Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pT alpha, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in TALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of TALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumor

WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia

open12siLoss of function mutations and deletions in Wilms tumor 1 (WT1) gene are present in about 10% of T-cell acute lymphoblastic leukemia. Clinically, (WT1) mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here, we demonstrate that WT1 plays a critical role in DNA damage response in T-cell leukemia. (WT1) loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, (WT1) loss positively affected the expression of the X-linked inhibitor of apoptosis protein (XIAP), and genetic or chemical inhibition with Embelin, a XIAP inhibitor, significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient xenografts. These results unveil an important role of (WT1) tumor suppressor gene in DNA damage response, and support a role for anti-XIAP targeted therapies in the treatment of (WT1)-mutant T-cell leukemia.openBordin, Fulvio; Piovan, Erich; Masiero, Elena; Ambesi-Impiombato, Alberto; Minuzzo, Sonia; Bertorelle, Roberta; Sacchetto, Valeria; Pilotto, Giorgia; Basso, Giuseppe; Zanovello, Paola; Amadori, Alberto; Tosello, ValeriaBordin, Fulvio; Piovan, Erich; Masiero, Elena; Ambesi-Impiombato, Alberto; Minuzzo, Sonia; Bertorelle, Roberta; Sacchetto, Valeria; Pilotto, Giorgia; Basso, Giuseppe; Zanovello, Paola; Amadori, Alberto; Tosello, Valeri

Static and dynamic retinal fixation stability in microperimetry

Objective: To compare static (during a pure fixation task) versus dynamic (during microperimetry) quantification of fixation stability using microperimetry in normal and pathologic eyes, by means of 2 available (clinical and bivariate contour ellipse area [BCEA]) classification methods. Design: Prospective comparative observational study. Participants: One hundred and forty-nine eyes (110 patients) with different macular diseases and 171 normal eyes (109 subjects). Methods: In all eyes studied, fixation stability was acquired during an isolated fixation task (static fixation) and during microperimetry (dynamic fixation). All fixation data were analyzed and compared by means of a clinical classification and by means of BCEA quantification. Results: Pathologic eyes were classified as follows: 41 eyes with diabetic macular edema (DME group), 13 eyes with vitreoretinal interface disease, 60 eyes with age-related macular degeneration (AMD group), and 35 eyes with primary open-angle glaucoma. Fixation stability was not uniform among groups according to clinical classification in both static and dynamic modalities (p < 0.0001). AMD group showed larger BCEA areas compared with all other groups (p < 0.0001). All pathologic groups showed more unstable fixation in dynamic fashion according to both clinical and BCEA methods (p < 0.0001). The variation of fixation stability of control group in dynamic task was highlighted only by BCEA analysis (p < 0.0001). A deterioration of retinal fixation according to clinical method matches a significant increase in BCEA areas (p < 0.0001). Conclusions: The detection of clinical fixation stability changes improves when acquired in the dynamic modality. BCEA analysis provides more accurate evaluation of fixation stability and may detect minimal quantitative changes of the fixation area. However, a standard clinical classification can also detect changes in fixation stability in pathologic eyes. Both methods are useful tools in the evaluation of fixation stability. \ua9 2013 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved

Autophagy inhibition reduces chemoresistance and tumorigenic potential of human ovarian cancer stem cells

Epithelial ovarian cancer (EOC) is one of the most malignant gynecological tumors with a high mortality rate owing to tumor relapse after anticancer therapies. It is widely accepted that a rare tumor cell population, known as cancer stem cells (CSC), is responsible for tumor progression and relapse; intriguingly, these cells are able to survive nutrient starvation (such as in vitro culture in the absence of glucose) and chemotherapy treatment. Recent data also indicated that chemotherapy resistance is associated with autophagy activation. We thus decided to investigate both in vitro and in vivo the autophagic activity and the effects of the perturbation of this pathway in CSC isolated from EOC ascitic effusions. Ovarian CSC, identified according to their CD44/CD117 co-expression, presented a higher basal autophagy compared with the non-stem counterpart. Inhibition of this pathway, by in vitro chloroquine treatment or CRISPR/Cas9 ATG5 knockout, impaired canonical CSC properties, such as viability, the ability to form spheroidal structures in vitro, and in vivo tumorigenic potential. In addition, autophagy inhibition showed a synergistic effect with carboplatin administration on both in vitro CSC properties and in vivo tumorigenic activity. On the whole, these results indicate that the autophagy process has a key role in CSC maintenance; inhibition of this pathway in combination with other chemotherapeutic approaches could represent a novel effective strategy to overcome drug resistance and tumor recurrence

Comparison of the Genomic Profile of Cancer Stem Cells and Their Non-Stem Counterpart: The Case of Ovarian Cancer

The classical cancer stem cell (CSC) model places CSCs at the apex of a hierarchical scale, suggesting different genetic alterations in non-CSCs compared to CSCs, since an ill-defined number of cell generations and time intervals separate CSCs from the more differentiated cancer cells that form the bulk of the tumor. Another model, however, poses that CSCs should be considered a functional state of tumor cells, hence sharing the same genetic alterations. Here, we review the existing literature on the genetic landscape of CSCs in various tumor types and as a case study investigate the genomic complexity of DNA obtained from matched CSCs and non-CSCs from five ovarian cancer patients, using a genome-wide single-nucleotide polymorphism (SNP) microarray

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages.

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location

NOTCH3 SIGNALING REGULATES MUSASHI-1 EXPRESSION IN METASTATIC COLORECTAL CANCER CELLS.

Musashi-1 (Msi-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the Notch pathway through inactivation of Numb, a Notch signalling repressor. To date, the mechanisms of regulation of Msi-1 levels remain largely unknown. Here we investigated the regulation of Msi-1 by Notch signalling in the colorectal cancer cell lines MICOL-14tum and LoVo and in primary cultures of colorectal cancer (CRC) metastases. Stimulation by the Notch ligand DLL4 was associated with an increase of Msi-1 mRNA and protein levels, and this phenomenon was prevented by the addition of antibody neutralizing Notch2/3 but not Notch1. Moreover, forced expression of activated Notch3 increased Msi-1 levels, whereas silencing of Notch3 by shRNA reduced Msi-1 levels in both CRC cells and tumor xenografts. Consistent with these findings, forced Notch3 expression or stimulation by DLL4 increased levels of activated Notch1 in MICOL-14tum and LoVo cells. Finally, treatment of CRC cells with anti-Notch2/3 antibody increased Numb protein while significantly reducing formation of spheroids in both MICOL-14tum cells and primary tumor cultures. This novel feed-forward circuit involving DLL4, Notch3, Msi-1, Numb and Notch1 may be relevant for regulation of Notch signalling in physiological processes as well as in tumor development. With regard to therapeutic implications, Notch3-specific drugs could represent a valuable strategy to limit Notch signaling in the context of colorectal cancers overexpressing this recepto