Helena Pereira

Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer

BackgroundMonocarboxylate transporters (MCTs) are transmembrane proteins involved in the transport of monocarboxylates across the plasma membrane, which appear to play an important role in solid tumours, however the role of MCTs in prostate cancer is largely unknown. The aim of the present work was to evaluate the clinico-pathological value of monocarboxylate transporters (MCTs) expression, namely MCT1, MCT2 and MCT4, together with CD147 and gp70 as MCT1/4 and MCT2 chaperones, respectively, in prostate carcinoma.MethodsProstate tissues were obtained from 171 patients, who performed radical prostatectomy and 14 patients who performed cystoprostatectomy. Samples and clinico-pathological data were retrieved and organized into tissue microarray (TMAs) blocks. Protein expression was evaluated by immunohistochemistry in neoplastic (n = 171), adjacent non-neoplastic tissues (n = 135), PIN lesions (n = 40) and normal prostatic tissue (n = 14). Protein expression was correlated with patients clinicopathologic characteristics.ResultsIn the present study, a significant increase of MCT2 and MCT4 expression in the cytoplasm of tumour cells and a significant decrease in both MCT1 and CD147 expression in prostate tumour cells was observed when compared to normal tissue. All MCT isoforms and CD147 were expressed in PIN lesions. Importantly, for MCT2 and MCT4 the expression levels in PIN lesions were between normal and tumour tissue, which might indicate a role for these MCTs in the malignant transformation. Associations were found between MCT1, MCT4 and CD147 expressions and poor prognosis markers; importantly MCT4 and CD147 overexpression correlated with higher PSA levels, Gleason score and pT stage, as well as with perineural invasion and biochemical recurrence.ConclusionsOur data provides novel evidence for the involvement of MCTs in prostate cancer. According to our results, we consider that MCT2 should be further explored as tumour marker and both MCT4 and CD147 as markers of poor prognosis in prostate cancer.

Modulation of Mitochondrial Outer Membrane Permeabilization and Apoptosis by Ceramide Metabolism

The yeast Saccharomyces cerevisiae undergoes a mitochondrial-dependent programmed cell death in response to different stimuli, such as acetic acid, with features similar to those of mammalian apoptosis. However, the upstream signaling events in this process, including those leading to mitochondrial membrane permeabilization, are still poorly characterized. Changes in sphingolipid metabolism have been linked to modulation of apoptosis in both yeast and mammalian cells, and ceramides have been detected in mitochondria upon apoptotic stimuli. In this study, we aimed to characterize the contribution of enzymes involved in ceramide metabolism to apoptotic cell death induced by acetic acid. We show that isc1Δ and lag1Δ mutants, lacking inositol phosphosphingolipid phospholipase C and ceramide synthase, respectively, exhibited a higher resistance to acetic acid that was associated with lower levels of some phytoceramide species. Consistently, these mutant cells displayed lower levels of ROS production and reduced mitochondrial alterations, such as mitochondrial fragmentation and degradation, and decreased translocation of cytochrome c into the cytosol in response to acetic acid. These results suggest that ceramide production contributes to cell death induced by acetic acid, especially through hydrolysis of complex sphingolipids catalyzed by Isc1p and de novo synthesis catalyzed by Lag1p, and provide the first in vivo indication of its involvement in mitochondrial outer membrane permeabilization in yeast.

CD147 immunohistochemistry discriminates between reactive mesothelial cells and malignant mesothelioma.

Malignant mesothelioma (MM) is a rare form of cancer. Its histopathological diagnosis is very difficult, as it exhibits a number of different appearances that can be misinterpreted as metastatic invasion or atypical hyperplasia. Thus, there is an urgent need to identify adequate markers to distinguish between benign and malignant cells, allowing the implementation of appropriate therapies and, possibly, specific directed therapies. MM, like other tumors, show an increase in glucose uptake, due to high rates of glycolysis, inducing an intracellular overload of acids. In this context, monocarboxylate transporters (MCTs) emerge as important players, by mediating the transmembranar co‐transport of lactate with a proton, thereby, regulating pH and allowing continuous glycolysis. Importantly, proper MCT expression and activity depend on its co‐expression with a chaperone, CD147, which is associated with poor prognosis in cancer. Twenty‐two samples including reactive mesothelial cells, MM, and atypical mesothelial hyperplasias were evaluated for immunoexpression of MCT1, MCT4, and CD147. Expression of these proteins was compared with GLUT1 as a new promising marker for MM. Although MCT isoforms were not differentially expressed in the two types of cytological specimens, CD147, as GLUT1, was almost exclusively expressed in MM. Both MCT1 and MCT4 are not able to discriminate between mesothelial reactive cells and mesothelial malignant cells, while CD147 was able to distinguish these two proliferations. If confirmed, besides being a good marker for identification of MM, CD147 may also be a target for therapeutical strategies in this rare type of tumor. Diagn. Cytopathol. 2012;40:478–483.

Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil. For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU. Here, we demonstrated that MCT inhibition using CHC (α-cyano-4-hydroxycinnamic acid), DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing. These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.

A novel exonic rearrangement affecting MLH1 and the contiguous LRRFIP2 is a founder mutation in Portuguese Lynch syndrome families

Purpose: Although Lynch syndrome is characterized by marked genetic heterogeneity, some specific mutations are observed at high frequency in well-defined populations or ethnic groups due to founder effects.Methods: Genomic breakpoint identification, haplotype analysis, and mutation age determination were performed in 14 unrelated patients and 95 family members presenting the same MLH1 exonic rearrangement, among a series of 84 Lynch syndrome families with germline mutations in MLH1, MSH2, or MSH6.Results: All 14 probands harbored an identical deletion, comprising exons 17–19 of the MLH1 gene and exons 26–29 of the LRRFIP2 gene, corresponding to the MLH1 mutation c.1896 + 280_oLRRFIP2:c.1750-678del. This mutation represents 17% of all deleterious mismatch repair mutations in our series. Haplotype analysis showed a conserved region of approximately 1 Mb, and the mutation age was estimated to be 283 ± 78 years. All 14 families are originated from the Porto district countryside.Conclusion: We have identified a novel MLH1 exonic rearrangement that is a common founder mutation in Lynch syndrome families, indicating that screening for this rearrangement as a first step may be cost-effective during genetic testing of Lynch syndrome suspects of Portuguese ancestry, especially those originating from the Porto district.

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

The protective role of yeast Cathepsin D in acetic acid-induced apoptosis depends on ANT (Aac2p) but not on the voltage-dependent channel (Por1p)

We have previously shown that the yeast Cathepsin D (CatD) Pep4p translocates from the vacuole to the cytosol during acetic acid‐induced apoptosis and is required for efficient mitochondrial degradation, though its specific role in this process is still elusive. Here, we show that the protective role of Pep4p in acetic acid‐induced apoptosis depends on its catalytic activity and is independent of the yeast voltage‐dependent anion channel Por1p (which has no role on mitochondrial degradation) but dependent on AAC proteins, the yeast adenine nucleotide translocator. Our results demonstrate a differential interplay between yeast vacuolar CatD and mitochondrial proteins involved in apoptosis regulation.

Yeast as a tool to explore cathepsin D function

Cathepsin D has garnered increased attention in recent years, mainly since it has been associated with several human pathologies. In particular, cathepsin D is often overexpressed and hypersecreted in cancer cells, implying it may constitute a therapeutic target. However, cathepsin D can have both anti- and pro-survival functions depending on its proteolytic activity, cellular context and stress stimulus. Therefore, a more detailed understanding of cathepsin D regulation and how to modulate its apoptotic functions is clearly needed. In this review, we provide an overview of the role of cathepsin D in physiological and pathological scenarios. We then focus on the opposing functions of cathepsin D in apoptosis, particularly relevant in cancer research. Emphasis is given to the role of the yeast protease Pep4p, the vacuolar counterpart of cathepsin D, in life and death. Finally, we discuss how insights from yeast cathepsin D and its role in regulated cell death can unveil novel functions of mammalian cathepsin D in apoptosis and cancer.

The Role of Diet Related Short-Chain Fatty Acids in Colorectal Cancer Metabolism and Survival: Prevention and Therapeutic Implications.

Colorectal cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of short chain fatty acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will cover the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed.