BackgroundNeural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it's estimated to be 2.5 to 3%.Methodology/Principal FindingsWe investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of β-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.Conclusions/SignificanceThese findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson's disease, multiple sclerosis, and Alzheimer's disease.

The basement membrane (BM) is a layer of specialized extracellular matrix that surrounds normal prostate glands and preserves tissue integrity. Lack or discontinuity of the BM is a prerequisite for tumor cell invasion into interstitial spaces, thus favoring metastasis. Therefore, BM maintenance represents a barrier against cancer development and progression. In the study, we show that miR-205 participates in a network involving DNp63a, which is essential for maintenance of the BM in prostate epithelium. At the molecular level, DNp63a is able to enhance miR-205 transcription by binding to its promoter, whereas the microRNA can post-transcriptionally limit the amount of DNp63a protein, mostly by affecting DNp63a proteasomal degradation rather than through a canonical miRNA/target interaction. Functionally, miR-205 is able to control the deposition of laminin-332 and its receptor integrin-b4. Hence, pathological loss of miR-205, as widely observed in prostate cancer, may favor tumorigenesis by creating discontinuities in the BM. Here we demonstrate that therapeutic replacement of miR-205 in prostate cancer (PCa) cells can restore BM deposition and 3D organization into normal-like acinar structures, thus hampering cancer progression. MicroRNAs (miRNAs) are short, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. 1 By hybridizing to at least partially complementary regions on target mRNAs, miRNAs can induce mRNA degradation or translation inhibition, thus finely tuning protein expression in a variety of biological processes. 1 Consequently, aberrant miRNA expression and function have been linked to the pathogenesis of human diseases, including cancer, where specific miRNAs have been proven to act as oncogenes or tumor suppressors. 2 We previously showed that miR-205 is downregulated in prostate cancer (PCa) compared with adjacent non-neoplastic tissue. 3 This finding was then confirmed by several independent studies (reviewed in Gandellini et al. 4 ; Schaefer et al. 5 ), and miR-205 recognized as the best single miRNA able to correctly distinguish prostate tumor from normal tissue. 6 We also reported that miR-205 acts as a tumor suppressor in human prostate, as its reintroduction in PCa cells reverts epithelial-to-mesenchymal transition (EMT), 3 thus suggesting that miR-205 reduction may drive the progression toward a cell phenotype with enhanced invasive properties and favor metastasis. Accordingly, tumors from patients with lymph node dissemination show lower miR-205 expression than those from node-negative patients. 3 However, evidence of a downregulation of the miRNA in clinically localized carcinomas 4 suggests that loss of miR-205 in PCa may anticipate disease progression. To gain insight into this early loss of the miRNA and into the mechanisms of PCa development, we investigated the physiological role of miR-205 in normal prostate.Prostatic epithelium is characterized by three different cell layers: (i) an outer, androgen-independent basal layer, lying on a basement m...

There is accumulating evidence that breast cancer may arise from mutated mammary stem/progenitor cells which have been termed breast cancer-initiating cells (BCIC). BCIC identified in clinical specimens based on membrane phenotype (CD44+/CD24−/low and/or CD133+ expression) or enzymatic activity of aldehyde dehydrogenase 1 (ALDH1+), have been demonstrated to have stem/progenitor cell properties, and are tumorigenic when injected in immunocompromized mice at very low concentrations. BCIC have also been isolated and in vitro propagated as non-adherent spheres of undifferentiated cells, and stem cell patterns have been recognized even in cancer cell lines. Recent findings indicate that aberrant regulation of self renewal is central to cancer stem cell biology. Alterations in genes involved in self-renewal pathways, such as Wnt, Notch, sonic hedgehog, PTEN and BMI, proved to play a role in breast cancer progression. Hence, targeting key elements mediating the self renewal of BCIC represents an attractive option, with a solid rationale, clearly identifiable molecular targets, and adequate knowledge of the involved pathways. Possible concerns are related to the poor knowledge of tolerance and efficacy of inhibiting self-renewal mechanisms, because the latter are key pathways for a variety of biological functions and it is unknown whether their interference would kill BCIC or simply temporarily stop them. Thus, efforts to develop BCIC-targeted therapies should not only be focused on interfering on self-renewal, but could seek to identify additional molecular targets, like those involved in regulating EMT-related pathways, in reversing the MDR phenotype, in inducing differentiation and controlling cell survival pathways.

We reported that the splice variant of human HER2 lacking exon 16 (delta16HER2) represents a highly penetrating HER2 oncogenic alteration identified in human primary breast tumor specimens and is able to influence the response to Trastuzumab. This HER2 variant forms covalent cysteine bonds that generate constitutively active homodimers, thereby activating multiple oncogenic downstream signaling pathways that we recently found to be mediated through activated Src kinase. To examine the ability of delta16HER2 to transform mammary epithelium in vivo and to monitor delta16HER2-driven tumorigenesis in live mice, we generated a FVB transgenic mouse model for the human delta16HER2 isoform. Transgenic female mice developed multifocal mammary tumors with a rapid onset starting at about 12 weeks of age and progressively thereafter, clearly pointing to the candidacy of the delta16HER2 isoform as the transforming form of the human HER2 oncoprotein. Histological and immunohistochemical analysis (IHC) of primary mammary nodules revealed a population of polygonal cells with classical epithelia-like aspects distinctly expressing HER2 and also a population of smaller spindle-shaped cells arranged in fascicles with lower levels of HER2 expression, suggesting the onset of the epithelial-to-mesenchymal transition (EMT). Consistent with these findings, FACS analysis of delta16HER2-positive tumor cells immunomagnetically purified from disaggregated transgenic primary tumors indicated the increased mean fluorescence intensity of HER2 staining with increasing tumor cell size. IHC analysis of the lung metastases that had formed in the majority of female mice revealed monomorphic and classical epithelial tumor cells homogeneously expressing high levels of delta16HER2. FACS and IHC analyses confirmed the lower binding efficacy of Trastuzumab to delta16HER2-overexpressing primary tumor cells cultured both under bidimensional (2D) and tridimensional (3D) conditions as compared to monoclonal reagents directed to different HER2 extracellular domain epitopes. Experiments in both primary and metastatic in vitro and in vivo delta16HER2-positive models are in progress to determine whether delta16HER2-driven tumor aggressiveness and Trastuzumab susceptibility depend not only on genetic changes intrinsic to the tumor cell, i.e., the EMT process, but also on extrinsic tumor surrounding microenvironment-related factors such as an imbalance between extracellular and intracellular pH, redox state and hypoxia. Preliminary FACS and IHC analyses indicate that delta16HER2-positive primary tumor cells are reactive for known epithelial markers as EpCAM, E-cadherin- and ck-18 and, a small subset of these mammary tumor cells, also stain positive for the mesenchymal differentiation markers vimentin, N-cadherin and ck14 significantly indicating an active EMT program. Supported by AIRC and Ministry of Health Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916. doi:1538-7445.AM2012-916

Background: Human Neural Stem Cells (hNSCs) are responsible for brain development at prenatal and postnatal stages and for tissue homeostasis and repair after injury on adulthood. The identification of hNSC specific markers is still a debated argument. GFAP-δ, the delta isoform of Glial Fibrillary Acidic Protein (GFAP), is particularly expressed in the Subventricular Zone (SVZ) of the brain and its expression has been related to long-term quiescent NSC. On the other hand, another cytoskeletal protein, gelsolin, is expressed in Neural Progenitor Cells (NPCs) migrating from the SVZ. Methods: In this study, we intended to investigate by immunocytochemistry the co-expression of GFAP-δ and gelsolin in different sources of hNSCs, and hNPCs, with particular emphasis on Good Manifacture Procedures (GMPs) grade hNSC currently used in two clinical trials on Amyotrophic Lateral Sclerosis (ALS) and Multiple Sclerosis (MS) patients. Results: We found that the two proteins are co-expressed by undifferentiated GFAP+ cells but tend to localize in different sub-cellular compartments and to segregate in divergent GFAP+ progenies along with differentiation. Interestingly, we proved that, after transplantation into the brain of rodent models of focal demyelination or transient global ischemia, hNSC integrating in the SVZ still retain the co-expression of GFAP-δ and gelsolin, indicating that hNSC intrinsically co-express the two proteins at the stem stage. Conclusion: These findings suggest that GFAP-δ and gelsolin may represent two candidate markers to distinguish NSC from NSC-deriving divergent astroglial phenotypes.

Almost 90% of human primary breast cancers (BCs) express 4-9% of total wild-type (WT) HER2 as a splice variant lacking exon 16 (d16HER2). Consequent in-frame activating deletion of 2 cysteine residues causes an imbalanced conformation resulting in receptor constitutive homodimerization, enhanced signaling activity, transformation and altered trastuzumab (T) binding. We recently produced a human d16HER2 transgenic (tg) mouse characterized by a rapid multifocal mammary tumors onset and the expression of active d16HER2 dimers on tumor cells, whose downstream signaling was found coupled to multiple activated nodes that include Src kinase. In order to dissect d16HER2 role in the aggressiveness and in the susceptibility to anti-HER2 therapy, we focused on the generation of stable d16HER2-expressing mammary tumor cell lines to be used in different bioassays. An immunomagnetic purification procedure was applied to generate primary homogeneously d16HER2-expressing cell cultures. These purified tumor cell lines were analyzed by flow cytometry and immunofluorescence and their migration/invasion ability was assessed through Boyden chamber test. d16HER2 downstream signaling was evaluated by western blot and T, Lapatinib (L) and Dasatinib (D) drugs sensitivity was measured with WST-1 and soft-agar bioassays. As controls, we compared in vitro d16HER2-models oncogenic features to those of the human BC BT474, which also co-expresses a low amount of d16HER2 transcript (4%), and to a murine mammary carcinoma cell line (wtHER2), derived from a primary mammary tumor developed in human WT HER2 tg mice. We found that d16HER2 in vitro models expressed high levels of stable homodimers combined with the recruitment of activated Src, STAT3, MAPK and Akt, as in vivo primary mammary tumors. Both in bidimensional (2D) and matrigel-cultured tumor cells, we confirmed the T lower binding capability for d16HER2 than other anti-HER2 MAbs directed to different extracellular domain epitopes. d16HER2 tumor cells had an enhanced migratory and invasive capacity compared to wtHER2 and BT474 cells and, notably, were completely resistant to T treatment and less responsive to L. In virtue of a highly activated Src kinase expression in d16HER2-positive models, we tested in 2D the therapeutic effects of D and observed that d16HER2-cells were significantly more sensitive than wtHER2 and BT474 cells. Preliminary 3D-drug susceptibility assays showed that the sensitivity of d16HER2 cells increased for all the tested drugs, if assessed in a suitable environment such as soft-agar. Our findings further indicate that the constitutive expression of d16HER2 variant identifies an aggressive tumor phenotype and confirm, at least in vitro in 2D conditions, that this isoform is resistant to T and L, whereas is sensitive to D. Further analyses are ongoing to analyze in vivo drug sensitivity of d16 in comparison to WT HER2 model. Supported by AIRC and Ministry of Health Citation Format: Gaia C. Ghedini, Arianna Palladini, Valentina Ciravolo, Lorenzo Castagnoli, Giulia Marzano, Roberta Zappasodi, Guido Santilli, Augusto Amici, Alessia Lamolinara, Manuela Iezzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. Role of d16HER2 splice variant in HER2-positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2637. doi:10.1158/1538-7445.AM2014-2637

No abstract

No abstract