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.

Purpose: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors. Experimental Design: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings. Results: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P = 0.0001) when compared with primary tumors.The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR. Conclusions: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.

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The authors report the clinicopathologic features of 105 carcinomas arising within fibroadenomas (FAs) of the breast. The mean age of the patients was 44 years. The presentation and gross characteristics of these tumors rarely differed from those of uncomplicated FAs. Carcinoma in situ (CIS) was the predominant type of malignancy (95%) found to arise in FAs, and lobular and ductal types occurred with equal frequency. Nine of ten FAs harboring an invasive carcinoma also contained CIS supporting the origin of the infiltrative component in the FAs. CIS within FAs was associated with in situ malignancy in surrounding breast tissue in 21% of cases. Age, fibroadenoma size, and type and extent of CIS were similar in patients with disease limited to the FA and in those with associated malignant disease in the remainder of the breast. Axillary nodal metastases were not detected. Sixty-three patients were observed for a mean period of 8.4 years. Only one of 26 patients with CIS within an FA who was treated conservatively developed an ipsilateral carcinoma. None of the 26 developed contralateral carcinoma; however, 3 of 23 with similar lesions, who were treated by mastectomy, did so. The contralateral carcinomas were invasive in two patients, one of whom died with distant metastases. Seven patients with FAs harboring lobular CIS underwent bilateral mastectomy. Their postoperative course was uneventful. None of seven patients with invasive carcinoma arising in an FA, two of whom were treated conservatively, succumbed to disease. However, one developed contralateral carcinoma. The authors recommend breast-conserving therapy for CIS arising in an FA.