Raf-1 oncogenic signaling is linked to activation of mesenchymal to epithelial transition pathway in metastatic breast cancer cells.

Aberrant activation of the Raf/MEK/MAPK pathway plays a key role in breast cancer development and progression. Dysregulation of Raf/MEK/MAPK oncogenic signaling often results from overexpression of the HER-2/Neu tyrosine kinase receptor leading to chemoendocrine resistance, development of distant metastases and ultimately poor prognosis in breast cancer patients. HER-2/Neu overexpression is also linked to activation of the epithelial to mesenchymal transition (EMT) pathway, loss of adhesion molecules and metastasis. Recently, it has been demonstrated that cancer cells that undergo EMT acquire a CD44+/CD24-/low basal cancer stem cell-like phenotype and are characterized by activation of HER-2/Neu and TGFß oncogenic signaling pathways with increased capacity of self-renewal, drug resistance, invasion and distant metastases. Following metastatic dissemination, cancer cells re-activate certain epithelial properties through mesenchymal to epithelial transition (MET) to establish neoplastic lesions at secondary sites, although the molecular mechanisms regulating MET remain elusive. In this study we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces HER-2/Neu overexpression leading to the development of distant metastases in ERα+ MCF-7 breast cancer xenografts. Importantly, development of distant metastases in xenograft models was linked to activation of the MET pathway characterized by reduced expression of EMT inducer genes (TGFB2, TWIST1 and FOXC1) and overexpression of BMB7, CXCR7 and EGR family of transcription factors. In summary, our results demonstrate for the first time that amplification of Raf/MEK/MAPK oncogenic signaling during tumor growth promotes the genesis of metastatic lesions from primary tumors by activating the mesenchymal epithelial transition.

Abrogation of p53 function leads to metastatic transcriptome networks that typify tumor progression in human breast cancer xenografts.

Development of chromosomal instability (CIN) and consequent phenotypic heterogeneity represent common events during breast cancer progression. Breast carcinomas harboring extensive chromosomal aberrations display a more aggressive behavior characterized by chemoresistance and the propensity to give rise to distant metastases. The tumor suppressor p53 plays a key role in the maintenance of chromosomal stability and tissue homeostasis through activation of cell cycle checkpoints following DNA damage and control of centrosome duplication that ensures equal chromosome segregation during cell division. Furthermore, p53 suppresses CD44 expression and the acquisition of stem cell-like properties responsible for epithelial to mesenchymal transition (EMT) and metastasis. In this study we employed MCF-7 breast cancer cells with endogenous wild-type p53, an engineered MCF-7 variant (vMCF-7(DNP53)) overexpressing a dominant negative p53val135 mutant, and cells re-cultured from vMCF-7(DNP53) tumor xenografts. We carried out an integrative transcriptome and cytogenetic analysis to characterize the mechanistic linkage between loss of p53 function, EMT and consequent establishment of invasive gene signatures during breast cancer progression. We demonstrate that abrogation of p53 function drives the early transcriptome changes responsible for cell proliferation, EMT and survival, while further transcriptome changes that occur during in vivo tumor progression are mechanistically linked to the development of CIN leading to a more invasive and metastatic breast cancer phenotype. Here we identified distinct novel non-canonical transcriptome networks involved in cell proliferation, EMT, chemoresistance and invasion that arise following abrogation of p53 function in vitro and development of CIN in vivo. These studies also have important translational implications since some of the nodal genes identified here are 'druggable' making them appropriate molecular targets for the treatment of breast carcinomas displaying mutant p53, EMT, CIN and high metastatic potential.

Role of active drug transporters in refractory multiple myeloma.

Drug resistance is a major drawback for cancer chemotherapy protocols and previous studies have demonstrated the overexpression of the P-glycoprotein (P-gp) as mechanism by which myeloma cells develop multidrug resistance (MDR). However, other molecules may apparently promote MDR in multiple myeloma (MM). They include both lung resistance-related protein (LRP) and p53 activation. The inhibition of P-gp in MM patients treated with melphalan (PAM) has been associated to increased toxicity, whereas defective apoptosis due to down-modulation of the NF-kB is a feature of MDR+ myeloma cells. On the contrary, clinical trials with proteasome inhibitors have been successfully carried out to overcome MDR despite their toxicity profile. Recently, sigma receptors (sigmaR)(S), namely sigmaR(1) and sigmaR(2), have been found to be overexpressed in breast cancer cells. In addition, their levels correlate with both P-gp upregulation and MDR development. By contrast, selective inhibitors of sigmaR(S) as PB28, disrupt the P-gp signals and restore the apoptosis machinery in malignant cells. We have reviewed the major pathogenetic events promoting MDR in MM and focused on the sigmaR(S) as potential mechanism driving this function. We demonstrate that MDR+ myeloma cells overexpress the sigmaR(2) and that the treatment with PB28 induces P-gp down-modulation through the activation of the caspases enrolled in both extrinsic and intrinsic apoptotic pathways. Thus, sigmaR(2) inhibitors may be tentatively proposed for the treatment of PAM-resistant MM patients.

Glomerular accumulation of plasmacytoid dendritic cells in active lupus nephritis: role of interleukin-18.

OBJECTIVE: Defective circulating dendritic cells (DCs) have been described in systemic lupus erythematosus (SLE) and correlated with high levels of interferon-alpha (IFNalpha). DCs are differentiated as being either myeloid or plasmacytoid, according to chemokine expression and the tendency to migrate toward inflamed tissue. We investigated the potential role of interleukin-18 (IL-18) in driving the glomerular migration of DCs in lupus nephritis (LN) and in affecting the ability of DCs to induce an imbalance in the Th1:Th2 ratio. METHODS: DC subsets were characterized by flow cytometry and defined as either myeloid or plasmacytoid according to the expression of CD11c/blood dendritic cell antigen 1 (BDCA-1) and CD123/BDCA-2, respectively. The serum Th1:Th2 profile was studied by enzyme-linked immunosorbent assay. IL-18 receptor (IL-18R) and other chemokine receptors were analyzed by flow cytometry. Glomerular levels of IL-18/IL-18R and the presence of plasmacytoid DCs and myeloid DCs were investigated by immunohistochemical analysis. RESULTS: The number of peripheral plasmacytoid DCs was decreased in patients with SLE compared with control subjects, and this defect in the number of DCs was correlated with LN. Patients with LN showed a prevalent Th1 response, with high production of IL-18, IL-12 and IFNgamma. Only plasmacytoid DCs expressed IL-18R. Patients with severe LN showed a high accumulation of IL-18 within glomeruli in association with the presence of plasmacytoid DCs, whereas myeloid DCs were almost absent. CONCLUSION: A deficient number of peripheral plasmacytoid DCs correlated with high levels of Th1 cytokines and was associated with LN. Both serum and glomerular IL-18 were increased in LN. It is suggested that the high level of expression of IL-18R by peripheral plasmacytoid DCs allows the DCs to relocate within glomeruli under IL-18 stimulation and triggers the resident T cells, thus promoting renal damage.

Expression and function of the calcitonin receptor by myeloma cells in their osteoclast-like activity in vitro.

Malignant plasma cells exert osteoclast-like activity in vitro. We investigated the function of the calcitonin (CT) receptor (R) on myeloma cells from patients and in myeloma cell lines. Primary myeloma cells expressed high CTR levels whereas the cell lines uniformly exposed the CTR-2 variant expressed by osteoclasts. Treatment of myeloma cell lines with CT modified the intracellular Ca(2+) and cAMP levels, suggesting the activation of both PKC and PKA pathways, and abrogated their bone resorptive property as erosive pits on osteologic substrates. Thus, the expression, sensitivity and function of CTR-2 in myeloma cells emphasize their osteoclast-like behavior in vitro.

Increased IL-18 production by dendritic cells in active inflammatory myopathies.

Inflammatory myopathies (IM) are chronic disorders characterized by muscular accumulation of inflammatory cells that promote cytotoxicity and tissue damage. Overexpression of chemokines and cytokines as well as imbalance of dendritic cells (DC) homeostasis have been postulated to exert a role in both dermatomyositis (DM) and polymyositis (PM). We studied the T helper (Th)-1 and Th2 cytokine levels by enzyme linked immunosorbent assay (ELISA) and the muscular expression of IL-18 and its receptor by both histochemistry (HIC) and in situ hybridization (ISH) in both patients and normal controls. Also, the cell populations infiltrating the muscles were investigated. We present evidence that DM and PM are characterized by a predominant Th1 immune response with high production of both interferon (IFN)-gamma and interleukin (IL)-18 in the presence of reduced levels of IL-4 and IL-6. IL-18 was also demonstrated in muscles and produced by both macrophages and DC surrounding either perivascular and perimysium areas or endomysium. IL-18R was highly expressed by T cells and DC as well as by endothelial cells (EC) and smooth muscle cells (SMC). High concentrations of serum and muscular IL-18 suggest that deregulated IL-18/IL-18R pathway may be pathogenetic in IM and measurement of IL-18 might be predictive of the disease activity.

Deregulated expression of monocyte chemoattractant protein-1 (MCP-1) in arterial hypertension: role in endothelial inflammation and atheromasia.

OBJECTIVE: Arterial hypertension is recurrently associated with inflammation of the endothelium as an effect of the upregulation of functional molecules, including cytokines, adhesion molecules and chemokines. However, the role of monocyte chemoattractant protein-1 (MCP-1) in maintaining the inflammatory state of endothelial cells (EC) that leads to the progressive cardiovascular damage is unclear. DESIGN: Here, we investigated the expression of MCP-1, its major cell source as well as recurrence of a defined polymorphism (-2518 MCP-1) apparently linked to endothelial damage in several diseases. METHODS: Serum MCP-1 was measured by enzyme-linked immunosorbent assay (ELISA) in 740 hypertensive patients, subdivided according to their individual organ damage. Expression of both MCP-1 and its receptor CCR2 was evaluated in circulating ECs and macrophages by flow cytometry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), while gene variants of MCP-1 were revealed by PCR. RESULTS: Soluble MCP-1 was significantly elevated in patients with diffuse atheromasia. Furthermore, it was overexpressed by ECs activated to attract macrophages via the MCP-1/CCR2 pathway, whereas the -2518 MCP-1 polymorphism was correlated with atherosclerosis in most patients. CONCLUSIONS.: Overexpression of MCP-1 is predominant in hypertensive patients with atheromasia in the form of a defined polymorphism. Measurement of MCP-1 may thus reflect the degree of endothelial damage, while early detection of such a polymorphism may acquire a prognostic value in the development of atherosclerosis.

The interplay of chemokines and dendritic cells in the pathogenesis of lupus nephritis.

Lupus nephritis (LN) occurs in more that one-third of patients with systemic lupus erythematosus. Production of nephritogenic autoantibodies, glomerular immune complex deposition, and cytokine overproduction have been postulated to contribute to the pathogenesis of LN. However, overexpression of chemokines and imbalance of dendritic cell (DC) homeostasis may contribute to the development of nephritis in SLE. We present evidence that monocyte chemoattractant protein (MCP)-1 promotes renal disease in experimental glomerulonephritis, while its increased urinary levels reflect the severity of the disease in humans. Although macrophages are the prevalent infiltrating population within the kidney, it has been recently proposed that several chemokines and related receptors expressed by DCs may divide this cell population into myeloid (mDC) and plasmacytoid (pDC) subsets. However, the chemokine receptors expressed by pDCs are not functional, and other molecules are involved in the chemoattraction of these cells. We found increased expression of interleukin (IL)-18 in glomeruli of patients with active LN along with glomerular infiltration by pDCS. Since pDCs bear IL-18 receptor (IL-18R), it is conceivable that circulating pDCs may migrate toward glomeruli by IL-18/IL-18R interactions. Therefore, the relative depletion of circulating pDCs reflects the severity of inflammatory disease in LN.

Recent advances in understanding the pathogenesis of anemia in multiple myeloma.

Anemia is a prominent feature of multiple myeloma (MM) and is commonly associated with clinical progression of MM. In addition to being affected by a number of pathogenetic events, including imbalance of the cytokine network, inappropriate erythropoietin (EPO) levels, blood loss, and hemolysis, the erythroid matrix is chronically deteriorated by the malignant plasma cell clone that activates a cytotoxic mechanism directed at the erythroid progenitors. In particular, malignant plasma cells express very high levels of apoptogenic receptors, including both Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand, which trigger apoptosis of immature erythroblasts by stimulating specific death receptors, namely Fas and the complex DR4/DR5. Erythroid cells also weakly express the transcription factor GATA-1, which drives erythroblast maturation by inhibiting apoptosis through antiapoptotic molecules such as EPO and Bcl-xL. This newly discovered pathogenetic mechanism of anemia in MM is based on persistent erythroblast cytotoxicity within the bone marrow that leads to progressive destruction of the erythroid matrix.