André Filipe Vieira

Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype

Background and Aim The study of CD44/CD24 and ALDH1 expression is the most accurate method to identify cancer stem cells (CSC) from breast cancer populations. However, the overlap between CD44+CD24−/low and ALDH1high CSC phenotypes in breast cancer seems to be very small, as well as their distribution among intrinsic breast cancer subtypes. Due to this discrepancy, it is imperative to improve the understanding of breast CSC marker distribution. Methods 466 invasive breast carcinomas and eight breast cancer cell lines were analysed for the expression of CD44, CD24 and ALDH1, to evaluate their distribution among the distinct molecular subtypes. Results Basal-like tumours (76.5%) contained the higher percentage of cells with the CSC phenotype CD44+CD24−/low (p<0.0001). From ALDH1-positive cases, 39.4% were also basal-like tumours (p<0.0001). The analysis of breast cancer cell lines indicated that luminal cell lines are mainly enriched in a CD44−/lowCD24+ cell population, basal/mesenchymal breast cancer cell lines are enriched in the CD44+CD24−/low phenotype, whereas the remaining basal/epithelial cell lines are mainly positive for both markers. ALDH1 activity was mainly found in HER-OE and basal/epithelial breast cancer cell. Conclusions CD44+CD24−/low and ALDH1+ phenotypes seem to identify CSC with distinct levels of differentiation. It seems that the paramount method and biomarkers that identify breast CSC within the distinct molecular subtypes need to be better explored, because it is pivotal to translate the CSC concept to clinical practice. In the future, the recognition of reliable markers to distinguish the CSC pool in each molecular subtype will be decisive for the development of specific target therapies.

Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells

Studies of the role of actin in tumour progression have highlighted its key contribution in cell softening associated with cell invasion. Here, using a human breast cell line with conditional Src induction, we demonstrate that cells undergo a stiffening state prior to acquiring malignant features. This state is characterized by the transient accumulation of stress fibres and upregulation of Ena/VASP-like (EVL). EVL, in turn, organizes stress fibres leading to transient cell stiffening, ERK-dependent cell proliferation, as well as enhancement of Src activation and progression towards a fully transformed state. Accordingly, EVL accumulates predominantly in premalignant breast lesions and is required for Src-induced epithelial overgrowth in Drosophila. While cell softening allows for cancer cell invasion, our work reveals that stress fibre-mediated cell stiffening could drive tumour growth during premalignant stages. A careful consideration of the mechanical properties of tumour cells could therefore offer new avenues of exploration when designing cancer-targeting therapies.

P‐Cadherin Is Coexpressed with CD44 and CD49f and Mediates Stem Cell Properties in Basal‐like Breast Cancer

Although the luminal progenitor cell of the normal mammary gland hierarchy has been proposed as the cell‐of‐origin for basal‐like breast cancers, finding the cancer stem cell (CSC) phenotype for this malignancy has proven a difficult task, mostly due to the lack of specific markers. Recently, basal‐like sporadic and familial cases of breast cancer have been linked to BRCA1 gene inactivation, which enables the upregulation of the target‐repressed CDH3/P‐cadherin gene, an important biomarker of basal‐like breast carcinomas. Previously, we demonstrated that P‐cadherin overexpression can mediate aggressive behavior in these tumors. Thus, our aim was to test whether P‐cadherin mediates stem cell properties in basal‐like breast carcinomas. Using a series of breast cancer cell lines and primary tumors, we showed that P‐cadherin was directly associated with the expression of the breast stem markers CD44, CD49f, and aldehyde dehydrogenase 1 in the basal subtype. Moreover, cell population enriched for P‐cadherin expression comprised increased in vitro mammosphere‐forming efficiency and capacity to grow colonies in three‐dimensional cultures as well as greater tumorigenicity. Importantly, an association was found with stem‐/progenitor‐like phenotypes of the breast, including the luminal progenitor population, CD49f+CD24+. Additionally, P‐cadherin expression conferred resistance to x‐ray‐induced cell death, sustaining a role for this molecule in another stem cell property. In summary, we demonstrated, for the first time, that P‐cadherin mediates stem cell properties, which could be explored in order to better define the CSC phenotype of basal‐like breast tumors and the cell‐of‐origin of this malignancy. STEM CELLS 2012;30:854–864

Engagement of Toll-like receptor 2 in mouse macrophages infected with Mycobacterium avium induces non-oxidative and TNF-independent anti-mycobacterial activity

Toll‐like receptor (TLR) 2 plays an important role in the immune response to mycobacterial infections, being required for optimal immunity against certain virulent Mycobacterium avium strains. Here we analyzed the role of TLR2 in the intra‐macrophagic growth of M. avium, using macrophages from TLR2‐deficient mice. We found that the engagement of TLR2/TLR6 and/or TLR2/TLR1 receptors induced bacteriostasis of M. avium inside bone marrow‐derived macrophages in a MyD88‐dependent way. Additionally, lipoproteins from the cell envelope of M. avium with a molecular mass of 20–25 kDa triggered this TLR2 pathway, leading to a decrease in the growth of the mycobacteria. Although TLR2 engagement induced the production of TNF, this cytokine as well as nitric oxide and superoxide molecules were not necessary for TLR2‐mediated bacteriostasis. Finally, TLR ligation did not induce the expression of the 47‐kDa guanosine triphosphatase (LRG‐47) but it promoted an increased maturation of the phagosome with regards to acquisition of LAMP1. Our data show that triggering TLR2 inhibited M. avium growth by an as‐yet‐unknown mechanism that may involve increased phagosome maturation.

P-cadherin and the journey to cancer metastasis

P-cadherin is a classical cell-to-cell adhesion molecule with a homeostatic function in several normal tissues. However, its behaviour in the malignant setting is notably dependent on the cellular context. In some tumour models, such as melanoma and oral squamous cell carcinoma, P-cadherin acts as a tumour suppressor, since its absence is associated with a more aggressive cancer cell phenotype; nevertheless, the overexpression of this molecule is linked to significant tumour promoting effects in the breast, ovarian, prostate, endometrial, skin, gastric, pancreas and colon neoplasms. Herein, we review the role of P-cadherin in cancer cell invasion, as well as in loco-regional and distant metastatic dissemination. We focus in P-cadherin signalling pathways that are activated to induce invasion and metastasis, as well as cancer stem cell properties. The signalling network downstream of P-cadherin is notably dependent on the cellular and tissue context and includes the activation of integrin molecules, receptor tyrosine kinases, small molecule GTPases, EMT transcription factors, and crosstalk with other cadherin family members. As new oncogenic molecular pathways mediated by P-cadherin are uncovered, putative therapeutic options can be tested, which will allow for the targeting of invasion or metastatic disease, depending on the tumour model.

Differential sensitivities to lactate transport inhibitors of breast cancer cell lines

The tumour microenvironment is known to be acidic due to high glycolytic rates of tumour cells. Monocarboxylate transporters (MCTs) play a role in extracellular acidification, which is widely known to be involved in tumour progression. Recently, we have described the upregulation of MCT1 in breast carcinomas and its association with poor prognostic variables. Thus, we aimed to evaluate the effect of lactate transport inhibition in human breast cancer cell lines. The effects of α-cyano-4-hydroxycinnamate, quercetin and lonidamine on cell viability, metabolism, proliferation, apoptosis, migration and invasion were assessed in a panel of different breast cancer cell lines. MCT1, MCT4 and CD147 were differently expressed among the breast cancer cell lines and, as expected, different sensitivities were observed for the three inhibitors. Interestingly, in the most sensitive cell lines, lactate transport inhibition induced a decrease in cell proliferation, migration and invasion, as well as an increase in cell death. Results were validated by silencing MCT1 expression using siRNA. The results obtained here support targeting of lactate transport as a strategy to treat breast cancer, with a special emphasis on the basal-like subtype, which so far does not have a specific molecular therapy.

Cancer stem cell markers in breast neoplasias: their relevance and distribution in distinct molecular subtypes.

The identification and characterization of cancer stem cells might lead to more effective control of neoplastic disease, by directing therapies to the most aggressive cells. For that reason, the identification of cancer stem cells (CSCs) in breast tumours is one of the priorities in breast cancer research, which has resulted in many studies attempting to identify their presence based on the expression of specific molecular markers. In this review, we describe the main molecular markers that have been identified as being able to recognise CSCs in breast carcinomas, the major molecular pathways that regulate CSCs and their association with the different molecular subtypes.

OXPHOS dysfunction regulates integrin-β1 modifications and enhances cell motility and migration

Mitochondria are central organelles for cellular metabolism. In cancer cells, mitochondrial oxidative phosphorylation (OXPHOS) dysfunction has been shown to promote migration, invasion, metastization and apoptosis resistance. With the purpose of analysing the effects of OXPHOS dysfunction in cancer cells and the molecular players involved, we generated cybrid cell lines harbouring either wild-type (WT) or mutant mitochondrial DNA (mtDNA) [tRNAmut cybrids, which harbour the pathogenic A3243T mutation in the leucine transfer RNA gene (tRNAleu)]. tRNAmut cybrids exhibited lower oxygen consumption and higher glucose consumption and lactate production than WT cybrids. tRNAmut cybrids displayed increased motility and migration capacities, which were associated with altered integrin-β1 N-glycosylation, in particular with higher levels of β-1,6-N-acetylglucosamine (GlcNAc) branched N-glycans. This integrin-β1 N-glycosylation pattern was correlated with higher levels of membrane-bound integrin-β1 and also with increased binding to fibronectin. When cultured in vitro, tRNAmut cybrids presented lower growth rate than WT cybrids, however, when injected in nude mice, tRNAmut cybrids produced larger tumours and showed higher metastatic potential than WT cybrids. We conclude that mtDNA-driven OXPHOS dysfunction correlates with increased motility and migration capacities, through a mechanism that may involve the cross talk between cancer cell mitochondria and the extracellular matrix.

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, β4 and β1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin in P-cadherin overexpression breast cancer models.

The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype

BackgroundCancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia.MethodsImmunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting.ResultsWe demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population.ConclusionsOur results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.