Hara Polioudaki

Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer

The CD44(+)/CD24(-/low) and ALDH1(+) cell phenotypes are associated with stemness and enhanced tumorigenic potential in breast cancer. We assessed the expression of CD44, CD24 and ALDH1 on tumor cells circulating in the peripheral blood (CTCs) of patients with metastatic breast cancer using triple-marker immunofluorescence microscopy. Among a total of 1439 CTCs identified in 20 (66.7%) out of 30 patients, 35.2% had the stem-like/tumorigenic phenotype CD44(+)/CD24(-/low), whereas 17.7% of the CTCs analyzed in seven patients, were ALDH1(high)/CD24(-/low). In conclusion, we report the existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer.

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation‐dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub‐set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross‐link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB‐binding protein, revealing a new mechanism for anchoring domains of under‐acetylated chromatin to the inner nuclear membrane.

Mitotic phosphorylation of histone H3 at threonine 3.

Nuclear envelope‐peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino‐terminal sequencing show that one of these activities co‐isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post‐translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio‐temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.

Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer

BackgroundCTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. However, no information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. We investigated the correlation between keratin expression of CTCs and patients’ outcome and characterized the EMT status of CTCs via the establishment of a numerical “ratio” value of keratin and vimentin expression levels on a single cell basis.MethodsKeratin expression was evaluated in 1262 CTCs from 61 CTC-positive patients with metastatic breast cancer, using analysis of images obtained through the CellSearch System. For the determination of vimentin/keratin (vim/K) ratios, expression levels of keratin and vimentin were measured in cytospin preparations of luminal (MCF-7 and T47D) and basal (MDA.MB231 and Hs578T) breast cancer cell lines and 110 CTCs from 5 CTC-positive patients using triple immunofluorescence laser scanning microscopy and image analysis.ResultsMCF-7 and T47D displayed lower vim/K ratios compared to MDA.MB231 and Hs578T cells, while MCF-7 cells that had experimentally undergone EMT were characterized by varying intermediate vim/K ratios. CTCs were consisted of an heterogeneous population presenting variable vim/K values with 46% of them being in the range of luminal breast cancer cell lines. Keratin expression levels of CTCs detected by the CellSearch System correlated with triple negative (p = 0.039) and ER-negative (p = 0.025) breast cancer, and overall survival (p = 0.038).ConclusionsKeratin expression levels of CTCs correlate with tumor characteristics and clinical outcome. Moreover, CTCs display significant heterogeneity in terms of the degree of EMT phenotype that probably reflects differential invasive potential. The assessment of the vim/K ratios as a surrogate marker for the EMT status of CTCs merits further investigation as a prognostic tool in breast cancer.

Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells

We have investigated the subcellular distribution and dynamics of soluble tubulin in unperturbed and transfected HeLa cells. Under normal culture conditions, endogenous α/β tubulin is confined to the cytoplasm. However, when the soluble pool of subunits is elevated by combined cold-nocodazole treatment and when constitutive nuclear export is inhibited by leptomycin B, tubulin accumulates in the cell nucleus. Transfection assays and FRAP experiments reveal that GFP-tagged β-tubulin shuttles between the cytoplasm and the cell nucleus. Nuclear import seems to occur by passive diffusion, whereas exit from the nucleus appears to rely on nuclear export signals (NESs). Several such motifs can be identified by sequence criteria along the β-tubulin molecule and mutations in one of these (NES-1) cause a significant accumulation in the nuclear compartment. Under these conditions, the cells are arrested in the G0-G1 phase and eventually die, suggesting that soluble tubulin interferes with important nuclear functions. Consistent with this interpretation, soluble tubulin exhibits stoichiometric binding to recombinant, normally modified and hyper-phosphorylated/acetylated histone H3. Tubulin-bound H3 no longer interacts with heterochromatin protein 1 and lamin B receptor, which are known to form a ternary complex under in vitro conditions. Based on these observations, we suggest that nuclear accumulation of soluble tubulin is part of an intrinsic defense mechanism, which tends to limit cell proliferation under pathological conditions. This readily explains why nuclear tubulin has been detected so far only in cancer or in transformed cells, and why accumulation of this protein in the nucleus increases after treatment with chemotherapeutic agents.

Cancer Stem Cells in Solid and Liquid Tissues of Breast Cancer Patients: Characterization and Therapeutic Perspectives

Breast cancer stem cells (BCSCs) represent a heterogeneous subpopulation of rare cells within breast cancer tumors, displaying an enhanced tumor initiating capability and underlying disease progression and therapy resistance. Unraveling their phenotypic, biological and functional profile is a major challenge in the context of diminishing patient mortality. In this review, following a brief description on how cancer stem cells (CSCs) and their microenvironment contribute to tumor preservation and heterogeneity, we summarize the current literature regarding the molecular signature of BCSCs either localized in the primary tumor or circulating in the blood of breast cancer patients. We present recent data on specific stem and epithelial-to-mesenchymal transition (EMT) markers designating the BCSC subpopulation and underline their pathogenic significance. The molecular characterization of BCSCs has promoted the design of novel therapeutic approaches targeting the BCSC subpopulation which are currently being experimentally and clinically evaluated. We highlight recent advances on the development of novel BCSC-targeting therapeutic strategies including the inhibition of cell signaling pathways, differentiation therapy, metabolic interference and nucleotide-, bio- and nano-technology based approaches. Eliminating the chemo- and radio-resistance properties of breast cancer tumor cells via BCSC-directed therapies, combined to conventional therapeutic approaches, will augment the effectiveness of breast cancer treatment and improve the clinical outcome of breast cancer patients.

Differential nuclear shape dynamics of invasive andnon-invasive breast cancer cells are associated with actin cytoskeleton organization and stability.

Cancer cells often exhibit characteristic aberrations in their nuclear architecture, which are indicative of their malignant potential. In this study, we have examined the nuclear and cytoskeletal composition, attachment configuration dynamics, and osmotic or drug treatment response of invasive (Hs578T and MDA-MB-231) and non-invasive (MCF-10A and MCF-7) breast cancer cell lines. Unlike MCF-10A and MCF-7, Hs578T and MDA-MB-231 cells showed extensive nuclear elasticity and deformability and displayed distinct kinetic profiles during substrate attachment. The nuclear shape of MCF-10A and MCF-7 cells remained almost unaffected upon detachment, hyperosmotic shock, or cytoskeleton depolymerization, while Hs578T and MDA-MB-231 revealed dramatic nuclear contour malformations following actin reorganization.

Differential detection of nuclear envelope autoantibodies in primary biliary cirrhosis using routine and alternative methods.

BackgroundDetection of autoantibodies giving nuclear rim pattern by immunofluorescence (anti-nuclear envelope antibodies - ANEA) in sera from patients with primary biliary cirrhosis (PBC) is a useful tool for the diagnosis and prognosis of the disease. Differences in the prevalence of ANEA in PBC sera so far reported have been attributed to the methodology used for the detection as well as to ethnic/geographical variations. Therefore, we evaluated the prevalence of ANEA in sera of Greek patients with PBC by using methods widely used by clinical laboratories and a combination of techniques and materials.MethodsWe screened 103 sera by immunoblotting on nuclear envelopes and indirect immunofluorescence (IIF) using cells and purified nuclei. Reactivities against specific autoantigens were assessed using purified proteins, ELISA, immunoprecipitation and mass spectrometry.ResultsWe found higher prevalence of ANEA when sera were assayed by IIF on purified nuclei or cultured cells (50%) compared to Hep2 commercially available slides (15%). Anti-gp210 antibodies were identified in 22.3% and 33% of sera using ELISA for the C-terminal of gp210 or both ELISA and immunoprecipitation, respectively. Immunoblotting on nuclear envelopes revealed that immunoreactivity for the 210 kDa zone is related to anti-gp210 antibodies (p < 0.0001). Moreover, we found that sera had antibodies for lamins A (6.8%), B (1%) and C (1%) and LBR (8.7%), whereas none at all had detectable anti-p62 antibodies.ConclusionsThe prevalence of ANEA or anti-gp210 antibodies is under-estimated in PBC sera which are analyzed by conventional commercially available IIF or ELISA, respectively. Therefore, new substrates for IIF and ELISA should be included by clinical laboratories in the analysis of ANEA in autoimmune sera.

Stem cell technology in breast cancer: current status and potential applications

Breast cancer, the leading cause of cancer among females, is supported by the presence of a rare subset of undifferentiated cells within the tumor, identified as breast cancer stem cells (BCSCs). BCSCs underlie the mechanisms of tumor initiation and sustenance and are implicated in the dissemination of the primary tumor to metastatic sites, as they have been found circulating in the blood of breast cancer patients. The discovery of BCSCs has generated a great amount of interest among the scientific community toward their isolation, molecular characterization, and therapeutic targeting. In this review, after summarizing the literature on molecular characterization of BCSCs and methodologies used for their isolation, we will focus on recent data supporting their molecular and functional heterogeneity. Additionally, following a synopsis of the latest approaches for BCSC targeting, we will specifically emphasize on the therapeutic use of naïve or engineered normal stem cells in the treatment of breast cancer and present contradictory findings challenging their safety.

BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness

Recent advances in cancer immunology revealed immune-related properties of cancer cells as novel promising therapeutic targets. The two TNF superfamily members, APRIL (TNFSF13), and BAFF (TNFSF13B), which are type II membrane proteins, released in active forms by proteolytic cleavage and are primarily involved in B-lymphocyte maturation, have also been associated with tumor growth and aggressiveness in several solid tumors, including breast cancer. In the present work we studied the effect of APRIL and BAFF on epithelial to mesenchymal transition, migration, and stemness of breast cancer cells. Our findings show that both molecules increase epithelial to mesenchymal transition and migratory capacity of breast cancer cells, as well as cancer stem cell numbers, by increasing the expression of pluripotency genes such as ALDH1A1, KLF4, and NANOG. These effects are mediated by their common receptor BCMA (TNFRSF17) and the JNK signaling pathway. Interestingly, transcriptional data analysis from breast cancer cells and patients revealed that androgens can increase APRIL transcription and subsequently, in an autocrine/paracrine manner, enhance its pluripotency effect. In conclusion, our data suggest a possible role of APRIL and BAFF in breast cancer disease progression and provide evidence for a new possible mechanism of therapy resistance, that could be particularly relevant in aromatase inhibitors-treated patients, were local androgen is increased.