Age-dependent association between protein expression of the embryonic stem cell marker Cripto-1 and survival of glioblastoma patients

Exploring the re-emergence of embryonic signaling pathways may reveal important information for cancer biology. Nodal is a transforming growth factor-β (TGF-β)-related morphogen that plays a critical role during embryonic development. Nodal signaling is regulated by the Cripto-1 co-receptor and another TGF-β member, Lefty. Although these molecules are poorly detected in differentiated tissues, they have been found in different human cancers. Poor prognosis of glioblastomas justifies the search for novel signaling pathways that can be exploited as potential therapeutic targets. Because our intracranial glioblastoma rat xenograft model has revealed importance of gene ontology categories related to development and differentiation, we hypothesized that increased activity of Nodal signaling could be found in glioblastomas. We examined the gene expressions of Nodal, Cripto-1, and Lefty in microarrays of invasive and angiogenic xenograft samples developed from four patients with glioblastoma. Protein expression was evaluated by immunohistochemistry in 199 primary glioblastomas, and expression levels were analyzed for detection of correlations with available clinical information. Gene expression ofNodal, Lefty, and Cripto-1 was detected in the glioblastoma xenografts. Most patient samples showed significant levels of Cripto-1 detected by immunohistochemistry, whereas only weak to moderate levels were detected for Nodal and Lefty. Most importantly, the higher Cripto-1 scores were associated with shorter survival in a subset of younger patients. These findings suggest for the first time that Cripto-1, an important molecule in developmental biology, may represent a novel prognostic marker and therapeutic target in categories of younger patients with glioblastoma.publishedVersio

The Stem Cell Phenotype of Aggressive Breast Cancer Cells

Aggressive cancer cells are characterized by their capacity to proliferate indefinitely and to propagate a heterogeneous tumor comprised of subpopulations with varying degrees of metastatic propensity and drug resistance properties. Particularly daunting is the challenge we face in the field of oncology of effectively targeting heterogeneous tumor cells expressing a variety of markers, especially those associated with a stem cell phenotype. This dilemma is especially relevant in breast cancer, where therapy is based on traditional classification schemes, including histological criteria, differentiation status, and classical receptor markers. However, not all patients respond in a similar manner to standard-of-care therapy, thereby necessitating the need to identify and evaluate novel biomarkers associated with the difficult-to-target stem cell phenotype and drug resistance. Findings related to the convergence of embryonic and tumorigenic signaling pathways have identified the embryonic morphogen Nodal as a promising new oncofetal target that is reactivated only in aggressive cancers, but not in normal tissues. The work presented in this paper confirms previous studies demonstrating the importance of Nodal as a cancer stem cell molecule associated with aggressive breast cancer, and advances the field by providing new findings showing that Nodal is not targeted by standard-of-care therapy in breast cancer patients. Most noteworthy is the linkage found between Nodal expression and the drug resistance marker ATP-binding cassette member 1 (ABCA1), which may provide new insights into developing combinatorial approaches to overcome drug resistance and disease recurrence

The Stem Cell Phenotype of Aggressive Breast Cancer Cells

Aggressive cancer cells are characterized by their capacity to proliferate indefinitely and to propagate a heterogeneous tumor comprised of subpopulations with varying degrees of metastatic propensity and drug resistance properties. Particularly daunting is the challenge we face in the field of oncology of effectively targeting heterogeneous tumor cells expressing a variety of markers, especially those associated with a stem cell phenotype. This dilemma is especially relevant in breast cancer, where therapy is based on traditional classification schemes, including histological criteria, differentiation status, and classical receptor markers. However, not all patients respond in a similar manner to standard-of-care therapy, thereby necessitating the need to identify and evaluate novel biomarkers associated with the difficult-to-target stem cell phenotype and drug resistance. Findings related to the convergence of embryonic and tumorigenic signaling pathways have identified the embryonic morphogen Nodal as a promising new oncofetal target that is reactivated only in aggressive cancers, but not in normal tissues. The work presented in this paper confirms previous studies demonstrating the importance of Nodal as a cancer stem cell molecule associated with aggressive breast cancer, and advances the field by providing new findings showing that Nodal is not targeted by standard-of-care therapy in breast cancer patients. Most noteworthy is the linkage found between Nodal expression and the drug resistance marker ATP-binding cassette member 1 (ABCA1), which may provide new insights into developing combinatorial approaches to overcome drug resistance and disease recurrence

Age-dependent association between protein expression of the embryonic stem cell marker Cripto-1 and survival of glioblastoma patients

Exploring the re-emergence of embryonic signaling pathways may reveal important information for cancer biology. Nodal is a transforming growth factor-β (TGF-β)-related morphogen that plays a critical role during embryonic development. Nodal signaling is regulated by the Cripto-1 co-receptor and another TGF-β member, Lefty. Although these molecules are poorly detected in differentiated tissues, they have been found in different human cancers. Poor prognosis of glioblastomas justifies the search for novel signaling pathways that can be exploited as potential therapeutic targets. Because our intracranial glioblastoma rat xenograft model has revealed importance of gene ontology categories related to development and differentiation, we hypothesized that increased activity of Nodal signaling could be found in glioblastomas. We examined the gene expressions of Nodal, Cripto-1, and Lefty in microarrays of invasive and angiogenic xenograft samples developed from four patients with glioblastoma. Protein expression was evaluated by immunohistochemistry in 199 primary glioblastomas, and expression levels were analyzed for detection of correlations with available clinical information. Gene expression ofNodal, Lefty, and Cripto-1 was detected in the glioblastoma xenografts. Most patient samples showed significant levels of Cripto-1 detected by immunohistochemistry, whereas only weak to moderate levels were detected for Nodal and Lefty. Most importantly, the higher Cripto-1 scores were associated with shorter survival in a subset of younger patients. These findings suggest for the first time that Cripto-1, an important molecule in developmental biology, may represent a novel prognostic marker and therapeutic target in categories of younger patients with glioblastoma

Cleavage of Histone 3 by Cathepsin D in the Involuting Mammary Gland

<div><p>The post-lactational regression of mammary gland is a complex multi-step process designed to conserve the biological function of the gland for next pregnancy. This developmental stage is a biological intrigue with great relevance to breast cancer research, and thus has been the subject of intensive scrutiny. Multipronged studies (microarray, proteomics profiling, animal knock-out models) have provided a repertoire of genes critical to involution. However, the caveat of these approaches remains in their failure to reveal post-translational modification(s), an emerging and critical aspect of gene regulation in developmental processes and mammary gland remodeling. The massive surge in the lysosomal enzymes concurrent with the onset of involution has been known for decades, and considered essential for “clearance” purposes. However, functional significance of these enzymes in diverse biological processes distinct from their proteolytic activity is just emerging. Studies from our laboratory had indicated specific post-translational modifications of the aspartyl endopeptidase Cathepsin D (CatD) at distinct stages mammary gland development. This study addresses the biological significance of these modifications in the involution process, and reveals that post-translational modifications drive CatD into the nucleus to cleave Histone 3. The cleavage of Histone 3 has been associated with cellular differentiation and could be critical instigator of involution process. From functional perspective, deregulated expression and increased secretion of CatD are associated with aggressive and metastatic phenotype of breast cancer. Thus unraveling CatD’s physiological functions in mammary gland development will bridge the present gap in understanding its pro-tumorigenic/metastatic functions, and assist in the generation of tailored therapeutic approaches.</p></div

Interferon Regulatory Factor 6 Promotes Cell Cycle Arrest and Is Regulated by the Proteasome in a Cell Cycle-Dependent Manner▿

Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors. IRF6 has not been linked to the regulatory pathways or functions associated with other IRF family members, and the regulation and function of IRF6 remain unknown. We recently identified a protein interaction between IRF6 and the tumor suppressor maspin. To gain insight into the biological significance of the maspin-IRF6 interaction, we examined the regulation and function of IRF6 in relation to maspin in normal mammary epithelial cells. Our results demonstrate that in quiescent cells, IRF6 exists primarily in a nonphosphorylated state. However, cellular proliferation leads to rapid IRF6 phosphorylation, resulting in proteasome-dependent IRF6 degradation. These data are supported in situ by the increased expression of IRF6 in quiescent, differentiated lobuloalveolar cells of the lactating mammary gland compared to its expression in proliferating ductal and glandular epithelial cells during pregnancy. Furthermore, the reexpression of IRF6 in breast cancer cells results in cell cycle arrest, and the presence of maspin augments this response. These data support a model in which IRF6, in collaboration with maspin, promotes mammary epithelial cell differentiation by facilitating entry into the G0 phase of the cell cycle

CatD purified from involuting mouse mammary tissue induces morphological changes in normal mammary epithelial cells.

<p>(A). SDS-PAGE (12.5% gel) and Western blot analysis of purified mCatD from different stages of development. (B–F). Phase contrast images of normal mammary epithelial cells following treatment with mCatD purified from distinct stages of mouse mammary gland development. Involution-derived mCatD induces the generation of few large cells with scant cytoplasm (arrowhead in E &F) in cultured epithelial cells. Recombinant hCatD and lactation-derived mCatD fail to induce comparable changes (C–D). The arrow in E points to cells presumably fusing. Original magnification:10×. (G). Western blot analysis of total cell lysates from treated cells indicated minimal effect of exogenously added mCatD on HMEpC’s endogenous CatD expression (probed by anti-human CatD, and seen as single chain ∼43 kDa and ∼32 kDa mature enzyme). GAPDH was used as loading control.</p

Confocal imaging and Western blot analysis of HMEpCs ± treatment with mCatD derived from lactation and involution stages.

<p>(A–F) Confocal images of HMEpCs ± treatment with Alexa 594 tagged mCatD preparations indicated involution-derived mCatD was transported mostly to the cytosol and often intensely localized to the nucleus (D & E, white arrow). It also promoted the process of entosis (cells engulfing other cells, D & E). Different stages of engulfment are captured in Fig. 2E (yellow arrow indicates an engulfed cell with intact membrane, the red arrow points to a cell with two nuclei). Occasionally, fragmenting nuclei of an engulfed cell could also be seen, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103230#pone.0103230.s003" target="_blank">Fig. S3</a>). Endogenous hCatD is highlighted in (A) by anti-hCatD, followed by Alexa Fluor 660 secondary, red fluorescent in the image). Additionally, positional differences between endogenous CatD (green fluorescence) and ID2-derived mCatD (red fluorescence) are depicted in Fig. 2F. Please note a similar pattern of distribution for r-hCatD (B), and L3-derived mCatD (C) with the endogenous CatD (mostly lysosomal, A&F). β- Catenin is depicted by Alexa Fluor 488 (green fluorescent) and the nucleus is stained with DAPI. Original magnifications: A–C &F: 40x, D&E:100x Images A–F are also presented as split images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103230#pone.0103230.s002" target="_blank">Fig. S2</a>. Scale bar represents 20 µm. Total cell lysates (G) as well as cytosolic and nuclear fractions (H) of HMEpCs ± mCatD treatments were subjected to Western blot analysis to determine intracellular localization of mCatD compared to untreated control. Only involution-derived mCatD (from day two onwards) translocated into the nucleus, cleaved H3, the H3 cleavage was inhibited by pepstatin, but not by CatL inhibitor Z-FY-CHO. GAPDH and Lamin B were used as loading controls with PCNA depicting changes in the proliferation following the treatment. GAPDH and Lamin B were also used as a quality control to confirm the absence of contaminating cytosolic or nuclear proteins in the nuclear and cytosolic fractions respectively. (I). Cytosolic (20 µg) and nuclear fractions (60 µg) from HMEpCs ± NOC12 were subjected to SDS-PAGE (4–20% gel) and Western blot analysis to determine the effect of nitration on CatD processing and cellular distribution. (J). Cytosolic and nuclear associated CatD were immunoprecipitated and subjected to Western blot analysis using anti-nitro tyrosine antibody.</p

Onset of involution prompts nuclear translocation of CatD and cleavage of H3 in mouse mammary gland.

<p>(A). Western blot analysis of cytosolic and nuclear fractions from lactation and involution stages of mouse mammary gland reveals nuclear association of CatD at involution days 2 and 3. Cleavage of H3 occurs following the onset of involution. The cleavage of H3 may occur at multiple sites as indicated by the presence of several cleavage products of H3. H3 is also detected in the cytosolic fraction of the mammary gland at lactation and involution stages (soluble form) and is lysine ²³ acetylated (AcK²³H3) at involution day 1. The AcK²³ could also be detected in the nuclear H3 but at a considerably lower abundance. (B–F). Immunofluorescence and confocal microscopy analysis of the formalin fixed, paraffin embedded mouse mammary tissue. Antibodies used were anti-mouse CatD and β-catenin followed by treatment with Alexa Fluor 488 secondary antibody for β-catenin (green) and Alexa Fluor 660 for mCatD (red). The nucleus was stained with DAPI. At involution day 3, CatD was detected in the nucleus (white arrows, Fig. 3D and specifically 3E), and sporadic multinucleated cells could be seen in the gland (white arrowhead, Fig. 3D). In addition, intense CatD immunostaining was noted in the structures reminiscent of phagosomes (Fig. 3E yellow arrows and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103230#pone.0103230.s005" target="_blank">Fig. S5</a>). By day 7 of involution, the gland is mostly populated with adipocytes (Fig. 3F). Original magnifications: B–F 63x, scale bar represents 20 µm.</p

The observed H3 cleavage site (depicted by green color in A) by involution-derived mCatD.

<p>Theoretically predicted cleavage sites are also indicated (red in A). The in vitro cleavage of the recombinant H3.3 protein by ID2-derived mCatD and CatL is also indicated in Fig. 4B, with the total cell lysate of the mouse mammary gland at involution day 2 included as reference. The NH2 terminal sequence of H3 cleavage product (marked by *) is depicted in C.</p