Identification of a pharmacological approach to reduce ACE2 expression and development of an in vitro COVID-19 viral entry model.

Because of rapid emergence and circulation of the SARS-CoV-2 variants, especially Omicron which shows increased transmissibility and resistant to antibodies, there is an urgent need to develop novel therapeutic drugs to treat COVID-19. In this study we developed an in vitro cellular model to explore the regulation of ACE2 expression and its correlation with ACE2-mediated viral entry. We examined ACE2 expression in a variety of human cell lines, some of which are commonly used to study SARS-CoV-2. Using the developed model, we identified a number of inhibitors which reduced ACE2 protein expression. The greatest reduction of ACE2 expression was observed when CK869, an inhibitor of the actin-related protein 2/3 (ARP2/3) complex, was combined with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of sodium-hydrogen exchangers (NHEs), after treatment for 24 h. Using pseudotyped lentivirus expressing the SARS-CoV-2 full-length spike protein, we found that ACE2-dependent viral entry was inhibited in CK869 + EIPA-treated Calu-3 and MDA-MB-468 cells. This study provides an in vitro model that can be used for the screening of novel therapeutic candidates that may be warranted for further pre-clinical and clinical studies on COVID-19 countermeasures.

Comparative Characterization of Different Molecular Formats of Bispecific Antibodies Targeting EGFR and PD-L1.

We generated two IgG1-like bispecific antibodies (BsAbs) with different molecular formats, symmetrical DVD-Ig and asymmetrical knob-in-hole (KIH), targeting the same antigens, EGFR and PD-L1 (designated as anti-EGFR/PD-L1). We performed the physiochemical and biological characterization of these two formats of anti-EGFR/PD-L1 BsAbs and compared some key quality attributes and biological activities of these two formats of BsAbs. Physiochemical binding characterization data demonstrated that both formats bound EGFR and PD-L1. However, the binding affinity of the KIH format was weaker than the DVD-Ig format in Biacore binding assays. In contrast, both DVD-Ig and KIH BsAbs had similar ELISA and cell surface binding activities, comparable to mAbs. Triple-negative breast cancer (TNBC) cells and a xenograft model were used to test the potency of BsAbs and other biological activities. Results showed that anti-EGFR/PD-L1 BsAbs exhibited in vitro and in vivo antitumor proliferation activity, but there was a difference in the potencies of the respective BsAb formats (DVD-Ig and KIH) when different cells or assays were used. This study provides evidence that the potency of the BsAbs targeting the same antigens can be affected by the respective molecular features, and selection of appropriate cell lines and assays is critically important for the assay development and potency testing of BsAbs.

Mechanisms contributing to ado-trastuzumab emtansine-induced toxicities: a gateway to better understanding of ADC-associated toxicities.

In order to improve the safety of novel therapeutic drugs, better understanding of the mechanisms of action is important. Ado-trastuzumab emtansine (also known as T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive breast cancer. While the treatment with T-DM1 results in significant efficacy in the selected patient population, nonetheless, there are concerns with side effects such as thrombocytopenia and hepatotoxicity. While current understanding of the mechanism of T-DM1-mediated side effects is still incomplete, there have been several reports of HER2-dependent and/or -independent mechanisms that could be associated with the T-DM1-induced adverse events. This review highlights the importance of HER2-independent mechanism of T-DM1 to induce hepatotoxicity, which offers a new insight into a role for CKAP5 in the overall maytansinoid-based ADC (DM1 and DM4)-mediated cytotoxicity. This discovery provides a molecular basis for T-DM1-induced off-target toxicity and opens a new avenue for developing the next generation of ADCs.

A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action.

Both EGFR and VEGFR2 frequently overexpress in TNBC and cooperate with each other in autocrine and paracrine manner to enhance tumor growth and angiogenesis. Therapeutic mAbs targeting EGFR (cetuximab) and VEGFR2 (ramucirumab) are approved by FDA for numerous cancer indications, but none of them are approved to treat breast cancers. TNBC cells secrete VEGF-A, which mediates angiogenesis on endothelial cells in a paracrine fashion, as well as promotes cancer cell growth in autocrine manner. To disrupt autocrine/paracrine loop in TNBC models in addition to mediating anti-EGFR tumor growth signaling and anti-VEGFR2 angiogenic pathway, we generated a BsAb co-targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb), using publicly available sequences in which cetuximab IgG backbone is connected to the single chain variable fragment (scFv) of ramucirumab via a glycine linker. Physiochemical characterization data shows that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 in a similar binding affinity comparable to parental antibodies. Anti-EGFR/VEGFR2 BsAb demonstrates in vitro and in vivo anti-tumor activity in TNBC models. Mechanistically, anti-EGFR/VEGFR2 BsAb not only directly inhibits both EGFR and VEGFR2 in TNBC cells but also disrupts autocrine mechanism in TNBC xenograft mouse model. Furthermore, anti-EGFR/VEGFR2 BsAb inhibits ligand-induced activation of VEGFR2 and blocks paracrine pathway mediated by VEGF secreted from TNBC cells in endothelial cells. Collectively, our novel findings demonstrate that anti-EGFR/VEGFR2 BsAb inhibits tumor growth via multiple mechanisms of action and warrants further investigation as a targeted antibody therapeutic for the treatment of TNBC.

Trastuzumab Blocks the Receiver Function of HER2 Leading to the Population Shifts of HER2-Containing Homodimers and Heterodimers.

HER2, a member of the Erythroblastosis Protein B/Human Epidermal Growth Factor Receptor (ErbB/HER) family of receptor tyrosine kinase, is overexpressed in 20~30% of human breast cancers. Trastuzumab, a HER2-targeted therapeutic monoclonal antibody, was developed to interfere with the homodimerization of HER2 in HER2-overexpressing breast cancer cells, which attenuates HER2-mediated signaling. Trastuzumab binds to the domain IV of the HER2 extracellular domain and does not directly block the dimerization interface of HER2-HER2 molecules. The three-dimensional structures of the tyrosine kinase domains of ErbB/HER family receptors show asymmetrical packing of the two monomers with distinct conformations. One monomer functions as an activator, whereas the other acts as a receiver. Once activated, the receiver monomer phosphorylates the activator or other proteins. Interestingly, in our previous work, we found that the binding of trastuzumab induced phosphorylation of HER2 with the phosphorylation pattern of HER2 that is different from that mediated by epidermal growth factor (EGF) in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Binding of trastuzumab to HER2 promoted an allosteric effect of HER2, in both tyrosine kinase domain and ectodomain of HER2 although details of allosteric regulation were missing. In this study, we utilized molecular dynamics (MD) simulations to model the allosteric consequences of trastuzumab binding to HER2 homodimers and heterodimers, along with the apo forms as controls. We focused on the conformational changes of HER2 in its monomeric and dimeric forms. The data indicated the apparent dual role of trastuzumab as an antagonist and an agonist. The molecular details of the simulation provide an atomic level description and molecular insight into the action of HER2-targeted antibody therapeutics.

Cell proliferation and invasion are regulated differently by EGFR and MRP1 in T-DM1-resistant breast cancer cells.

We recently reported that T-DM1-resistant JIMT1 (T-DM1R-JIMT1) cells exhibited high invasive activity via EGFR and integrin cooperated pathways and gained cross-resistance to doxorubicin. Here, we show that EGFR positively coordinates with MRP1 in T-DM1R-JIMT1 cells to contribute to cross-resistance to doxorubicin. Downregulating EGFR and MRP1 inhibits T-DM1R-JIMT1 cell growth and re-sensitizes T-DM1R cells to doxorubicin, suggesting that dual targeting EGFR and MRP1 could serve as a therapeutic approach to overcome T-DM1 resistance. However, it increases cell invasion activity of T-DM1R-JIMT1 cells with molecular and cellular phenotypes similar to the breast cancer cells that express low levels of HER2 (MDA-MB-231 and BT-549 cells). Importantly, the invasion activity of MDA-MB-231 and BT-549 cells is also significantly increased after chronically exposed to T-DM1 although cell growth of MDA-MB-231 and BT-549 cells is not inhibited by T-DM1. These results highlight the importance of HER2 heterogenicity in HER-positive breast cancers treated with T-DM1. Our study also provides evidence demonstrating that proliferation and invasion activities of T-DM1R-JIMT1, and MDA-MB-231 and BT-549 cells are regulated by different mechanisms and that different aspects of cancer cell behaviors affected by targeted-therapeutics should be fully characterized in order to overcome T-DM1-resistant disease and to prevent cancer metastasis.

Atezolizumab potentiates Tcell-mediated cytotoxicity and coordinates with FAK to suppress cell invasion and motility in PD-L1+ triple negative breast cancer cells.

Immune check point inhibitors targeting programmed cell death protein-1 (PD-1) and its ligand (PD-L1) have shown clinical success in treatment of human malignancies. Triple negative breast cancer (TNBC), which is primarily characterized by high heterogeneity and presence of tumor infiltrating lymphocytes, remains therapeutic challenge due to unavailability of approved targeted therapy. Therapeutic potential of immune check point inhibitors for TNBC patients is under active clinical investigation. In this study, we show that FDA-approved anti-PD-L1 antibody, atezolizumab (ATE), potentiates T cell-mediated cytotoxicity and apoptosis of TNBC cells that express higher levels of PD-L1, but does not have significant effect on TNBC cells expressing low levels of PD-L1. PD-L1 knockdown further confirmed that ability of ATE to promote T cell-induced cytotoxicity is PD-L1 expression dependent. Combination of ATE with PD-L1 upregulating agents, such as HDAC, proteasomal, and lysosomal inhibitors, further augmented cytotoxic activity of T cells toward TNBC cells. Based on analysis of breast cancer tissue samples deposited in The Cancer Genome Atlas (TCGA), we found a positive correlation between PD-L1 and focal adhesion kinase (FAK) mRNA expression in PD-L1-positive (PD-L1+) TNBC, suggesting a functional association of FAK and immune checkpoints. We further demonstrate that ATE dramatically downregulates phosphorylation status of FAK, an important regulator of cell invasion and migration, and significantly enhances FAK inhibitor mediated inhibition of cell motility and invasion of PD-L1+ TNBC cells independent of T cells. Taken together, our data suggest that ATE shows promising anti-tumor activity in PD-L1+ TNBC via both T cell-dependent and -independent mechanisms.

Trastuzumab-mediated cardiotoxicity: current understanding, challenges, and frontiers.

Trastuzumab, an epidermal growth factor receptor 2 (HER2) targeting humanized monoclonal antibody, has been approved for the treatment HER2-positive breast cancer and HER2-positve metastatic gastric cancer. However, cardiotoxicity associated with its clinical application poses challenges for clinicians and patients, mechanisms of which are still evolving. This review will summarize the current mechanistic understanding of trastuzumab-mediated cardiotoxicity, discuss the novel role of DNA topoisomerase IIB as a shared target for enhanced cardiotoxicity induced by trastuzumab and anthracyclines-based combination regimens, and speculate the potential impact of trastuzumab intervention in immune checkpoint inhibitors-based therapies.

T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways.

Ado-trastuzumab emtansine (Kadcyla®; T-DM1) is an antibody-drug conjugate developed to treat trastuzumab-resistant disease. Despite initial favorable outcomes, most patients eventually cease to respond due to developing acquired resistance to T-DM1. Currently, there is no targeted therapy to treat T-DM1-resistant disease. To explore novel therapeutic targets to improve therapeutic efficacy of T-DM1, we generated T-DM1-resistant cells using trastuzumab-resistant JIMT1 cells. We found that the loss of human epidermal growth factor receptor 2 confers T-DM1 resistance, which in turn activates a compensatory mechanism that increases epidermal growth factor receptor (EGFR) expression. Upregulation of EGFR increases the protein levels of α5ß1 and αVß3 integrins, resulting in enhanced motility and invasion of T-DM1-resistant cells. This study delineates previously unappreciated relationships between α5ß1 and αVß3 and suggests that specific integrins should be carefully selected as therapeutic targets to treat T-DM1-resistant disease. Specifically, silencing ß1 integrin expression by siRNA in T-DM1-resistant cells destabilizes α5, but increases expression of αV, a critical integrin mediating the invasion and metastases in many different cancers. As a consequence, T-DM1-resistant cells gain metastatic potential and become more invasive. This finding is underscored by the fact that ß1 integrin blockage induced by an inhibitory antibody, MAB 13, significantly increases invasion of T-DM1-resistant cells. However, the increased cell invasion induced by ß1 integrin blockage can be significantly reduced by either EGFR inhibitor or specific siRNA against αV integrin. The discovery of functional cooperation between EGFR and αV integrin in regulating cell growth and invasion provides an opportunity to develop novel therapeutic strategy by dual-targeting EGFR and specific integrin to overcome T-DM1 resistance.

Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity.

Despite heightened risk of cardiotoxicity associated with combination therapy of anthracyclines and trastuzumab in HER2-positive breast cancer patients, little research effort has been invested in exploring the molecular mechanisms of cardiotoxicity induced by this combination therapy. In this study, we demonstrate that trastuzumab downregulates both gene and protein expressions of type IIB DNA topoisomerase/DNA topoisomerase IIB (TOP2B), a major intracellular target mediating doxorubicin-induced cardiotoxicity, in human primary cardiomyocytes. This in turn induces DNA damage activity and DNA double strand breaks, which is indicated by the enhanced phosphorylation of H2AX (γH2AX) and ataxia telangiectasia and Rad3-related protein (ATR pS428) in trastuzumab-treated cardiomyocytes. Furthermore, concurrent or sequential treatment of doxorubicin and trastuzumab significantly increases the downregulation of the protein levels of TOP2B, enhances apoptosis and cell growth inhibition, and promotes production of reactive oxidative and nitrative species in human cardiomyocytes as compared to either trastuzumab or doxorubicin treatment, indicating augmentation of cardiotoxicity in combination therapy. Additionally, our data reveal that doxorubicin treatment increases the levels of ErbB2/HER2 expression in human cardiomyocytes as compared with that in cells not treated with doxorubicin, leading to the enhanced activity downstream of HER2 signaling. Consequently, this may render the cardiomyocytes to become addicted to HER2 signaling for survival under stressed conditions. Enhanced HER2 protein expression leaves cardiomyocytes more sensitive to trastuzumab treatment after doxorubicin exposure. This study provides molecular basis for significantly increased cardiotoxicity in cancer patients who are treated with anthracyclines and trastuzumab-based combination regimens.

Payload of T-DM1 binds to cell surface cytoskeleton-associated protein 5 to mediate cytotoxicity of hepatocytes.

Off-target toxicity is a major cause of dose-limiting toxicity for antibody-drug conjugates (ADCs), mechanisms of which remain poorly understood. Here, we demonstrate that cytoskeleton-associated protein 5 (CKAP5) serves as a cell surface target for T-DM1 and that binding of T-DM1 to CKAP5 is mediated by payload (DM1). This study introduces a novel molecular mechanism of ADC payload-mediated interaction with cell surface molecules to induce cytotoxicity. Upon binding to CKAP5, T-DM1 causes cell membrane damage and leads to calcium influx into the cells, resulting in disorganized microtubule network and apoptosis. While binding of T-DM1 with HER2 is critical for killing HER2-positive tumor cells, our data suggest that cytotoxicity induced by T-DM1 interaction with CKAP5 may preferentially damage normal cells/tissues where HER2 expression is low or missing to cause off-target toxicity. This study provides molecular basis of ADC-induced off-target cytotoxicity and opens a new avenue for developing next generation of ADCs.

Cardiotoxicity of ErbB2-targeted therapies and its impact on drug development, a spotlight on trastuzumab.

INTRODUCTION: Trastuzumab, a therapeutic monoclonal antibody directed against ErbB2, is often noted as a successful example of targeted therapy. Trastuzumab improved outcomes for many patients with ErbB2-positive breast and gastric cancers, however, cardiac side effects [e.g., left ventricular dysfunction and congestive heart failure (CHF)] were reported in the early phase clinical studies. This finding, subsequently corroborated by multiple clinical studies, raised concerns that the observed cardiotoxicity induced by trastuzumab might adversely impact the clinical development of other therapeutics targeting ErbB family members. Areas covered: In this review we summarize both basic research and clinical findings regarding trastuzumab-induced cardiotoxicity and assess if there has been an impact of trastuzumab-induced cardiotoxicity on the development of other agents targeting ErbB family members. Expert opinion: There are a number of scientific gaps that are critically important to address for the continued success of HER2-targeted agents. These include: 1) elucidating the molecular mechanisms contributing to cardiotoxicity; 2) developing relevant preclinical testing systems for predicting cardiotoxicity; 3) developing clinical strategies to identify patients at risk of cardiotoxicity; and 4) enhancing management of clinical symptoms of cardiotoxicity.

VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation.

VPS34 is reported to activate S6K1 and is implicated in regulating cell growth, the mechanisms of which remain elusive. Here, we describe novel mechanisms by which VPS34 upregulates mTOR/S6K1 activity via downregulating TSC2 protein and activating RheB activity. Specifically, upregulation of VPS34 lipid kinase increases local production of ptdins(3)p in the plasma membrane, which recruits PIKFYVE, a FYVE domain containing protein, to ptdins(3)p enriched regions of the plasma membrane, where VPS34 forms a protein complex with PIKFYVE and TSC1. This in turn disengages TSC2 from the TSC1/TSC2 heterodimer, leading to TSC2 ubiquitination and degradation. Downregulation of TSC2 promotes the activation of RheB and mTOR/S6K1. When VPS34 lipid kinase activity is increased by introduction of an H868R mutation, ptdins(3)p production at the plasma membrane is dramatically increased, which recruits more PIKFYVE and TSC1 molecules to the plasma membrane. This results in the enhanced TSC2 ubiquitination and degradation, and subsequent activation of RheB and mTORC1/S6K1, leading to oncogenic transformation. The role played by VPS34 in regulating mTOR/S6K1 activity and cellular transformation is underscored by the fact that the VPS34 kinase dead mutant blocks VPS34-induced recruitment of PIKFYVE and TSC1 to the plasma membrane. This study provides mechanistic insight into the cellular function of VPS34 in regulating oncogenic transformation and important indications for identifying VPS34 specific mutations in human cancers.

Trastuzumab, but Not Pertuzumab, Dysregulates HER2 Signaling to Mediate Inhibition of Autophagy and Increase in Reactive Oxygen Species Production in Human Cardiomyocytes.

Dysregulation of autophagy has been implicated in various cardiovascular diseases. Trastuzumab, a humanized monoclonal antibody, binds to HER2 domain IV and is approved for the treatment of HER2-positive breast cancer. Trastuzumab therapy is associated with considerable cardiotoxicity, the mechanism of which remains unclear. HER2 signaling plays a pivotal role in cardiomyocyte development and survival and is essential for the prevention of cardiomyopathy. However, a direct link has not been confirmed between trastuzumab-induced cardiomyopathy and impaired HER2 signaling. Our data reveal a novel mechanism by which trastuzumab dysregulates HER2 signaling and impairs basal autophagic process in human primary cardiomyocytes. Specifically, trastuzumab treatment leads to the phosphorylation of HER1-Y845 and HER2-Y1248 and the activation of Erk. This in turn results in upregulation of mTOR signaling pathway and subsequently inhibition of autophagy in primary cardiomyocytes and C57BL/6 mice. Trastuzumab-induced downregulation of autophagy is further supported by the fact that trastuzumab treatment reduces protein levels of autophagosome-associated signaling molecules such as Atg 5-12, Atg 7, Atg 14, and Beclin 1. We further demonstrated that trastuzumab-mediated inhibition of autophagy resulted in the increased production of reactive oxygen species (ROS) in cardiomyocytes. Pertuzumab, another anti-HER2 therapeutic mAb binding to HER2 domain II, fails to modulate HER2 signaling and is unable to inhibit autophagy and to increase ROS production in cardiomyocytes. This study provides novel mechanistic insights into trastuzumab-induced cardiotoxicity, which may assist in formulating novel approaches for clinical management of trastuzumab-induced cardiomyopathy. Mol Cancer Ther; 15(6); 1321-31. ©2016 AACR.

Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity.

Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2-positive metastatic breast cancer. It consists of trastuzumab, a humanized mAb directed against HER2, and a microtubule inhibitor, DM1, conjugated to trastuzumab via a thioether linker. Hepatotoxicity is one of the serious adverse events associated with T-DM1 therapy. Mechanisms underlying T-DM1-induced hepatotoxicity remain elusive. Here, we use hepatocytes and mouse models to investigate the mechanisms of T-DM1-induced hepatotoxicity. We show that T-DM1 is internalized upon binding to cell surface HER2 and is colocalized with LAMP1, resulting in DM1-associated cytotoxicity, including disorganized microtubules, nuclear fragmentation/multiple nuclei, and cell growth inhibition. We further demonstrate that T-DM1 treatment significantly increases the serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in mice and induces inflammation and necrosis in liver tissues, and that T-DM1-induced hepatotoxicity is dose dependent. Moreover, the gene expression of TNFα in liver tissues is significantly increased in mice treated with T-DM1 as compared with those treated with trastuzumab or vehicle. We propose that T-DM1-induced upregulation of TNFα enhances the liver injury that may be initially caused by DM1-mediated intracellular damage. Our proposal is underscored by the fact that T-DM1 induces the outer mitochondrial membrane rupture, a typical morphologic change in the mitochondrial-dependent apoptosis, and mitochondrial membrane potential dysfunction. Our work provides mechanistic insights into T-DM1-induced hepatotoxicity, which may yield novel strategies to manage liver injury induced by T-DM1 or other ADCs.

Quantitative optical inspection of contact lenses immersed in wet cell using swept source OCT.

We demonstrate swept source optical coherence tomography (OCT) imaging of contact lenses (CLs) in a wet cell and comprehensive quantitative characterization of CLs from volumetric OCT datasets. The approach is based on a technique developed for lens autopositioning and autoleveling enabled by lateral capillary interactions between the wet cell wall and the lens floating on the liquid surface. The demonstrated OCT imaging has enhanced contrast due to the application of a scattering medium and it improves visualization of both CL interfaces and edges. We also present precise and accurate three-dimensional metrology of soft and rigid CLs based on the OCT data. The accuracy and precision of the extracted lens parameters are compared with the manufacturer's specifications. The presented methodology facilitates industrial inspection methods of the CLs.

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1.

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models.

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.

Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice.

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.