Vera Miranda-Gonçalves

Monocarboxylate transporters (MCTs) in gliomas: Expression and exploitation as therapeutic targets

BACKGROUND Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas. METHODS MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models. RESULTS MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed. CONCLUSIONS This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.

Downregulation of RKIP Is Associated with Poor Outcome and Malignant Progression in Gliomas

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients.

RKIP Inhibition in cervical cancer Is associated with higher tumor aggressive behavior and resistance to cisplatin therapy

Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (∼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.

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.

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

Background Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods Expression of hypoxia- and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (α-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.

The metabolic microenvironment of melanomas: prognostic value of MCT1 and MCT4

ABSTRACT BRAF mutations are known drivers of melanoma development and, recently, were also described as players in the Warburg effect, while this reprogramming of energy metabolism has been identified as a possible strategy for treating melanoma patients. Therefore, the aim of this work was to evaluate the expression and prognostic value of a panel of glycolytic metabolism-related proteins in a series of melanomas. The immunohistochemical expression of MCT1, MCT4, GLUT1, and CAIX was evaluated in 356 patients presenting melanoma and 20 patients presenting benign nevi. Samples included 20 benign nevi, 282 primary melanomas, 117 lymph node and 54 distant metastases samples. BRAF mutation was observed in 29/92 (31.5%) melanoma patients and 17/20 (85%) benign nevi samples. NRAS mutation was observed in 4/36 (11.1%) melanoma patients and 1/19 (5.3%) benign nevi samples. MCT4 and GLUT1 expression was significantly increased in metastatic samples, and MCT1, MCT4 and GLUT1 were significantly associated with poor prognostic variables. Importantly, MCT1 and MCT4 were associated with shorter overall survival. In conclusion, the present study brings new insights on metabolic aspects of melanoma, paving the way for the development of new-targeted therapies.

The monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamic acid disrupts rat lung branching.

Background/Aims: The human embryo develops in a hypoxic environment. In this way, cells have to rely on the glycolytic pathway for energy supply, leading to an intracellular accumulation of monocarboxylates such as lactate and pyruvate. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for the maintenance of intracellular pH homeostasis. This transport is mediated by a family of transporters, designated by monocarboxylate transporters (MCTs), namely isoforms 1 and 4. MCT1/4 expression is regulated by the ancillary protein CD147.The general aim of this study was to characterize the expression pattern of MCT1/4, CD147 and the glucose transporter GLUT1 during human fetal lung development and elucidate the role of MCTs in lung development. Methods: The expression pattern of MCT1/4 and GLUT1 was characterized by immunohistochemistry and fetal lung viability and branching were evaluated by exposing rat fetal lung explants to CHC, an inhibitor of MCT activity. Results: Our findings show that all the biomarkers are differently expressed during fetal lung development and that CHC appears to have an inhibitory effect on lung branching and viability, in a dose dependent way. Conclusion: We provide evidence for the role of MCTs in embryo lung development, however to prove the dependence of MCT activity further studies are waranted.

HER Family Receptors are Important Theranostic Biomarkers for Cervical Cancer: Blocking Glucose Metabolism Enhances the Therapeutic Effect of HER Inhibitors

Persistent HPV infection alone is not sufficient for cervical cancer development, which requires additional molecular alterations for tumor progression and metastasis ultimately leading to a lethal disease. In this study, we performed a comprehensive analysis of HER family receptor alterations in cervical adenocarcinoma. We detected overexpression of HER protein, mainly HER2, which was an independent prognostic marker for these patients. By using in vitro and in vivo approaches, we provided evidence that HER inhibitors, allitinib and lapatinib, were effective in reducing cervical cancer aggressiveness. Furthermore, combination of these drugs with glucose uptake blockers could overcome the putative HIF1-α-mediated resistance to HER-targeted therapies. Thus, we propose that the use of HER inhibitors in association with glycolysis blockers can be a potentially effective treatment option for HER-positive cervical cancer patients.

Metabolic alterations underlying Bevacizumab therapy in glioblastoma cells

Anti-VEGF therapy with Bevacizumab is approved for glioblastoma treatment, however, it is known that tumors acquired resistance and eventually became even more aggressive and infiltrative after treatment. In the present study we aimed to unravel the potential cellular mechanisms of resistance to Bevacizumab in glioblastoma in vitro models. Using a panel of glioblastoma cell lines we found that Bevacizumab is able to block the secreted VEGF by the tumor cells and be internalized to the cytoplasm, inducing cytotoxicity in vitro. We further found that Bevacizumab increases the expression of hypoxic (HIF-1α and CAIX) and glycolytic markers (GLUT1 and MCT1), leading to higher glucose uptake and lactate production. Furthermore, we showed that part of the consumed glucose by the tumor cells can be stored as glycogen, hampering cell dead following Bevacizumab treatment. Importantly, we found that this change on the glycolytic metabolism occurs independently of hypoxia and before mitochondrial impairment or autophagy induction. Finally, the combination of Bevacizumab with glucose uptake inhibitors decreased in vivo tumor growth and angiogenesis and shift the expression of glycolytic proteins. In conclusion, we reported that Bevacizumab is able to increase the glucose metabolism on cancer cells by abrogating autocrine VEGF in vitro. Define the effects of anti-angiogenic drugs at the cellular level can allow us to discover ways to revert acquired resistance to this therapeutic approaches in the future.

Alginate hydrogel improves anti-angiogenic bevacizumab activity in cancer therapy

Graphical abstract Figure. No Caption available. Abstract Anti‐vascular endothelial growth factor (anti‐VEGF) therapy applied to solid tumors is a promising strategy, yet, the challenge to deliver these agents at high drug concentrations together with the maintenance of therapeutic doses locally, at the tumor site, minimizes its benefits. To overcome these obstacles, we propose the development of a bevacizumab‐loaded alginate hydrogel by electrostatic interactions to design a delivery system for controlled and anti‐angiogenic therapy under tumor microenvironmental conditions. The tridimensional hydrogel structure produced provides drug stability and a system able to be introduced as a flowable solution, stablishing a depot after local administration. Biological performance by the chick embryo chorioallantoic membrane (CAM) assay indicated a pH‐independent improved anti‐angiogenic activity (˜50%) compared to commercial available anti‐VEGF drug. Moreover, there was a considerable regression in tumor size when treated with this system. Immunohistochemistry highlighted a reduced number and disorganization of microscopic blood vessels resulting from applied therapy. These results suggest that the developed hydrogel is a promising approach to create an innovative delivery system that offers the possibility to treat different solid tumors by intratumoral administration.