Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome.

BACKGROUND: (18)Fluor-deoxy-glucose PET-scanning of glycolytic metabolism is being used for staging in many tumors however its impact on prognosis has never been studied in breast cancer. METHODS: Glycolytic and hypoxic markers: glucose transporter (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 and 4 (MCT1, 4), MCT accessory protein basigin and lactate-dehydrogenase A (LDH-A) were assessed by immunohistochemistry in two cohorts of breast cancer comprising 643 node-negative and 127 triple negative breast cancers (TNBC) respectively. RESULTS: In the 643 node-negative breast tumor cohort with a median follow-up of 124 months, TNBC were the most glycolytic (≈70%), followed by Her-2 (≈50%) and RH-positive cancers (≈30%). Tumoral MCT4 staining (without stromal staining) was a strong independent prognostic factor for metastasis-free survival (HR=0.47, P=0.02) and overall-survival (HR=0.38, P=0.002). These results were confirmed in the independent cohort of 127 cancer patients. CONCLUSION: Glycolytic markers are expressed in all breast tumors with highest expression occurring in TNBC. MCT4, the hypoxia-inducible lactate/H(+) symporter demonstrated the strongest deleterious impact on survival. We propose that MCT4 serves as a new prognostic factor in node-negative breast cancer and can perhaps act soon as a theranostic factor considering the current pharmacological development of MCT4 inhibitors.

The asparaginyl hydroxylase factor-inhibiting HIF is essential for tumor growth through suppression of the p53-p21 axis.

We showed previously that factor-inhibiting hypoxia-inducible factor HIF (FIH) monitors the expression of a spectrum of genes that are dictated by the cell's partial oxygen pressure. This action is mediated by the C-TAD, one of two transactivation domains (TADs) of the hypoxia-inducible factor. Here, we questioned: (1) the function of FIH as a HIF-1 modulator of gene expression in the context of a physiological oxygen gradient occurring in three-dimensional cultures and in tumors and (2) the role of FIH as a modulator of the growth of human tumor cells. We first showed that the expression pattern of HIF target genes that depend on the C-TAD, such as carbonic anhydrase IX, was spacially displaced to more oxygenated areas when FIH was silenced, whereas overexpression of FIH restricted this pattern to more hypoxic areas. Second, we showed that silencing fih severely reduced in vitro cell proliferation and in vivo tumor growth of LS174 colon adenocarcinoma and A375 melanoma cells. Finally, silencing of fih significantly increased both the total and phosphorylated forms of the tumor suppressor p53, leading to an increase in its direct target, the cell cycle inhibitor p21. Moreover, p53-deficient or mutant cells were totally insensitive to FIH expression. Thus, FIH activity is essential for tumor growth through the suppression of the p53-p21 axis, the major barrier that prevents cancer progression.

Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms: relationships with 5-fluorouracil sensitivity.

The relationship of thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms on 5-fluorouracil (FU) sensitivity was tested on 19 human cancer cell lines (head and neck, breast, digestive tract) in the absence and presence of folinic acid (FA) supplementation. Thymidylate synthase polymorphisms in the 5' promoter region (double or triple tandem repeats) and 3' untranslated region (6-bp deletion) were analysed by PCR. The C677T and A1298C MTHFR polymorphisms were determined by melting curve analyses (LightCycler). Thymidylate synthase activity and intracellular concentration of the reduced folate 5-10 methylenetetrahydrofolate (CH(2)FH(4)) were measured (biochemical assays). Thymidylate synthase activity was significantly different according to 5' TS genotype, heterozygous cell lines (2R/3R) exhibiting higher TS activities than homozygous ones (P=0.05). However, whether in the absence or presence of FA, FU sensitivity was not statistically associated with either 5' or 3' TS polymorphism. Basal CH(2)FH(4) cellular concentrations were lowest in C677T homozygous wild-type (wt) (C/C) cell lines. FU sensitivity was not linked to C677T polymorphism. In contrast, there was a marked trend for a greater FU efficacy in mutated A1298C variants (C/C+A/C) as compared to wt homozygous cell lines (A/A) (P=0.055 and 0.085 without and with FA supplementation, respectively). These results suggest for the first time a potential role of A1298C MTHFR polymorphism on fluoropyrimidine sensitivity.

Expression of a prenylation-deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-kinase and protein kinase B.

The small GTPase Rab4 has been shown to participate in the subcellular distribution of GLUT4 under both basal and insulin-stimulated conditions in adipocytes. In the present work, we have characterized the effect of Rab4 DeltaCT, a prenylation-deficient and thus cytosolic form of Rab4, in this process. We show that the expression of Rab4 DeltaCT in freshly isolated adipocytes inhibits insulin-induced GLUT4 translocation, but only when this protein is in its GTP-bound active form. Further, it not only blocks the effect of insulin, but also that of a hyperosmotic shock, but does not interfere with the effect of zinc ions on GLUT4 translocation. Rab4 DeltaCT was then shown to prevent GLUT4 translocation induced by the expression of an active form of phosphatidylinositol 3-kinase or of protein kinase B, without altering the activities of the enzymes. Our results are consistent with a role of Rab4 DeltaCT acting as a dominant negative protein towards Rab4, possibly by binding to Rab4 effectors.

RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH.

Activation of the RET protooncogene through somatic rearrangements represents the most common genetic alteration in papillary thyroid carcinoma (PTC). Three main rearranged forms of RET have been described: RET/PTC1 and RET/PTC3, which arise from a paracentric inversion of the long arm of chromosome 10, and RET/PTC2, which originates from a 10;17 translocation. We have developed a dual-color FISH approach to detect RET/PTC rearrangements in interphase nuclei of thyroid lesions. By using a pool of three cosmids encompassing the RET chromosome region and a chromosome 10 centromeric probe, we could discriminate between the presence of an inversion (RET/PTC1 and RET/PTC3) or a translocation (RET/PTC2). We have investigated a series of thyroid tissue samples from Italian and French patients corresponding to a total of 69 PTCs and 22 benign lesions. Among PTCs, 13 (18.8%) showed a RET rearrangement, and 11 (15.9%) of these carried an inversion (RET/PTC1 or RET/PTC3) in more than 10% of the nuclei examined. Activated forms of RET were also observed in three adenomas. RT-PCR analysis on the same samples confirmed the presence and the type of rearrangement predicted using FISH analysis. An interesting difference in the frequency and type of RET rearrangements was detected between the Italian and the French patients. Furthermore, we identified a putative novel type of rearrangement in at least one PTC sample. Several PTCs carried a significant number of cells characterized by a trisomy or a tetrasomy of chromosome 10. Overall, the FISH approach in interphase nuclei represents a powerful tool for detecting, at the single cell level, RET/PTC rearrangements and other anomalies involving the RET chromosome region.

Cytotoxic effects of two gamma linoleic salts (lithium gammalinolenate or meglumine gammalinolenate) alone or associated with a nitrosourea: an experimental study on human glioblastoma cell lines.

Gamma linoleic acid (GLA) salts may exert a direct antiproliferative activity on tumor cells. The cytotoxicity is linked to the generation of conjugated dienes, peroxyl radicals and superoxide radicals. Lithium gammalinolenate (LiGLA) and meglumine gammalinolenate (MeGLA) have been recently developed for enhancing the water solubility of these compounds. MeGLA or LiGLA (10(-5) to 10(-4) mol/l) and fotemustine (Fote) (2 x 10(-6) to 2 x 10(-4) mol/l) were applied, alone or in combination, for up to 9 days to two human glioblastoma cell lines A172 and U373MG. Fote was applied first followed by LiGLA and/or MeGLA. Cytotoxicity was evaluated by the MTT test, and the effects of drug combinations were analyzed by the isobolographic representation according to the Chou and Talalay method (combination indexes). For both GLA salts, cytotoxicity was manifested after 4 days of cell exposure and with very sharp dose-response curves. Comparison of IC50 values indicated that MeGLA was more active than LiGLA. There was a constant reduction in IC50 values following an increase in exposure time for A172 cells: between 4 and 9 days of cell exposure, IC50 changed from 73 to 46 microM for LiGLA and from 49 to 31 microM for MeGLA (p<0.05). With U373MG cells, there was no influence of exposure duration on IC50 values. Combination index values indicated that association between Fote and GLA salts globally resulted in slightly antagonistic effects. These results may be useful for further development of GLA salts at the clinical level.