EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.

Molecular Targeting Therapy against EGFR Family in Breast Cancer: Progress and Future Potentials.

：The epidermal growth factor receptor (EGFR) family contains four transmembrane tyrosine kinases (EGFR1/ErbB1, Her2/ErbB2, Her3/ErbB3 and Her4/ErbB4) and 13 secreted polypeptide ligands. EGFRs are overexpressed in many solid tumors, including breast, pancreas, head-and-neck, prostate, ovarian, renal, colon, and non-small-cell lung cancer. Such overexpression produces strong stimulation of downstream signaling pathways, which induce cell growth, cell differentiation, cell cycle progression, angiogenesis, cell motility and blocking of apoptosis.The high expression and/or functional activation of EGFRs correlates with the pathogenesis and progression of several cancers, which make them attractive targets for both diagnosis and therapy. Several approaches have been developed to target these receptors and/or the EGFR modulated effects in cancer cells. Most approaches include the development of anti-EGFRs antibodies and/or small-molecule EGFR inhibitors. This review presents the state-of-the-art and future prospects of targeting EGFRs to treat breast cancer.

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking.

Serotonin receptors 5-HT(1A) and 5-HT(7) are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT(1A) and 5-HT(7) receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT(1A) or 5-HT(7) receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT(7)-5-HT(7) homodimers, followed by the 5-HT(7)-5-HT(1A) heterodimers and 5-HT(1A)-5-HT(1A) homodimers. Functionally, heterodimerization decreases 5-HT(1A)-receptor-mediated activation of G(i) protein without affecting 5-HT(7)-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT(1A) receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT(1A) receptor internalization and also enhances the ability of the 5-HT(1A) receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT(7) receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT(1A)-5-HT(7) heterodimers and, consequently, their functional importance undergoes pronounced developmental changes.

Intercellular transport of Oct4 in mammalian cells: a basic principle to expand a stem cell niche?

BACKGROUND: The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We report on the ability of STRO-1(+) deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and α-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1(+) DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) under the control of an Oct4 responsive element is only expressed in the presence of Oct4. GFP expression in Oct4-GFP cells started after 24 hours of co-culture providing evidence of Oct4 transfer from STRO-1(+) DaMSCs to Oct4-GFP MEF target cells. CONCLUSIONS: Our findings indicate a possible mechanism for the expansion of a resident stem cell niche by induction of pluripotency in surrounding non-niche cells via transfer of transcription factors through intercellular connections. This provides a new approach to explain the rapid annual antler regrowth.

An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions.

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.

Evolutionary spread and recombination of porcine endogenous retroviruses in the suiformes.

Different Suiformes with increasing phylogenetic distance to the common pig (Sus scrofa) were assayed for the presence of porcine endogenous retroviruses (PERV) in general (pol gene), while the distribution of long terminal repeat (LTR) types (with or without repeats in U3) and env genes (classes A, B, and C) were determined in detail. PERV was not detectable in the most distantly related species, while classes PERV-A and PERV-B are present in Suiformes originating in the Pliocene epoch, and class PERV-C was detectable only in S. scrofa and in closely related species originating in the Holocene epoch. This distribution pattern of PERV classes is in line with our previous study on the age of PERV (45) and suggests an African origin of about 7.5 million years ago (MYA) and a gradual spread of PERV through the Suiformes. It seems likely that PERV-C originated more recently (1.5 to 3.5 MYA) by recombination with a homologue of unknown descent, while the origin of the repeatless LTR was a separate event approximately 3.5 MYA.

Detection of porcine endogenous retrovirus (PERV) using highly specific antisera against Gag and Env.

Porcine endogenous retroviruses (PERV) are considered an obstacle to the safe use of cells, tissues, and organs from pigs in the course of xenotransplantation. Thus, the detection of viral proteins and of a potential PERV infection is of major interest. Recently, we have published the generation of a highly specific antiserum directed against the nucleocapsid (p10) of PERV (Xenotransplantation 7 (2000), 221). Here we present new peptide-antisera specific to the capsid protein (p30) and the surface molecule of PERV class B (SU, gp70(B)) as well as the transmembrane moiety of the envelope protein (TM, p15E) of PERV which showed functionality in several immunological assays, such as immunoblots, immunofluorescence, and immunogold staining. Thus, these antisera can be used as tools for the identification of viral proteins in basic research as well as clinical trials.

Retroviral safety: analyses of phylogeny, prevalence and polymorphisms of porcine endogenous retroviruses.

Porcine endogenous retroviruses (PERV) are discussed as putative infectious agents in xenotransplantation (XTx). PERV classes A, B, and C harboring different envelope proteins and two types of long terminal repeat (LTR) structures exist. One type of LTR contains a distinct repeat structure in U3, while the other is repeat-less and confers poor transcriptional activity. As the different LTR structures were found to be distributed unequally among the proviruses, we were interested in determining which LTR is the ancestor. Since replication-competent PERV can still be found today suggesting an evolutionary recent origin, we investigated the distribution and prevalence of six well-characterized and chromosomally assigned PERV in individuals of five different pig (Suidae, Sus scrofa) subspecies. Our studies revealed a heterogenous distribution of replication-competent PERV among individuals as well as among subspecies. The age of PERV was calculated to be 7.6 x 10(6) years, whereby the repeat-less LTR type evolved approximately 3.4 x 10(6) years ago being the phylogenetically younger structure. The age correlates with the time of separation between pigs and their closest relatives, american-borne peccaries (Tayassuidae, Pecari tajacu), 7.4 x 10(6) years ago.