Fabrice Journé

Increased Dickkopf-1 expression in breast cancer bone metastases

The aim of this study was to determine whether Dickkopf-1 (Dkk-1) expression in breast cancer was associated with bone metastases. We first analysed Dkk-1 expression by human breast cancer cell lines that induce osteolytic or osteoblastic lesions in animals. Dickkopf-1 levels were then measured in the bone marrow aspirates of hind limbs from eight NMRI mice inoculated with breast cancer cells that induced bone metastases and 11 age-matched non-inoculated control animals. Finally, Dkk-1 was measured in the serum of 17 women with breast cancer in complete remission, 19 women with breast cancer and bone metastases, 16 women with breast cancer and metastases at non-bone sites and 16 healthy women. Only breast cancer cells that induce osteolytic lesions in animals produced Dkk-1. There was a six-fold increase in Dkk-1 levels in the bone marrow from animals inoculated with MDA-B02 cells when compared with that of control non-inoculated animals (P=0.003). Median Dkk-1 levels in the serum of patients with breast cancer and bone metastases were significantly higher than levels of patients in complete remission (P=0.016), patients with breast cancer having metastases at non-bone sites (P<0.0001) and healthy women (P=0.047), although there was a large overlap in individual levels between the different groups. In conclusion, Dkk-1 is secreted by osteolytic human breast cancer cells lines and increased circulating levels are associated with the presence of bone metastases in patients with breast cancer. Measurements of circulating Dkk-1 levels may be useful for the clinical investigation of patients with breast cancer and bone metastases.

Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading. Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event. This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively. Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors. Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma. Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.

Role of the proteasome in the regulation of estrogen receptor α turnover and function in MCF-7 breast carcinoma cells

Estrogen receptor alpha (ER) turnover in MCF-7 cells was assessed by pulse chase analysis and measurement of ER steady-state level. In untreated cells, degradation of (35)S-labeled ER was characterized by a slow phase followed by a more rapid decline. Without ligand, ER elimination was totally compensated by synthesis which maintained receptor homeostasis. Estradiol (E(2)) and the pure antiestrogen RU 58,668 abolished the slow phase of ER breakdown and enhanced the degradation of neosynthesized ER, producing a low ER steady-state level. By contrast, the partial antiestrogen OH-Tam was ineffective in this respect and caused ER accumulation. Regardless of the conditions, ER breakdown was abolished by proteasome inhibition (MG-132). ER ligands decreased cell capacity to bind [(3)H]E(2), even in the presence of MG-132, indicating that the regulation of ER level and E(2) binding capacity occurs through distinct mechanisms. MG-132 partially blocked the basal transcription of an ERE-dependent reporter gene and modified the ability of E(2) to induce the expression of the latter: the hormone was unable to restore the transactivation activity measured without MG-132. RU 58,668 and OH-Tam failed to enhance the inhibitory action of MG-132, suggesting that a loss of basal ER-mediated transactivation mainly affects the stimulatory effect of estrogens. Overall, our findings reveal that ER steady state level, ligand binding capacity and transactivation potency fit in a complex regulatory scheme involving distinct mechanisms, which may be dissociated from each other under various treatments.

The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

BackgroundThe proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined.MethodsWe evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]).ResultsDasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity.ConclusionsOur results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism.

Mechanisms governing the accumulation of estrogen receptor alpha in MCF-7 breast cancer cells treated with hydroxytamoxifen and related antiestrogens.

This study aimed at a better understanding of estrogen receptor alpha (ER) up regulation induced by partial estrogen antagonists. Effect of treatment with hydroxytamoxifen (OH-Tam) on ER level in MCF-7 cells was investigated by an approach combining ER measurement (enzyme immunoassay) and morphological demonstration (immunofluorescence). Furthermore, the influence of drug exposure on the rates of ER synthesis and degradation was assessed by determining [35S]methionine incorporated into the receptor in different experimental conditions (measurement of synthesis or pulse-chase experiments). ER up regulation was already induced by a 1-h pulse treatment with OH-Tam, thus a continuous exposure was not required. This process appeared reversible (i.e. ER accumulation due to OH-Tam rapidly vanished upon subsequent exposure to 17beta-estradiol (E2) or the pure antiestrogen RU 58668). While OH-Tam did not affect the rate of [35S]methionine incorporation into ER, it clearly caused an impairment of ER degradation (pulse-chase experiments) indicating that up regulation results from a stabilization of the receptor associated with the maintenance of its synthesis. Various tamoxifen derivatives, as well as a few related partial antiestrogens, were compared on the basis of binding ability and propensity to induce ER up regulation. A close relationship was found between both properties. Structure-activity analysis revealed that the capacity of these compounds to induce ER up regulation is associated with characteristics of their aminoalkyle side-chain, similar to those required for antiestrogenicity.

Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation

Farnesoid X receptor (FXR) is a metabolic nuclear receptor expressed in the liver and traditionally considered as a bile acid sensor. Yet, FXR has been recently demonstrated in other tissues and cells, such as the kidneys, the adrenals, and arterial smooth muscle cells. Immunohistochemical data reported in this study point to the expression of FXR in human breast cancer. In addition, FXR expression was also found by Western blotting and immunofluorescence microscopy in breast-cancer-derived cell lines MCF-7 (estrogen receptor [ER]-positive) and MDA-MB-231 (ER-negative). The FXR activator farnesol, a mevalonate pathway intermediate, exerts a mitogenic effect on MCF-7 cells. The growth stimulation is completely suppressed by antiestrogens. In contrast, MDA-MB-231 cells appear farnesol-insensitive, suggesting an involvement of ER in farnesol mitogenicity. In accordance with this interpretation, farnesol induces in MCF-7 cells a decrease of ER level, consistent with a phenomenon of receptor downregulation. Farnesol also increases progesterone receptor (PgR) expression in MCF-7 cells and stimulates ER-mediated gene transactivation in MVLN cells (MCF-7 cells stably transfected with an ER reporter gene). Of note, both effects of farnesol on ER expression and activity are completely suppressed by antiestrogens. In addition, farnesol-induced PgR is markedly reduced by FXR gene silencing (siRNA), demonstrating the involvement of FXR in the estrogenic effects of farnesol. Finally, coimmunoprecipitation experiments (FXR immunoprecipitation followed by Western blot analysis of ER in the immunoprecipitate) produced definite evidence that FXR interacts with ER. Altogether, these observations reveal the hitherto unreported presence of FXR in breast cancer and show that the latter receptor functionally interacts with ER. The occurrence of such a crosstalk calls for some caution regarding the pharmacological use of FXR agonists.

Characterization and Clinical Evaluation of CD10+ Stroma Cells in the Breast Cancer Microenvironment

Purpose: There is growing evidence that interaction between stromal and tumor cells is pivotal in breast cancer progression and response to therapy. Based on earlier research suggesting that during breast cancer progression, striking changes occur in CD10+ stromal cells, we aimed to better characterize this cell population and its clinical relevance. Experimental Design: We developed a CD10+ stroma gene expression signature (using HG U133 Plus 2.0) on the basis of the comparison of CD10 cells isolated from tumoral (n = 28) and normal (n = 3) breast tissue. We further characterized the CD10+ cells by coculture experiments of representative breast cancer cell lines with the different CD10+ stromal cell types (fibroblasts, myoepithelial, and mesenchymal stem cells). We then evaluated its clinical relevance in terms of in situ to invasive progression, invasive breast cancer prognosis, and prediction of efficacy of chemotherapy using publicly available data sets. Results: This 12-gene CD10+ stroma signature includes, among others, genes involved in matrix remodeling (MMP11, MMP13, and COL10A1) and genes related to osteoblast differentiation (periostin). The coculture experiments showed that all 3 CD10+ cell types contribute to the CD10+ stroma signature, although mesenchymal stem cells have the highest CD10+ stroma signature score. Of interest, this signature showed an important role in differentiating in situ from invasive breast cancer, in prognosis of the HER2+ subpopulation of breast cancer only, and potentially in nonresponse to chemotherapy for those patients. Conclusions: Our results highlight the importance of CD10+ cells in breast cancer prognosis and efficacy of chemotherapy, particularly within the HER2+ breast cancer disease. Clin Cancer Res; 18(4); 1004–14. ©2012 AACR.

Role of farnesoid X receptor (FXR) in the process of differentiation of bone marrow stromal cells into osteoblasts

Bone tissue contains bile acids which accumulate from serum and which can be released in large amounts in the bone microenvironment during bone resorption. However, the direct effects of bile acids on bone cells remain largely unexplored. Bile acids have been identified as physiological ligands of the farnesoid X receptor (FXR, NR1H4). In the present study, we have examined the effects of FXR activation/inhibition on the osteoblastic differentiation of human bone marrow stromal cells (BMSC). We first demonstrated the expression of FXR in BMSC and SaOS2 osteoblast-like cells, and observed that FXR activation by chenodeoxycholic acid (CDCA) or by farnesol (FOH) increases the activity of alkaline phosphatase and the calcification of the extracellular matrix. In addition, we observed that FXR agonists are able to stimulate the expression of osteoblast marker genes [bone sialoprotein (BSP), osteocalcin (OC), osteopontin (OPN) and alkaline phosphatase (ALP)] (FXR involvement validated by shRNA-induced gene silencing), as well as the DNA binding activity of the bone transcription factor RUNX2 (EMSA and ChIP assay). Importantly, we observed that nitrogen-containing bisphosphonates (BPs) inhibit the basal osteoblastic differentiation of BMSC, possibly through suppression of endogenous FOH production, independently of their effects on protein prenylation. Likewise, we found that the FXR antagonist guggulsterone (GGS) inhibits ALP activity, calcium deposition, DNA binding of RUNX2, and bone marker expression, indicating that GGS interferes with osteoblastic differentiation. Furthermore, GGS induced the appearance of lipid vesicles in BMSC and stimulated the expression of adipose tissue markers (peroxisome proliferator activated receptor-gamma (PPARγ), adipoQ, leptin and CCAAT/enhancer-binding protein-alpha (C/EBPα)). In conclusion, our data support a new role for FXR in the modulation of osteoblast/adipocyte balance: its activation stimulates RUNX2-mediated osteoblastic differentiation of BMSC, whereas its inhibition leads to an adipocyte-like phenotype.

Pharmacological inhibition of macrophage migration inhibitory factor interferes with the proliferation and invasiveness of squamous carcinoma cells

Recent clinical observations and experimental studies of our group indicate that macrophage migration inhibitory factor (MIF) may contribute to tumor progression in head and neck squamous cell carcinomas (HNSCC). The present study was undertaken to examine the effects of the irreversible MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) on proliferation and invasiveness of the squamous carcinoma cell line SCCVII. Cell counting, crystal violet assay and flow cytometry were used to analyze the effects of 4-IPP on SCCVII cell growth. The impact of 4-IPP on cell invasiveness was assessed by Boyden chamber assay. Knockdown of the MIF receptor CD74 was achieved by transduction with lentiviral vectors encoding anti-CD74 shRNAs. As shown by immunofluorescence staining, SCCVII cells express both MIF and CD74. Decreased MIF immunoreactivity as a result of exposure to 4-IPP suggested a covalent modification of the cytokine. 4-IPP inhibited SCCVII cell proliferation and invasiveness. Moreover, the cytostatic effect of 4-IPP was enhanced by CD74 knockdown. The inhibitory effects of 4-IPP on cell proliferation and invasiveness strongly suggest that MIF is involved in proliferative activity and invasive properties of squamous carcinoma cells. In conclusion, MIF inhibition may open possibilities for target-directed treatment of head and neck squamous cell carcinoma.

Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells.

Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.