Yulian Niu

Psoriasin (S100A7) Expression and Invasive Breast Cancer

Alteration of psoriasin (S100A7) expression has previously been identified in association with the transition from preinvasive to invasive breast cancer. In this study we have examined persistence of psoriasin mRNA and protein expression in relation to prognostic factors in a cohort of 57 invasive breast tumors, comprising 34 invasive ductal carcinomas and 23 other invasive tumor types (lobular, mucinous, medullary, tubular). We first developed an IgY polyclonal chicken antibody and confirmed specificity for psoriasin by Western blot in transfected cells and tumors. The protein was localized by immunohistochemistry predominantly to epithelial cells, with both nuclear and cytoplasmic staining, as well as occasional stromal cells in psoriatic skin and breast tumors; however, in situ hybridization showed that psoriasin mRNA expression was restricted to epithelial cells. In breast tumors, higher levels of psoriasin measured by reverse transcriptase-polymerase chain reaction and Western blot (93% concordance) were significantly associated with estrogen and progesterone receptor-negative status (P < 0.0001, P = 0.0003), and with nodal metastasis in invasive ductal tumors (P = 0. 035), but not with tumor type or grade. Psoriasin expression also correlated with inflammatory infiltrates (all tumors excluding medullary, P = 0.0022). These results suggest that psoriasin may be a marker of aggressive behavior in invasive tumors and are consistent with a function as a chemotactic factor.

Phospho-serine-118 estrogen receptor-alpha expression is associated with better disease outcome in women treated with tamoxifen.

Purpose: The purpose of this research was to determine whether estrogen receptor α specifically phosphorylated at Ser118 is associated with clinical outcome in primary breast tumors from estrogen receptor-positive and node-negative breast cancer patients. Experimental Design: Estrogen receptor α specifically phosphorylated at Ser118 was determined by immunohistochemistry in 117 primary breast tumors from node-negative patients who were subsequently treated with adjuvant tamoxifen. The relationship of estrogen receptor α specifically phosphorylated at Ser118 expression to disease-free survival and overall survival was determined. Results: Estrogen receptor α specifically phosphorylated at Ser118 was limited to estrogen receptor α ligand binding assay-positive tumors and among this subset was expressed in 70 (62%) of these tumors. Estrogen receptor α specifically phosphorylated at Ser118 expression was more frequently observed in progesterone receptor-positive tumors compared with progesterone receptor-negative tumors (χ2 test, P = 0.012, n = 113). A significant correlation was also seen between estrogen receptor α specifically phosphorylated at Ser118 and progesterone receptor levels (Spearman r = 0.236, P = 0.0118, n = 113). Kaplan-Meier outcome analysis showed that patients whose primary tumors expressed estrogen receptor α specifically phosphorylated at Ser118 had a longer disease-free survival (P = 0.0018, n = 113) and a trend toward better overall survival, but this was not statistically significant. Among the subset of progesterone receptor-positive tumors, progesterone receptor-positive/estrogen receptor α specifically phosphorylated at Ser118-positive patients had a significantly longer disease-free survival that progesterone receptor-positive/estrogen receptor α specifically phosphorylated at Ser118-negative patients (P = 0.0041). Conclusions: Our data suggest that estrogen receptor α specifically phosphorylated at Ser118 is a marker of a functional, intact ligand-dependent estrogen receptor signaling pathway in breast cancer and that estrogen receptor α specifically phosphorylated at Ser118 status has the potential to provide a more precise biomarker of responsiveness to endocrine therapy in conjunction with estrogen receptor α and progesterone receptor status.

Necrosis and hypoxia in invasive breast carcinoma

To investigate the relation between necrosis and hypoxia in breast cancer we examined the expression of hypoxia-associated markers HIF1, CA IX and GLUT1 by immunohistochemistry in 97 invasive ductal carcinomas. This selected series comprised 48 tumors with extensive necrosis and 49 control tumors without necrosis. Over 90% of necrotic and 30% of non-necrotic tumors expressed at least one hypoxia marker. We also observed expression of hypoxia associated markers in tumor stroma. Examination of primary human breast fibroblasts in vitro confirmed that CA IX mRNA and protein can be induced by hypoxia. Survival analysis of 53 cases found that the subset of tumors with stromal hypoxia exhibit better prognosis (p = 0.027). Our results indicate that necrosis is often accompanied by hypoxia but that hypoxia without necrosis may also be a frequent occurrence. The use of several hypoxia markers may identify a continuum of hypoxia in tumors, which can be sub-classified by different co-expression patterns. We conclude that stromal and epithelial hypoxia may have different biological backgrounds and that stromal hypoxia may affect survival.

Estrogen Receptor-α Phosphorylated at Ser118 Is Present at the Promoters of Estrogen-Regulated Genes and Is Not Altered Due to HER-2 Overexpression

Detection of estrogen receptor (ER)-alpha phosphorylated at Ser(118) (P-Ser(118)-ER-alpha) may be an indicator of an intact ligand-dependent ER-alpha in breast tumors in vivo and may predict responsiveness to endocrine therapy. The current study addresses whether P-Ser(118)-ER-alpha is functionally involved in ER target gene transcription and if this is modulated by HER-2 overexpression. Using chromatin immunoprecipitation analysis, P-Ser(118)-ER-alpha was found associated with the promoters of several estrogen-regulated genes in MCF-7 breast cancer cells 30 minutes following estrogen treatment. Coactivators AIB1 and p300 were coimmunoprecipitated with P-Ser(118)-ER-alpha following estrogen treatment. The overexpression of HER-2 protein in MCF-7 cells did not affect estrogen induction of phosphorylation of Ser(118) or its presence at the promoters of several estrogen-regulated genes. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) pathway, had no effect on P-Ser(118)-ER-alpha. The lack of effect of HER-2 overexpression on P-Ser(118)-ER-alpha expression in cell models is supported by similar levels of expression of P-Ser(118)-ER-alpha in ER(+)/HER-2-overexpressing and ER(+)/HER-2(-) breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], and IkappaB kinase-alpha (IKK-alpha; BAY-11-7082), we show that IKK-alpha, but not Cdk7, is at least in part involved in estrogen-mediated phosphorylation at Ser(118) in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser(118)-ER-alpha in estrogen-regulated signaling and do not support the hypothesis that resistance of breast tumors to tamoxifen therapy involves ligand independent activation of ER-alpha due to constitutive phosphorylation of Ser(118) by constitutive activation of MAPK pathway.

The S100A7-c-Jun Activation Domain Binding Protein 1 Pathway Enhances Prosurvival Pathways in Breast Cancer

S100A7 is among the most highly expressed genes in preinvasive breast cancer, is a marker of poor survival when expressed in invasive disease, and promotes breast tumor progression in experimental models. To explore the mechanism of action, we examined the role of S100A7 in cell survival and found that overexpression of S100A7 in MDA-MB-231 cell lines promotes survival under conditions of anchorage-independent growth. This effect is paralleled by increased activity of nuclear factor-kappaB (3-fold) and phospho-Akt (4-fold), which are known to mediate prosurvival pathways. S100A7 and phospho-Akt are also correlated in breast tumors examined by immunohistochemistry (n = 142; P < 0.0001; r = 0.34). To explore the underlying mechanism, we examined the role of a putative c-Jun activation domain-binding protein 1 (Jab1)-binding domain within S100A7 using a panel of MDA-MB-231 breast cell lines stably transfected with either S100A7 or S100A7 mutated at the Jab1 domain. Structural analysis by three-dimensional protein modeling, immunoprecipitation, and yeast two-hybrid assay and functional analysis using transfected reporter gene and Western blot assays revealed that the in vitro effects of S100A7 on phospho-Akt and the nuclear factor-kappaB pathway are dependent on the Jab1-binding site and the interaction with Jab1. Enhanced epidermal growth factor receptor signaling was also found to correlate with the increased phospho-Akt. Furthermore, the Jab1-binding domain is also necessary for the enhanced tumorigenicity conferred by S100A7 expression in murine xenograft tumors in vivo. We conclude that the S100A7-Jab1 pathway acts to enhance survival under conditions of cellular stress, such as anoikis, which may promote progression of breast cancer.

Intermittent Hypoxia Induces Proteasome-Dependent Down-Regulation of Estrogen Receptor α in Human Breast Carcinoma

Purpose: Hypoxia may influence gene expression to promote malignancy, and acute hypoxia has been shown to transiently repress estrogen receptor (ER)-α expression in breast cell lines. However, the effect of intermittent hypoxia, which is likely more prevalent in breast cancers, remains to be determined. Experimental Design: ER-α expression was assessed by Western blot and immunohistochemistry in a selected cohort of 51 ER-α–positive breast carcinomas, in relation to markers of hypoxia. The effect of acute and intermittent hypoxia on ER-α expression was also determined in MCF7 and ZR-75 breast cell lines, together with the role of proteasome function with the proteasome inhibitor bortezomib. Results: Regional loss of ER-α expression occurs in breast tumors and is consistently present in hypoxic regions defined by the proximity of necrosis and induction of hypoxia-induced genes carbonic anhydrase IX (CA-IX) and glucose transporter 1 (Glut-1), in both in situ (n = 29; P < 0.0001) and invasive (n = 20; P = 0.0001) carcinomas. In MCF7 and ZR-75 cells, ER-α is transiently down-regulated by acute hypoxia and rapidly restored by reoxygenation. However, intermittent, acute hypoxia can cause a similar down-regulation of ER-α that is not attributable to decreased mRNA and persists in MCF7 cells despite reoxygenation for up to 14 days. This effect occurs with no change in cell viability but a corresponding reduction in growth response to estradiol. However, ER-α expression can be restored by bortezomib. Conclusions: Intermittent hypoxia can cause persistent changes in proteasome function that may contribute to reduced ER-α expression in breast tumors and consequently to diminished response and development of resistance to endocrine therapy.

Phospho-serine-118 estrogen receptor-α detection in human breast tumors in vivo

Purpose: To determine whether estrogen receptor (ER)-α specifically phosphorylated at Ser118 is detectable in multiple human breast cancer biopsy samples. To gain insight into possible roles for P-Ser118-ERα in human breast cancer in vivo. Experimental Design: A specific antibody for P-Ser118-ERα was validated for immunohistochemistry (IHC), and Western blot analysis confirmed IHC results. IHC was used to determine the relationship of P-Ser118-ERα to known prognostic markers and active mitogen-activated protein kinase (MAPK; erk1/2) expression. Results: P-Ser118-ERα was significantly correlated with the expression of total ER, determined by ligand binding assay (r = 0.442, P = 0.002), but not with progesterone receptor expression or nodal status. P-Ser118-ERα was inversely correlated with histological grade (r = −0.34, P = 0.023), reflecting a similar trend for total ER (r = −0.287, P = 0.056). Categorical contingency analysis confirmed that P-Ser118-ERα was more frequently associated with lower than higher grade breast tumors (P = 0.038). In addition P-Ser118-ERα was significantly associated with detection of active MAPK (Erk1/2; Spearman r = 0.649, P < 0.0001; Fisher’s exact test, P = 0.0004). Conclusions: P-Ser118-ERα detection is associated with a more differentiated phenotype and other markers of good prognosis in human breast cancer. P-Ser118-ERα is correlated with active MAPK in human breast tumor biopsies, suggesting the possibility that active MAPK either directly or indirectly has a role in the regulation of P-Ser118-ERα expression in vivo. These data provide evidence for a role of P-Ser118-ERα in human breast cancer in vivo.

Potential role of estrogen receptor α (ERα) phosphorylated at Serine118 in human breast cancer in vivo

Post-translational modifications of proteins are known to be important in protein activity and ERalpha is known to be phosphorylated at multiple sites within the protein. The exact function of site-specific phosphorylation in ERalpha is unknown, although several hypotheses have been developed using site-directed mutagenesis and cell culture models. Targeting the ERalpha at the level of such post-translational modification pathways would be a new and exciting approach to endocrine therapy in breast cancer, but adequate knowledge is lacking with regard to the relevance of site-specific phosphorylation in ERalpha in human breast cancer in vivo. Recently, antibodies to P-Serine(118)-ERalpha and P-Serine(167)-ERalpha, two major sites of phosphorylation in ERalpha, have become available and some in vivo data are now available to complement studies in cells in culture. However, the in vivo data are somewhat contradictory and limited by the small cohorts used and the lack of standard well-characterized reagents and protocols.

Expression analysis of the mouse S100A7/psoriasin gene in skin inflammation and mammary tumorigenesis

BackgroundThe human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis.MethodsOn the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7.ResultsAlthough mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene.ConclusionThese observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression.