Lipids and cholesterol in particular, have long been associated with breast cancer (BC) onset and progression. However, the causative effects of elevated lipid levels and breast cancer remain largely undisclosed and were the subject of the present study.We took advantage of well-established in vitro and in vivo models of cholesterol enrichment to exploit the mechanism involved in LDL-cholesterol favouring BC growth and invasiveness. We analyzed its effects in models that mimic different BC subtypes and stages.Our data show that LDL-cholesterol (but not HDL-cholesterol) promotes BC cells proliferation, migration and loss of adhesion, hallmarks of the epithelial to mesenchymal transition. In vivo studies modeling cholesterol levels showed that breast tumors are consistently larger and more proliferative in hypercholesterolemic mice, which also have more frequently lung metastases. Microarray analysis revealed an over expression of intermediates of Akt and ERK pathways suggesting a survival response induced by LDL, confirmed by WB analyses. Gene expression analysis also evidenced an activation of ErbB2 signaling pathway and decreased expression of adhesion molecules (cadherin-related family member3, CD226, Claudin 7 and Ocludin) in the cells exposed to LDL.Together, the present work shows novel mechanistic evidence that high LDL-cholesterol levels promote BC progression. These data provide rationale for the clinical control of cholesterol levels in BC patients.

Bone marrow contains endothelial progenitor cells (EPCs) that, upon pro-angiogenic stimuli, migrate and differentiate into endothelial cells (ECs) and contribute to re-endothelialization and neo-vascularization. There are currently no reliable markers to characterize EPCs, leading to their inaccurate identification. In the past, we showed that, in a panel of tumors, some cells on the vessel wall co-expressed CD14 (monocytic marker) and CD31 (EC marker), indicating a putative differentiation route of monocytes into ECs. Herein, we disclosed monocytes as potential EPCs, using in vitro and in vivo models, and also addressed the cancer context. Monocytes acquired the capacity to express ECs markers and were able to be incorporated into blood vessels, contributing to cancer progression, by being incorporated in tumor neo-vasculature. Reactive oxygen species (ROS) push monocytes to EC differentiation, and this phenotype is reverted by cysteine (a scavenger and precursor of glutathione), which indicates that angiogenesis is controlled by the interplay between the oxidative stress and the scavenging capacity of the tumor microenvironment.Over the last decade, different studies reported EPCs as essential in restoring injured vessels. EPCs belong to a subset of cells, arising from hematopoietic stem cells in bone marrow; upon pro-angiogenic stimuli, they proliferate, migrate, and differentiate into endothelial cells (ECs) [4][5][6]. Some reports addressing EPCs and disease, such as systemic sclerosis, showed contradictory and discrepant results about EPCs mobilization and differentiation; in part, because there is a lack of a precise panel of cell surface markers used for the characterization of this subset of cells [4][5][6][7][8][9][10]. In mouse embryonic vascular endothelium, erythro-myeloid progenitors (EMPs) can differentiate into ECs [11] and in a mouse model of carotid injury, monocytes (CD14 + cells) are capable of improving re-endothelialization [12]. In vivo and in vitro targeting of Tie2-monocytes decreases angiogenesis by abrogating EC proliferation [13][14][15] and an in vivo CCR2 (chemokine (C-C motif) receptor 2) knockout impairs monocytes recruitment and VEGFA (also named VEGF, vascular endothelial growth factor) expression, accompanied by a reduction in the angiogenesis rate [16]. The release of cytokines and pro-angiogenic factors (e.g., VEGFA, VEGFC, and VEGFD, TNFα (tumor necrosis factor α), IL-8 (interleukin-8), and FGF-2 (fibroblast growth factor-2), and extracellular matrix (ECM) modifying proteins (e.g., metalloproteinase-9 (MMP-9)) by macrophages enhances the tissue's ability to support capillary sprouting and vascular density [17,18]. The precise mechanism by which monocytes influence angiogenesis in tissue development, homeostasis, and diseases is not fully understood. However, different studies, have shown that under in vitro pro-angiogenic pressure, blood mononuclear cells can acquire endothelial markers and morphology [19][20][21]. In addition, in a previous study, we showed that some ECs sim...

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Uterine cervix cancer is the second most common malignancy in women worldwide with human papillomavirus (HPV) as the etiologic factor. The two main histological variants, squamous cell carcinomas (SCC) and adenocarcinomas (AC), resemble the cell morphology of exocervix and endocervix, respectively. Cancer metabolism is a cancer hallmark conditioned by the microenvironment. As uterine cervix homeostasis is dependent on lactate, we hypothesized lactate plays a role in uterine cervix cancer progression. Using in vitro (SiHa-SCC and HeLa-AC) and BALB-c/SCID models, we demonstrated that lactate metabolism is linked to histological types, with SCC predominantly consuming and AC producing lactate. MCT1 is a key factor, allowing lactate consumption and being regulated in vitro by lactate through the FOXM1:STAT3 pathway. In vivo models showed that SCC (SiHa) expresses MCT1 and is dependent on lactate to grow, whereas AC (HeLa) expresses MCT1 and MCT4, with higher growth capacities. Immunohistochemical analysis of tissue microarrays (TMA) from human cervical tumors showed that MCT1 expression associates with the SCC type and metastatic behavior of AC, whereas MCT4 expression concomitantly increases from in situ SCC to invasive SCC and is significantly associated with the AC type. Consistently, FOXM1 expression is statistically associated with MCT1 positivity in SCC, whereas the expression of FOXO3a, a FOXM1 functional antagonist, is linked to MCT1 negativity in AC. Our study reinforces the role of the microenvironment in the metabolic adaptation of cancer cells, showing that cells that retain metabolic features of their normal counterparts are positively selected by the organ's microenvironment and will survive. In particular, MCT1 was shown to be a key element in uterine cervix cancer development; however, further studies are needed to validate MCT1 as a suitable therapeutic target in uterine cervix cancer.

Ovarian cancer is the leading cause of death from gynaecological. There is an urgent need for testing novel tumour markers and therapeutic agents. HDAC inhibitors are a new class of target anticancer agents, which block deacetylation function, causing cell cycle arrest, differentiation and/or apoptosis. Vorinostat is an inhibitor of class I and II HDACs. Butyric acid is a small molecular weight carboxylates that are class I HDAC inhibitors. Our objective was to evaluate cell and molecular alterations induced by HDACs inhibitors in ovarian cancer cells. For that, we exposed serous ovarian carcinoma cell lines, ES2 and OVCAR3, and clear cells carcinoma cell line, OV90, to butyric acid and to vorinostat. Cell death, apoptosis and necrosis, was evaluated by FACS using Annexin V and 7AAD staining. By RQ-PCR we evaluated the expression of Notch downstream target genes, the expression of TP53 and HNF1, which is responsible for the clear cells carcinoma morphological phenotype. We observed that butyric acid is more effective as an inducer of cell death than vorinostat, in all cell lines. However, apoptotic levels induced by vorinostat are higher in OV90 cells than in ES2 and OVCAR3. We also observed that Notch pathway is activated in all ovarian cell lines by butyric acid, since Notch downstream targets HES1, HEY1 and 2 are more expressed. P53 expression is slightly decreased in all cell lines after exposure to butyric acid. Interestingly, HNF1 expression decreases in OV90 whereas it increases in ES2 and OVCAR3. We conclude that butyric acid induces cell death in all cell lines; and the induction of apoptosis in OV90 by vorinostat may be through the downregulation of HNF1.

Majority of metastasis in breast cancer occur via the dissemination of tumor cells through the bloodstream. How tumor cells enter the blood (intravasation) is, however, a poorly understood mechanism at the cellular and molecular levels. Particularly uncharacterized is how intravasation is affected by systemic factors. High levels of systemic LDL-cholesterol have been shown to contribute to breast cancer progression and metastasis in various models, but the mechanisms involved are still undisclosed. Here we show that a high cholesterol diet promotes intravasation in two mouse models of breast cancer and that this could be reverted by blocking LDL binding to LDLR in tumor cells. Moreover, we show that LDL promotes vascular invasion in vitro and a phenotypic change resembling VM, and augments the expression of Serpine2, previously shown to be required for both VM and intravasation. Finally, we show that blocking the binding of LDL to LDLR on tumor cells prevents the increase in lung metastasis promoted by a high cholesterol diet. Overall, our manuscript unravels novel mechanisms by which systemic hypercholesterolemia may affect the onset of metastatic breast cancer by favoring phenotypic changes in breast cancer cells and increasing intravasation.

Cerebral cavernous malformations (CCMs) are vascular malformations characterized by the abnormal growth of vascular structures in the central nervous system. However, the precise mechanism(s) responsible for the development of CCM vascular abnormalities remain poorly understood. Although the mechanisms of action of propranolol in CCM have not yet been fully explored it is not commonly prescribed, it has been shown to be effective in children and appears to play a protective role in the prevention of CCM-derived hemorrhage in adults. The present study performed in vitro and ex vivo assays in order to examine the effects of propranolol on endothelial cells (ECs). The percentage of CD14 + /CD31 + cells and the levels of VEGF in the peripheral blood (PB) of a child patient with CCM, with recurrent seizures and hemorrhages, who was maintained under propranolol therapy, were also analyzed. In addition to the effects of propranolol on differentiated ECs, and the decrease angiogenic-related features in vitro and ex vivo, it was observed that in the PB of this patient, propranolol administration decreased the percentage of circulating cells sharing monocytic and EC features (CD14 + /CD31 + cells), as well as the VEGF levels; this was concomitant with a good prognosis and with the reversion of CCM lesions. A decrease in VEGF levels by propranolol may also be involved in the impairment of the recruitment of CD14 + /CD31 + monocytes functioning as endothelial progenitor cells to sustain the vascular lesion. On the whole, the present study demonstrates that propranolol impairs angiogenesis in vitro and may thus be a useful tool for the clinical management of CCM. Moreover, the present study highlights the monitorization of the levels of CD14 + /CD31 + monocytes and VEGF levels as a useful tool for predicting the clinical efficacy of propranolol in patients with CCM.

Here we hypothesize that systemic cholesterol favors metastasis by increasing blood vessel endothelial permeability. The majority of metastatic dissemination occurs through the hematogenous route and factors that alter blood vessel properties can affect the entry of cancer cells in circulation. It is generally accepted that high levels of systemic cholesterol, in particular low density lipoprotein (LDL), have a negative impact in endothelial function. This has been demonstrated mainly in large arteries but less is known about how LDL affects the microvasculature of different organs. High cholesterol has also been associated with poor prognosis in several cancers. In our lab, using orthotopic mouse models of breast cancer, we were able to show that mice in which increased LDL in circulation is induced by a high cholesterol diet, have more metastasis. Here we also show in an “in vitro” system, that LDL increases endothelial permeability and that this can be reverted by blocking the interaction of LDL with the LDL-receptor. Additionally we observe that this higher endothelial permeability is due to cholesterol enrichment of endothelial cells, as nystatin, a cholesterol sequestering agent is also able to revert the phenotype. Back to “in vivo”, using the same orthotopic breast cancer model, we are able to show that high systemic cholesterol increases the number of circulating cancer cells in the blood. We are now testing the hypothesis of whether this is due to increased permeability of tumor blood vessels or of the vessels surrounding the tumor. Citation Format: Ana Magalhaes, Catarina R. Santos, Germana Domingues, Tãnia Carvalho, Sergio Dias. High systemic cholesterol increases blood vessel endothelial permeability and favors metastasis: Are these events related? [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C24.