Abstract4-Methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility, and invasion of DU145, PC3-ML, LNCaP, C4-2B, and/or LAPC-4 cells. At IC 50 for HA synthesis (0.4 mmol/L), 4-MU induced >3-fold apoptosis in prostate cancer cells, which could be prevented by the addition of HA. 4-MU induced caspase-8, caspase-9, and caspase-3 activation, PARP cleavage, upregulation of Fas-L, Fas, FADD and DR4, and downregulation of bcl-2, phosphorylated bad, bcl-XL, phosphorylated Akt, phosphorylated IKB, phosphorylated ErbB2, and phosphorylated epidermal growth factor receptor. At IC 50 , 4-MU also caused >90% inhibition of NF-κB reporter activity, which was prevented partially by the addition of HA. With the exception of caveolin-1, HA reversed the 4-MU-induced downregulation of HA receptors (CD44 and RHAMM), matrix-degrading enzymes (MMP-2 and MMP-9), interleukin-8, and chemokine receptors (CXCR1, CXCR4, and CXCR7) at the protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU-induced apoptosis and inhibition of cell growth and interleukin-8, RHAMM, HAS2, CD44, and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (>3-fold) when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry, or body weight. Tumors from 4-MU-treated animals showed reduced microvessel density (∼3-fold) and HA expression but increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells and expression of apoptosisrelated molecules. Therefore, the anticancer effects of 4-MU, an orally bioavailable and relatively nontoxic agent, are primarily mediated by inhibition of HA signaling. Cancer Res; 70(7); 2613-23. ©2010 AACR.

BackgroundRecent advances have revealed a significant contribution of chemokines and their receptors in tumor growth, survival after chemotherapy, and organ-specific metastasis. The CXC chemokine receptor-7 (CXCR7) is the latest chemokine receptor implicated in cancer. Although over expressed in breast cancer cell lines and tumor tissues, its mechanism of action in breast cancer (BrCa) growth and metastasis is unclear. Studies in other cancers have implicated CXCR7 in cell proliferation, anti-apoptotic activity and cell-cell adhesion. The present study was initiated to examine the pattern of CXCR7 expression and its role in regulation of growth signaling in breast cancer.MethodsThe contribution of CXCR7 in BrCa cell proliferation was investigated in representative cell lines using real time quantitative PCR (q-PCR), proliferation assays, immunohistochemistry and immunoblotting. Phenotypic changes were examined after CXCR7 specific cDNA and siRNA transfection and expression levels were monitored by q-PCR. Further, the association of CXCR7 with epidermal growth factor receptor (EGFR) and modulation of its activity were investigated by western blotting, immunofluorescence, and in-situ proximity ligation assays in human BrCa cells and tissues.ResultsCXCR7 was expressed in both, estrogen receptor (ER) positive and negative BrCa cell lines. CXCR7 was also expressed unevenly in normal breast tissues and to a much higher extent in ER + cancer tissues. Depletion of CXCR7 in MCF7 BrCa cells by RNA interference decreased proliferation and caused cell cycle arrest. Further, proximity ligation assay (PLA) revealed colocalization of CXCR7 with EGFR in cancer tissues and cancer cell lines. CXCR7 depletion reduced levels of phospho-EGFR at Tyrosine1110 after EGF-stimulation and also reduced phosphorylation of ERK1/2, indicating a potentially direct impact on mitogenic signaling in MCF7 cells. Using siRNA to knockdown β-arrestin2 in cells with EGFR over expression we were able to nearly deplete the CXCR7-EGFR colocalization events, suggesting that β-arrestin2 acts as a scaffold to enhance CXCR7 dependent activation of EGFR after EGF stimulation.ConclusionsThese results demonstrate coupling of CXCR7 with EGFR to regulate proliferation of BrCa cells and suggest an important ligand-independent role of CXCR7 in BrCa growth. Thus, the CXCR7-EGFR axis is a promising target for breast cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-198) contains supplementary material, which is available to authorized users.

There is growing interest in developing non-pharmacological treatments that could boost natural defenses against cancer and contribute to primary and secondary cancer prevention. Recent studies have shown that gentle daily stretching for 10 minutes can reduce local connective tissue inflammation and fibrosis. Because mechanical factors within the stroma can influence the tumor microenvironment, we hypothesized that stretching would reduce the growth of tumors implanted within locally stretched tissues and tested this hypothesis in a mouse orthotopic breast cancer model. Female FVB mice (N = 66) underwent bilateral injection of p53/PTEN double-null primary mouse mammary tumor cells into the third mammary fat pad. Mice were randomized to stretch vs. no stretch, and treated for 10 minutes once a day, for four weeks. Tumor volume at end-point was 52% smaller in the stretch group, compared to the no-stretch group (p < 0.001) in the absence of any other treatment. Cytotoxic immune responses were activated and levels of Specialized Pro-Resolving Mediators were elevated in the stretch group. These results suggest a link between immune exhaustion, inflammation resolution and tumor growth. Stretching is a gentle, non-pharmacological intervention that could become an important component of cancer treatment and prevention.