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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ren, Z P
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, F
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, J
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.1998In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 17, no 14Article in journal (Refereed)
    Abstract [en]

    Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

  • 2. Alvarez-Baron, Claudia P
    et al.
    Jonsson, Philip
    Thomas, Christoforos
    Dryer, Stuart E
    Williams, Cecilia
    University of Houston.
    The two-pore domain potassium channel KCNK5: induction by estrogen receptor alpha and role in proliferation of breast cancer cells.2011In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 25, no 8, p. 1326-36Article in journal (Refereed)
    Abstract [en]

    The growth of many human breast tumors requires the proliferative effect of estrogen acting via the estrogen receptor α (ERα). ERα signaling is therefore a clinically important target for breast cancer prevention and therapeutics. Although extensively studied, the mechanism by which ERα promotes proliferation remains to be fully established. We observed an up-regulation of transcript encoding the pH-sensitive two-pore domain potassium channel KCNK5 in a screen for genes stimulated by 17β-estradiol (E2) in the ERα(+) breast cancer cell lines MCF-7 and T47D. KCNK5 mRNA increased starting 1 h after the onset of E2 treatment, and protein levels followed after 12 h. Estrogen-responsive elements are found in the enhancer region of KCNK5, and chromatin immunoprecipitation assays revealed binding of ERα to the KCNK5 enhancer in E2-treated MCF-7 cells. Cells treated with E2 also showed increases in the amplitude of pH-sensitive potassium currents, as assessed by whole-cell recordings. These currents are blocked by clofilium. Although confocal microscopy suggested that most of the channels are located in intracellular compartments, the increase in macroscopic currents suggests that E2 treatment increases the number of active channels at the cell surface. Application of small interfering RNA specific for KCNK5 decreased pH-sensitive potassium currents and also reduced the estrogen-induced proliferation of T47D cells. We conclude that E2 induces the expression of KCNK5 via ERα(+) in breast cancer cells, and this channel plays a role in regulating proliferation in these cell lines. KCNK5 may therefore represent a useful target for treatment, for example, of tamoxifen-resistant breast cancer.

  • 3. Andersson, Sandra
    et al.
    Sundberg, Marten
    Pristovsek, Nusa
    Ibrahim, Ahmed
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Natl Res Ctr, Egypt.
    Jonsson, Philip
    Katona, Borbala
    Clausson, Carl-Magnus
    Zieba, Agata
    Ramstrom, Margareta
    Soderberg, Ola
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Univ Houston, USA; Karolinska Inst, Sweden.
    Asplund, Anna
    Insufficient antibody validation challenges oestrogen receptor beta research2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 15840Article in journal (Refereed)
    Abstract [en]

    The discovery of oestrogen receptor beta (ER beta/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER alpha (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER beta antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER beta in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER beta protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

  • 4. Aydoğdu, Eylem
    et al.
    Katchy, Anne
    Tsouko, Efrosini
    Lin, Chin-Yo
    Haldosén, Lars-Arne
    Helguero, Luisa
    Williams, Cecilia
    University of Houston.
    MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer.2012In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 33, no 8, p. 1502-11Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

  • 5.
    Berg, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hedrum, A
    Holmberg, A
    Pontén, F
    Uhlén, M
    Lundeberg, J
    Direct solid-phase sequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides.1995In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 41, no 10, p. 1461-6Article in journal (Refereed)
    Abstract [en]

    Among the candidate cancer-prognostic genes is the p53 tumor suppressor gene, which, when mutated, plays an important role in the development of many types of cancers. To facilitate robust large-scale DNA analysis of microdissected tumor biopsies, we describe a multiplex/nested PCR approach for a simultaneous outer amplification of exons 4-9 of the human p53 gene with parallel amplification of the HLA-DQB1 locus, involving a total of 14 primers. This approach reduces the required number of cells for analysis and avoids any variation in the amplifications of the individual p53 exons during the common outer amplification step. The HLA sequencing allows sample identification because the DQB1 locus is highly polymorphic and is thereby patient-specific. The p53 and HLA amplicons are analyzed by solid-phase sequencing in a semiautomated format. To improve the DNA sequence quality, we used 2'-deoxyribonucleoside 5'-O-1-thiotriphosphates in the sequencing reactions.

  • 6. Berger, Ashton C
    et al.
    Korkut, Anil
    Kanchi, Rupa S
    Hegde, Apurva M
    Lenoir, Walter
    Liu, Wenbin
    Liu, Yuexin
    Fan, Huihui
    Shen, Hui
    Ravikumar, Visweswaran
    Rao, Arvind
    Schultz, Andre
    Li, Xubin
    Sumazin, Pavel
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Mestdagh, Pieter
    Gunaratne, Preethi H
    Yau, Christina
    Bowlby, Reanne
    Robertson, A Gordon
    Tiezzi, Daniel G
    Wang, Chen
    Cherniack, Andrew D
    Godwin, Andrew K
    Kuderer, Nicole M
    Rader, Janet S
    Zuna, Rosemary E
    Sood, Anil K
    Lazar, Alexander J
    Ojesina, Akinyemi I
    Adebamowo, Clement
    Adebamowo, Sally N
    Baggerly, Keith A
    Chen, Ting-Wen
    Chiu, Hua-Sheng
    Lefever, Steve
    Liu, Liang
    MacKenzie, Karen
    Orsulic, Sandra
    Roszik, Jason
    Shelley, Carl Simon
    Song, Qianqian
    Vellano, Christopher P
    Wentzensen, Nicolas
    Weinstein, John N
    Mills, Gordon B
    Levine, Douglas A
    Akbani, Rehan
    A Comprehensive Pan-Cancer Molecular Study of Gynecologic and Breast Cancers.2018In: Cancer Cell, ISSN 1535-6108, E-ISSN 1878-3686, Vol. 33, no 4, p. 690-705.e9, article id S1535-6108(18)30119-3Article in journal (Refereed)
    Abstract [en]

    We analyzed molecular data on 2,579 tumors from The Cancer Genome Atlas (TCGA) of four gynecological types plus breast. Our aims were to identify shared and unique molecular features, clinically significant subtypes, and potential therapeutic targets. We found 61 somatic copy-number alterations (SCNAs) and 46 significantly mutated genes (SMGs). Eleven SCNAs and 11 SMGs had not been identified in previous TCGA studies of the individual tumor types. We found functionally significant estrogen receptor-regulated long non-coding RNAs (lncRNAs) and gene/lncRNA interaction networks. Pathway analysis identified subtypes with high leukocyte infiltration, raising potential implications for immunotherapy. Using 16 key molecular features, we identified five prognostic subtypes and developed a decision tree that classified patients into the subtypes based on just six features that are assessable in clinical laboratories.

  • 7. Bondesson, Maria
    et al.
    Hao, Ruixin
    Lin, Chin-Yo
    Williams, Cecilia
    University of Houston, United States.
    Gustafsson, Jan-Åke
    Estrogen receptor signaling during vertebrate development2015In: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, ISSN 1874-9399, E-ISSN 1876-4320, Vol. 1849, no 2, p. 142-151Article, review/survey (Refereed)
    Abstract [en]

    Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans.

  • 8. Dahlman-Wright, Karin
    et al.
    Qiao, Yichun
    Jonsson, Philip
    Gustafsson, Jan-Åke
    Williams, Cecilia
    University of Houston.
    Zhao, Chunyan
    Interplay between AP-1 and estrogen receptor α in regulating gene expression and proliferation networks in breast cancer cells2012In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 33, no 9, p. 1684-91Article in journal (Refereed)
    Abstract [en]

    Estrogen receptor α (ERα) is a ligand-dependent transcription factor that plays an important role in breast cancer. Estrogen-dependent gene regulation by ERα can be mediated by interaction with other DNA-binding proteins, such as activator protein-1 (AP-1). The nature of such interactions in mediating the estrogen response in breast cancer cells remains unclear. Here we show that knockdown of c-Fos, a component of the transcription factor AP-1, attenuates the expression of 37% of all estrogen-regulated genes, suggesting that c-Fos is a fundamental factor for ERα-mediated transcription. Additionally, knockdown of c-Fos affected the expression of a number of genes that were not regulated by estrogen. Pathway analysis reveals that silencing of c-Fos downregulates an E2F1-dependent proproliferative gene network. Thus, modulation of the E2F1 pathway by c-Fos represents a novel mechanism by which c-Fos enhances breast cancer cell proliferation. Furthermore, we show that c-Fos and ERα can cooperate in regulating E2F1 gene expression by binding to regulatory elements in the E2F1 promoter. To start to dissect the molecular details of the cross talk between AP-1 and estrogen signaling, we identify a novel ERα/AP-1 target, PKIB (cAMP-dependent protein kinase inhibitor-β), which is overexpressed in ERα-positive breast cancer tissues. Knockdown of PKIB results in robust growth suppression of breast cancer cells. Collectively, our findings support c-Fos as a critical factor that governs estrogen-dependent gene expression and breast cancer proliferation programs.

  • 9.
    Danielsson, Frida
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fasterius, Erik
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Sullivan, Devin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hases, Linnea
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute, Huddinge, Sweden.
    Sanli, Kemal
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Huss, M.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Hørsholm, Denmark.
    Williams, Cecilia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute, Huddinge, Sweden.
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Transcriptome profiling of the interconnection of pathways involved in malignant transformation and response to hypoxia2018In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 9, no 28, p. 19730-19744Article in journal (Refereed)
    Abstract [en]

    In tumor tissues, hypoxia is a commonly observed feature resulting from rapidly proliferating cancer cells outgrowing their surrounding vasculature network. Transformed cancer cells are known to exhibit phenotypic alterations, enabling continuous proliferation despite a limited oxygen supply. The four-step isogenic BJ cell model enables studies of defined steps of tumorigenesis: the normal, immortalized, transformed, and metastasizing stages. By transcriptome profiling under atmospheric and moderate hypoxic (3% O2) conditions, we observed that despite being highly similar, the four cell lines of the BJ model responded strikingly different to hypoxia. Besides corroborating many of the known responses to hypoxia, we demonstrate that the transcriptome adaptation to moderate hypoxia resembles the process of malignant transformation. The transformed cells displayed a distinct capability of metabolic switching, reflected in reversed gene expression patterns for several genes involved in oxidative phosphorylation and glycolytic pathways. By profiling the stage-specific responses to hypoxia, we identified ASS1 as a potential prognostic marker in hypoxic tumors. This study demonstrates the usefulness of the BJ cell model for highlighting the interconnection of pathways involved in malignant transformation and hypoxic response.

  • 10. Dey, Prasenjit
    et al.
    Jonsson, Philip
    Hartman, Johan
    Williams, Cecilia
    Ström, Anders
    Gustafsson, Jan-Åke
    Estrogen receptors β1 and β2 have opposing roles in regulating proliferation and bone metastasis genes in the prostate cancer cell line PC32012In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 26, no 12, p. 1991-2003Article in journal (Refereed)
    Abstract [en]

    The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.

  • 11. Dey, Prasenjit
    et al.
    Velazquez-Villegas, Laura A.
    Faria, Michelle
    Turner, Anthony
    Jonsson, Philp
    Webb, Paul
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. University of Houston, United States.
    Gustafsson, Jan-Åke
    Ström, Anders M.
    Estrogen Receptor beta 2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1 alpha in Prostate Cancer2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 5, article id e0128239Article in journal (Refereed)
    Abstract [en]

    The estrogen receptor (ER) beta variant ER beta 2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ER beta 2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ER beta 2 interacts with and stabilizes HIF-1 alpha protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1 alpha is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ER beta 2 interacts with HIF-1 alpha and piggybacks to the HIF-1 alpha response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ER beta 2 is mediated by HIF-1 alpha and that targeting of this ER beta 2 -HIF-1 alpha interaction may be a strategy to treat prostate cancer.

  • 12. Dória, M Luisa
    et al.
    Ribeiro, Ana S
    Wang, Jun
    Cotrim, Cândida Z
    Domingues, Pedro
    Williams, Cecilia
    University of Houston.
    Domingues, M Rosário
    Helguero, Luisa A
    Fatty acid and phospholipid biosynthetic pathways are regulated throughout mammary epithelial cell differentiation and correlate to breast cancer survival2014In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 10, p. 4247-64Article in journal (Refereed)
    Abstract [en]

    This work combined gene and protein expression, gas chromatography-flame ionization detector, and hydrophilic interaction liquid chromatography-tandem mass spectrometry to compare lipid metabolism changes in undifferentiated/proliferating vs. functionally differentiated mammary epithelial cells (MECs) and to study their correlation to breast cancer survival. Sixty-eight genes involved in lipid metabolism were changed in MEC differentiation. Differentiated cells showed induction of Elovl6 (2-fold), Scd1 (4-fold), and Fads2 (2-fold), which correlated with increased levels of C16:1 n-7 and C18:1 n-9 (1.5-fold), C20:3 n-6 (2.5-fold), and C20:4 n-6 (6-fold) fatty acids (FAs) and more phospholipids (PLs) containing these species. Further, increased expression (2- to 3-fold) of genes in phosphatidylethanolamine (PE) de novo biosynthesis resulted in a 20% PE increase. Proliferating/undifferentiated cells showed higher C16:0 (1.7-fold) and C18:2 n-6 (4.2-fold) levels and more PLs containing C16:0 FAs [PC(16:0/16:1), PG(16:0/18:2), PG(16:0/18:1), and SM(16:0/18:0)]. Kaplan-Meier analysis of data from 3455 patients with breast cancer disclosed a positive correlation for 59% of genes expressed in differentiated MECs with better survival. PE biosynthesis and FA oxidation correlated with better prognosis in patients with breast cancer, including the basal-like subtype. Therefore, genes involved in mammary gland FA and PL metabolism and their resulting molecular species reflect the cellular proliferative ability and differentiation state and deserve further studies as potential markers of breast cancer progression

  • 13. Edvardsson, Karin
    et al.
    Nguyen-Vu, Trang
    Kalasekar, Sharanya M
    Pontén, Fredrik
    Gustafsson, Jan-Åke
    Williams, Cecilia
    University of Houston.
    Estrogen receptor β expression induces changes in the microRNA pool in human colon cancer cells2013In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 34, no 7, p. 1431-41Article in journal (Refereed)
    Abstract [en]

    There is epidemiological, animal and in vitro evidence that estrogen receptor β (ERβ) can mediate protective effects against colon cancer, but the mechanism is not completely understood. Previous research has indicated critical pathways whereby ERβ acts in an antitumorigenic fashion. In this study, we investigate ERβ's impact on the microRNA (miRNA) pool in colon cancer cells using large-scale genomic approaches, bioinformatics and focused functional studies. We detect and confirm 27 miRNAs to be significantly changed following ERβ expression in SW480 colon cancer cells. Among these, the oncogenic miR-17-92 cluster and miR-200a/b are strongly downregulated. Using target prediction and anticorrelation to gene expression data followed by focused mechanistic studies, we demonstrate that repression of miR-17 is a secondary event following ERβ's downregulatory effect on MYC. We show that re-introduction of miR-17 can reverse the antiproliferative effects of ERβ. The repression of miR-17 also influences cell death upon DNA damage and mediates regulation of NCOA3 (SRC-3) and CLU in colon cancer cells. We further determine that the downregulation of miR-200a/b mediates increased ZEB1 while decreasing E-cadherin levels in ERβ-expressing colon cancer cells. Changes in these genes correspond to significant alterations in morphology and migration. Our work contributes novel data of ERβ and miRNA in the colon. Elucidating the mechanism of ERβ and biomarkers of its activity has significant potential to impact colon cancer prevention and treatment.

  • 14. Edvardsson, Karin
    et al.
    Ström, Anders
    Jonsson, Philip
    Gustafsson, Jan-Åke
    Williams, Cecilia
    University of Houston.
    Estrogen receptor β induces antiinflammatory and antitumorigenic networks in colon cancer cells.2011In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 25, no 6, p. 969-79Article in journal (Refereed)
    Abstract [en]

    Several studies suggest estrogen to be protective against the development of colon cancer. Estrogen receptor β (ERβ) is the predominant estrogen receptor expressed in colorectal epithelium and is the main candidate to mediate the protective effects. We have previously shown that expression of ERβ reduces growth of colorectal cancer in xenografts. Little is known of the actions of ERβ and its effect on gene transcription in colon cancers. To dissect the processes that ERβ mediates and to investigate cell-specific mechanisms, we reexpressed ERβ in three colorectal cancer cell lines (SW480, HT29, and HCT-116) and conducted genome-wide expression studies in combination with gene-pathway analyses and cross-correlation to ERβ-chromatin-binding sites. Although induced gene regulation was cell specific, overrepresentation analysis of functional classes indicated that the same biological themes, including apoptosis, cell differentiation, and regulation of the cell cycle, were affected in all three cell lines. Novel findings include a strong ERβ-mediated down-regulation of IL-6 and downstream networks with significant implications for inflammatory mechanisms involved in colon carcinogenesis. We also discovered cross talk between the suggested nuclear receptor coregulator PROX1 and ERβ, demonstrating that ERβ both regulates and shares target genes with PROX1. The influence of ERβ on apoptosis was further explored using functional studies, which suggested an increased DNA-repair capacity. We conclude that reexpression of ERβ induces transcriptome changes that, through several parallel pathways, converge into antitumorigenic capabilities in all three cell lines. We propose that enhancing ERβ action has potential as a novel therapeutic approach for prevention and/or treatment of colon cancer.

  • 15. Ehrlund, Anna
    et al.
    Jonsson, Philip
    Vedin, Lise-Lotte
    Williams, Cecilia
    University of Houston.
    Gustafsson, Jan-Åke
    Treuter, Eckardt
    Knockdown of SF-1 and RNF31 affects components of steroidogenesis, TGFβ, and Wnt/β-catenin signaling in adrenocortical carcinoma cells.2012In: PloS one, ISSN 1932-6203, Vol. 7, no 3, p. e32080-Article in journal (Refereed)
    Abstract [en]

    The orphan nuclear receptor Steroidogenic Factor-1 (SF-1, NR5A1) is a critical regulator of development and homeostasis of the adrenal cortex and gonads. We recently showed that a complex containing E3 ubiquitin ligase RNF31 and the known SF-1 corepressor DAX-1 (NR0B1) interacts with SF-1 on target promoters and represses transcription of steroidogenic acute regulatory protein (StAR) and aromatase (CYP19) genes. To further evaluate the role of SF-1 in the adrenal cortex and the involvement of RNF31 in SF-1-dependent pathways, we performed genome-wide gene-expression analysis of adrenocortical NCI-H295R cells where SF-1 or RNF31 had been knocked down using RNA interference. We find RNF31 to be deeply connected to cholesterol metabolism and steroid hormone synthesis, strengthening its role as an SF-1 coregulator. We also find intriguing evidence of negative crosstalk between SF-1 and both transforming growth factor (TGF) β and Wnt/β-catenin signaling. This crosstalk could be of importance for adrenogonadal development, maintenance of adrenocortical progenitor cells and the development of adrenocortical carcinoma. Finally, the SF-1 gene profile can be used to distinguish malignant from benign adrenocortical tumors, a finding that implicates SF-1 in the development of malignant adrenocortical carcinoma.

  • 16. Hartman, Johan
    et al.
    Edvardsson, Karin
    Lindberg, Karolina
    Zhao, Chunyan
    Williams, Cecilia
    Karolinska Institutet.
    Ström, Anders
    Gustafsson, Jan-Åke
    Tumor repressive functions of estrogen receptor beta in SW480 colon cancer cells2009In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 69, no 15, p. 6100-6Article in journal (Refereed)
    Abstract [en]

    Estrogen receptor beta (ERbeta) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERbeta expression is reduced in colorectal cancer. Our hypothesis is that ERbeta inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERbeta in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERbeta expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERbeta resulted in inhibition of proliferation and G(1) phase cell cycle arrest and this effect was dependent on a functional DNA-binding region. c-Myc is overexpressed in an overwhelming majority of colorectal tumors. By Western blot and real-time PCR, we found c-Myc to be down-regulated in the ERbeta-expressing cells. Furthermore, the c-Myc target gene p21((Waf1/Cip1)) was induced and Cdc25A was reduced by ERbeta at the transcriptional level. The second cdk2-inhibitor, p27(Kip1), was induced by ERbeta, but this regulation occurred at the posttranscriptional level, probably through ERbeta-mediated repression of the F-box protein p45(Skp2). Expression of the ERbeta-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells +/- ERbeta transplanted into severe combined immunodeficient/beige mice; after three weeks of ERbeta-expression, a 70% reduction of tumor volume was seen. Our results show that ERbeta inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERbeta-mediated inhibition of cell-cycle pathways. Furthermore, this ERbeta-mediated cell cycle repression is dependent on functional ERE binding.

  • 17. Jonsson, Philip
    et al.
    Coarfa, Cristian
    Mesmar, Fahmi
    Raz, Tal
    Rajapakshe, Kimal
    Thompson, John F
    Gunaratne, Preethi H
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single-Molecule Sequencing Reveals Estrogen-Regulated Clinically Relevant lncRNAs in Breast Cancer2015In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 29, no 11Article in journal (Refereed)
    Abstract [en]

    Estrogen receptor (ER)α-positive tumors are commonly treated with ERα antagonists or inhibitors of estrogen synthesis, but most tumors develop resistance, and we need to better understand the pathways that underlie the proliferative and tumorigenic role of this estrogen-activated transcription factor. We here present the first single-molecule sequencing of the estradiol-induced ERα transcriptome in the luminal A-type human breast cancer cell lines MCF7 and T47D. Sequencing libraries were prepared from the polyadenylated RNA fraction after 8 hours of estrogen or vehicle treatment. Single-molecule sequencing was carried out in biological and technical replicates and differentially expressed genes were defined and analyzed for enriched processes. Correlation analysis with clinical expression and survival were performed, and follow-up experiments carried out using time series, chromatin immunoprecipitation and quantitative real-time PCR. We uncovered that ERα in addition to regulating approximately 2000 protein-coding genes, also regulated up to 1000 long noncoding RNAs (lncRNAs). Most of these were up-regulated, and 178 lncRNAs were regulated in both cell lines. We demonstrate that Long Intergenic Non-protein Coding RNA 1016 (LINC01016) and LINC00160 are direct transcriptional targets of ERα, correlate with ERα expression in clinical samples, and show prognostic significance in relation to breast cancer survival. We show that silencing of LINC00160 results in reduced proliferation, demonstrating that lncRNA expression have functional consequences. Our findings suggest that ERα regulation of lncRNAs is clinically relevant and that their functions and potential use as biomarkers for endocrine response are important to explore.

  • 18. Jonsson, Philip
    et al.
    Katchy, Anne
    Williams, Cecilia
    Support of a bi-faceted role of estrogen receptor β (ERβ) in ERα-positive breast cancer cells.2014In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 21, no 2, p. 143-160Article in journal (Refereed)
    Abstract [en]

    The expression of estrogen receptor α (ERα) in breast cancer identifies patients most likely to respond to endocrine treatment. The second ER, ERβ, is also expressed in breast tumors, but its function and therapeutic potential need further study. Although in vitro studies have established that ERβ opposes transcriptional and proliferative functions of ERα, several clinical studies report its correlation with proliferative markers and poorer prognosis. The data demonstrate that ERβ opposes ERα are primarily based on transient expression of ERβ. Here, we explored the functions of constitutively expressed ERβ in ERα-positive breast cancer lines MCF7 and T47D. We found that ERβ, under these conditions heterodimerized with ERα in the presence and absence of 17β-estradiol, and induced genome-wide transcriptional changes. Widespread anti-ERα signaling was, however, not observed and ERβ was not antiproliferative. Tamoxifen antagonized proliferation and ER-mediated gene regulation both in the presence and absence of ERβ. In conclusion, ERβ's role in cells adapted to its expression appears to differ from its role in cells with transient expression. Our study is important because it provides a deeper understanding of ERβ's role in breast tumors that coexpress both receptors and supports an emerging bi-faceted role of ERβ.

  • 19. Katchy, Anne
    et al.
    Edvardsson, Karin
    Aydogdu, Eylem
    Williams, Cecilia
    University of Houston, United States .
    Estradiol-activated estrogen receptor α does not regulate mature microRNAs in T47D breast cancer cells.2012In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 128, no 3-5, p. 145-53Article in journal (Refereed)
    Abstract [en]

    Breast cancers are sensitive to hormones such as estrogen, which binds to and activates estrogen receptors (ER) leading to significant changes in gene expression. microRNAs (miRNA) have emerged as a major player in gene regulation, thus identification of miRNAs associated with normal or disrupted estrogen signaling is critical to enhancing our understanding of the diagnosis and prognosis of breast cancer. We have previously shown that 17β-estradiol (E2) induced activation of ERα in T47D cells results in significant changes in the expression of protein-coding genes involved in cell cycle, proliferation, and apoptosis. To identify miRNAs regulated by E2-activated ERα, we analysed their expression in T47D cells following E2-activation using both dual-color microarrays and TaqMan Low Density Arrays, and validations were carried out by real-time PCR. Although estrogen treatment results in altered expression of up to 900 protein-coding transcripts, no significant changes in mature miRNA expression levels could be confirmed. Whereas previous studies aiming to elucidate the role of miRNA in ER-positive breast cancers cell lines have yielded conflicting results, the work presented here represents a thorough investigation of and significant step forward in our understanding of ERα mediated miRNA regulation.

  • 20. Katchy, Anne
    et al.
    Pinto, Caroline
    Jonsson, Philip
    Nguyen-Vu, Trang
    Pandelova, Marchela
    Riu, Anne
    Schramm, Karl-Werner
    Samarov, Daniel
    Gustafsson, Jan-Åke
    Bondesson, Maria
    Williams, Cecilia
    University of Houston, United States .
    Coexposure to phytoestrogens and bisphenol a mimics estrogenic effects in an additive manner2014In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 138, no 1, p. 21-35Article in journal (Refereed)
    Abstract [en]

    Endocrine-disrupting chemicals (EDC) are abundant in our environment. A number of EDCs, including bisphenol A (BPA) can bind to the estrogen receptors (ER), ERα and ERβ, and may contribute to estrogen-linked diseases such as breast cancer. Early exposure is of particular concern; many EDCs cross the placenta and infants have measurable levels of, eg, BPA. In addition, infants are frequently fed soy-based formula (SF) that contains phytoestrogens. Effects of combined exposure to xeno- and phytoestrogens are poorly studied. Here, we extensively compared to what extent BPA, genistein, and an extract of infant SF mimic estrogen-induced gene transcription and cell proliferation. We investigated ligand-specific effects on ER activation in HeLa-ERα and ERβ reporter cells; on proliferation, genome-wide gene regulation and non-ER-mediated effects in MCF7 breast cancer cells; and how coexposure influenced these effects. The biological relevance was explored using enrichment analyses of differentially regulated genes and clustering with clinical breast cancer profiles. We demonstrate that coexposure to BPA and genistein, or SF, results in increased functional and transcriptional estrogenic effects. Using statistical modeling, we determine that BPA and phytoestrogens act in an additive manner. The proliferative and transcriptional effects of the tested compounds mimic those of 17β-estradiol, and are abolished by cotreatment with an ER antagonist. Gene expression profiles induced by each compound clustered with poor prognosis breast cancer, indicating that exposure may adversely affect breast cancer prognosis. This study accentuates that coexposure to BPA and soy-based phytoestrogens results in additive estrogenic effects, and may contribute to estrogen-linked diseases, including breast cancer.

  • 21. Katchy, Anne
    et al.
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Department of Biology and Biochemistry, University of Houston, Houston.
    Expression Profiles of Estrogen-Regulated MicroRNAs in Breast Cancer Cells2016In: Methods in Molecular Biology: The Estrogen Receptors / [ed] Kathleen M. Eyster, Springer, 2016, Vol. 1366Chapter in book (Other academic)
    Abstract [en]

    Molecular signaling through both estrogen and microRNAs are critical for breast cancer development and growth. The activity of estrogen is mediated by transcription factors, the estrogen receptors. Here we describe a method for robust characterization of estrogen-regulated microRNA profiles. The method details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, microRNA large-scale profiling, and subsequent confirmations.

  • 22. Katchy, Anne
    et al.
    Williams, Cecilia
    Profiling of estrogen-regulated microRNAs in breast cancer cells.2014In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 84, article id e51285Article in journal (Refereed)
    Abstract [en]

    Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

  • 23. Krementsov, Dimitry N
    et al.
    Katchy, Anne
    Case, Laure K
    Carr, Frances E
    Davis, Barbara
    Williams, Cecilia
    University of Houston, United States .
    Teuscher, Cory
    Studies in experimental autoimmune encephalomyelitis do not support developmental bisphenol a exposure as an environmental factor in increasing multiple sclerosis risk2013In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 135, no 1, p. 91-102Article in journal (Refereed)
    Abstract [en]

    Multiple sclerosis (MS), a demyelinating immune-mediated central nervous system disease characterized by increasing female penetrance, is the leading cause of disability in young adults in the developed world. Epidemiological data strongly implicate an environmental factor, acting at the population level during gestation, in the increasing incidence of female MS observed over the last 50 years, yet the identity of this factor remains unknown. Gestational exposure to bisphenol A (BPA), an endocrine disruptor used in the manufacture of polycarbonate plastics since the 1950s, has been reported to alter a variety of physiological processes in adulthood. BPA has estrogenic activity, and we hypothesized that increased gestational exposure to environmental BPA may therefore contribute to the increasing female MS risk. To test this hypothesis, we utilized two different mouse models of MS, experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice (chronic progressive) and in SJL/J mice (relapsing-remitting). Dams were exposed to physiologically relevant levels of BPA in drinking water starting 2 weeks prior to mating and continuing until weaning of offspring. EAE was induced in adult offspring. No significant changes in EAE incidence, progression, or severity were observed with BPA exposure, despite changes in cytokine production by autoreactive T cells. However, endocrine disruption was evidenced by changes in testes development, and transcriptomic profiling revealed that BPA exposure altered the expression of several genes important for testes development, including Pdgfa, which was downregulated. Overall, our results do not support gestational BPA exposure as a significant contributor to the increasing female MS risk.

  • 24. Ling, G
    et al.
    Ahmadian, A
    Persson, A
    Undén, A B
    Afink, G
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, M
    Toftgård, R
    Lundeberg, J
    Pontén, F
    PATCHED and p53 gene alterations in sporadic and hereditary basal cell cancer.2001In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 20, no 53Article in journal (Refereed)
    Abstract [en]

    It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

  • 25. Ma, Ran
    et al.
    Karthik, Govindasamy-Muralidharan
    Lövrot, John
    Haglund, Felix
    Rosin, Gustaf
    Katchy, Anne
    Zhang, Xiaonan
    Viberg, Lisa
    Frisell, Jan
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden; Univ Houston, Dept Biol & Biochem, Ctr Nucl Receptors & Cell Signaling, Houston, TX USA.
    Linder, Stig
    Fredriksson, Irma
    Hartman, Johan
    Estrogen Receptor β as a Therapeutic Target in Breast Cancer Stem Cells.2017In: Journal of the National Cancer Institute, ISSN 0027-8874, E-ISSN 1460-2105, Vol. 109, no 3, p. 1-14Article in journal (Refereed)
    Abstract [en]

    Background: Breast cancer cells with tumor-initiating capabilities (BSCs) are considered to maintain tumor growth and govern metastasis. Hence, targeting BSCs will be crucial to achieve successful treatment of breast cancer.

    Methods: We characterized mammospheres derived from more than 40 cancer patients and two breast cancer cell lines for the expression of estrogen receptors (ERs) and stem cell markers. Mammosphere formation and proliferation assays were performed on cells from 19 cancer patients and five healthy individuals after incubation with ER-subtype selective ligands. Transcriptional analysis was performed to identify pathways activated in ERβ-stimulated mammospheres and verified using in vitro experiments. Xenograft models (n = 4 or 5 per group) were used to study the role of ERs during tumorigenesis.

    Results: We identified an absence of ERα but upregulation of ERβ in BSCs associated with phenotypic stem cell markers and responsible for the proliferative role of estrogens. Knockdown of ERβ caused a reduction of mammosphere formation in cell lines and in patient-derived cancer cells (40.7%, 26.8%, and 39.1%, respectively). Gene set enrichment analysis identified glycolysis-related pathways (false discovery rate < 0.001) upregulated in ERβ-activated mammospheres. We observed that tamoxifen or fulvestrant alone was insufficient to block proliferation of patient-derived BSCs while this could be accomplished by a selective inhibitor of ERβ (PHTPP; 53.7% in luminal and 45.5% in triple-negative breast cancers). Furthermore, PHTPP reduced tumor initiation in two patient-derived xenografts (75.9% and 59.1% reduction in tumor volume, respectively) and potentiated tamoxifen-mediated inhibition of tumor growth in MCF7 xenografts.

    Conclusion: We identify ERβ as a mediator of estrogen action in BSCs and a novel target for endocrine therapy.

  • 26. Monteiro, Fátima Liliana
    et al.
    Vitorino, Rui
    Wang, Jun
    Cardoso, Hugo
    Laranjeira, Hugo
    Simões, Joana
    Caldas, Margarida
    Henrique, Rui
    Amado, Francisco
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jerónimo, Carmen
    Helguero, Luisa A
    The histone H2A isoform Hist2h2ac is a novel regulator of proliferation and epithelial-mesenchymal transition in mammary epithelial and in breast cancer cells.2017In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, article id S0304-3835(17)30171-4Article in journal (Refereed)
    Abstract [en]

    Proliferation and differentiation are controlled through chromatin remodelling. Therefore, there is an enormous biological significance and clinical value in understanding how specific signalling pathways are affected by histone replacement in the nucleosome. In this work, mass spectrometry was used to screen HC11 mammary epithelial cells for changes in histone levels throughout cell differentiation. The canonical histone isoform Histone H2A type 2-C (Hist2h2ac) was found only in undifferentiated/proliferating cells. Hist2h2ac mRNA was induced by EGF, specifically in the CD24+/CD29hi/DC44hi cell subpopulation. Hist2h2ac mRNA was increased by MEK(1/2) or PI3-K activation in HC11 and EpH4 mammary epithelial cells, and in MC4-L2 and T47-D breast cancer cells. Hist2h2ac silencing inhibited EGF-induced Zeb-1 expression and E-cadherin down-regulation, and this effect was reverted by Hist2h2ac re-expression. Notably, silencing of Hist2h2ac increased EGFR, ERBB2, and ERK(1/2) activation but did not allow EGF-induced proliferation. HIST2H2AC was expressed in all breast cancer molecular subtypes and found altered in 17% breast cancers, being 16.8% of the cases related to HIST2H2AC gene amplification and/or mRNA upregulation. In summary, this is the first study that identifies a canonical histone isoform -Hist2h2ac-downstream of the EGFR pathway, regulating oncogenic signalling and thereby contributing to deregulation of target genes.

  • 27. Nguyen-Vu, Trang
    et al.
    Vedin, Lise-Lotte
    Liu, Ka
    Jonsson, Philip
    Lin, Jean Z
    Candelaria, Nicholes R
    Candelaria, Lindsay P
    Addanki, Sridevi
    Williams, Cecilia
    University of Houston.
    Gustafsson, Jan-Åke
    Steffensen, Knut R
    Lin, Chin-Yo
    Liver × receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism2013In: Breast Cancer Research, ISSN 1465-5411, E-ISSN 1465-542X, Vol. 15, no 3, article id R51Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION: Liver × receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors and have established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment.

    METHODS: To further determine the effects of LXR ligands and identify their potential mechanisms of action in breast cancer cells, we carried out microarray analysis of gene expression in four breast cancer cell lines following treatments with the synthetic LXR ligand GW3965. Differentially expressed genes were further subjected to gene ontology and pathway analyses, and their expression profiles and associations with disease parameters and outcomes were examined in clinical samples. Response of E2F target genes were validated by real-time PCR, and the posited role of E2F2 in breast cancer cell proliferation was tested by RNA interference experiments.

    RESULTS: We observed cell line-specific transcriptional responses as well as a set of common responsive genes. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes. Hierarchical clustering of breast cancer patients based on the expression profiles of the commonly downregulated LXR ligand-responsive genes showed a strong association of these genes with patient survival.

    CONCLUSIONS: Taken together, these results indicate that LXR ligands target gene networks, including those regulated by E2F family members, are critical for tumor biology and disease progression and merit further consideration as potential agents in the prevention and treatment of breast cancers.

  • 28. Nguyen-Vu, Trang
    et al.
    Wang, Jun
    Mesmar, Fahmi
    Mukhopadhyay, Srijita
    Saxena, Ashish
    McCollum, Catherine W.
    Gustafsson, Jan-Ake
    Bondesson, Maria
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Estrogen receptor beta reduces colon cancer metastasis through a novel miR-205-PROX1 mechanism2016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 27, p. 42159-42171Article in journal (Refereed)
    Abstract [en]

    Colon cancer is a common cause of cancer death in the Western world. Accumulating evidence supports a protective role of estrogen via estrogen receptor beta (ER beta) but the mechanism of action is not known. Here, we elucidate a molecular mechanism whereby ER beta represses the oncogenic prospero homebox 1 (PROX1) through the upregulation of miR-205. We show that PROX1 is a potential target of miR-205 and that in clinical specimens from The Cancer Genome Atlas data, ER beta and miR-205 are decreased in colorectal cancer tissue compared to non-tumorous colon, while PROX1 levels are increased. Through mechanistic studies in multiple colorectal cancer cell lines, we show that ER beta upregulates miR-205, and that miR-205 targets and represses PROX1 through direct interaction with its 3' UTR. Through the generation of intestine-specific ER beta knockout mice, we establish that this pathway is correspondingly regulated in normal intestinal epithelial cells in vivo. Functionally, we demonstrate that miR-205 decreases cell proliferation and decreases migratory and invasive potential of colon cancer cells, leading to a reduction of micrometastasis in vivo. In conclusion, ER beta in both normal and cancerous colon epithelial cells upregulates miRNA-205, which subsequently reduces PROX1 through direct interaction with its 3' UTR. This results in reduced proliferative and metastatic potential of the cells. Our study proposes a novel pathway that may be exploited using ER beta-selective agonists and/or miR-205-replacement therapy in order to improve preventive and therapeutic approaches against colon cancer.

  • 29. Odeberg, J
    et al.
    Ahmadian, A
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, M
    Pontén, F
    Lundeberg, J
    Context-dependent Taq-polymerase-mediated nucleotide alterations, as revealed by direct sequencing of the ZNF189 gene: implications for mutation detection.1999In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 235, no 1-2Article in journal (Refereed)
    Abstract [en]

    We have recently reported on the genetic organisation of a novel Krüppel-like zinc finger, ZNF189, located to 9q22-q31. In that study we found no mutations in the coding sequence when using ZNF189 as a candidate gene for sporadic basal cell cancer and squamous cell cancer. Here, by direct sequencing of the proximal promotor of ZNF189, mutations were found to appear in a small hot-spot region in over 50% of analysed tumour samples, the majority being G to A substitutions. The hot-spot region spans a 24bp G-rich region. Repeated analyses of the original sample lysates fail to confirm each of these mutations; and frequently new mutations appear at neighbouring positions. Subsequent analysis with serial dilutions of genomic DNA and a cosmid harbouring the wild-type ZNF189 gene demonstrate that these sequence-specific alterations arise in the outer PCR-amplification when 50 copies or less of template are used. Although the mechanism of how these context-specific alterations arise is not proven, the results demonstrate a previously unreported type of PCR-mediated sequence-specific alteration that easily could have been interpreted as being of clinical relevance. The phenomena observed show that mutations detected by direct sequencing can be caused by PCR-introduced alterations. Consequently, this should be of general caution in mutation analysis of disease gene candidates when using small amounts of template, such as microdissected biopsies.

  • 30. Persson, A E
    et al.
    Ling, G
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Bäckvall, H
    Pontén, J
    Pontén, F
    Lundeberg, J
    Analysis of p53 mutations in single cells obtained from histological tissue sections.2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 287, no 1Article in journal (Refereed)
    Abstract [en]

    We have previously reported on direct sequence analysis of the p53 gene in laser-dissected single cells from tissue sections, where each allele of two fragments (exons 7 and 8) could be accurately analyzed in only 14% of the cells due to the high frequency of exon and allele dropout. Here in an effort to minimize this problem, we have investigated various approaches for sample preparation and gene amplification. By pinpointing some critical steps in the procedure, we could increase the number of investigated exons and substantially improve the genetic analysis of single cells obtained from histochemically stained frozen tissue sections. The biggest improvement was achieved by minimizing DNA degradation using EDTA as a nuclease inhibitor in all sample preparation steps. Efforts to increase primer annealing, by increasing the concentration of template and primers, in addition to prolonging the annealing and extension times, also improved the amplification efficiency. With these measures we can now amplify six individual exons of the p53 gene (exons 4-9) in 70% of the cells and in 50% of these cells both alleles are amplified. This allows application of the method in various investigations such as within the field of tumor pathology.

  • 31. Persson, Asa E
    et al.
    Edström, Desiree W
    Bäckvall, Helena
    Lundeberg, Joakim
    Pontén, Fredrik
    Ros, Anne-Marie
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    The mutagenic effect of ultraviolet-A1 on human skin demonstrated by sequencing the p53 gene in single keratinocytes.2002In: Photodermatology, Photoimmunology & Photomedicine, ISSN 0905-4383, E-ISSN 1600-0781, Vol. 18, no 6Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Sun exposure is accepted as the major risk factor for developing skin cancer, the most common cancer in the western world. Ultraviolet-B (UV-B) radiation is considered the causative agent, but recently several findings suggest a role also for ultraviolet-A (UV-A) radiation. Repeated suberythemal doses of ultraviolet-A1 (UV-A1) on healthy human skin induce an increase of p53 immunoreactive cells in epidermis, which may indicate cell cycle arrest and/or occurrence of p53 mutations.

    METHODS: We have investigated the possible mutagenic effect of UV-A1 on skin by sequencing exons 4-11 and adjacent intron sequence of the p53 gene in immunoreactive single cells from three healthy individuals. Previously unexposed buttock skin was irradiated three times a week for 2 weeks with physiological fluences (40 J/cm2) of UV-A1. Punch biopsies were taken before and at different time-points after the exposure, and from these single p53 immunoreactive cells were isolated by using laser-assisted microdissection.

    RESULTS: Three mutations--all being indicative of oxidative damage and most likely related to UV-A exposure--were found among the 37 single cells from exposed skin, whereas no mutations were found in the 22 single cells taken before exposure.

    CONCLUSIONS: The findings indicate a mutagenic effect of low-dose UV-A1 on healthy human skin, which further demonstrates the importance of considering UV-A when taking protective measures against skin cancer.

  • 32. Pontén, F
    et al.
    Berg, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ahmadian, A
    Ren, Z P
    Nistér, M
    Lundeberg, J
    Uhlén, M
    Pontén, J
    Molecular pathology in basal cell cancer with p53 as a genetic marker.1997In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 15, no 9Article in journal (Refereed)
    Abstract [en]

    Human basal cell cancer (BCC) has unique growth characteristics with virtual inability to metastasize. We investigated clonality and genetic progression using p53 mutations as marker. Sampling was done through microdissection of frozen immunohistochemically stained 16 microm slices of tumors. From 11 BCC tumors 78 samples were analysed. Direct DNA sequencing of exons 5-8 was performed, haplotypes were determined after cloning of p53 exons and loss of heterozygosity (LOH) ascertained by microsatellite analysis. All tumors had p53 mutations and in a majority both p53 alleles were affected, commonly through missense mutations. Microdissection of small parts (50-100 cells) of individual tumors showed BCC to be composed of a dominant cell clone and prone to genetic progression with appearance of subclones with a second and even third p53 mutation. Samples from normal immunohistochemically negative epidermis always showed wild type sequence, except for a case of previously unknown germline p53 mutation. Our analysis also included p53 immunoreactive patches i.e. morphologically normal epidermis with a compact pattern of p53 immunoreactivity. Mutations within those were never the same as in the adjacent BCC. This detailed study of only one gene thus uncovered a remarkable heterogeneity within a tumor category famous for its benign clinical behavior.

  • 33. Pontén, F
    et al.
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ling, G
    Ahmadian, A
    Nistér, M
    Lundeberg, J
    Pontén, J
    Uhlén, M
    Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy.1997In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 382, no 1-2Article in journal (Refereed)
    Abstract [en]

    In this study, we show that direct mutational analysis of genomic DNA can be performed on single somatic cells extracted from a frozen, immunohistochemically stained tissue section using laser-assisted capture microscopy. Eighty-nine single tumor cells were separately dissected from one case of human basal cell cancer (BCC) and p53 mutations were analyzed by direct semi-automated sequencing of PCR fragments. Amplification was obtained for at least one of the two analyzed exons from approximately 50% of the single tumor cells. Identical p53 mutations were found in widely spread areas of the tumor, suggesting a clonal proliferation originating from one cell. Interestingly, comparison between results of immunohistochemistry and genetic analysis of the single cells revealed the same p53 mutations irrespective of the p53 immunoreactivity. We propose that this approach has a great potential to allow investigation of genotypic differences in single cells and more specifically to resolve important and fundamental questions determining cancer heterogeneity.

  • 34. Reins, Rose Y.
    et al.
    Mesmar, Fahmi
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    McDermott, Alison M.
    Microarray Analysis of Vitamin D Treated Human Corneal Epithelial Cells2016In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 57, no 12Article in journal (Refereed)
  • 35. Reins, Rose Y.
    et al.
    Mesmar, Fahmi
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    McDermott, Alison M.
    Vitamin D Induces Global Gene Transcription in Human Corneal Epithelial Cells: Implications for Corneal Inflammation2016In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 57, no 6, p. 2689-2698Article in journal (Refereed)
    Abstract [en]

    PURPOSE. Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we further examined the timing of the cytokine response to combined vitamin D-TLR treatment and used genome-wide microarray analysis to examine the effect of vitamin D on corneal gene expression. METHODS. Telomerase-immortalized HCEC (hTCEpi) were stimulated with polyinosinicpolycytidylic acid (poly[I: C]) and 1,25-dihydroxyvitamin D-3 (1,25D(3)) for 2 to 24 hours and interleukin (IL)-8 expression was examined by quantitative (q) PCR and ELISA. Telomeraseimmortalized HCEC and SV40-HCEC were treated with 1,25D(3) and used in genome-wide microarray analysis. Expression of target genes was validated using qPCR in both cell lines and primary HCEC. For confirmation of IjBa protein, hTCEpi were treated with 1,25D(3) for 24 hours and cell lysates used in an ELISA. RESULTS. Treatment with 1,25D(3) increased poly(I: C)-induced IL-8 mRNA and protein expression after 2 to 6 hours. However, when cells were pretreated with 1,25D(3) for 24 hours, 1,25D(3) decreased cytokine expression. For microarray analysis, 308 genes were differentially expressed by 1,25D(3) treatment in hTCEpi, and 69 genes in SV40s. Quantitative (q) PCR confirmed the vitamin D-mediated upregulation of target genes, including nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (I kappa B alpha). In addition to increased transcript levels, I kappa B alpha protein was increased by 28% following 24 hours of vitamin D treatment. CONCLUSIONS. Microarray analysis demonstrates that vitamin D regulates numerous genes in HCEC and influences TLR signaling through upregulation of I kappa B alpha. These findings are important in dissecting the role of vitamin D at the ocular surface and highlight the need for further research into the functions of vitamin D and its influence on corneal gene expression.

  • 36. Ren, Z. P.
    et al.
    Ahmadian, A.
    Pontén, F.
    Nistér, M.
    Berg, Cecilia
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Lundeberg, J.
    Uhlén, M.
    Pontén, J.
    Benign clonal keratinocyte patches with p53 mutations show no genetic link to synchronous squamous cell precancer or cancer in human skin1997In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 150, no 5, p. 1791-803Article in journal (Refereed)
    Abstract [en]

    Ultraviolet light, which is the major etiology of human skin cancer, will cause mutations in the p53 gene. We and others have found that such mutations occur in more than one-half of non-melanoma squamous cell cancer and precancer. Immunostaining for p53 has disclosed a characteristic compact pattern not only in cancer/precancer but also in areas of microscopically normal epidermis termed p53 patches. By microdissection, sequence analysis of the p53 gene, and analysis of loss of heterozygosity (LOH) at the site of this gene, we have now extended previous data to ascertain whether these p53 patches are precursors of simultaneously present squamous cell cancer or its morphologically recognized precancerous stages (dysplasia, carcinoma in situ). In none of 11 instances with co-existence of a p53 patch with dysplasia or in situ or invasive cancer were the mutations identical. We conclude that p53 patches, estimated to be approximately 100,000 times as common as dysplasia, have a very small or even no precancerous potential. Their common presence demonstrates that human epidermis contains a large number of p53 mutations apparently without detrimental effect. The only result of the mutation may be a clandestine benign clonal keratinocyte proliferation. The importance of p53 mutations for such benign cell multiplication on one band and malignant transformation on the other is unclear. Although the spectrum, type, and multiplicity of mutations were similar in both types of proliferative responses, there was a clear difference with respect to LOH. No LOH was found in 17 p53 patches. By contrast 11 of 30 precancers/cancers had LOH.

  • 37. Richter, Karin
    et al.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO).
    Dahl, Lina
    Bruce, Sara
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Carlsson, Leif
    Williams, Cecilia
    KTH, School of Biotechnology (BIO).
    Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression2006In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, p. 75-Article in journal (Refereed)
    Abstract [en]

    Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.

    Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.

    Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

  • 38. Simon, Marisa
    et al.
    Mesmar, Fahmi
    Helguero, Luisa
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome-wide effects of MELK-inhibitor in triple-negative breast cancer cells indicate context-dependent response with p53 as a key determinant2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 2, article id e0172832Article in journal (Refereed)
    Abstract [en]

    Triple-negative breast cancer (TNBC) is an aggressive, highly recurrent breast cancer subtype, affecting approximately one-fifth of all breast cancer patients. Subpopulations of treatment-resistant cancer stem cells within the tumors are considered to contribute to disease recurrence. A potential druggable target for such cells is the maternal embryonic leucine-zipper kinase (MELK). MELK expression is upregulated in mammary stem cells and in undifferentiated cancers, where it correlates with poor prognosis and potentially mediates treatment resistance. Several MELK inhibitors have been developed, of which one, OTSSP167, is currently in clinical trials. In order to better understand how MELK and its inhibition influence TNBC, we verified its anti-proliferative and apoptotic effects in claudin-low TNBC cell lines MDA-MB-231 and SUM-159 using MTS assays and/or trypan blue viability assays together with analysis of PARP cleavage. Then, using microarrays, we explored which genes were affected by OTSSP167. We demonstrate that different sets of genes are regulated in MDA-MB-231 and SUM-159, but in both cell lines genes involved in cell cycle, mitosis and protein metabolism and folding were regulated. We identified p53 (TP53) as a potential upstream regulator of the regulated genes. Using western blot we found that OTSSP167 downregulates mutant p53 in all tested TNBC cell lines (MDA-MB-231, SUM-159, and BT-549), but upregulates wild-type p53 in the luminal A subtype MCF-7 cell line. We propose that OTSSP167 might have context-dependent or off-target effects, but that one consistent mechanism of action could involve the destabilization of mutant p53.

  • 39. Svensson, Per
    et al.
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ryden, Patrik
    Bergqvist, Ingela
    Edlund, Helena
    Gene array identification of Ipf1/Pdx1(-/-) regulated genes in pancreatic progenitor cells2007In: BMC Developmental Biology, ISSN 1471-213X, E-ISSN 1471-213X, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Background: The homeodomain transcription factor IPF1/PDX1 exerts a dual role in the pancreas; Ipf1/Pdx1 global null mutants fail to develop a pancreas whereas conditional inactivation of Ipf1/Pdx1 in beta-cells leads to impaired beta-cell function and diabetes. Although several putative target genes have been linked to the beta-cell function of Ipf1/Pdx1, relatively little is known with respect to genes regulated by IPF1/PDX1 in early pancreatic progenitor cells. Results: Microarray analyses identified a total of III genes that were differentially expressed in e10.5 pancreatic buds of Ipf1/Pdx1(-/-) embryos. The expression of one of these, Spondin 1, which encodes an extracellular matrix protein, has not previously been described in the pancreas. Quantitative real-time RT-PCR analyses and immunohistochemical analyses also revealed that the expression of FgfR2IIIb, that encodes the receptor for FGF10, was down-regulated in Ipf1/Pdx1(-/-) pancreatic progenitor cells. Conclusion: This microarray analysis has identified a number of candidate genes that are differentially expressed in Ipf1/Pdx1(-/-) pancreatic buds. Several of the differentially expressed genes were known to be important for pancreatic progenitor cell proliferation and differentiation whereas others have not previously been associated with pancreatic development.

  • 40. Tsouko, Efrosini
    et al.
    Wang, Jun
    Frigo, Daniel E.
    Aydogdu, Eylem
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    miR-200a inhibits migration of triple-negative breast cancer cells through direct repression of the EPHA2 oncogene2015In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 36, no 9, p. 1051-1060Article in journal (Refereed)
    Abstract [en]

    Triple-negative breast cancer (TNBC) is characterized by aggressiveness and affects 10-20% of breast cancer patients. Since TNBC lacks expression of ER alpha, PR and HER2, existing targeted treatments are not effective and the survival is poor. In this study, we demonstrate that the tumor suppressor microRNA miR-200a directly regulates the oncogene EPH receptor A2 (EPHA2) and modulates TNBC migration. We show that EPHA2 expression is correlated with poor survival specifically in basal-like breast cancer and that its expression is repressed by miR-200a through direct interaction with the 3'UTR of EPHA2. This regulation subsequently affects the downstream activation of AMP-activated protein kinase (AMPK) and results in decreased cell migration of TNBC. We establish that miR-200a directs cell migration in a dual manner; in addition to regulating the well-characterized E-cadherin pathway it also regulates a EPHA2 pathway. The miR-200a-EPHA2 axis is a novel mechanism highlighting the possibility of utilizing miR-200a delivery to target TNBC metastases.

  • 41.
    Wang, Jun
    et al.
    University of Houston, United States .
    Tsouko, Efrosini
    University of Houston, United States .
    Jonsson, Philip
    University of Houston, United States .
    Bergh, Jonas
    Karolinska Institutet and University Hospital, Stockholm, Sweden .
    Hartman, Johan
    Karolinska Institutet and University Hospital, Stockholm, Sweden .
    Aydogdu, Eylem
    University of Houston, United States .
    Williams, Cecilia
    University of Houston, United States .
    miR-206 inhibits cell migration through direct targeting of the actin-binding protein coronin 1C in triple-negative breast cancer2014In: Molecular oncology, ISSN 1878-0261, Vol. 8, no 8, p. 1690-702Article in journal (Refereed)
    Abstract [en]

    Patients with triple-negative breast cancer (TNBC) have an overall poor prognosis, which is primarily due to a high metastatic capacity of these tumors. Novel therapeutic approaches to target the signaling pathways that promote metastasis are desirable, in order to improve the outcome for these patients. A loss of function of a microRNA, miR-206, is related to increased metastasis potential in breast cancers but the mechanism is not known. In this study, we show that miR-206 was decreased in TNBC clinical tumor samples and cell lines whereas one of its predicted targets, actin-binding protein CORO1C, was increased. Expression of miR-206 significantly reduced proliferation and migration while repressing CORO1C mRNA and protein levels. We demonstrate that miR-206 interacts with the 3'-untranslated region (3'-UTR) of CORO1C and regulates this gene post-transcriptionally. This post-transcriptional regulation was dependent on two miR-206-binding sites within the 3'-UTR of CORO1C and was relieved by mutations of corresponding sites. Further, silencing of CORO1C reduced tumor cell migration and affected the actin skeleton and cell morphology, similar to miR-206 expression, but did not reduce proliferation. In accordance with this, overexpression of CORO1C rescued the inhibitory effect of miR-206 on cell migration. Our findings suggest that miR-206 represses tumor cell migration through direct targeting of CORO1C in TNBC cells which modulates the actin filaments. This pathway is a novel mechanism that offers a mechanistic basis through which the metastatic potential of TNBC tumors could be targeted.

  • 42.
    Williams, Cecilia
    et al.
    University of Houston, United States.
    Bondesson, Maria
    Krementsov, Dimitry N.
    Teuscher, Cory
    Gestational bisphenol A exposure and testis development2014In: Endocrine disruptors, ISSN 2327-3747, Vol. 2, no 1, article id e29088Article, review/survey (Refereed)
    Abstract [en]

    Virtually all humans are exposed to bisphenol A (BPA). Since BPA can act as a ligand for estrogen receptors, potential hazardous effects of BPA should be evaluated in the context of endogenous estrogenic hormones. Because estrogen is metabolized in the placenta, developing fetuses are normally exposed to very low endogenous estrogen levels. BPA, on the other hand, passes through the placenta and might have distinct adverse consequences during the sensitive stages of fetal development. Testicular gametogenesis and steroidogenesis begin early during fetal development. These processes are sensitive to estrogens and play a role in determining the number of germ stem cells, sperm count, and male hormone levels in adulthood. Although studies have shown a correlation between BPA exposure and perturbed reproduction, a clear consensus has yet to be established as to whether current human gestational BPA exposure results in direct adverse effects on male genital development and reproduction. However, studies in animals and in vitro have provided direct evidence for the ability of BPA exposure to influence male reproductive development. This review discusses the current knowledge of potential effects of BPA exposure on male reproductive health and whether gestational exposure adversely affects testis development.

  • 43.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab. University of Houston, USA; Karolinska Institutet, Sweden.
    DiLeo, Alfredo
    Niv, Yaron
    Gustafsson, Jan-Ake
    Estrogen receptor beta as target for colorectal cancer prevention2016In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 372, no 1, p. 48-56Article, review/survey (Refereed)
    Abstract [en]

    Colorectal cancer (CRC) is a leading cause of death in the United States. Despite its slow development and the capacity for early diagnosis, current preventive approaches are not sufficient. However, a role for estrogen has been demonstrated in multiple epidemiologic studies, which may benefit CRC prevention. A large body of evidence from preclinical studies indicates that expression of the estrogen receptor beta (ER beta/ESR2) demonstrates an inverse relationship with the presence of colorectal polyps and stage of tumors, and can mediate a protective response. Natural compounds, including phytoestrogens, or synthetic ER beta selective agonists, can activate or upregulate ER beta in the colon and promote apoptosis in preclinical models and in clinical experience. Importantly, this activity has been associated with a reduction in polyp formation and, in rodent models of CRC, has been shown to lower incidence of colon adenocarcinoma. Collectively, these findings indicate that targeted activation of ER beta may represent a novel clinical approach for management of colorectal adenomatous polyps and prevention of colorectal carcinoma in patients at risk for this condition. In this review, we discuss the potential of new chemopreventive or dietary approaches based on estrogen signaling.

  • 44.
    Williams, Cecilia
    et al.
    Karolinska Institutet.
    Edvardsson, K
    Lewandowski, S A
    Ström, A
    Gustafsson, J-A
    A genome-wide study of the repressive effects of estrogen receptor beta on estrogen receptor alpha signaling in breast cancer cells.2008In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 27, no 7, p. 1019-1032Article in journal (Refereed)
    Abstract [en]

    Transcriptional effects of estrogen result from its activation of two estrogen receptor (ER) isoforms; ERalpha that drives proliferation and ERbeta that is antiproliferative. Expression of ERbeta in xenograft tumors from the T47D breast cancer cell line reduces tumor growth and angiogenesis. If ERbeta can halt tumor growth, its introduction into cancers may be a novel therapeutic approach to the treatment of estrogen-responsive cancers. To assess the complete impact of ERbeta on transcription, we have made a full transcriptome analysis of ERalpha- and ERbeta-mediated gene regulation in T47D cell line with Tet-Off regulated ERbeta expression. Of the 35 000 genes and transcripts analysed, 4.1% (1434) were altered by ERalpha activation. Tet withdrawal and subsequent ERbeta expression inhibited the ERalpha regulation of 998 genes and, in addition, altered expression of 152 non-ERalpha-regulated genes. ERalpha-induced and ERbeta-repressed genes were involved in proliferation, steroid/xenobiotic metabolism and ion transport. The ERbeta repressive effect was further confirmed by proliferation assays, where ERbeta was shown to completely oppose the ERalpha-E2 induced proliferation. Additional analysis of ERbeta with a mutated DNA-binding domain revealed that this mutant, at least for a quantity of genes, antagonizes ERalpha even more strongly than ERbeta wt. From an examination of the genes regulated by ERalpha and ERbeta, we suggest that introduction of ERbeta may be an alternative therapeutic approach to the treatment of certain cancers.

  • 45.
    Williams, Cecilia
    et al.
    University of Houston, United States; Karolinska Institutet, Sweden .
    Helguero, Luisa
    Karolinska Institutet, Sweden .
    Edvardsson, Karin
    University of Houston, United States; Karolinska Institutet, Sweden .
    Haldosén, Lars-Arne
    Karolinska Institutet, Sweden .
    Gustafsson, Jan-Ake
    University of Houston, United States; Karolinska Institutet, Sweden .
    Gene expression in murine mammary epithelial stem cell-like cells shows similarities to human breast cancer gene expression.2009In: Breast cancer research : BCR, ISSN 1465-542X, Vol. 11, no 3, p. R26-Article in journal (Refereed)
    Abstract [en]

    INTRODUCTION: Mammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized. Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, a detailed characterization of the transcriptome in mammary stem cells is essential.

    METHODS: We have used a murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique. Subsequently, we have performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development.

    RESULTS: Our analysis of mammary stem-like and stepwise cell differentiation, and an in-depth description of our findings in a breast cancer perspective provide a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. We correlate the alterations to in vivo mammary gland differentiation, and describe novel changes in nuclear receptor gene expression. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression.

    CONCLUSIONS: The transcriptional characterization of these mammary stem-like cells and their differentiation-induced gene expression patterns is here made widely accessible and provides a basis for research on mammary stem-like cells. Our comparisons suggest that some tumors are more stem-like than others, with a corresponding worse prognosis. This information would, if established, be important for treatment decisions. We also suggest several marker candidates valuable to investigate further.

  • 46.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. University of Houston, United States .
    Lin, Chin-Yo
    University of Houston, United States .
    Oestrogen receptors in breast cancer: basic mechanisms and clinical implications2013In: ecancermedicalscience, ISSN 1754-6605, E-ISSN 1754-6605, Vol. 7, no 1, article id 370Article, review/survey (Refereed)
    Abstract [en]

    Since the discovery of the connection between ovarian hormones and breast cancer, endocrine therapy has been an integral adjuvant treatment for patients with hormone-dependent breast cancers. Oestrogen receptor (ER) plays a central role in mediating the effects of endogenous hormones and therapeutic agents. ER serves as a prognostic marker for responsiveness to endocrine therapy and is targeted either directly by selective oestrogen receptor modulators (SERMs) and pure antagonists or indirectly by aromatase inhibitors (AIs) that block oestrogen production. A significant number of ER-positive patients, however, fail to respond to therapy or develop resistance over time. This review focuses on the current understanding of ER functions and recent advances in genomic technologies and research that have provided a global perspective on hormone and ER activity and led to a number of significant discoveries, including the roles of co-regulatory factors and non-coding RNAs. Mechanistic insights into normal ER functions and therapeutic actions of SERMs and AIs will enable the development of better predictive markers and more effective target mechanisms and ultimately facilitate improvements in disease outcomes and patient survival.

  • 47.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Norberg, T
    Ahmadian, A
    Pontén, F
    Bergh, J
    Inganäs, M
    Lundeberg, J
    Uhlén, M
    Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets.1998In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 44, no 3Article in journal (Refereed)
    Abstract [en]

    The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.

  • 48.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, F
    Ahmadian, A
    Ren, Z P
    Ling, G
    Rollman, O
    Ljung, A
    Jaspers, N G
    Uhlén, M
    Lundeberg, J
    Pontén, J
    Clones of normal keratinocytes and a variety of simultaneously present epidermal neoplastic lesions contain a multitude of p53 gene mutations in a xeroderma pigmentosum patient.1998In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 58, no 11Article in journal (Refereed)
    Abstract [en]

    A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.

  • 49.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pontén, F
    Moberg, C
    Söderkvist, P
    Uhlén, M
    Pontén, J
    Sitbon, G
    Lundeberg, J
    A high frequency of sequence alterations is due to formalin fixation of archival specimens.1999In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 155, no 5Article in journal (Refereed)
    Abstract [en]

    Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

  • 50.
    Williams, Cecilia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology.
    Meletis, Konstantinos
    Wikström, Lilian
    Carlsson, Leif
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Catalog of gene expression in adult neural stem cells and their in vivo microenvironment2006In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 10, p. 1798-1812Article in journal (Refereed)
    Abstract [en]

     Stem cells generally reside in a stem cell micro environment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique sternness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.

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