Repressing notch signaling and expressing TNFα are sufficient to mimic retinal regeneration by inducing Müller glial proliferation to generate committed progenitor cells.

Retinal damage in teleosts, unlike mammals, induces robust Müller glia-mediated regeneration of lost neurons. We examined whether Notch signaling regulates Müller glia proliferation in the adult zebrafish retina and demonstrated that Notch signaling maintains Müller glia in a quiescent state in the undamaged retina. Repressing Notch signaling, through injection of the γ-secretase inhibitor RO4929097, stimulates a subset of Müller glia to reenter the cell cycle without retinal damage. This RO4929097-induced Müller glia proliferation is mediated by repressing Notch signaling because inducible expression of the Notch Intracellular Domain (NICD) can reverse the effect. This RO4929097-induced proliferation requires Ascl1a expression and Jak1-mediated Stat3 phosphorylation/activation, analogous to the light-damaged retina. Moreover, coinjecting RO4929097 and TNFα, a previously identified damage signal, induced the majority of Müller glia to reenter the cell cycle and produced proliferating neuronal progenitor cells that committed to a neuronal lineage in the undamaged retina. This demonstrates that repressing Notch signaling and activating TNFα signaling are sufficient to induce Müller glia proliferation that generates neuronal progenitor cells that differentiate into retinal neurons, mimicking the responses observed in the regenerating retina.

A nanotherapeutic approach to selectively eliminate metastatic breast cancer cells by targeting cell surface GRP78.

Here, rational engineering of doxorubicin prodrug loaded peptide-targeted liposomal nanoparticles to selectively target metastatic breast cancer cells in vivo is described. Glucose-regulated protein 78 (GRP78), a heat shock protein typically localized in the endoplasmic reticulum in healthy cells, has been identified to home to the cell surface in certain cancers, and thus has emerged as a promising therapeutic target. Recent reports indicated GRP78 to be expressed on the cell surface of an aggressive subpopulation of stem-like breast cancer cells that exhibit metastatic potential. In this study, a targeted nanoparticle formulation with a GRP78-binding peptide (Kd of 7.4 ± 1.0 µM) was optimized to selectively target this subpopulation. In vitro studies with breast cancer cell lines showed the targeted nanoparticle formulation (TNPGRP78pep) achieved enhanced cellular uptake, while maintaining selectivity over the control groups. In vivo, TNPGRP78pep loaded with doxorubicin prodrug was evaluated using a lung metastatic mouse model and demonstrated inhibition of breast cancer cell seeding to lungs down at the level of negative control groups. Combined, this study established that specific-targeting of surface GRP78 expressing a subpopulation of aggressive breast cancer cells was able to inhibit breast cancer metastasis to lungs, and underpinned the significance of GRP78 in breast cancer metastasis.

Cell surface GRP78 and Dermcidin cooperate to regulate breast cancer cell migration through Wnt signaling.

The heat shock protein GRP78 typically resides in the endoplasmic reticulum in normal tissues, but it has been shown to be expressed on the cell surface of several cancer cells, and some stem cells, where it can act as a signaling molecule by not-yet-fully defined mechanisms. Although cell surface GRP78 (sGRP78) has emerged as an attractive chemotherapeutic target, understanding how sGRP78 is functioning in cancer has been complicated by the fact that sGRP78 can function in a cell-context dependent manner, with a diverse array of reported binding partners, to regulate a variety of cellular responses. We had previously shown that sGRP78 was important in regulating pluripotent stem cell (PSC) functions, and hypothesized that embryonic-like mechanisms of GRP78 were critical to regulating aggressive breast cancer cell functions. Here, using proteomics we identify Dermcidin (DCD) as a novel sGRP78 binding partner common to both PSCs and breast cancer cells. We show that GRP78 and DCD cooperate to regulate stem cell and cancer cell migration that is dependent on the cell surface functions of these proteins. Finally, we identify Wnt/ß-catenin signaling, a critical pathway in stem cell and cancer cell biology, as an important downstream intermediate in regulating this migration phenotype.

Cell surface GRP78 promotes stemness in normal and neoplastic cells.

Reliable approaches to identify stem cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly acquired by cancer cells has been challenging. We harnessed the power of reprogramming to examine GRP78, a chaperone protein generally restricted to the endoplasmic reticulum in normal tissues, but which is expressed on the cell surface of human embryonic stem cells and many cancer types. We have discovered that (1) cell surface GRP78 (sGRP78) is expressed on iPSCs and is important in reprogramming, (2) sGRP78 promotes cellular functions in both pluripotent and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a CD24-/CD44+ tumor initiating cell (TIC) population (4) sGRP78+ breast cancer cells are enriched for stemness genes and appear to be a subset of TICs (5) sGRP78+ breast cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective findings show that GRP78 has important functions in regulating both pluripotency and oncogenesis, and suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo.

Clonal origin of metastatic testicular teratomas.

PURPOSE: Testicular teratomas in adult patients are histologically diverse tumors that frequently coexist with other germ cell tumor (GCT) components. These mixed GCTs often metastasize to retroperitoneal lymph nodes where multiple GCT elements are frequently present in the same metastatic lesion. Neither the genetic relationships among the different components in metastatic lesions nor the relationships between primary and metastatic GCT components have been elucidated. EXPERIMENTAL DESIGN: We examined metastases from 31 patients who underwent primary retroperitoneal lymph node dissection for metastatic testicular GCT. All patients had metastatic mature teratoma with one or more other GCT components. This study included a total of 72 metastatic GCT components and 16 primary GCT components from 31 patients. Genomic DNA samples from each component were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for seven microsatellite polymorphic markers on chromosomes 1p36 (D1S1646), 9p21 (D9S171 and IFNA), 9q21 (D9S303), 13q22-q31 (D13S317), 18q22 (D18S543), and 18q21 (D18S60) were done to assess clonality. RESULTS: Twenty-nine of 31 (94%) cases showed allelic loss in one or more components of the metastatic GCTs. Twenty-nine of 31 mature teratomas showed allelic loss in at least one of seven microsatellite polymorphic markers analyzed. The frequency of allelic loss in informative cases of metastatic mature teratoma was 27% (8 of 30) with D1S1646, 34% (10 of 29) with D9S171, 37% (10 of 27) with IFNA, 27% (8 of 30) with D9S303, 46% (13 of 28) with D13S317, 26% (7 of 27) with D18S543, and 36% (10 of 28) with D18S60. Completely concordant allelic loss patterns between the mature teratoma and all of the other metastatic GCT components were seen in 26 of 29 cases in which the mature teratoma component showed LOH. Nearly identical allelic loss patterns were seen in the three remaining cases. In six cases analyzed, LOH patterns of each metastatic component were compared with each GCT component of the primary testicular tumor. In all six cases, each primary and metastatic component showed an identical pattern of allelic loss. CONCLUSION: Our data support the common clonal origin of metastatic mature teratomas with other components of metastatic testicular GCTs and with each component of the primary tumor.

Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics.

Induced pluripotent stem cells (iPSCs) show variable methylation patterns between lines, some of which reflect aberrant differences relative to embryonic stem cells (ESCs). To examine whether this aberrant methylation results from genetic variation or non-genetic mechanisms, we generated human iPSCs from monozygotic twins to investigate how genetic background, clone, and passage number contribute. We found that aberrantly methylated CpGs are enriched in regulatory regions associated with MYC protein motifs and affect gene expression. We classified differentially methylated CpGs as being associated with genetic and/or non-genetic factors (clone and passage), and we found that aberrant methylation preferentially occurs at CpGs associated with clone-specific effects. We further found that clone-specific effects play a strong role in recurrent aberrant methylation at specific CpG sites across different studies. Our results argue that a non-genetic biological mechanism underlies aberrant methylation in iPSCs and that it is likely based on a probabilistic process involving MYC that takes place during or shortly after reprogramming.