Breast Neoplasms: Zhao C

Shah SP, Köbel M, Senz J, Morin RD, Clarke BA, Wiegand KC, Leung G, Zayed A, Mehl E, Kalloger SE, Sun M, Giuliany R, Yorida E, Jones S, Varhol R, Swenerton KD, Miller D, Clement PB, Crane C, Madore J, Provencher D, Leung P, DeFazio A, Khattra J, Turashvili G, Zhao Y, Zeng T, Glover JN, Vanderhyden B, Zhao C, Parkinson CA, Jimenez-Linan M, Bowtell DD, Mes-Masson AM, Brenton JD, Aparicio SA, Boyd N, Hirst M, Gilks CB, Marra M, Huntsman DG. · Centre for Translational and Applied Genomics, British Columbia Cancer Agency, Vancouver, Canada. · N Engl J Med. · Pubmed #19516027 No free full text.

Abstract: BACKGROUND: Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery. METHODS: We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays. RESULTS: All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors. CONCLUSIONS: Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Zhao C, Raza A, Martin SE, Pan J, Greaves TS, Cobb CJ. · Department of Pathology, Los Angeles County + University of Southern California Medical Center, Los Angeles, CA, USA. · Cancer Cytopathol. · Pubmed #19365832 No free full text.

Abstract: BACKGROUND: The fine-needle aspiration (FNA) diagnosis of proliferative breast lesion is an indeterminate category. The aim of this correlative study was to determine whether a subcategory of "proliferative breast lesion with atypia" was achievable and whether this subcategory has management utility. METHODS: Breast FNA cases from 2000 through 2005 diagnosed as proliferative breast lesion and proliferative breast lesion with atypia were retrieved. Both cytologic and surgical slides of these cases were reviewed blindly. A cytologic diagnosis of proliferative breast lesion (without atypia) or proliferative breast lesion with atypia was used if the findings of the proliferative breast lesion did not fit a more specific category. RESULTS: Of the 3934 breast FNAs performed on palpable breast masses from January 2000 to December 2005 at the LAC + USC Medical Center, 317 (8.1%) were diagnosed cytologically as proliferative breast lesion with atypia, without atypia or without mention of atypia. There was subsequent histopathology on 201 of these cases. After the cytologic smears were reviewed, 29 cases were excluded from this study. Of the 172 remaining cases, 21 (12.2%) were found to be malignant and the remaining 151 (87.8%) were found to be benign on histology. Of the malignant cases, 90% had an FNA diagnosis of proliferative breast lesion with atypia; of the benign cases, 78% were interpreted as proliferative breast lesion without atypia. CONCLUSIONS: Proliferative breast lesion with atypia was clinically significant because it was associated with a significantly increased likelihood of malignancy compared with proliferative breast lesion without atypia. Most of the malignancies had hypocellularity or low nuclear grade on the FNA smears. Fibroadenoma accounted for most of the benign lesions in both proliferative breast lesion and proliferative breast lesion with atypia.

Li J, Shen Y, Liu A, Wang X, Zhao C. · Department of Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, People's Republic of China. · Oncol Rep. · Pubmed #18636195 No free full text.

Abstract: D-amino acid oxidase (DAAO) can catalyze the dehydrogenation of D-amino acids, such as D-alanine, to the corresponding amino acids and is then reoxidized by molecular oxygen to yield hydrogen peroxide, a reactive oxygen species, which reacts with DNA, lipids and protein, inducing cell death. This study investigated whether rat glioma 9L cells infected with the recombinant retrovirus containing the DAAO cDNA fragment can be induced in order to undergo cytotoxic oxidative stress by D-alanine. The recombinant retroviral vector, plzrus-DAAO-FLAG-GFP (pl-Dfg), was constructed and used to transfect packaged phoenix cells. The supernatant containing recombinant retroviral particles from the transfected phoenix cells was harvested and utilized to infect target 9L cells. The cytotoxic oxidative stress of infected 9L cells was induced by the DAAO substrate, D-alanine. The plasmid pl-Dfg was successfully constructed. The high titer retroviral supernatant was obtained from the transfected phoenix cells. Infected 9L cells were less viable after exposure to D-alanine compared to the control group. Anti-apoptotic proteins significantly inhibited cell death. The DAAO/D-alanine system has a potential utility for gene therapy and may be an effective strategy for the treatment of brain cancer and other malignant tumors.

Garcia A, Zheng Y, Zhao C, Toschi A, Fan J, Shraibman N, Brown HA, Bar-Sagi D, Foster DA, Arbiser JL. · Department of Biological Sciences, Hunter College of The City University of New York, New York 10021, USA. · Clin Cancer Res. · Pubmed #18594009 links to  free full text

Abstract: PURPOSE: Elevated phospholipase D (PLD) activity provides a survival signal in several human cancer cell lines and suppresses apoptosis when cells are subjected to the stress of serum withdrawal. Thus, targeting PLD survival signals has potential to suppress survival in cancer cells that depend on PLD for survival. Honokiol is a compound that suppresses tumor growth in mouse models. The purpose of this study was to investigate the effect of honokiol on PLD survival signals and the Ras dependence of these signals. EXPERIMENTAL DESIGN: The effect of honokiol upon PLD activity was examined in human cancer cell lines where PLD activity provides a survival signal. The dependence of PLD survival signals on Ras was investigated, as was the effect of honokiol on Ras activation. RESULTS: We report here that honokiol suppresses PLD activity in human cancer cells where PLD has been shown to suppress apoptosis. PLD activity is commonly elevated in response to the stress of serum withdrawal, and, importantly, the stress-induced increase in PLD activity is selectively suppressed by honokiol. The stress-induced increase in PLD activity was accompanied by increased Ras activation, and the stress-induced increase in PLD activity in MDA-MB-231 breast cancer cells was dependent on a Ras. The PLD activity was also dependent on the GTPases RalA and ADP ribosylation factor. Importantly, honokiol suppressed Ras activation. CONCLUSION: The data provided here indicate that honokiol may be a valuable therapeutic reagent for targeting a large number of human cancers that depend on Ras and PLD for their survival.

Zhao C, Fu P, Sheng Y, Li Z. · Department of Nuclear Medicine, Union Hospital, Tongji Medicial College, Huazhong University of Science and Technology, Wuhan 430022, China. · J Exp Ther Oncol. · Pubmed #18038763 No free full text.

Abstract: To investigate the effect of radiolabed mouse double minute 2 (MDM2) antisense oligonucleotide on gene expression in human breast cancer MCF-7 cells, an antisense oligonucleotide (ASON) targeting MDM2 mRNA was synthesized and radiolabeled with 99Tcm. The labeling efficiency, radiochemical purity, and the ability of labeled ASON to hybridize to the sense oligonucleotides (SON) were investigated. To study whether the antisense probe hybridizes to respective sequence on MDM2 mRNA strand after radiolabeling, cells were incubated with radiolabeling oligonucleotides antisense oligonucleotide (0, 100, 500 nm/L) or mismatch oligonucleotide (ASONM) (500 nm/L) for 24 h, in the presence of Lipofectin 2000. RT-PCR and Western blotting was carried out to measure the MDM2 mRNA and protein levels. The antisense oligonucleotide was radiolabeled with the bifunctional chelator HYNIC at the labeling efficiency of 57.2 +/- 2.98% (n = 5) and the mismatch oligonucleotide was 56.3 +/- 3.01% (n = 5). The radiochemical purity was above 95% and labeled antisense oligonucleotide has the ability to hybridize to the sense oligonucleotide. The levels of mRNA and protein have significant differences in different concentration groups. The oligonucleotide can be successfully radiolabeled, and specially hybridized to the MDM2 mRNA and inhibit gene expression intensively as compared to mismatch oligonucleotide. This method will be very useful in the in vivo investigation of tumor targeting.

Zhao C, Matthews J, Tujague M, Wan J, Ström A, Toresson G, Lam EW, Cheng G, Gustafsson JA, Dahlman-Wright K. · Department of Biosciences and Nutrition, Novum, Karolinska Institutet, Huddinge, Sweden. · Cancer Res. · Pubmed #17440111 links to  free full text

Abstract: Estrogens, by binding to and activating two estrogen receptors (ERalpha and ERbeta), are critically involved in the development of the mammary gland and breast cancer. An isoform of ERbeta, ERbeta2 (also called ERbetacx), with an altered COOH-terminal region, is coexpressed with ERalpha in many human breast cancers. In this study, we generated a stable cell line from MCF7 breast cancer cells expressing an inducible version of ERbeta2, along with endogenous ERalpha, and examined the effects of ERbeta2 on the ERalpha protein levels and function. We showed that ERbeta2 inhibited ERalpha-mediated transactivation via estrogen response element and activator protein-1 sites of reporter constructs as well as the endogenous genes pS2 and MMP-1. Chromatin immunoprecipitation assays revealed that ERbeta2 expression caused a significant reduction in the recruitment of ERalpha to both the pS2 and MMP-1 promoters. Furthermore, ERbeta2 expression induced proteasome-dependent degradation of ERalpha. The inhibitory effects of ERbeta2 on ERalpha activity were further confirmed in HEK293 cells that lack functional endogenous ERs. We also showed that ERbeta2 can interact with ERalpha both in vitro and in mammalian cells, which is compatible with a model where ERbeta2/ERalpha heterodimers are targeted to the proteasome. Finally, in human breast cancer samples, we observed that expression of ERbeta2 significantly correlated with ERalpha-negative phenotype. Our data suggest that ERbeta2 could influence ERalpha-mediated effects relevant for breast cancer development, including hormone responsiveness.

Zhao C, Bratthauer GL, Barner R, Vang R. · Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Washington, DC, USA. · Am J Surg Pathol. · Pubmed #17255771 No free full text.

Abstract: The main neoplasms in the differential diagnosis for primary ovarian tumors with a tubule-rich pattern are pure Sertoli cell tumor, endometrioid tumors (including borderline tumor, well-differentiated carcinoma, and the sertoliform variant of endometrioid carcinoma), and carcinoid tumor. Because traditional immunohistochemical markers [pan-cytokeratin (pan-CK), low molecular weight cytokeratin (CK8/18), epithelial membrane antigen (EMA), inhibin, calretinin, CD99, chromogranin, and synaptophysin] can occasionally have diagnostic limitations, the goal of this study was to determine whether or not any alternative markers [cytokeratin 7 (CK7), estrogen receptor (ER), progesterone receptor (PR), CD10, and CD56] have better diagnostic utility when compared with traditional markers for this differential diagnosis. Immunohistochemical stains for alternative, as well as traditional, markers were performed on the following primary ovarian tumors: pure Sertoli cell tumor (n = 40), endometrioid borderline tumor (n = 38), sertoliform endometrioid carcinoma (n = 13), well-differentiated endometrioid carcinoma (n = 27), and carcinoid tumor (n = 42). Extent and intensity of immunostaining were semiquantitatively scored. In addition, immunohistochemical composite scores (ICSs) in positive cases were calculated on the basis of the combination of extent and intensity scores. Cytokeratin 7 (CK7) was positive in 97% of endometrioid tumors, 13% of Sertoli cell tumors, and 24% of carcinoid tumors. The differences in the mean ICSs for endometrioid tumors versus Sertoli cell tumor or carcinoid tumor were statistically significant (P values ranging from <0.001 to 0.018). ER and PR were positive in 87% and 86% of endometrioid tumors, 8% and 13% of Sertoli cell tumors, and 2% each of carcinoid tumors, respectively. The differences in the mean ICSs for endometrioid tumors versus Sertoli cell tumor were statistically significant (P values ranging from <0.001 to 0.012). Among the epithelial markers, EMA seemed to be the most discriminatory but only slightly better than CK7, ER, or PR. Pan-CK and CK8/18 were not helpful. CD10 showed overlapping patterns of expression in all categories of tumors. Among the sex cord markers, CD10 was markedly less useful than inhibin or calretinin; CD99 was not discriminatory. CD56 showed overlapping patterns of expression in all categories of tumors. Among the neuroendocrine markers, CD56 was less useful than chromogranin or synaptophysin. When traditional immunohistochemical markers are problematic for the differential diagnosis of ovarian Sertoli cell tumor versus endometrioid tumors versus carcinoid tumor, adding CK7, ER, and/or PR to a panel of markers can be helpful. Endometrioid tumors more frequently express CK7, ER, and PR and show a greater extent of immunostaining in contrast to Sertoli cell tumor and carcinoid tumor. Compared with traditional epithelial markers, CK7, ER, and PR are nearly as advantageous as EMA. Inhibin is the most discriminatory sex cord marker, and CD10 is not helpful in the differential diagnosis. Chromogranin and synaptophysin are excellent discriminatory markers for carcinoid tumor, and CD56 is neither sufficiently sensitive nor specific enough for this differential diagnosis to warrant its use in routine practice.

Zhao C, Cai M, Zhang Y, Liu Y, Sun R, Zhang N. · Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology and State Key Laboratory of Molecular Dynamic and Stable Structures, College of Chemistry, Peking University, Beijing 100871, China. · Anal Biochem. · Pubmed #17240347 No free full text.

Abstract: Protein kinase Czeta (PKCzeta) plays a critical role in cancer cell chemotaxis. Upon activation induced by epidermal growth factor (EGF) or chemoattractant SDF-1alpha, PKCzeta redistributes from cytosol to plasma membrane. Based on this property, we developed a rapid cell-based assay for inhibitors of ligand-induced PKCzeta activation. PKCzeta green fluorescent protein (GFP) was transfected into human breast cancer cells, MDA-MB-231, to establish a stable cell line, PKCzeta-GFP/MDA-MB-231. PKCzeta-GFP/MDA-MB-231 maintained phenotypes, such as chemotaxis, adhesion, and cell migration, similar to those of its parental cell line. Therefore it could be used as a representative cancer cell line. EGF induced translocation of PKCzeta-GFP to plasma membrane in a pattern similar to that of endogenous PKCzeta, indicative of activation of PKCzeta Translocation of PKCzeta-GFP could be easily and directly recorded by an inverted fluorescence microscope. Inhibitors of chemotaxis also impaired the translocation of PKCzeta-GFP, which further validated the biological relevance of our assay. Taken together, we have developed a simple, rapid, and reliable assay to detect the ligand-induced activation of PKCzeta in human cancer cells. This assay can be used in screening for inhibitors of PKCzeta activation, which is critically required for cancer cell chemotaxis.

Zhao C, Bratthauer GL, Barner R, Vang R. · Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Washington, DC, USA. · Int J Gynecol Pathol. · Pubmed #17197889 No free full text.

Abstract: The distinction of ovarian Sertoli cell tumor from other tumors in the histological differential diagnosis, particularly endometrioid carcinoma and carcinoid tumor, may be difficult. Many immunohistochemical markers have been studied for this differential diagnosis, but currently available markers are neither 100% sensitive nor specific. Sox9 is a transcription factor involved in Sertoli cell differentiation in the testis. The role that this molecule plays in the pathogenesis of ovarian Sertoli cell tumors and the potential use as an immunohistochemical marker for differential diagnosis have not been investigated. Immunohistochemical staining for Sox9 was performed in 152 ovarian tumors: pure Sertoli cell tumor (n = 36), endometrioid borderline tumor (n = 38), well-differentiated endometrioid carcinoma (n = 26), sertoliform endometrioid carcinoma (n = 13), and carcinoid tumor (n = 39). Nuclear expression was considered positive. Extent and intensity of staining were semiquantitatively scored. In addition, immunohistochemical composite scores in positive cases (ranging from 1 to 12) were calculated based on the extent score multiplied by the intensity score. Sox9 was expressed in 44% of Sertoli cell tumors, 55% of endometrioid borderline tumors, 65% of well-differentiated endometrioid carcinomas, 39% of sertoliform endometrioid carcinomas, and 10% of carcinoid tumors. The mean Sox9 immunohistochemical composite scores in positive cases were 6.3 for Sertoli cell tumor, 5.3 for endometrioid borderline tumor, 8.0 for well-differentiated endometrioid carcinoma, 2.8 for sertoliform endometrioid carcinoma, and 6.8 for carcinoid tumor. The differences in the mean Sox9 composite scores between Sertoli cell tumor and the other tumor categories were not statistically significant (p values ranged from 0.092 to 0.523). We conclude that Sox9 is variably expressed in ovarian Sertoli cell tumor and other tumors that are in the differential diagnosis and, thus, is not helpful for immunohistochemical distinction. Understanding the role of Sox9 in the pathogenesis of ovarian Sertoli cell tumor requires further study.

Man YG, Zhao C, Chen X. · Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC 20306-6000, USA. · Pathol Res Pract. · Pubmed #16842934 No free full text.

Abstract: Immunohistochemical staining for cytokeratin (CK) 34ssE12 has been routinely used to elucidate prostate basal cells for differentiation between non-invasive and invasive lesions. Our previous studies, however, revealed that some morphologically distinct basal cells observed on H&E-stained sections completely lacked CK34ssE12 expression. Our current study attempted to assess whether these basal cells would also lack the expression of other phenotypic markers, and whether basal cell alterations would affect the proliferation status of the associated tumor cells. Consecutive sections from prostate tumors with large basal cell clusters that were morphologically distinct in H&E sections but were completely negative for CK 34ssE12 were morphologically and immunohistochemically assessed with a panel of basal cell phenotypic and other markers. In addition to CK 34ssE12, these basal cells also completely lacked the expression of other phenotypic markers, including CK5, CK14, p63, and maspin, in contrast to adjacent basal cells, which were strongly positive for these markers. Tumors surrounded by basal cell layers that lack the expression of basal cell phenotypic markers showed a significantly higher rate of cell proliferation and mast cell infiltration than their counterparts. These findings suggest that basal cells might be targets of a variety of pathological alterations, which could significantly impact biological presentations of associated tumor cells.

Koller E, Propp S, Zhang H, Zhao C, Xiao X, Chang M, Hirsch SA, Shepard PJ, Koo S, Murphy C, Glazer RI, Dean NM. · Department of Functional Genomics, Isis Pharmaceuticals, Carlsbad, California 92008, USA. · Cancer Res. · Pubmed #16489005 links to  free full text

Abstract: A library of 2'-methoxyethyl-modified antisense oligonucleotides (2'MOE ASO) targeting 1,510 different genes has been developed, validated, and used to identify cell cycle regulatory genes. The most effective molecular target identified was Eg5 (kinesin-like-1), which when inhibited gave the largest increase in 4N DNA in various tumor cells. The Eg5 ASO reduced Eg5 levels, inhibited proliferation, increased apoptosis, and altered the expression of other cell cycle proteins, including survivin and Aurora-A. To examine the therapeutic utility of the Eg5 ASO, the compound was also evaluated in xenograft models. Treatment with Eg5 ASO produced a statistically significant reduction of tumor growth, reduction in Eg5 expression in the tumors, and changes in histone phosphorylation, consistent with a loss of Eg5 protein expression. These data show, for the first time, the utility of a 2'MOE ASO library for high-throughput cell culture-based functional assays and suggest that an Eg5 ASO also has potential in a therapeutic strategy.

Wei B, Bu H, Zhu CR, Guo LX, Chen HJ, Zhao C, Zhang P, Chen DY, Tang Y, Jiang Y. · Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, China. · Sichuan Da Xue Xue Bao Yi Xue Ban. · Pubmed #15573772 No free full text.

Abstract: OBJECTIVE: The aim of this study involving general pathologists was to assess interobserver reproducibility in the pathologic diagnosis of borderline ductal proliferative breast diseases. METHODS: Ten general pathologists independently reviewed 43 specimens chosen to represent the spectrum of borderline ductal proliferative breast lesions. All slides were blindly reviewed without given standardized criteria, and were classified as either mild usual hyperplasia, moderate-severe usual hyperplasia, mild atypical hyperplasia, moderate-severe atypical hyperplasia, ductal carcinoma in situ, or ductal carcinoma in situ with invasion. According to the years of training, these 10 general pathologists were divided into the experienced group and the less trained group. Interobserver agreement was statistically analyzed using Kappa statistic. Then, by comparing all the diagnoses of individual pathologist with the consensus opinion confirmed by two breast pathologists in terms of Page standard, we acquired the diagnostic accuracy and ascertained the undue diagnosis. RESULTS: The ten general pathologists' interobserver reproducibility in the diagnosis of borderline ductal proliferative breast diseases was rather low, especially that in their diagnosis of atypical hyperplasia. The reproducibility and accuracy were slightly higher in the experienced pathologists than in the less trained pathologists. Some pathologists made over-diagnosis or under-diagnosis of the lesions to different degrees. However, when the categories of diagnostic terms were simplified, the interobserver reproducibility increased. CONCLUSION: The use of standardized criteria is an important approach to increasing the diagnostic reproducibility and accuracy.

Zhao C, Wacnik PW, Tall JM, Johns DC, Wilcox GL, Meyer RA, Raja SN. · Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. · J Pain. · Pubmed #15042518 No free full text.

Abstract: Bone is a common metastatic site for prostate and breast cancer, and bone cancer is usually associated with severe pain. Traditional treatments for cancer pain can sometimes be ineffective or associated with side effects. Thus an increasing number of patients seek alternative therapies. In this study we investigated the analgesic effects of a soy diet on 3 experimental models of bone cancer pain. Mice were fed a diet in which the protein source was either soy or casein. After 1 week on the diet, sarcoma cells (NCTC 2472) were injected into the medullary cavity of the humeri, femur, or calcaneus. Experimenters blinded to diet of the animal assessed the pain behavior in these animals, forelimb grip force in the humerus model and paw withdrawal frequency to mechanical stimuli in the calcaneus and femur models. The effect of morphine on cancer-induced pain behavior was investigated in calcaneus and femur models. In addition, in the femur model, the effects of soy on tumor size and bone destruction were studied. The soy diet reduced secondary mechanical hyperalgesia in the femur model but had no effect on primary mechanical hyperalgesia in the calcaneus model or on movement-related hyperalgesia in the humerus model. No dietary impact was discerned in measurements of tumor size, bone destruction, and body weight in the femur model, suggesting that the soy diet had no effect on cancer growth. Morphine dose-dependently reduced hyperalgesia with no diet-based difference. These results suggest that a soy diet might provide analgesia in certain forms of hyperalgesia associated with bone cancer. PERSPECTIVE: The study raises the possibility of dietary supplements influencing aspects of cancer pain. Further research will help determine if use of nutritional supplements, such as soy proteins, can reduce opioid analgesic use in chronic pain states and help minimize the side effects associated with long term use of opioids.

Zhao C, Lam EW, Sunters A, Enmark E, De Bella MT, Coombes RC, Gustafsson JA, Dahlman-Wright K. · Department of Biosciences at Novum, Karolinska Institutet, S-14157 Huddinge, Sweden. · Oncogene. · Pubmed #14576822 No free full text.

Abstract: Two novel estrogen receptor beta (ERbeta) mRNA isoforms that diverge in their 5'-untranslated regions, ERbeta mRNA (0K-1) and ERbeta mRNA (0N-1), have recently been identified. This indicates that transcription of the human ERbeta gene occurs from at least two different promoters, named promoter 0K and promoter 0N. The aim of this study was to investigate the expression of ERbeta isoforms in primary cultures of normal breast epithelial cells, a panel of breast cancer cell lines and in normal breast and breast cancer tissues; and to examine whether methylation of the two ERbeta promoters is involved in regulation of ERbeta gene expression. Using quantitative real-time PCR techniques, we found that ERbeta mRNA levels were significantly lower in breast cancer cell lines than in primary cultures of normal breast epithelial cells. Bisulfite genomic sequencing analysis revealed that two promoters of the ERbeta gene exhibit distinct methylation patterns. Promoter 0N was unmethylated in normal breast epithelial cells, but extensively methylated in breast cancer cell lines. In contrast, promoter 0K was unmethylated in both normal and malignant breast epithelial cells. We demonstrated a significant correlation between promoter 0N hypermethylation and loss of ERbeta mRNA expression in breast cancer cell lines. Treatment of breast cancer cells with demethylating agent effectively reactivated the expression of ERbeta mRNA. The observations from the cell lines were also reflected in primary breast cancer tumors. Thus, expression of ERbeta mRNA in breast tumors was found to be inversely associated with the degree of methylation of promoter 0N. Our results suggest that a decreased level of ERbeta mRNA may be associated with breast tumorigenesis, and that DNA methylation is an important mechanism for ERbeta gene silencing in breast cancer.

Försti A, Zhao C, Israelsson E, Dahlman-Wright K, Gustafsson JA, Hemminki K. · Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden. · Breast Cancer Res Treat. · Pubmed #12846425 No free full text.

Abstract: Polymorphisms in the estrogen receptor beta (ERbeta) gene may influence the cellular growth regulating effects of estradiol. In this first association study about breast cancer risk and polymorphisms in the ERbeta gene we have screened 219 Finnish sporadic breast cancer cases and 248 ethnically matched male controls. No difference in the allele distribution of the six studied polymorphisms was found between the breast cancer and control groups.

Zhao C, Yasui K, Lee CJ, Kurioka H, Hosokawa Y, Oka T, Inazawa J. · Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. · Cancer. · Pubmed #12833450 links to  free full text

Abstract: BACKGROUND: Amplification of DNA in certain chromosomal regions plays a crucial role in the development and progression of human malignancies, specifically when protooncogenic target genes within those amplicons are overexpressed. Comparative genomic hybridization studies have revealed frequent amplification at 20q in primary breast tumors. The aim of the current study was to identify specific genes in the 20q amplicon that were likely to have clinical significance. METHODS: The authors examined 38 primary breast tumors by using a quantitative real-time reverse transcription-polymerase chain reaction assay to determine expression levels of 18 potential targets for amplification events involving 20q. Potential correlations between elevated expression of the genes in question and clinicopathologic parameters or clinical outcomes were analyzed. RESULTS: Elevated expression of NABC1 was significantly associated with positive estrogen (P < 0.001) and progesterone (P = 0.027) receptors in breast tumors, and high expression of PTK6 was significantly correlated with positive estrogen receptor status (P = 0.022) and postmenopausal status (P = 0.008). Patients whose tumors showed elevated expression of NCOA3 (AIB1) had significantly shorter disease-free (P = 0.017) and overall (P = 0.0021) survival times after surgery than did other patients with breast tumors. Reduced disease-free survival, but not reduced overall survival, was associated with high expression of TOP1 (P = 0.035) and TFAP2C (P = 0.035). CONCLUSIONS: TOP1, TFAP2C, and (particularly) NCOA3 may be prognostic indicators for patients with breast tumors.

Kumar R, Höglund L, Zhao C, Försti A, Snellman E, Hemminki K. · Department of Biosciences, Center for Nutrition and Toxicology, Karolinska Institute, Novum, Huddinge, Sweden. · Int J Cancer. · Pubmed #12494477 No free full text.

Abstract: In this study we determined the effect of single nucleotide polymorphisms in the XPG gene on DNA repair and breast cancer susceptibility. Ninety individuals, with previously studied DNA repair rate at 24 hr of 2 types of UV-specific cyclobutane pyrimidines dimers (CPDs) in skin were genotyped for XPG polymorphism at codon 1104 (exon 15 G>C; Asp > His). The repair rate of TT=C dimer was similar in both wild-type GG homozygotes and GC heterozygotes, whereas, for TT=T, dimer repair was non-significantly (Student's t-test, p = 0.34) lower in GC heterozygotes than wild-type GG homozygotes. Genotyping of 220 breast cancer cases and 308 controls for the same single nucleotide polymorphism in exon 15 of the XPG gene exhibited marginally significant increased frequency of the variant allele (chi(2) 3.84, p = 0.05; OR 1.33, 95% CI 1.0-1.8) in cases (C-allele 0.29) compared to controls (C-allele 0.24). Combined heterozygote and variant homozygote genotype frequency was also higher in cases than controls (chi(2) 4.79, p = 0.03; OR 1.50, 95%CI 1.04-2.16).

Zhao C, Fang Q, Tan K, Lu X. · Department of Breast Surgery, Changzhou First People's Hospital, Changzhou 213003, China. · Zhonghua Yi Xue Za Zhi. · Pubmed #12425800 No free full text.

Abstract: OBJECTIVE: To investigate the relationship among breast cancer and negative life event and cell immunity. METHODS: A questionnaire survey using the Life Event by YANG Desen to investigate the family life problems, work problems, and social and other problems was conducted among 115 patients with breast cancer diagnosed by pathology and cytology and 115 gender, age, profession, education, and life habit-matched patients with benign breast diseases as controls. Fasting blood was drawn from all patients. Fluorescent-labeled bodies were added. Flow cytometry was made to count the immunocytes. RESULTS: The negative life event rate was 87% in the breast cancer group, higher than that in the control group (55%, P < 0.01). The rate of family problems in the breast cancer group was 63%, higher than that in the control group (40%). The total score of negative events was 31.5 +/- 9.7 in the breast cancer group, higher than that in the control group (17.3 +/- 5.6, P < 0.01). The percentage of CD(3) (total T cell) in the breast cancer group was 58.8 +/- 12.2%, significantly lower than that in the control group (63.9 +/- 9.9%, P < 0.01). There was no difference in the percentages of CD(4), CD(8), CD(4)/CD(8), and the percentage of natural killer cells (NK) between the two groups. CONCLUSION: Breast cancer is closely correlated with negative life events, especially family problems concerning marriage and children. The negative life events are related to the decrease of total T cells, and unrelated to the percentages of other cells.

Sano M, Kikuchi K, Zhao C, Kobayashi M, Nakanishi Y, Nemoto N. · No affiliation provided · Virchows Arch. · Pubmed #15014987 No free full text.

This publication has no abstract.

Glaros S, Atanaskova N, Zhao C, Skafar DF, Reddy KB. · Department of Pathology, Wayne State University School of Medicine, 540 East Canfield Avenue, and The Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA. · Mol Endocrinol. · Pubmed #16455819 links to  free full text

Abstract: The antiestrogen tamoxifen has been widely used for decades as selective estrogen receptor (ER) modulator for ERalpha-positive breast tumors. Tamoxifen significantly reduces tumor recurrence by binding to the activation function-2 (AF-2) domain of the ER. Acquired resistance to tamoxifen in breast cancer patients is a serious therapeutic problem. Antiestrogen-resistant breast cancer often shows increased expression of the epidermal growth factor receptor (EGFR) family members, EGFR and ErbB2. In this report we now show that overexpression of EGFR or activated AKT-2 in MCF-7 cells leads to phosphorylation of Ser167 in the AF-1 domain of ERalpha, enhanced ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of tamoxifen, and resistance to tamoxifen. In contrast, transfection of activated MAPK kinase, an immediate upstream activator of MAPK (ERK 1 and 2) into MCF-7 cells leads to phosphorylation of Ser118 in the AF-1 domain of ERalpha, inhibition of ER-amplified in breast cancer 1 (ER:AIB1) interaction in the presence of Tam, and maintenance of sensitivity to tamoxifen. Inhibition of AKT by short inhibitory RNA blocked Ser167 phosphorylation in ER and restored tamoxifen sensitivity. However, maximum sensitivity to tamoxifen was observed when both AKT and MAPK were inhibited. Taken together, these data demonstrate that different phosphorylation sites in the AF-1 domain of ERalpha regulate the agonistic and antagonistic actions of tamoxifen in human breast cancer cells.