Deborah A. Zajchowski

Protein fold analysis of the B30.2-like domain.

The B30.2‐like domain occurs in some members of a diverse and growing family of proteins containing zinc‐binding B‐box motifs, whose functions include regulation of cell growth and differentiation. The B30.2‐like domain is also found in proteins without the zinc‐binding motifs, such as butyrophilin (a transmembrane glycoprotein) and stonustoxin (a secreted cytolytic toxin). Currently, the function for the B30.2‐like domain is not clear and the structure of a protein containing this domain has not been solved. The secondary structure prediction methods indicate that the B30.2‐like domain consists of fifteen or fewer β‐strands. Fold recognition methods identified different structural topologies for the B30.2‐like domains. Secondary structure prediction, deletion and lack of local sequence identity at the C‐terminal region for certain members of the family, and packing of known core structures suggest that a structure containing two beta domains is the most probable of these folds. The most C‐terminal sequence motif predicted to be a β‐strand in all B30.2‐like domains is a potential subdomain boundary based on the sequence‐structure alignments. Models of the B30.2‐like domains were built based on immunoglobulin‐like folds identified by the fold recognition methods to evaluate the possibility of the B30.2 domain adopting known folds and infer putative functional sites. The SPRY domain has been identified as a subdomain within the B30.2‐like domain. If the B30.2‐like domain is a subclass of the SPRY domain family, then this analysis would suggest that the SPRY domains are members of the immunoglobulin superfamily. Proteins 1999;35:235–249.

Different estrogen receptor structural domains are required for estrogen- and tamoxifen-dependent anti-proliferative activity in human mammary epithelial cells expressing an exogenous estrogen receptor

Estrogen (E) inhibits the growth of both non-tumorigenic, immortal human mammary epithelial cells (HMEC) and breast cancer cells which stably express exogenous estrogen receptors (ER). The anti-estrogenic compounds 4-hydroxy-tamoxifen (HT) and ICI 164384 (ICI) have different effects on the growth of the ER-transfectants. HT is a potent growth inhibitor, while ICI has no effect by itself but is able to block the anti-proliferative effects of E and HT. In order to elucidate the mechanism by which E or HT-bound ER inhibit cell growth, we have evaluated the effects of these compounds on the growth of HMEC stably expressing ER with mutations or deletions in the N-terminal A/B domain, the DNA-binding domain (DBD), and the C-terminal ligand-binding domain. These studies revealed that E and HT require different structural domains of the ER for their anti-proliferative activities. The N-terminal A/B domain is required for HT-, but not E-dependent growth inhibition. The DNA-binding domain of the ER is not essential for HT-mediated anti-proliferative effects, but is important for E-dependent activity. The effect of ER mutations on the ligand-inducible expression of the endogenous progesterone receptor (PR) and pS2 genes was also evaluated. Neither gene was induced in the cells containing the ER mutated in the DBD, even though cell growth was inhibited. These results suggest that E and HT use different pathways to elicit their anti-proliferative effects and that this occurs via modulation of genes that are controlled by mechanisms different from those important for activation of the PR and pS2 genes.

Abstract 5077: Molecular profiling in recurrent ovarian cancer patients: Considerations for the design of clinical studies to validate profiling for therapy selection

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Therapies for recurrent, advanced stage ovarian cancer are often empirically selected and associated with similarly poor response rates and short progression-free survivals. Our ultimate goal is to improve patient outcomes by enabling a more rational selection of chemotherapies based upon individual tumor molecular profiles. To identify molecular markers to include in such profiling, we performed a literature search for evidence supporting the association between chemotherapy response markers and in vivo responses to drugs currently employed in ovarian cancer treatment. We found multiple reports that demonstrated a correlation between expression levels of RRM1, TOPO1, TOPO2, ERCC1, TS, MGMT, SPARC, MRP1, MDR1, or BCRP and clinical responses to gemcitabine, topoisomerase 1 and 2 inhibitors, platinum, fluoropyrimidines, temozolamide, nab-paclitaxel, taxanes, and other drugs. However, few studies addressed the correlation between these biomarkers and therapy responses for ovarian cancer patients. We therefore measured expression of these markers in formalin-fixed, paraffin-embedded tumor blocks (obtained following informed patient consent) by immunohistochemical analyses at CLIA-certified laboratories to establish expression characteristics of these proteins in epithelial ovarian carcinomas. Protein expression data were recorded as histoscores (% tumor cells stained x intensity). We profiled fifty-nine advanced stage ovarian, peritoneal, or fallopian tube cancers of which 42 were of either serous papillary histology or adenocarcinomas (25 primary and 17 recurrent serous ovarian tumor specimens). The first-line response to platinum-taxane therapy for most of these patients (23 sensitive, 7 resistant, 2 refractory) and BRCA1 and 2 mutation status (7 BRCA1 mutation carriers) were also known. Most of the marker proteins were heterogeneously expressed both within the tumor sample and across the cohort of patient tumors. The exceptions were TOPO2 and MDR1, which were poorly detected in most tumors. Interestingly, primary specimens had significantly lower expression levels of MDR1 and higher expression of RRM1 and TOPO1 than recurrent lesions underscoring the potential importance of determining the molecular profile of recurrent specimens. Six of the seven BRCA1 mutation carriers were sensitive to platinum-taxane treatment, but no significant association with platinum responsiveness was observed for expression of any of the measured proteins. These data provide a basis for establishing expression cutoff points for each marker to test hypotheses that correlate individual or combined marker expression profiles with responses to chemotherapy in prospective analyses. Profiling of recurrent specimens is recommended for future clinical trials to evaluate this approach to individualize cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5077. doi:10.1158/1538-7445.AM2011-5077

Abstract A74: Estrogen receptor α‐regulated growth and transcription is dependent on X‐box binding protein‐1 in breast cancer cells

This study was designed to investigate the impact of X‐Box Binding protein 1 (Xbp1) mRNA knockdown on the estrogen‐responsive growth and gene expression in an estrogen receptor α (ERα) positive breast cancer cell line model. Xbp1 is a basic leucine zipper‐containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by unfolded or misfolded proteins in the endoplasmic reticulum. MCF7 cells, a model of ERα positive breast cancer, express high levels of Xbp1 and are dependent on estrogen for growth and ERα regulated transcription. Previous studies have revealed that further overexpression of Xbp1 in ERα positive breast cancer cells leads to estrogen independent ERα regulated transcription and anti‐estrogen resistance, suggesting that Xbp1 expression influences ERα regulated transcription. Unknown is whether ERα requires Xbp1 as a cofactor for transcription. Here we show that Xbp1 mRNA knockdown by specific siRNAs prevents estrogen responsive growth of MCF7 cells as well as ERα regulated gene expression, while non‐specific control siRNAs had little effect. These results, together with the high expression of Xbp1 seen in ERα positive breast cancers, but not in normal mammary ductal cells, suggests that high levels of Xbp1 enable ERα tumor cells to use estrogen as a growth factor. Thus, high levels of Xbp1 expression could represent an early transformation step leading to ERα positive breast cancer, and could explain why normal ERα positive breast ductal cells have not been established as cell lines. Citation Information: Cancer Res 2009;69(23 Suppl):A74.