FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange.

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

C-X-C chemokine receptor 7: a functionally associated molecular marker for bladder cancer.

BACKGROUND: C-X-C chemokine receptor 4 (CXCR4) and CXCR7 are 7-transmembrane chemokine receptors of the stroma-derived factor (SDF-1). CXCR4, but not CXCR7, has been examined in bladder cancer (BCa). This study examined the functional and clinical significance of CXCR7 in BCa. METHODS: CXCR4 and CXCR7 levels were measured in BCa cell lines, tissues (normal = 25; BCa = 44), and urine specimens (n = 186) by quantitative polymerase chain reaction and/or immunohistochemistry. CXCR7 function in BCa cells were examined by transient transfections using a CXCR7 expression vector or small interfering RNA. RESULTS: In BCa cell lines, CXCR7 messenger RNA levels were 5- to 37-fold higher than those for CXCR4. Transient overexpression of CXCR7 in BCa cell lines promoted growth and chemotactic motility. CXCR7 colocalized and formed a functional complex with epidermal growth factor receptor, phosphoinositide 3-kinase/Akt, Erk, and src and induced their phosphorylation. CXCR7 also induced up-regulation of cyclin-D1 and bcl-2. Suppression of CXCR7 expression reversed these effects and induced apoptosis. CXCR7 messenger RNA levels and CXCR7 staining scores were significantly (5- to 10-fold) higher in BCa tissues than in normal tissues (P < .001). CXCR7 expression independently associated with metastasis (P = .019) and disease-specific mortality (P = .03). CXCR7 was highly expressed in endothelial cells in high-grade BCa tissues when compared to low-grade BCa and normal bladder. CXCR7 levels were elevated in exfoliated urothelial cells from high-grade BCa patients (P = .0001; 90% sensitivity; 75% specificity); CXCR4 levels were unaltered. CONCLUSIONS: CXCR7 promotes BCa cell proliferation and motility plausibly through epidermal growth factor receptor receptor and Akt signaling. CXCR7 expression is elevated in BCa tissues and exfoliated cells and is associated with high-grade and metastasis.

Dietary supplement hymecromone and sorafenib: a novel combination for the control of renal cell carcinoma.

PURPOSE: Current treatments for metastatic renal cell carcinoma do not extend survival beyond a few months. Sorafenib is a targeted drug approved for metastatic renal cell carcinoma but it has modest efficacy. Hymecromone is a nontoxic dietary supplement with some antitumor activity at high doses of 450 to 3,000 mg per day. Hymecromone inhibits the synthesis of hyaluronic acid, which promotes tumor growth and metastasis. We recently noted that the hyaluronic acid receptors CD44 and RHAMM are potential predictors of metastatic renal cell carcinoma. In the current study we examined the antitumor properties of hymecromone, sorafenib and the combination in renal cell carcinoma models. MATERIALS AND METHODS: Using proliferation, clonogenic and apoptosis assays, we examined the effects of hymecromone (0 to 32 µg/ml), sorafenib (0 to 3.2 µg/ml) and hymecromone plus sorafenib in Caki-1, 786-O, ACHN and A498 renal cell carcinoma cells, and HMVEC-L and HUVEC endothelial cells. A Boyden chamber was used for motility and invasion assays. Apoptosis indicators, hyaluronic acid receptors, epidermal growth factor receptor and c-Met were evaluated by immunoblot. The efficacy of hymecromone, sorafenib and hymecromone plus sorafenib was assessed in the sorafenib resistant Caki-1 xenograft model. RESULTS: Hymecromone plus sorafenib synergistically inhibited proliferation (greater than 95%), motility/invasion (65%) and capillary formation (76%) in renal cell carcinoma and/or endothelial cells, and induced apoptosis eightfold (p <0.001). Hymecromone plus sorafenib inhibited hyaluronic acid synthesis and adding hyaluronic acid reversed the cytotoxicity of hymecromone plus sorafenib. Hymecromone plus sorafenib up-regulated pro-apoptotic indicators and down-regulated Mcl-1, CD44, RHAMM, phospho-epidermal growth factor receptor and phospho-cMet. In all assays hymecromone and sorafenib alone were ineffective. Oral administration of hymecromone (50 to 200 mg/kg) plus sorafenib (30 mg/kg) eradicated Caki-1 tumor growth without toxicity. Hymecromone and sorafenib alone were ineffective. CONCLUSIONS: To our knowledge this is the first study to show that the combination of sorafenib and the nontoxic dietary supplement hymecromone is highly effective for controlling renal cell carcinoma.

Targeting hyaluronidase for cancer therapy: antitumor activity of sulfated hyaluronic acid in prostate cancer cells.

The tumor cell-derived hyaluronidase (HAase) HYAL-1 degrades hyaluronic acid (HA) into proangiogenic fragments that support tumor progression. Although HYAL-1 is a critical determinant of tumor progression and a marker for cancer diagnosis and metastasis prediction, it has not been evaluated as a target for cancer therapy. Similarly, sulfated hyaluronic acid (sHA) has not been evaluated for biological activity, although it is an HAase inhibitor. In this study, we show that sHA is a potent inhibitor of prostate cancer. sHA blocked the proliferation, motility, and invasion of LNCaP, LNCaP-AI, DU145, and LAPC-4 prostate cancer cells, and induced caspase-8-dependent apoptosis associated with downregulation of Bcl-2 and phospho-Bad. sHA inhibited Akt signaling including androgen receptor (AR) phosphorylation, AR activity, nuclear factor κB (NFκB) activation, and VEGF expression. These effects were traced to a blockade in complex formation between phosphoinositide 3-kinase (PI3K) and HA receptors and to a transcriptional downregulation of HA receptors, CD44, and RHAMM, along with PI3K inhibition. Angiogenic HA fragments or overexpression of myristoylated Akt or HA receptors blunted these effects of sHA, implicating a feedback loop between HA receptors and PI3K/Akt signaling in the mechanism of action. In an animal model, sHA strongly inhibited LNCaP-AI prostate tumor growth without causing weight loss or apparent serum-organ toxicity. Inhibition of tumor growth was accompanied by a significant decrease in tumor angiogenesis and an increase in apoptosis index. Taken together, our findings offer mechanistic insights into the tumor-associated HA-HAase system and a preclinical proof-of-concept of the safety and efficacy of sHA to control prostate cancer growth and progression.