BRAF(V600E) melanoma cells secrete factors that activate stromal fibroblasts and enhance tumourigenicity.

BACKGROUND: Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer deaths and is highly metastatic, readily spreading to the lymph nodes or metastasising to other organs. The frequent genetic mutation found in metastatic melanoma, BRAF(V600E), results in constitutive activation of the mitogen-activated protein kinase pathway. METHODS: In this study, we utilised genetically engineered melanoma cell lines and xenograft mouse models to investigate how BRAF(V600E) affected cytokine (IL-1ß, IL-6, and IL-8) and matrix metalloproteinase-1 (MMP-1) expression in tumour cells and in human dermal fibroblasts. RESULTS: We found that BRAF(V600E) melanoma cells expressed higher levels of these cytokines and of MMP-1 than wild-type counterparts. Further, conditioned medium from the BRAF(V600E) melanoma cells promoted the activation of stromal fibroblasts, inducing expression of SDF-1 and its receptor CXCR4. This increase was mitigated when the conditioned medium was taken from melanoma cells treated with the BRAF(V600E) specific inhibitor, vemurafenib. CONCLUSIONS: Our findings highlight the role of BRAF(V600E) in activating the stroma and suggest a mechanistic link between BRAF(V600E) and MMP-1 in mediating melanoma progression and in activating adjacent fibroblasts in the tumour microenvironment.

Stereoselective inhibition of cholesterol side chain cleavage by enantiomers of aminoglutethimide.

Because aminoglutethimide is a potentially important drug in the treatment of certain maligancies as well as fertility control, its stereoisomers were studied for binding to corpus luteum mitochondrial cytochrome P-450 and inhibition of cholesterol side chain cleavage. The binding affinity, determined from induced spectral changes, is 2.6 times greater for the d- than for the l-isomer. In the enzyme assay, the d-isomer is 2.5 times more potent as an inhibitor of cholesterol side chain cleavage than is the l-isomer. The extent of inhibition and the change in the absorptivity of the P-450-inhibitor complex are linearly related for both chiral and racemic forms. Thus, the active center of the enzyme is stereoselective for the enantiomers of aminoglutethimide.

Comparison of luteolytic potencies of aminoglutethimide enantiomers in the rabbit and rat.

The luteolytic potency of D- and L-aminoglutethimide (D- and L-AG) was compared in in vivo assays in the rat and rabbit. By assay of plasma progesterone depletion in the rabbit, the potency of L-AG relative to D-AG was 0.21. By the plasma procedure in the rat, the relative potencies of L-AG and of the racemic mixture to D-AG were 0.04 and 0.37, respectively. By the ovarian progesterone depletion method, the L-form had very little activity and the DL-mixture was half as active as the D-isomer. Thus, in both species, almost all of the activity of the racemic mixture results from the content of D-AG. Interpretation of paradoxical data implies that in the rat, L-AG may inhibit liver degradation of progesterone at levels which do not modify secretion from the corpus luteum.

Effects of cholesterol side chain cleavage inhibitors on 11 beta-hydroxylation: linkage of mitochondrial oxygenase systems.

Potent inhibitors of cholesterol side chain cleavage were tested for inhibition of 11 beta-hydroxylation of 11-deoxycortisol by bovine adrenal cortex mitochondria. Compounds which inhibited 11 beta-hydroxylation were metyrapone, 4-phenylimidazole, 1-benzylimidazole, 17 beta-ureido - 1, 4- androstadien-3-one. SU-8000, 4-methylaminoglutethimide, and 20 alpha-hydroxycholestrol. Compounds which did not inhibit 11 beta-hydroxylation at concentrations of 0.5 mM were d-aminoglutethimide tartrate, 1-aminoglutethimide tartrate, N-methylaminoglutethimide, 16 alpha-methylpregnenolone, 16 beta-methylpregnenolone, 20-tolylpregnenediol, 16 alpha-chloropregnenolone-3-acetate, 16 alpha-benzyloxpregnenolone-3-acetate and cyanoketone. The results obtained indicate that aminoglutethimide and its congeners, the 16-halogenated and 16-benzoylated derivatives of pregnenolone and cyanoketone are specific inhibitors of cholesterol side chain cleavage enzyme. The two mitochondrial steroid oxyganase systems are linked through their competition for a single electron source.

A KrasG12D-driven genetic mouse model of pancreatic cancer requires glypican-1 for efficient proliferation and angiogenesis.

Pancreatic ductal adenocarcinomas (PDACs) exhibit multiple molecular alterations and overexpress heparin-binding growth factors (HBGFs) and glypican-1 (GPC1), a heparan sulfate proteoglycan that promotes efficient signaling by HBGFs. It is not known, however, whether GPC1 has a role in genetic mouse models of PDAC. Therefore, we generated a GPC1 null mouse that combines pancreas-specific Cre-mediated activation of oncogenic Kras (Kras(G12D)) with deletion of a conditional INK4A/Arf allele (Pdx1-Cre;LSL-Kras(G12D);INK4A/Arf(lox/lox);GPC1(-/-) mice). By comparison with Pdx1-Cre;LSL-Kras(G12D);INK4A/Arf(lox/lox) mice that were wild type for GPC1, the Pdx1-Cre;LSL-Kras(G12D);INK4A/Arf(lox/lox);GPC1(-/-) mice exhibited attenuated pancreatic tumor growth and invasiveness, decreased cancer cell proliferation and mitogen-activated protein kinase activation. These mice also exhibited suppressed angiogenesis in conjunction with decreased expression of messenger RNAs encoding several pro-angiogenic factors and molecules, including vascular endothelial growth factor-A (VEGF-A), SRY-box containing gene (SOX17), chemokine C-X3-C motif ligand 1 (CX3CL1) and integrin ß3 (ITGB3). Moreover, pancreatic cancer cells isolated from the tumors of GPC1(-/-) mice were not as invasive in response to fibroblast growth factor-2 (FGF-2) as cancer cells isolated from wild-type mice, and formed smaller tumors that exhibited an attenuated metastatic potential. Similarly, VEGF-A and FGF-2 did not enhance the migration of hepatic endothelial cells and immortalized murine embryonic fibroblasts isolated from GPC1 null mice. These data demonstrate in an oncogenic Kras-driven genetic mouse model of PDAC that tumor growth, angiogenesis and invasion are enhanced by GPC1, and suggest that suppression of GPC1 may be an important component of therapeutic strategies in PDAC.