Laura B. Todaro

Neural cell adhesion molecule in human serum. Increased levels in dementia of the Alzheimer type

Memory impairment is a process associated with alterations in neuronal plasticity, synapses formation, and stabilization. As the neural cell adhesion molecule (NCAM) plays a key role in synaptic bond stabilization, we analyzed the usefulness of soluble NCAM isoforms in the diagnosis of patients with dementia of the Alzheimer type (DAT). NCAM was measured in the sera of 70 control subjects and 43 DAT patients (with different severity of cognitive impairment, GDS), employing Western blot and densitometric quantification. LMW-NCAM bands (100-130 kDa) decreased significantly with age independently of sex. DAT patients presented values of LMW-NCAM and HMW-NCAM significantly higher than healthy controls of similar age (higher than 130 kDa). Only LMW-NCAM was associated with GDS. Our results suggest that NCAM could be involved in the pathogenesis of DAT disorder and that serum NCAM levels could be useful as differential diagnostic markers of the disease.

Autophagy: Friend or Foe in Breast Cancer Development, Progression, and Treatment

Autophagy is a catabolic process responsible for the degradation and recycling of long-lived proteins and organelles by lysosomes. This degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. Thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. On the other hand, this process also functions as a protective cell-survival mechanism against environmental stress causing resistance to antineoplastic therapies. Although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis-resistant cancers, indicating that autophagy induction may also be used as a therapy. This paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti‐NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti‐NCAM‐treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM‐140 knocked‐down by siRNA in LP07 cells pretreated or not with anti‐NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti‐NCAM treatment could be mimicking homophilic trans‐interactions and NCAM‐140 knocked‐down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression. Mol. Carcinog.

Involvement of PKC delta (PKCδ) in the resistance against different doxorubicin analogs

Doxorubicin is an anti-tumor antibiotic widely used in the management of cancer patients. Its main mechanism of action involves the generation of DNA damage and the inhibition of topoisomerase II, promoting apoptosis. AD 198 is a novel doxorubicin analog devoid of DNA binding and topoisomerase II inhibitory capacities. It has been proposed that AD 198 induces apoptosis by activating protein kinase C delta (PKCδ); a PKC isoform described as growth inhibitory in a large number of cell types. We have previously demonstrated that PKCδ overexpression in NMuMG cells induced the opposite effect, promoting proliferation and cell survival. In this study, we found that PKCδ overexpression confers an enhanced cell death resistance against AD 198 cytotoxic effect and against AD 288, another doxorubicin analog that preserves its mechanism of action. These resistances involve PKCδ-mediated activation of two well-known survival pathways: Akt and NF-κB. While the resistance against AD 198 could be abrogated upon the inhibition of either Akt or NF-κB pathways, only NF-κB inhibition could revert the resistance to AD 288. Altogether, our results indicate that PKCδ increases cell death resistance against different apoptosis inductors, independently of their mechanism of action, through a differential modulation of Akt and NF-κB pathways. Our study contributes to a better understanding of the mechanisms involved in PKCδ-induced resistance and may greatly impact in the rationale design of isozyme-specific PKC modulators as therapeutic agents.

Opposite effects of protein kinase C beta1 (PKCβ1) and PKCε in the metastatic potential of a breast cancer murine model

In this paper we investigated whether protein kinase C (PKC) β1 and PKCε, members of the classical and novel PKC family, respectively, induce phenotypic alterations that could be associated with tumor progression and metastatic dissemination in a murine model of breast cancer. Stable overexpression of PKCβ1 in LM3 cells altered their ability to proliferate, adhere, and survive, and impaired their tumorigenicity and metastatic capacity. Moreover, PKCβ1 induced the re-expression of fibronectin, an extracellular matrix glycoprotein which loss has been associated with the acquisition of a transformed phenotype in different cell models, and exerted an important inhibition on proteases production, effects that probably impact on LM3 invasiveness and dissemination. Conversely, PKCε overexpression enhanced LM3 survival, anchorage-independent growth, and caused a significant increase in spontaneous lung metastasis. Our results suggest PKCβ1 functions as an inhibitory protein for tumor growth and metastasis dissemination whereas PKCε drives metastatic dissemination without affecting primary tumor growth.

Is the Epithelial-to-Mesenchymal Transition Clinically Relevant for the Cancer Patient?

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process by which a fully differentiated epithelial cell acquires mesenchymal traits, and therefore, mesenchymal abilities such as motility and invasiveness. It is a pivotal physiological process involved in embryogenesis (Type 1 EMT) and in wound healing and tissue remodeling (Type 2 EMT), which, some authors claim, but there are still some controversies, has also been co-opted by tumor cells to increase their malignant potential (Type 3 EMT). Many biomarkers of Type 3 EMT have been characterized and classified into functional categories (i.e., extracellular proteins, cell surface molecules, cytoskeletal markers, transcriptional factors, and, recently, micro RNAs). The extra and intracellular signals that lead to EMT are only starting to be understood, but there is a consensus that Ras and TGF-beta signaling must converge with NF-κB in order to achieve a full EMT. The most classical experimental model is the induction of EMT by TGF-beta in cultures of epithelial cells. Other pathways involving GSK3b, and Wnt/beta-catenin, are also implicated. Ultimately, every EMT-inducing pathway will activate any of the E-cadherin transcriptional repressors (ZEB1, ZEB2, Twist, Snail or Slug). Although in the pre-clinical setting, EMT has also been related to an accelerated tumor progression and to an increased resistance to conventional chemotherapy. In this sense, several groups are beginning to use EMT as a predictive marker of response to treatment. Finally, two chemicals targeting TGF-beta are in clinical trials and many laboratories have initiated studies to use other EMT-related molecules as a therapeutic target for the cancer patient with some modest, but encouraging results.

PKCδ Inhibition Impairs Mammary Cancer Proliferative Capacity But Selects Cancer Stem Cells, Involving Autophagy

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone‐independent mammary cancer cell line LM38‐LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38‐LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic‐vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self‐renewal. Furthermore, Rottlerin pre‐treatment induced in CSC the development of a “grape‐like” morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self‐renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self‐renewal. J. Cell. Biochem. 117: 730–740, 2016.

Myoepithelial and luminal breast cancer cells exhibit different responses to all-trans retinoic acid

PurposeBreast cancer is the leading cause of death among women worldwide. The exact role of luminal epithelial (LEP) and myoephitelial (MEP) cells in breast cancer development is as yet unclear, as also how retinoids may affect their behaviour. Here, we set out to evaluate whether retinoids may differentially regulate cell type-specific processes associated with breast cancer development using the bi-cellular LM38-LP murine mammary adenocarcinoma cell line as a model.Materials and methodsThe bi-cellular LM38-LP murine mammary cell line was used as a model throughout all experiments. LEP and MEP subpopulations were separated using inmunobeads, and the expression of genes known to be involved in epithelial to mysenchymal transition (EMT) was assessed by qPCR after all-trans retinoic acid (ATRA) treatment. In vitro invasive capacities of LM38-LP cells were evaluated using 3D Matrigel cultures in conjunction with confocal microscopy. Also, in vitro proliferation, senescence and apoptosis characteristics were evaluated in the LEP and MEP subpopulations after ATRA treatment, as well as the effects of ATRA treatment on the clonogenic, adhesive and invasive capacities of these cells. Mammosphere assays were performed to detect stem cell subpopulations. Finally, the orthotopic growth and metastatic abilities of LM38-LP monolayer and mammosphere-derived cells were evaluated in vivo.ResultsWe found that ATRA treatment modulates a set of genes related to EMT, resulting in distinct gene expression signatures for the LEP or MEP subpopulations. We found that the MEP subpopulation responds to ATRA by increasing its adhesion to extracellular matrix (ECM) components and by reducing its invasive capacity. We also found that ATRA induces apoptosis in LEP cells, whereas the MEP compartment responded with senescence. In addition, we found that ATRA treatment results in smaller and more organized LM38-LP colonies in Matrigel. Finally, we identified a third subpopulation within the LM38-LP cell line with stem/progenitor cell characteristics, exhibiting a partial resistance to ATRA.ConclusionsOur results show that the luminal epithelial (LEP) and myoephitelial (MEP) mammary LM38-P subpopulations respond differently to ATRA, i.e., the LEP subpopulation responds with increased cell cycle arrest and apoptosis and the MEP subpopulation responds with increased senescence and adhesion, thereby decreasing its invasive capacity. Finally, we identified a third subpopulation with stem/progenitor cell characteristics within the LM38-LP mammary adenocarcinoma cell line, which appears to be non-responsive to ATRA.

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT‐qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.

CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

Metabolic syndrome (MeS) has been identified as a risk factor for breast cancer. C-terminal binding protein 1 (CtBP1) is a co-repressor of tumor suppressor genes that is activated by low NAD+/NADH ratio. High fat diet (HFD) increases intracellular NADH. We investigated the effect of CtBP1 hyperactivation by HFD intake on mouse breast carcinogenesis. We generated a MeS-like disease in female mice by chronically feeding animals with HFD. MeS increased postnatal mammary gland development and generated prominent duct patterns with markedly increased CtBP1 and Cyclin D1 expression. CtBP1 induced breast cancer cells proliferation. Serum from animals with MeS enriched the stem-like/progenitor cell population from breast cancer cells. CtBP1 increased breast tumor growth in MeS mice modulating multiple genes and miRNA expression implicated in cell proliferation, progenitor cells phenotype, epithelial to mesenchymal transition, mammary development and cell communication in the xenografts. These results define a novel function for CtBP1 in breast carcinogenesis.