Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER+ Breast Cancer Cells.

BACKGROUND/AIMS: Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-α positive (ER+) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, H19, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER+ breast epithelial cells. We hypothesized that H19 expression is also important for the proliferation and survival of ETR cells. METHODS: Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of H19 function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered H19 expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). RESULTS: Here we report that treating ETR cells with Tam or Fulvestrant increases H19 expression and that it's decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, H19 expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an H19-dependent manner in these cells. Lastly, we demonstrate that H19 regulates ERα expression at the transcript and protein levels in the ETR cells and that H19 protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and H19 expression in the ETR cells. CONCLUSION: In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, H19 expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.

ERß splice variant expression in four large cohorts of human breast cancer patient tumors.

Though the role of Estrogen Receptor (ER)α in breast cancer has been studied extensively, there is little consensus about the role of alternative ER isoform ERß in breast cancer biology. ERß has significant sequence homology to ERα but is located on a different chromosome and maintains both overlapping and unique functional attributes. Five variants exist, resulting from alternative splicing of the C-terminal region of ERß. The relevance of ERß variants in breast cancer outcomes and response to therapy is difficult to assess because of conflicting reports in the literature, likely due to variable methods used to assess ERß in patient tumors. Here, we quantitatively assess expression of ERß splice variants on over 2,000 breast cancer patient samples. Antibodies against ERß variants were validated for staining specificity in cell lines by siRNA knockdown of ESR2 and staining reproducibility on formalin-fixed paraffin-embedded tissue by quantitative immunofluorescence (QIF) using AQUA technology. We found antibodies against splice variants ERß1 and ERß5, but not ERß2/cx, which were sensitive, specific, and reproducible. QIF staining of validated antibodies showed both ERß1 and ERß5 QIF scores, which have a normal (bell shaped) distribution on most cohorts assessed, and their expression is significantly associated with each other. Extensive survival analyses show that ERß1 is not a prognostic or predictive biomarker for breast cancer. ERß5 appears to be a context-dependent marker of worse outcome in HER2-positive and triple-negative patients, suggesting an unknown biological function in the absence of ERα.

Claudin 1 expression in basal-like breast cancer is related to patient age.

BACKGROUND: Defects in tight junctions, gate-keepers of the integrity of the epidermal barrier function, are known to contribute to cancer development. As such, enhancing our understanding of how the expression of proteins involved in these junctions is regulated in cancer, remains a priority. Although the expression of one of these proteins, claudin 1, is down regulated in most invasive human breast cancers (HBC), we have recently shown that high levels of claudin 1, characterized tumors belonging to the very aggressive basal-like breast cancer (BLBC) subtype. In these tumors, the claudin 1 protein, usually localized in the cell membrane, is often mislocalized to the cytoplasm. METHODS: To examine the clinical relevance of this observation, we have generated and analyzed an invasive HBC tissue microarray consisting of 151 breast tumor samples; 79 of which presented a basal-like phenotype (i.e. ER-ve, PR-ve HER2-ve, CK5/6 or EGFR+ve). We also interrogated the outcome of claudin 1 knockdown in a human BLBC cell line, BT-20. RESULTS: Immunohistochemical analysis of this patient cohort revealed a significant association between high claudin 1 expression and BLBCs in women 55 years of age and older. Interestingly, no significant association was found between claudin 1 and nodal involvement, tumor grade or tumor size. Regression analysis however, showed a significant positive association between claudin 1 and claudin 4, even though claudin 4 did not significantly correlate with patient age. Claudin 1 knockdown in BT-20 cells resulted in decreased cell migration. It also significantly altered the expression of several genes involved in epithelial-mesenchymal-transition (EMT); in particular, SERPINE 1 (PAI1) and SSP1 (osteopontin), known to inhibit EMT and cancer cell migration. Conversely, genes known to maintain EMT through their interaction, SNAIL2, TCF4 and FOXC2 were significantly down regulated. CONCLUSIONS: The association of high claudin 1 protein levels observed in tumors derived from older women with BLBC, suggests that claudin 1 has the potential to serve as a marker which can identify a specific subgroup of patients within the BLBC subtype and thus, further contribute to the characterization of these ill-defined breast cancers. More importantly, our studies strongly suggest that claudin 1 directly participates in promoting breast cancer progression, possibly through the alteration of expression of EMT genes.

Increasing the relative expression of endogenous non-coding Steroid Receptor RNA Activator (SRA) in human breast cancer cells using modified oligonucleotides.

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Student's t-test, P < 0.003) higher SRA-intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA-intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.

Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.

We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) with stable-isotope-labeled standard peptides. In contrast with classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one nonphosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated sample and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the percent phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ERalpha and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites of biological importance. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ERalpha protein revealed that the PPQ-MRM method is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount.

Steroid Receptor RNA Activator Protein (SRAP): a potential new prognostic marker for estrogen receptor-positive/node-negative/younger breast cancer patients.

INTRODUCTION: The steroid receptor RNA activator is a functional RNA suspected to participate in the mechanisms underlying breast tumor progression. This RNA is also able to encode for a protein, Steroid Receptor RNA Activator Protein (SRAP), whose exact function remains to be determined. Our aim was to assess, in a large breast cancer cohort, whether levels of this protein could be associated with outcome or established clinical parameters. METHODS: Following antibody validation, SRAP expression was assessed by tissue-microarray (TMA) analysis of 372 breast tumors. Clinical follow-up and parameters such as steroid receptor and node status were available for all the corresponding cases. Immunohistochemical scores were independently determined by three investigators and averaged. Statistical analyses were performed using standard univariate and multivariate tests. RESULTS: SRAP levels were significantly (Mann-Whitney rank sum test, P < 0.05) higher in estrogen receptor-alpha positive (ER+, n = 271), in progesterone receptor positive (PR+, n = 257) and in older patients (age > 64 years, n = 182). When considering ER+ tumors, PR+ tumors, or younger patients (< or = 64 years), cases with high SRAP expression had a significantly (Mantel-Cox test, P < 0.05) worse breast cancer specific survival (BCSS) than those with low SRAP levels. SRAP also appeared as a very powerful indicator of poor prognostic for BCSS in the subset of ER+, node negative and young breast cancer patients (Cox regression analysis, n = 60, BCSS Hazard Ratio = 8.61, P < 0.006). CONCLUSIONS: Our data suggest that SRAP levels might provide additional information on potential risk of recurrence and negative outcome in a specific set of patients with otherwise good prognosis when considering only estrogen receptor and nodal status.

Reactive oxygen species induce phosphorylation of serine 118 and 167 on estrogen receptor alpha.

Estrogen receptor alpha (ERalpha) is a well-known target for signaling pathways originating from growth factor receptors. Reactive oxygen species (ROS) can induce activation of extracellular response kinase 1/2 (Erk1/2) and protein kinase B (Akt). Both kinases have been implicated in the phosphorylation of serine 118 and serine 167 on ERalpha, respectively. This activity may lead either to ligand-independent activation of ERalpha or down-regulation of ERalpha and may contribute to development of the resistance to endocrine therapy. Treatment of MCF-7 human breast cancer cells with glucose oxidase (GO, 0.1 un/ml) induced transient phosphorylation of serine 118 and serine 167. The increase in expression of p-ser118-ERalpha was 355 +/- 98% (mean +/- SD) and of p-ser167-ERalpha was 632 +/- 355%. These effects were enhanced in Her2 over-expressing MCF7 cells. ERalpha expression declined to 63 +/- 20% within the first 90 min of treatment and was below 10% 24 h later. ROS induced phosphorylation of ERalpha resulted in decreased expression of pS2 and progesterone receptor. Activation of Erk1/2 and Akt was transient with highest levels of Erk1/2 being 595 +/- 143% and p-Akt 311 +/- 125%. Inhibition of Erk1/2 by U0126 (10 microM) decreased p-ser118-ERalpha by 51.7 +/- 8.5% and decreased p-ser167-ERalpha by 41.9 +/- 16.9% whereas inhibition of Akt by LY294002 (20 microM) and wortmannin (500 nM) or by siRNA knock-down, had no effect on p-ser167-ERalpha expression. Our data show for the first time that ROS can induce post-translational modifications of ERalpha at serine 118 and serine 167, and may lead to ERalpha down-regulation in human breast cancer cells. Both the phosphorylation and consequent down-regulation of ERalpha may be a mechanism associated with development of endocrine therapy resistance.

Immunohistochemical validation of multiple phospho-specific epitopes for estrogen receptor alpha (ERalpha) in tissue microarrays of ERalpha positive human breast carcinomas.

Estrogen receptor alpha (ERalpha) activity is regulated by phosphorylation at several sites. Recently several antibodies specific for individual phosphorylated sites within ERalpha have became available. Such antibodies potentially provide invaluable tools to gain insight into the relevance in vivo of phosphorylated ERalpha in human breast tumors. However, validation of these antibodies for immunohistochemistry in particular is necessary in the first instance. In this study we have investigated the usefulness of several antibodies generated to specific phosphorylated sites within ERalpha for immunohistochemistry of formalin-fixed, paraffin-embedded human breast cancer biopsy samples. As well, these data demonstrate for the first time, the detection of multiple phosphorylated ERalpha forms in breast cancer (P-S104/106-ERalpha, P-S118-ERalpha, P-S167-ERalpha, P-S282-ERalpha, P-S294-ERalpha, P-T311-ERalpha, and P-S559-ERalpha) suggesting the possibility that profiling of phosphorylated ERalpha isoforms might be useful in selecting subgroups of breast cancer patients that would benefit from endocrine therapy.

Targeted production of proprotein convertase PC1 enhances mammary development and tumorigenesis in transgenic mice.

Elevated production of proprotein convertases (PCs), proteolytic enzymes that posttranslationally modify the biological activities of diverse groups of cellular proteins, is a common occurrence in human breast carcinomas. A transgenic mouse model was developed to gain insight into the significance of PC production in breast development and neoplasia. Mammary epithelium-specific and early expression of PC1 was targeted by the use of the mouse mammary tumor virus promoter/enhancer. Whole-mount examinations revealed that the mammary glands of 83-day-old virgin PC1 transgenic mice exhibited an accelerated lobuloalveolar development compared with that of age-matched wild-type mice (p < 0.001). This phenotypic change was accompanied by extensive alterations in gene expression assessed by gene expression microarray analyses. Pathway analysis of PC1-induced alterations in gene expression has revealed possible mechanism of action of PC1 in the mammary gland. PC1 expression alone, however, did not promote spontaneous mammary tumorigenesis in the transgenic mice. PC1 transgene expression resulted in a significantly higher incidence (p = 0.008) and accelerated growth (p = 0.023) of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas. The present study therefore shows that PC1 expression can promote normal and neoplastic mammary development and growth and suggests that proprotein convertases may be important etiological factors in human breast neoplasia.

Paracrine Crosstalk between Fibroblasts and ER+ Breast Cancer Cells Creates an IL1ß-Enriched Niche that Promotes Tumor Growth.

Breast cancer-induced activated fibroblasts support tumor progression. However, the role of normal fibroblasts in tumor progression remains controversial. In this study, we used modified patient-derived organoid cultures and demonstrate that constitutively secreted cytokines from normal breast fibroblasts initiate a paracrine signaling mechanism with estrogen receptor-positive (ER+) breast cancer cells, which results in the creation of an interleukin (IL)-1ß-enriched microenvironment. We found that this paracrine signaling mechanism is shared between normal and activated fibroblasts. Interestingly, we observed that in reconstructed tumor microenvironment containing autologous ER+ breast cancer cells, activated fibroblasts, and immune cells, tamoxifen is more effective in reducing tumor cell proliferation when this paracrine signaling is blocked. Our findings then suggest that ER+ tumor cells could create a growth-promoting environment without activating stromal fibroblasts and that in breast-conserving surgeries, normal fibroblasts could be a significant modulator of tumor recurrence by enhancing the proliferation of residual breast cancer cells in the tumor-adjacent breast tissue.

Influence of evolution in tumor biobanking on the interpretation of translational research.

PURPOSE: Translational cancer research increasingly relies on human tissue biospecimens and this has coincided with a shift in tissue biobanking approach. Newer biobanks (post year 2000) deploy standard operating procedures to reduce variability around biospecimen collection. Because current translational research is based on pre-2000 and post-2000 era biospecimens, we consider whether the collection era may influence gene expression data. DESIGN: We compared the range of breast tumor collection times from pre-2000 and post-2000 era biobanks and compared estrogen receptor (ER) protein expression with collection time. We then collected 10 breast tumor biospecimens under a standardized protocol and examined whether the expression of c-myc and ER was influenced by storage on ice < or = 24 hours. RESULTS: The range of collection times achieved at a pre-2000 versus post-2000 era biobank differed. Thirty-two percent of biospecimens were cryopreserved within 30 minutes at the pre-2000 era biobank versus 76% at the post-2000 era biobank. Collection time and ER protein level was inversely correlated (r = -0.19, P = 0.025; n = 137). We observed a wide range in initial c-myc and ER mRNA levels (50- versus 130-fold). Although mRNA levels of both genes declined with increasing collection time, the rate of change differed because c-myc was significantly reduced after 24 hours (mean reduction to 79% of initial) versus ER (94% of initial). CONCLUSION: The overall shift in biobanking around the year 2000 is reflected in the ranges of collection times associated with pre-2000 and post-2000 era biobanks. Because collection time can differentially alter gene expression, the biospecimen collection era should be considered in gene expression studies.

Estrogen receptor alpha negative breast cancer patients: estrogen receptor beta as a therapeutic target.

Clinical management of breast cancer is increasingly guided by assessment of tumor phenotypic parameters. One of these is estrogen receptor (ER) status, currently defined by ERalpha expression. However with the discovery of a second ER, ERbeta and its variant isoforms, the definition of ER status is potentially more complex. In breast tumors there are two ERbeta expression cohorts. One where ERbeta is co-expressed with ERalpha and the other expressing ERbeta alone. In the latter subgroup of currently defined ER negative patients ERbeta has the potential to be a therapeutic target. Characterization of the nature and role of ERbeta in ERalpha negative tumors is essentially unexplored but available data suggest that the role of ERbeta may be different when co-expressed with ERalpha and when expressed alone. This review summarizes available data and explores the possibility that ERbeta signaling may be a therapeutic target in these tumors. Evidence so far supports the idea that the role of ERbeta in breast cancer is different in ERalpha negative compared to ERalpha positive tumors. However, cohort size and numbers of independent studies are small to date, and more studies are needed with better standardization of antibodies and protocols. Also, the ability to determine the role of ERbeta in ERalpha negative breast cancer and therefore assess ERbeta signaling pathways as therapeutic targets would be greatly facilitated by identification of specific downstream markers of ERbeta activity in breast cancer.

Estrogen receptor-alpha 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.

Recurrent copy number alterations in young women with breast cancer.

Breast cancer diagnosis in young women has emerged as an independent prognostic factor with higher recurrence risk and death than their older counterparts. We aim to find recurrent somatic copy number alteration (CNA) regions identified from breast cancer microarray data and associate the CNA status of the genes harbored in the regions to the survival of young women with breast cancer. By using the interval graph-based algorithm we developed, and the CNA data consisting of a Discovery set with 130 young women and a Validation set with 125 young women, we identified 38 validated recurrent CNAs containing 39 protein encoding genes. CNA gain regions encompassing genes CAPN2, CDC73 and ASB13 are the top 3 with the highest occurring frequencies in both the Discovery and Validation dataset, while gene SGCZ ranked top for the recurrent CNA loss regions. The mutation status of 9 of the 39 genes shows significant associations with breast cancer specific survival. Interestingly, the expression level of 2 of the 9 genes, ASB13 and SGCZ, shows significant association with survival outcome. Patients with CNA mutations in both of these genes had a worse survival outcome when compared to patients without the gene mutations. The mutated CNA status in gene ASB13 was associated with a higher gene expression, which predicted patient survival outcome. Together, identification of the CNA events with prognostic significance in young women with breast cancer may be used in genomic-guided treatment.

A robust cell culture system for large scale feeder cell-free expansion of human breast epithelial progenitors.

BACKGROUND: Normal human breast epithelial cells are maintained by the proliferation and differentiation of different human breast epithelial progenitors (HBEPs). However, these progenitor subsets can only be obtained at low frequencies, limiting their further characterization. Recently, it was reported that HBEPs can be minimally expanded in Matrigel cocultures with stromal feeder cells. However, variability of generating healthy feeder cells significantly impacts the effective expansion of HBEPs. METHODS: Here, we report a robust feeder cell-free culture system for large-scale expansion of HBEPs in two-dimensional cultures. RESULTS: Using this cell culture system HBEPs can be exponentially expanded as bulk cultures. Moreover, purified HBEP subtypes can also be separately expanded using our cell culture system. The expanded HBEPs retain their undifferentiated phenotype and form distinct epithelial colonies in colony forming cell assays. CONCLUSIONS: The availability of a culture system enabling the large-scale expansion of HBEPs facilitates their application to screening platforms and other cell-based assays.

Breast Cancers Activate Stromal Fibroblast-Induced Suppression of Progenitors in Adjacent Normal Tissue.

Human breast cancer cells are known to activate adjacent "normal-like" cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor ß. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.

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.

Is oestrogen receptor-beta a predictor of endocrine therapy responsiveness in human breast cancer?

The role of oestrogen receptor (ER) beta in human breast cancer remains unclear. However, it is now apparent that when considering ER beta in human breast cancer it is important to recognise two ER beta expressing groups, one in which ER beta is co-expressed with ER alpha and the other where ERbeta is expressed alone. Emerging data support different functions between ER beta when it is expressed alone and when it is co-expressed with ER alpha. With regard to the latter group (ER alpha +/ER beta +), there are now 9 out of 10 retrospective clinical outcome studies published, that support the hypothesis that increased expression of ER beta is associated with increased likelihood of response to endocrine therapy. The data strongly support undertaking prospective studies to determine if the addition of ERbeta to ER alpha is clinically beneficial and whether to include both ER beta and ER alpha when establishing clinically relevant cut-offs for defining ER status.

Targeting the EGFR pathway for cancer therapy.

Clinical studies have shown that HER-2/Neu is over-expressed in up to one-third of patients with a variety of cancers, including B-cell acute lymphoblastic leukemia (B-ALL), breast cancer and lung cancer, and that these patients are frequently resistant to conventional chemo-therapies. Additionally, in most patients with multiple myeloma, the malignant cells over-express a number of epidermal growth factor receptors (EGFR)s and their ligands, HB-EGF and amphiregulin, thus this growth-factor family may be an important aspect in the patho-biology of this disease. These and other, related findings have provided the rationale for the targeting of the components of the EGFR signaling pathways for cancer therapy. Below we discuss various aspects of EGFR-targeted therapies mainly in hematologic malignancies, lung cancer and breast cancer. Beside novel therapeutic approaches, we also discuss specific side effects associated with the therapeutic inhibition of components of the EGFR-pathways. Alongside small inhibitors, such as Lapatinib (Tykerb, GW572016), Gefitinib (Iressa, ZD1839), and Erlotinib (Tarceva, OSI-774), a significant part of the review is also dedicated to therapeutic antibodies (e.g.: Trastuzumab/Herceptin, Pertuzumab/Omnitarg/rhuMab-2C4, Cetuximab/Erbitux/IMC-C225, Panitumumab/Abenix/ABX-EGF, and also ZD6474). In addition, we summarize, both current therapy development driven by antibody-based targeting of the EGFR-dependent signaling pathways, and furthermore, we provide a background on the history and the development of therapeutic antibodies.

Prohibitin: a potential target for new therapeutics.

Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function and protection against senescence. There is considerable controversy concerning the function of nuclear-localized PHB. PHB has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. PHB might also function as a cell-surface receptor for an as-yet unidentified ligand. Cell-associated PHB in the gastrointestinal tract has been implicated in protection against infection and inflammation and the induction of apoptosis in other tissues. The diverse array of functions of PHB, together with the emerging evidence that its function can be modulated specifically in certain tissues, suggest that targeting PHB would be a useful therapeutic approach for the treatment of variety of disease states, including inflammation, obesity and cancer.