Interactions between rheumatoid arthritis antibodies are associated with the response to anti-tumor necrosis factor therapy.

BACKGROUND: Blocking of the Tumor Necrosis Factor (TNF) activity is a successful therapeutic approach for 50-60% of rheumatoid arthritis (RA) patients. However, there are yet no biomarkers to stratify patients for anti-TNF therapy. Rheumatoid factor (RF) and anti-cyclic-citrullinated antibodies (anti-CCP) have been evaluated as biomarkers of response but the results have shown limited consistency. Anti-carbamylated protein (anti-CarP) and anti-peptidylarginine deiminase type 4 (anti-PAD4) antibodies have been much less studied. Despite being linked to common immune processes, the interaction between these markers has not been evaluated yet. Our aim was to analyze the interaction between these four antibodies in relation to the response to anti-TNF therapy. METHODS: For this objective, a prospective cohort of n = 80 RA patients starting anti-TNF therapy was recruited. Serum determinations at baseline were performed for RF, anti-CCP, anti-CarP and anti-PAD4 antibodies using enzyme-linked immunosorbent assays (ELISA). The clinical response to anti-TNF therapy was determined at week 12 using the change in DAS28 score. Association was performed using multivariate linear regression adjusting for baseline DAS28, sex and age. RESULTS: The interaction between pairs of antibodies was tested by the addition of an interaction term. We found two highly significant antibody interactions associated with treatment response: anti-CarP with anti-PAD4 (p = 0.0062), and anti-CCP with RF (p = 0.00068). The latter antibody interaction was replicated in an independent retrospective cohort of RA patients (n = 199, p = 0.04). CONCLUSIONS: The results of this study suggest that antibody interaction effects are important factors in the response to anti-TNF therapy in RA.

The level of caveolin-1 expression determines response to TGF-ß as a tumour suppressor in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-ß) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-ß is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-ß in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-ß response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-ß-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-ß-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-ß-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-ß activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-ß from cytostatic to tumourigenic, which could have clinical meaning in patient stratification.

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

As part of its potential pro-tumorigenic actions, Transforming Growth Factor-(TGF)-ß 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-ß 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-ß/EMT/stem gene signature that would help in recognizing HCC patients as good candidates for anti-TGF-ß therapy. Results indicate that when TGF-ß 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-ß 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-ß 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.

Overactivation of the TGF-ß pathway confers a mesenchymal-like phenotype and CXCR4-dependent migratory properties to liver tumor cells.

UNLABELLED: Transforming growth factor-beta (TGF-ß) is an important regulatory suppressor factor in hepatocytes. However, liver tumor cells develop mechanisms to overcome its suppressor effects and respond to this cytokine by inducing other processes, such as the epithelial-mesenchymal transition (EMT), which contributes to tumor progression and dissemination. Recent studies have placed chemokines and their receptors at the center not only of physiological cell migration but also of pathological processes, such as metastasis in cancer. In particular, CXCR4 and its ligand, stromal cell-derived factor 1α (SDF-1α) / chemokine (C-X-C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we show that autocrine stimulation of TGF-ß in human liver tumor cells correlates with a mesenchymal-like phenotype, resistance to TGF-ß-induced suppressor effects, and high expression of CXCR4, which is required for TGF-ß-induced cell migration. Silencing of the TGF-ß receptor1 (TGFBR1), or its specific inhibition, recovered the epithelial phenotype and attenuated CXCR4 expression, inhibiting cell migratory capacity. In an experimental mouse model of hepatocarcinogenesis (diethylnitrosamine-induced), tumors showed increased activation of the TGF-ß pathway and enhanced CXCR4 levels. In human hepatocellular carcinoma tumors, high levels of CXCR4 always correlated with activation of the TGF-ß pathway, a less differentiated phenotype, and a cirrhotic background. CXCR4 concentrated at the tumor border and perivascular areas, suggesting its potential involvement in tumor cell dissemination. CONCLUSION: A crosstalk exists among the TGF-ß and CXCR4 pathways in liver tumors, reflecting a novel molecular mechanism that explains the protumorigenic effects of TGF-ß and opens new perspectives for tumor therapy.

Utility of cell-cycle modulators to predict vascular invasion and recurrence after surgical treatment of hepatocellular carcinoma.

BACKGROUND: The outcome of surgical treatment of hepatocellular carcinoma (HCC) could be improved by applying patient selection criteria based on tumoral aggressiveness. Here we analyzed the prognostic role of the expression of several genes involved in cell-cycle regulation in a group of patients with HCC. METHODS: We retrospectively studied 93 patients (67 transplanted and 26 resections) treated between 1996 and 2000. In micro-thick sections from paraffin-embedded tumoral tissues, the expression of p53, pRb, p16, and cyclin D1 was analyzed. A logistic regression model was used to detect factors related to vascular invasion. A Cox regression model was applied to identify pathologic and molecular factors with the capacity to predict the recurrence of HCC. RESULTS: Only tumor size>3 cm (odds ratio [OR]: 3.4; 95% CI: 1.2-9.9; P=0.019) and pRb expression (OR: 4.1; 95% CI: 1.02-17; P=0.053) were associated with an increased risk of vascular invasion. The regression model applied to the group of transplanted patients showed three factors that were independently related to recurrence: vascular invasion (OR 7.5; 95% CI: 1.1-51.8; P=0.039); pRb expression (OR: 11; 95% CI: 1.2-96.9; P=0.03); and p16 expression (OR: 69.7; 95% CI: 5.1-9448; P=0.001). In the group of resected patients, pRb expression was associated with higher risk of recurrence only in the univariate analysis (P=0.037). The multivariate analysis showed tumor size>3 cm (OR: 57.5; 95% CI: 1.1-51.8; P=0.039) and vascular invasion (OR: 6.1; 95% CI: 1.05-35.3; P=0.044) to be significantly associated with recurrence. CONCLUSIONS: Of the molecular factors studied, only pRb expression was useful as a predictive factor of vascular invasion in patients with HCC, and also of recurrence in transplanted patients with this carcinoma. pRb expression may be relevant to consider when selecting patients for resection and when identifying transplanted patients with a high risk of recurrence.