Angels Fabra

Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor

Overexpression of the transcription factor Snail in epithelial MDCK cells promotes the epithelial-mesenchymal transition (EMT) and the acquisition of an invasive phenotype. We report here that the expression of Snail is associated with an increase in the promoter activity and expression of the matrix metalloproteinase MMP-9. The effect of Snail silencing on MMP-9 expression corroborates this finding. Induced transcription of MMP-9 by Snail is driven by a mechanism dependent on the MAPK and phosphoinositide 3-kinase (PI3K) signalling pathways. Although other regions of the promoter were required for a complete stimulation by Snail, a minimal fragment (nucleotides -97 to +114) produces a response following an increased phosphorylation of Sp-1 and either Sp-1 or Ets-1 binding to the GC-box elements contained in this region. The expression of a dominant negative form of MEK decreased these complexes. A moderate increase in the binding of the nuclear factor κB (NFκB) to the upstream region (nucleotide -562) of the MMP-9 promoter was also observed in Snail-expressing cells. Interestingly, oncogenic H-Ras (RasV12) synergistically co-operates with Snail in the induction of MMP-9 transcription and expression. Altogether, these results indicate that MMP-9 transcription is activated in response to Snail expression and that it might explain, at least in part, the invasive properties of the Snail-expressing cells.

Transforming growth factor-β1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.

Role of CXCR4/SDF-1α in the migratory phenotype of hepatoma cells that have undergone epithelial–mesenchymal transition in response to the transforming growth factor-β

Treatment of FaO rat hepatoma cells with TGF-beta selects cells that survive to its apoptotic effect and undergo epithelial-mesenchymal transitions (EMT). We have established a cell line (T beta T-FaO, from TGF-beta-treated FaO) that shows a mesenchymal, de-differentiated, phenotype in the presence of TGF-beta and is refractory to its suppressor effects. In the absence of this cytokine, cells revert to an epithelial phenotype in 3-4 weeks and recover the response to TGF-beta. T beta T-FaO show higher capacity to migrate than that observed in the parental FaO cells. We found that FaO cells express low levels of CXCR4 and do not respond to SDF-1 alpha. However, TGF-beta up-regulates CXCR4, through a NF kappaB-dependent mechanism, and T beta T-FaO cells show elevated levels of CXCR4, which is located in the presumptive migration front. A specific CXCR4 antagonist (AMD3100) attenuates the migratory capacity of T beta T-FaO cells on collagen gels. Extracellular SDF-1 alpha activates the ERKs pathway in T beta T-FaO, but not in FaO cells, increasing cell scattering and protecting cells from apoptosis induced by serum deprivation. Targeted knock-down of CXCR4 with specific siRNA blocks the T beta T-FaO response to SDF-1 alpha. Thus, the SDF-1/CXCR4 axis might play an important role in mediating cell migration and survival after a TGF-beta-induced EMT in hepatoma cells.

Rac1 modulates TGF-β1-mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes

Transforming growth factor‐β1 (TGF‐β1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant‐negative N17TRac1 abolished TGF‐β1‐induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase‐9 (MMP9) production to levels comparable to those induced by TGF‐β1, while N17TRac1 was inhibitory. TGF‐β1‐mediated EMT involves the expression of the E‐cadherin repressor Snail1, regulated by the Rac1 and mitogen‐activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK‐dependent, as the MEK inhibitor PD98059 decreased TGF‐β1‐induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF‐β1‐mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.

Metastatic uveal melanoma: is there a role for conventional chemotherapy? - A single center study based on 58 patients.

Uveal melanoma metastases develop in 6.5–35% of patients, most commonly to the liver. Metastatic uveal melanoma (MUM) survival is poor, with 5–7 months of median survival. We reviewed retrospectively all patients with MUM diagnosed between January 1990 and December 2008 at our institution. We analyzed a total of 58 patients with a median age of 61 years (31–84 years). Median time for metastases development was 25.63 months (0.17–102.43 months). Fifty-six patients had hepatic involvement, 63.8% bilobar and 51.7% had more than or equal to five hepatic metastatic lesions. Sixteen patients (27.6%) had two or more organs involved. Six patients (10.71%) were treated with surgery, 25 patients (44.67%) received systemic chemotherapy, and 23 (41.07%) had best supportive care (BSC). The median overall survival (OS) for all the patients was 10.83 months [95% confidence interval (CI): 6.92–14.74]. Patients who had undergone chemotherapy presented 10.83 months (95% CI: 5.35–16.308) of median OS whereas the patients who did not undergo this treatment had an OS of 8.033 months (95% CI: 2.46–13.61). There were more patients with poor survival characteristics such as worse Eastern Cooperative Oncology Group performance status in the BSC group. OS was poor in treated and BSC patients. Differences in survival are more likely to be related to patient characteristics rather than to a chemotherapy effect. Patients with MUM should be included in clinical trials evaluating other options with newer agents.

Transforming growth factor-β-induced plasticity causes a migratory stemness phenotype in hepatocellular carcinoma

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-β induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells. Whether EMT induces changes in tumor cell plasticity has not been fully explored yet. Here, we analyze the effects of TGF-β on the EMT and stem-related properties of HCC cells and the potential correlation among those processes. The translational aim of the study was to propose a TGF-β/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-β therapy. Results indicate that when TGF-β induces EMT in HCC cells, a switch in the expression of stem genes is observed and their stemness potential and migratory/invasive capacity are enhanced. However, TGF-β may induce a partial EMT in some epithelial HCC cells, increasing the expression of mesenchymal genes and CD44, but maintaining epithelial gene expression. Epithelial cells show higher stemness potential than the mesenchymal ones, but respond to TGF-β increasing their migratory and invasive capacity. In HCC patient samples, TGFB1 expression most frequently correlates with a partial EMT, increase in mesenchymal genes and CD44 expression, as well as maintenance or over-expression of epithelial-related genes.

METASTATIC ABILITY OF MXT MOUSE MAMMARY SUBPOPULATIONS CORRELATES WITH CLONAL EXPRESSION AND/OR MEMBRANE-ASSOCIATION OF GELATINASE A

We have developed a novel murine mammary tumor system with variants representing different stages of tumor progression. The MXT‐s parental cell line was established from a urethane‐induced and hormone‐sensitive mammary tumor. MXT‐s parental cells are highly tumorigenic but poorly metastatic. MXT clones and variants were selected by either in vitro or in vivo procedures, and they differ in metastatic ability and 17β‐estradiol dependency for tumor growth. The MXT‐c1.1 and MXT‐B2 cell lines produced lung metastasis after intravenous injection into 100% of syngenic mice, but only MXT‐c1.1 cells were highly metastatic from intramammary tumors. The fingerprints obtained by arbitrarily primed‐polymerase chain reaction demonstrated that the metastatic variants and clones had a common genetic background and resulted from clonal selection from the parental cell line. We studied whether the matrix metalloproteinase (MMP) profile is correlated with tumor progression and metastatic ability in the MXT tumor system. Gelatinases A and B were assayed in the cells, both by enzyme activity and mRNA expression. Gelatinase A was expressed in MXT‐c1.1 cells, whereas MXT‐B2 cells did not express either MMP. In contrast, the mammary fat pad tumors expressed both gelatinases. Membrane Type 1‐MMP transcripts were also detected in MXT cells and tumors. Because the mRNA levels of gelatinase A were low in MXT‐B2 tumors, we suggested that exogenous gelatinase A bound the cell membranes of MXT‐B2 cells in vivo. Indirect evidence was obtained in vitro by treatment of MXT‐B2 cells with NIH/3T3 fibroblast‐conditioned medium. After this treatment, we detected a gelatinolytic activity at M, 68 000 in the cell‐membrane extract of MXT‐B2 cells and an increased in migratory ability through type IV collagen matrices. On the other hand, Ha‐ras gene dosage correlated positively with metastatic ability but not with either gelatinase A or gelatinase B expression. No significant differences were observed in the expression of stromelysin‐1 and tissue inhibitors of MMP. Thus, in the MXT tumor system, the expression of gelatinase A or its cell association and Ha‐ras gene dosage independently contribute to the metastatic phenotype. Mol. Carcinog. 19:54–66, 1997.

AURKA Overexpression Is Driven by FOXM1 and MAPK/ERK Activation in Melanoma Cells Harboring BRAF or NRAS Mutations: Impact on Melanoma Prognosis and Therapy

The cell cycle-related genes AURKA and FOXM1 are overexpressed in melanoma. We show here that AURKA overexpression is associated with poor prognosis in three independent cohorts of melanoma patients and correlates with the presence of genomic amplification of AURKA locus and BRAFV600E mutation. AURKA overexpression may also be driven by increased promoter activation through elements such as ETS and FOXM1 found within the 5 proximal promoter region. Activated MAPK/ERK signaling pathway mediates robust AURKA promoter activation, thereby knockdown of BRAFV600E and ERK inhibition results in reduced AURKA transcription and expression. We show a positive correlation between FOXM1 and AURKA expression in three independent cohorts of melanoma patients. FOXM1 silencing decreases expression of AURKA and late cell cycle genes in melanoma cells. We further found that FOXM1 expression levels are significantly higher in tumors carrying the BRAFV600E mutation compared with the wild-type BRAF (BRAFwt). Accordingly, the knockdown of BRAFV600E also reduces the expression of FOXM1 in BRAFV600E cells. Moreover, Aurora kinase A and FOXM1 inhibition by either genetic knockdown or pharmacologic inhibitors impair melanoma growth and survival both in culture and inxa0vivo, underscoring their therapeutic value for melanoma patients who fail to benefit from BRAF/MEK signaling inhibition.

Detection of differentially expressed gelatinase A in metastatic and non-metastatic subpopulations of tumor cells by target RNA arbitrarily primed polymerase chain reaction (TRAP-PCR)

We have developed a novel procedure called Targeted RNA AP-PCR (TRAP-PCR) to quantitatively measure specific mRNA expression. The target mRNA is reverse transcribed using a specific primer and PCR is performed under low stringency conditions to generate a rich fingerprint-type band pattern. In this situation multiple sequences are coamplified with the targeted sequence. The amplification is carried out in a competitive fashion and is, in consequence, quantitative. We have applied this technique to determine Gelatinase A (Gel A) mRNA expression in the MXT mouse mammary carcinoma system. TRAP-PCR analysis using primers for Gel A produced a reproducible fingerprint including one major band whose iden-tity was confirmed to be Gel A cDNA. Highly metastatic MXT subclones show an increased Gel A expres-sion. Results were confirmed by Northern blot and protein activity (gelatin zymography). TRAP-PCR is a simple, sensitive and specific technique to comparatively quantify mRNA expression and requires less template than conventional methods. ©Lippincott Williams & Wilkins