrane and intracellular kinase regions simultaneously is an eagerly pursued but still unmet challenge of structural biology. The Human Epidermal Growth Factor Receptor 3 (HER3) that has a catalytically inactive kinase domain (pseudokinase) forms a potent signaling complex upon binding of growth factor neuregulin 1ß (NRG1ß) and upon dimerization with a close homolog, the HER2 receptor. The HER2/HER3/NRG1ß complex is often referred to as an oncogenic driver in breast cancer and is an attractive target for anti-cancer therapies. After overcoming significant hurdles in isolating sufficient amounts of the HER2/HER3/NRG1ß complex for structural studies by cryo-electron microscopy (cryo-EM), we recently obtained the first high-resolution structures of the extracellular portion of this complex. Here we describe a step-by-step protocol for obtaining a stable and homogenous HER2/HER3/NRG1ß complex for structural studies and our recommendation for collecting and processing cryo-EM data for this sample. We also show improved EM density for the transmembrane and kinase domains of the receptors, which continue to evade structural determination at high resolution. The discussed strategies are tunable and applicable to other membrane receptor complexes.

Molecular docking and dynamic simulation to identify potential phytocompound inhibitors for EGFR and HER2 as anti-breast cancer agents.

Breast cancer is the most prevalent cancer in women worldwide. To treat human breast cancer by inhibiting EGFR and HER2 targets is an important therapeutic option. Phytochemicals are found to have beneficial health effects in treating various diseases. An effort has been made to virtually screen phytochemical inhibitor by molecular docking and dynamic simulation in the current studies. The docking scores analysis resulted in a common hit Panaxadiol ligand with a low dock score for EGFR and HER2 targets. The inhibitory action of the phytocompounds was also validated by comparing it with the reference compounds Erlotinib for EGFR and Neratinib for HER2. Molecular dynamic simulation of EGFR and HER2 lead complexes ensure the ligand's appropriate refinement in the dynamic system. The target and ligand complex interaction motif established a high affinity of lead candidates in a dynamic system similar to molecular docking results. This study reveals that Panaxadiol hit molecule can be developed as a novel multi-target EGFR and HER2 target inhibitor with greater potential and low toxicity.Communicated by Ramaswamy H. Sarma.

Structures of the HER2-HER3-NRG1ß complex reveal a dynamic dimer interface.

Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex1-3 upon binding of growth factor neuregulin-1ß (NRG1ß). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Here we isolated the NRG1ß-bound near full-length HER2-HER3 dimer and, using cryo-electron microscopy, reconstructed the extracellulardomain module, revealing unexpected dynamics at the HER2-HER3 dimerization interface. We show that the dimerization arm of NRG1ß-bound HER3 is unresolved because the apo HER2 monomer does not undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm-binding pocket. In a structure of the oncogenic extracellular domain mutant HER2(S310F), we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. Both HER2-HER3 and HER2(S310F)-HER3 retain the capacity to bind to the HER2-directed therapeutic antibody trastuzumab, but the mutant complex does not bind to pertuzumab. Our structure of the HER2(S310F)-HER3-NRG1ß-trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2-HER3 heterodimer. The unique features of a singly liganded HER2-HER3 heterodimer underscore the allosteric sensing of ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via a canonical active dimer interface.

Glycosylation promotes the cancer regulator EGFR-ErbB2 heterodimer formation - molecular dynamics study.

ErbB family of receptor tyrosine kinases play significant roles in cellular differentiation and proliferation. Mutation or overexpression of these receptors leads to several cancers in humans. The family has four homologous members including EGFR, ErbB2, ErbB3, and ErbB4. From which all except the ErbB2 bind to growth factors via the extracellular domain to send signals to the cell. However, dimerization of the ErbB receptor occurs in extracellular, transmembrane, and intracellular domains. The ErbB receptors are known to form homodimers and heterodimers in the active form. Heterodimerization increases the variety of identified ligands and signaling pathways that can be activated by these receptors. Furthermore, glycosylation of the ErbB receptors has shown to be critical for their stability, ligand binding, and dimerization. Here, atomistic molecular dynamics simulations on the glycosylated and unglycosylated heterodimer showed that the EGFR-ErbB2 heterodimer is more stable in its dynamical pattern compared to the EGFR-EGFR homodimer. This increased stability is regulated by maintaining the dimeric interface by the attached glycans. It was also shown that the presence of various glycosylation sites within the ErbB2 growth factor binding site leads to occlusion of this site by the glycans that inhibit ligand binding to ErbB2 and participate in further stabilization of the heterodimer construct. Putting together, glycosylation seems to promote the heterodimer formation within the ErbB family members as the dominant molecular mechanism of activation for these receptors.

Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer.

Human epidermal growth factor receptor 2 (HER2) is one of the key molecular targets in breast cancer pathogenesis. Overexpression and/or amplification of HER2 in approximately 15-20% of breast cancer patients is associated with high mortality and poor prognosis. Accumulating evidence shows that accurate and sensitive detection of HER2 improves the survival outcomes for HER2-positive breast cancer patients from targeted therapies. The current methods of clinical determination of HER2 expression levels are based on slide-based assays that rely on invasively collected primary tumours. Alternatively, ELISA-based detection of the shredded HER2 extracellular domain (HER2-ECD) of has been suggested as a surrogate method for monitoring disease progress and treatment response in breast cancer patients. In the past decade, biosensors have emerged as an alternative modality for the detection of circulating HER2-ECD in human serum samples. In particular, electrochemical biosensors based on nanomaterials and antibodies and aptamers have been increasingly developed as promising tools for rapid, sensitive, and cost-effective detection of HER2-ECD. These biosensors harness the high affinity and specificity of antibodies and aptamers, and unique conductive properties, biocompatibility, large surface area, and chemical stability of nanomaterials for selective and sensitive assessment of the HER2. This review provides an overview of the recent advances in the application of nanomaterials-based immunosensors and aptasensors for detection of circulating HER2-ECD. In particular, various electrochemical techniques, detection approaches, and nanomaterials are discussed. Further, analytical figures of merit of various HER2 immunosensors and aptasensors are compared. Finally, possible challenges and potential opportunities for biosensor-based detection of HER2-ECD are discussed.

Melatonin potentiates the cytotoxic effect of Neratinib in HER2+ breast cancer through promoting endocytosis and lysosomal degradation of HER2.

Complete blockade of the HER2 protein itself and HER signaling network is critical to achieving effective HER2-targeted therapies. Despite the success of HER2-targeted therapies, the diseases will relapse in a significant fraction of patients with HER2+ breast cancers. How to improve the therapeutic efficacy of existing HER2-targeted agents remains an unmet clinical need. Here, we uncover a role of Melatonin in diminishing HER2-mediated signaling by destruction of HER2 protein. Mechanistically, Melatonin treatment attenuated the protective effect of the HSP90 chaperone complex on its client protein HER2, triggering ubiquitylation and subsequent endocytic lysosomal degradation of HER2. The inhibitory effect of Melatonin on HER2 signaling substantially enhanced the cytotoxic effects of the pan-HER inhibitor Neratinib in HER2+ breast cancer cells. Lastly, we demonstrate that dual inhibition of HER2 by combined use of Melatonin and Neratinib effectively blocked the growth of HER2+ breast tumor xenografts in vivo. Our findings shed light on the potential use of Melatonin in a novel dual HER2 blockade strategy for HER2+ breast cancer treatment.

Topomer-CoMFA proposed as a tool to construct dual EGFR/HER-2 models.

Protein kinases (in this case, HER-2 and EGFR) are involved in cancer-related diseases. Some reports have shown unique CoMFA models using the sum of activities expressed as pIC50 (-log IC50), as the classical CoMFA technique would not be the best strategy to construct models for multitarget therapy considering that the molecular alignment will not be the same for different targets. An alternative for this problem is the use of Topomer-CoMFA, a variation of CoMFA, which does not require the alignment step in the generation of 3D models. In this study, we propose the combined use of the sum of activities and Topomer-CoMFA for the construction of a unique dual 3D model considering the inhibitory activities against EGFR and HER-2. For this, 88 compounds from the literature were divided into two groups: training (71) and test (17) sets. The biological activity of each compound, expressed as IC50 for EGFR and HER-2, was transformed into pIC50, summed, and used as the dependent variable in the Topomer-CoMFA analyses. The obtained model was considered statistically robust in the prediction of the dual activity of new compounds. Finally, based on the obtained model, we proposed structural modifications to some of the compounds used to improve the biological data. From the 3D model, we suggested new derivative compounds with improved biological activity for both targets. Therefore, the combination of the techniques proposed in this study proves to be a good strategy to construct better statistical models that can predict biological activities in multitarget systems.

Mig6 not only inhibits EGFR and HER2 but also targets HER3 and HER4 in a differential specificity: Implications for targeted esophageal cancer therapy.

The human EGF receptor family plays pivotal roles in physiology and cancer, which contains four closely-related members: HER1/EGFR, HER2, HER3 and HER4. Previously, it was found that the mitogen-inducible gene 6 (Mig6) protein is a negative regulator of EGFR and HER2 by using its S1 segment to bind at the kinase dimerization interface. However, it is still unclear whether the S1 segment can also effectively target HER3 and HER4? Here, we performed a systematic investigation to address this issue. The segment can bind to all the four HER kinases with a varying affinity and moderate selectivity; breaking of the segment into shorter hotspot peptides would largely impair the affinity and selectivity, indicating that the full-length sequence is required for the effective binding of S1 to these kinases. The hs2 peptide, which corresponds to the middle hotspot region of S1 segment, can partially retain the affinity to HER kinases, can moderately compete with S1 segment at the dimerization interfaces, and can mimic the biological function of Mig6 protein to suppress HER4+ esophageal cancer at cellular level. In addition, we also analyzed the binding potency of S1 segment and hs2 peptide to the kinase domains of other five widely documented growth factor receptors (GFRs). It was showed that both the S1 and hs2 cannot effectively interact with these receptors. Overall, the Mig6 is suggested as a specific pan-HER inhibitor, which can target and suppress HER family members with a broad selectivity, but exhibits weak or no activity towards other GFRs.

Co-occurring gain-of-function mutations in HER2 and HER3 modulate HER2/HER3 activation, oncogenesis, and HER2 inhibitor sensitivity.

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization.

Overexpression of the receptor tyrosine kinase HER2 plays a critical role in the development of various tumors. Biparatopic designed ankyrin repeat proteins (bipDARPins) potently induce apoptosis in HER2-addicted breast cancer cell lines. Here, we have investigated how the spatiotemporal receptor organization at the cell surface is modulated by these agents and is distinguished from other molecules, which do not elicit apoptosis. Binding of conventional antibodies is accompanied by moderate reduction of receptor mobility, in agreement with HER2 being dimerized by the bivalent IgG. In contrast, the most potent apoptosis-inducing bipDARPins lead to a dramatic arrest of HER2. Dual-color single-molecule tracking revealed that the HER2 "lockdown" by these bipDARPins is caused by the formation of HER2-DARPin oligomer chains, which are trapped in nanoscopic membrane domains. Our findings establish that efficient neutralization of receptor tyrosine kinase signaling can be achieved through intermolecular bipDARPin crosslinking alone, resulting in inactivated, locked-down bipDARPin-HER2 complexes.

Biochemistry, structure, and cellular internalization of a four nanobody-bearing Fc dimer.

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

Dimeric Her2-specific affibody mediated cisplatin-loaded nanoparticles for tumor enhanced chemo-radiotherapy.

BACKGROUND: Solid tumor hypoxic conditions prevent the generation of reactive oxygen species (ROS) and the formation of DNA double-strand breaks (DSBs) induced by ionizing radiation, which ultimately contributes to radiotherapy (RT) resistance. Recently, there have been significant technical advances in nanomedicine to reduce hypoxia by facilitating in situ O2 production, which in turn serves as a "radiosensitizer" to increase the sensitivity of tumor cells to ionizing radiation. However, off-target damage to the tumor-surrounding healthy tissue by high-energy radiation is often unavoidable, and tumor cells that are further away from the focal point of ionizing radiation may avoid damage. Therefore, there is an urgent need to develop an intelligent targeted nanoplatform to enable precise enhanced RT-induced DNA damage and combined therapy. RESULTS: Human epidermal growth factor receptor 2 (Her2)-specific dimeric affibody (ZHer2) mediated cisplatin-loaded mesoporous polydopamine/MnO2/polydopamine nanoparticles (Pt@mPDA/MnO2/PDA-ZHer2 NPs) for MRI and enhanced chemo-radiotherapy of Her2-positive ovarian tumors is reported. These NPs are biodegradable under a simulated tumor microenvironment, resulting in accelerated cisplatin release, as well as localized production of O2. ZHer2, produced using the E. coli expression system, endowed NPs with Her2-dependent binding ability in Her2-positive SKOV-3 cells. An in vivo MRI revealed obvious T1 contrast enhancement at the tumor site. Moreover, these NPs achieved efficient tumor homing and penetration via the efficient internalization and penetrability of ZHer2. These NPs exhibited excellent inhibition of tumor growth with X-ray irradiation. An immunofluorescence assay showed that these NPs significantly reduced the expression of HIF-1α and improved ROS levels, resulting in radiosensitization. CONCLUSIONS: The nanocarriers described in the present study integrated Her2 targeting, diagnosis and RT sensitization into a single platform, thus providing a novel approach for translational tumor theranostics.

Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.