Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Standard methods to measure recreational water quality require at least 24 hr to obtain results, making it impossible to assess the quality of water within a single day. Methods to measure recreational water quality in ≤ 2 hr have been developed. Application of rapid methods could give considerably more accurate and timely assessments of recreational water quality. We conducted a prospective study of beachgoers at two Great Lakes beaches to examine the association between recreational water quality, obtained using rapid methods, and gastrointestinal (GI) illness after swimming. Beachgoers were asked about swimming and other beach activities and 10–12 days later were asked about the occurrence of GI symptoms. We tested water samples for Enterococcus and Bacteroides species using the quantitative polymerase chain reaction (PCR) method. We observed significant trends between increased GI illness and Enterococcus at the Lake Michigan beach and a positive trend for Enterococcus at the Lake Erie beach. The association remained significant for Enterococcus when the two beaches were combined. We observed a positive trend for Bacteroides at the Lake Erie beach, but no trend was observed at the Lake Michigan beach. Enterococcus samples collected at 0800 hr were predictive of GI illness that day. The association between Enterococcus and illness strengthened as time spent swimming in the water increased. This is the first study to show that water quality measured by rapid methods can predict swimming-associated health effects.

Fig. 1. Apoptosis induction by lenalidomide is specific for 5q-deleted cells. (A) Cells from AML that evolved from MDS patients with (Center) or without (del) 5q and U937 cells (Left and Right) were exposed to lenalidomide, thalidomide, or vehicle (DMSO) at the concentrations indicated for 48 h before apoptosis was assessed by flow cytometry using Annexin-V/PI staining. Representative results are shown as the mean of the triplicate measurement ± SD from 1 patient. A total of 5 different MDS patients were tested. (B) Lenalidomide arrests 5q deleted cells in G 2 . Cells were treated with lenalidomide at the concentration of 1 μM for 48 h at 37°C and stained with propidium Iodide (PI) in BD Stain Buffer (10 6 /ml) before analysis on BD FACScan. (C) FISH analysis of haplo-deficiency of Cdc25C in (del)5q cells. A normal chromosome 5 showing FISH signals for D5S23/D5S721 (green) and Cdc25C (orange); the ideogram demonstrates the relative gene locations of EGR1 and Cdc25C in 5q31. Probes for EGR1 are commonly used to detect classical 5q deletions. The Right Inset illustrates D5S23/D5S721 and Cdc25C signal pattern for classical 5q deletions in interphase nuclei. (D) Reduced expression of Cdc25C and PP2Acα in bone marrow cells from patients with (del)5q by Q-PCR. RNA was purified from BM-MNC from patients with or without (del)5q as indicated. Relative expression levels of Cdc25C and PP2Acα were analyzed by Q-PCR to quantitate transcript level. Expression is normalized to the reference gene (GAPDH) and fold changes for Cdc25C and PP2Acα in patients are compared with the average data from non(del)5q cells (P < 0.001).

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