Ali Shidfar

RANKL expression in normal and malignant breast tissue responds to progesterone and is up-regulated during the luteal phase

The receptor activator of nuclear factor-κB ligand (RANKL) acts as a paracrine factor in progesterone-induced mammary epithelial proliferation and tumorigenesis. This evidence comes mainly from mouse models. Our aim was to examine whether RANKL expression in human normal and malignant breast is under the control of progesterone throughout the menstrual cycle. Breast epithelial samples were obtained by random fine needle aspiration (rFNA) of the contralateral unaffected breasts (CUB) of 18 breast cancer patients, with simultaneous serum hormone measurements. Genes correlated with serum progesterone levels were identified through Illumina microarray analysis. Validation was performed using qRT-PCR in rFNA samples from CUB of an additional 53 women and using immunohistochemistry in tissue microarrays of 61 breast cancer samples. Expression of RANKL, DIO2, and MYBPC1 was correlated with serum progesterone in CUB, and was significantly higher in luteal phase. RANKL and MYBPC1 mRNA expression were highly correlated between CUB and matched tumor samples. RANKL protein expression was also significantly increased in the luteal phase and highly correlated with serum progesterone levels in cancer samples, especially in hormone receptor positive tumors. The regulatory effects of progesterone on the expression of RANKL, DIO2, and MYBPC1 were confirmed in three-dimensional cultures of normal breast organoids. In normal breast and in breast cancer, RANKL mRNA and protein expression fluctuate with serum progesterone with highest levels in the luteal phase, suggesting that RANKL is a modulator of progesterone signaling in normal and malignant breast tissue and a potential biomarker of progesterone action and blockade.

Overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor-negative breast cancer

Risk biomarkers for estrogen receptor (ER)‐negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER‐negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre‐B‐cell leukemia homeobox‐1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser‐capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER‐positive cases, 28 ER‐negative cases, 28 healthy controls). Gene expression was quantitated by qRT‐PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA‐MB‐453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER‐negative versus ER‐positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER‐negative than ER‐positive tumors. PBX1 overexpression in MCF10A cells up‐regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA‐MB‐453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP‐qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER‐negative tumorigenesis.

Expression of miR-18a and miR-210 in normal breast tissue as candidate biomarkers of breast cancer risk

miRNAs are noncoding RNAs with abnormal expression in breast cancer; their expression in high-risk benign breast tissue may relate to breast cancer risk. We examined miRNA profiles in contralateral unaffected breasts (CUB) of patients with breast cancer and validated resulting candidates in two additional sample sets. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays in 30 breast cancer samples [15 estrogen receptor (ER)-positive and 15 ER-negative] and paired CUBs and 15 reduction mammoplasty controls. Pairwise comparisons identified miRNAs with significantly differential expression. Seven candidate miRNAs were examined using qRT-PCR in a second CUB sample set (40 cases, 20 ER+, 20 ER−) and 20 reduction mammoplasty controls. Further validation was performed in 80 benign breast biopsy (BBB) samples; 40 from cases who subsequently developed breast cancer and 40 from controls who did not. Logistic regression, using tertiles of miRNA expression, was used to discriminate cases from controls. Seven miRNAs were differentially expressed in tumors and CUBs versus reduction mammoplasty samples. Among them, miR-18a and miR-210 were validated in the second CUB set, showing significantly higher expression in tumor and CUBs than in reduction mammoplasty controls. The expression of miR-18a and miR-210 was also significantly higher in BBB cases than in BBB controls. When both miR-18a and miR-210 were expressed in the upper tertiles in BBB, OR for subsequent cancer was 3.20, P = 0.023. miR-18a and miR-210 are expressed at higher levels in CUBs of patients with breast cancer, and in BBB prior to cancer development, and are therefore candidate breast cancer risk biomarkers. Cancer Prev Res; 10(1); 89–97. ©2016 AACR.

Protein Biomarkers for Breast Cancer Risk Are Specifically Correlated with Local Steroid Hormones in Nipple Aspirate Fluid

The local endocrine environment of the breast may have stronger relations to breast cancer risk than systemic hormones. Nipple aspiration fluid (NAF) provides a window into this milieu. We hypothesized that the correlations between proteins and steroid hormones in NAF are stronger, and specific relationships may reveal links to breast cancer risk. NAF and blood samples were obtained simultaneously from 54 healthy women and from the contralateral unaffected breast of 60 breast cancer patients. The abundance of five proteins, superoxide dismutase (SOD1), C-reactive protein (CRP), chitinase-3-like protein 1 (YKL40), cathepsin D (CatD), and basic fibroblast growth factor (bFGF) in NAF was measured using ELISA. The NAF and serum concentrations of estradiol, estrone, progesterone, androstenedione, testosterone, and dehydroepiandrostrerone (DHEA) were measured using ELISA or RIA. The correlations between proteins and hormones revealed that NAF proteins correlated with each other: SOD1 with CRP (R = 0.276, P = 0.033) and CatD (R = 0.340, P = 0.0036), and bFGF with CRP (R = 0.343, P = 0.0021). NAF proteins displayed significant correlations with NAF steroids, but not with serum steroids: SOD1 with DHEA (R = 0.333, P = 0.019), YKL40 with testosterone (R = 0.389, P = 0.0012), and bFGF negatively correlated with testosterone (R = −0.339, P = 0.015). The regulation of YKL40 and bFGF by testosterone was confirmed in breast cancer cell lines. In summary, NAF proteins were more strongly related to local hormone levels than to systematic hormone levels. Some proteins were specifically correlated with different NAF steroids, suggesting that these steroids may contribute to breast cancer risk through different mechanisms.

Abstract P4-07-02: Expression of miR-18a and miR-210 in normal breast tissue as candidate markers of breast cancer risk

Purpose: miRNAs are non-coding RNAs that are abnormally expressed in breast cancer, with critical roles in cancer due to their regulation of large gene networks. miRNA expression in benign high-risk breast tissue has never been evaluated, but it may provide information about early dysregulation events that contribute to breast cancer risk. The contralateral unaffected breast (CUB) of women with unilateral breast cancer is in high-risk for the second primary cancer. Thus, we examined miRNA expression profiles in tumor and the matching CUBs to seek potential miRNA biomarkers for breast cancer risk. Methods: FFPE tissues of breast cancer and their matching CUB tissues were sectioned. The areas of tumor and normal ductal epithelium were outlined and then dissected using laser microdissection system. Total RNA was extracted for miRNA profiling studies. Expression profiles of 754 mature miRNAs were examined using TaqMan Low Density Arrays assays in 30 paired breast cancer and CUB samples (15 with ER+ tumors, 15 with ER- tumors) and 15 reduction mammoplasty (RM) controls, matched by age, race and menopausal status. ANOVA test was performed to examine the differential expression among groups and pairwise comparison with Sidak adjustment was used for multiple comparison. Seven candidate miRNAs were then examined in an independent CUB sample set (20 with ER+ tumors, 20 with ER- tumors) and 20 RM controls. Further independent validation was performed using qRT-PCR in 80 benign breast biopsy (BBB) samples: 40 from women who subsequently developed breast cancer (cases) and 40 from those who did not (controls). Logistic regression analysis and receiver operating characteristic (ROC) analysis were performed using combinations of the expression of multiple miRNAs to establish models discriminating cases from controls. Results: Seven miRNAs (miR-18a, miR-210, miR-214*, miR-124, miR-193a-3p, miR485-3p and miR-671-3p) were found to be differentially expressed in breast cancer and CUB samples vs. RM samples in the discovery sample set. Among them, miR-18a and miR-210 were validated in a second, independent CUB sample set. The expression of miR-18a and miR-210 were significantly higher in tumor (regardless ER status) compared to CUBs and RM controls. The expression levels in CUBs were significantly higher than in RM. We then examined miR expression in case BBB samples, and confirmed that expression of miR-18a and miR-210 were increased compared with controls. ROC analysis using miR-18a and miR-210 discriminated high-risk cases from standard-risk controls with OR 2.44, P = 0.022. Conclusion: The expression of miR-18a and miR-210 were elevated in breast cancer, in matched CUBs, and in BBB predating cancer diagnosis. These data provide strong support for the hypothesis that miR-18a and miR-210 expression in BBB is an indicator of increased risk of breast cancer. Given the high expression in tumors, they are also potential cancer detection biomarkers. Citation Format: Wang J, Shidfar A, Costa FF, Scholtens D, Bischof JM, Sullivan ME, Ivancic D, Soares MB, Khan SA. Expression of miR-18a and miR-210 in normal breast tissue as candidate markers of breast cancer risk [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-02.

Abstract 1985: PBX1 regulated lipid metabolism gene expression and epithelial-mesenchymal transition independent of estrogen receptor

Introduction: Pre-B-cell leukemia homeobox-1 (PBX1) is a member of the three amino acid loop extension (TALE) family of homeodomain proteins that bind to DNA and regulate gene transcription by forming heterodimeric transcription complexes with Meis and Prep1. PBX1 is involved in cell fate determination during organogenesis and contributes to oncogenic activity in breast cancer. As a pioneer factor, PBX1 was found to drive ER signaling in ER+ breast cancer by remodeling the chromatin and increasing DNA accessibility. But the role of PBX1 in benign breast and ER- cancer cells is not clear. In our previous studies, we identified and validated that the expression of a set of lipid metabolism genes was higher in the contralateral breast of ER- tumor. Bioinformatic analysis on lipid metabolism gene promoter regions and revealed that PBX1 may act as a potential transcription factor to co-regulate those genes. In this study, we further investigate the function of PBX1 in ER- cells. Methods: Among the ER- cell lines, we infected cell lines expressing low endogenous PBX1 (MCF10A and MDA-MB-231) with PBX1 gene in lentiviral vector. We also infected cell lines expressing high endogenous PBX1 (MDA-MB-453 and SK-BR-3) with PBX1-shRNA to knockdown PBX1. The expression of lipid metabolism genes was detected by qRT-PCR. Markers for epithelial-to-mesenchymal transition (EMT) including E-cadherin, vimentin, β-catenin and α-SMA were detected using Western blot. The effects of overexpression or knock-down of PBX1 on proliferation, migration, and invasion were measured using IncuCyte live cell imaging system. The expression of PBX1 protein was measured in benign contralateral breast and in the matching tumor using mmnunohistochemistry. Results: Over-expression of PBX1 in ER- cell lines (MCF10A and MDA-MB-231) up-regulated lipid metabolism genes and promoted cell migration and invasion by inducing EMT (increased vimentin and decreased E-cadherin and β-catenin). In contrast, knocking-down PBX1 using shRNA in ER- cell lines (MDA-MB-453 and SK-BR-3) suppressed lipid metabolism gene expression. PBX1 was more highly expressed in benign tissues associated with ER- tumors compared to ER+ tumors. In tumor tissue, on the contrary, ER+ tumors shower higher PBX1 expression levels than ER- tumors. Conclusion: PBX1 is a master regulator of lipid metabolism genes. PBX1 promoted cell migration and invasion by inducing EMT. PBX1 may play different roles and interact with different co-factors in ER+ tumors and in benign tissues associated with ER- tumors. Citation Format: Ali Shidfar, Liannian Liu, Vamsi Parini, MiRan Choi, David Ivancic, Megan E. Sullivan, Demirkan B. Gursel, Seema A. Khan, Jun Wang. PBX1 regulated lipid metabolism gene expression and epithelial-mesenchymal transition independent of estrogen receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2015-1985

Abstract P3-08-02: Expression of lipid metabolism genes in tumor and contralateral unaffected breast are conversely associated with tumor estrogen receptor status

Background: The identification of women at risk for ER- cancer would allow optimization of breast cancer prevention strategies by guiding their recruitment to studies of agents with efficacy against ER- cancer and sparing them the toxicity of prevention agents effective only against ER+ cancer. In our previous studies, we identified lipid metabolism (LiMe) gene set in rFNA samples from contralateral unaffected breast (CUB) that was associated with tumor ER status. In the current study, we further validate LiMe gene expression in tumor and CUB at the mRNA and protein levels. Methods: Tissue samples from 56 bilateral mastectomy cases (28 ER+ and 28 ER-) and 28 healthy reduction mammoplasty (RM) controls were used. The ER+ cases, ER- cases and controls were matched by age, race and menopausal status. We performed laser capture microdissection of epithelial cells in fresh frozen tissues from tumor and unaffected breast. Total RNA was extracted and LiMe genes were detected using Taqman low density gene expression arrays. The difference among groups was analyzed using ANOVA with Sidak multiple comparison adjustment. Three proteins (HMGCS2, ACSL3 and HPGD) were detected in FFPE sections of tumor and CUB tissues using immunohistochemistry. Results: Among the 13 LiMe genes, 6 genes (DHRS2, HMGCS2, UGT2B7, UGT2B11, UGT2B28 and GLYATL1) were significantly higher in CUB of ER- cases compared to CUB of ER+ cases (2.2-2.9 fold, P Conclusion: Differential expression of the LiMe genes in the CUB is associated with ER- index tumors and may characterize the environment leading to the development of ER- breast cancer. The converse patterns in tumor and CUB by ER status suggest that LiMe genes may be regulated by different mechanisms in benign and malignant tissues. These genes are potential risk biomarkers of ER- breast cancer and generate novel etiologic hypotheses regarding the development of ER- versus ER+ disease. Citation Format: Ali Shidfar, David Ivancic, Megan E Sullivan, Pranjal Patankar, Seema A Khan, Jun Wang. Expression of lipid metabolism genes in tumor and contralateral unaffected breast are conversely associated with tumor estrogen receptor status [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-08-02.

Abstract A2-03: Hotspot mutation in breast benign biopsy associated with subsequent progression to malignant breast cancer

Background: Breast cancer prevention is currently in a challenge due to the inability to identify high-risk women accurately only based on family history and pathologic evaluation of benign biopsy. Somatic molecular changes in clinically normal breasts have potential value for to improve the assessment of truly high-risk women. The identification of risk biomarkers in benign biopsy material will directly benefit the millions of women who undergo benign breast biopsy annually, adding precision to the risk implied by the histologic features of the benign biopsy. Therefore, we are pursuing robust biomarkers for breast cancer risk by sequencing pre-cancer benign biopsies, which may lead to discovery of genomic variation responsible for initiation and early progression of breast cancer. Methods: We are assembling a case-control set of benign breast biopsy matched by age, race, and duration of follow-up. The cases were the benign biopsies from women who subsequently developed breast cancer after at least one year, and the controls were the benign biopsy samples obtained from women who remain cancer free. 10-micron sections from formalin-fixed, paraffin-embedded tissue blocks are used for laser capture microdissection of selected epithelial areas and extraction of DNA. As a pilot experiment, we examined hotspot mutation in 4 cases and 4 control samples, using Ion AmpliSeq cancer hotspot panel consisting of 50 oncogenes/tumor suppressor and detecting 2855 hotspot mutation from COSMIC (Catalogue Of Somatic Mutations In Cancer). The average amplicon length was 154 (111-187) with average depth coverage of 1400x. SNP detection sensitivity was 98% for 5% variant frequency. Results: Among the 2855 hotspot mutation in 50 oncogenes/tumor suppressor detected, 153 mutation in 27 genes were identified in cases, and 62 mutations in 18 genes were found in controls. The most frequent mutanted gene was TP53 (71 mutation in cases, 24 mutation in controls). In 6 genes (CTNNB1, CDKN2A, ATM, KRAS, STK11 and SMARCB1), hotspot mutation was identified in at least two cases, but no hotspot mutation was identified in controls. Among the 4 cases, one cases had 95 hotspot mutation, much higher than the other 3 cases (5, 16, 37 mutation, respectively) and 4 controls (8, 11, 14, 29 mutation, respectively). This case progressed to triple negative (ER-, PR-, HER2-) breast cancer, a very aggressive subtype of cancer with poor prognosis. Conclusion: The pilot study of deep sequencing of breast benign biopsy suggested that the overall hotspot mutation frequency was higher in cases which progressed to malignant cancer compared to controls which did not progress to malignant cancer. Several genes were identified to be specifically associated with the progression. Citation Format: Jun Wang, Gang Feng, Nadereh Jafari, Ali Shidfar, David Ivanicic, Megan E. Sullivan, Seema A. Khan. Hotspot mutation in breast benign biopsy associated with subsequent progression to malignant breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-03.

Abstract P3-07-11: Nipple aspiration fluid yielders and non-yielders: Genetic characteristics

Background: Nipple aspiration fluid (NAF) is a noninvasively acquired biosample that can provide a window of observation into the breast environment, but NAF yield is variable across studies, and is related to a variety of demographic and reproductive factors. We have recently observed a positive correlation between serum prolactin level and NAF yield status. The genetic traits determining NAF yield are unknown, but data from the 1970s linked wet type ear wax to NAF yield, and recent studies point to single nucleotide polymorphisms (SNPs) in the ABCC11 gene as a determinant of earwax type. Further, prolactin and prolactin receptor SNPs are related to serum prolactin level. We have investigated associations between NAF yield and SNPs in prolactin, prolactin receptor and ABCC11 genes in a recently completed case-control study. Method: NAF and/or blood were collected from 916 women. Subjects were defined as yielder if NAF volume was equal or more than 2μL and non-yielder if it was less than 2μL. According to this definition, our subjects categorized as 557 yielders and 359 non-yielders. DNA was extracted from blood using Qiagen kit and 20 ng of DNA was used to amplify and genotype for ABCC11 (rs17822931), PRLR (rs37364), PRL (rs2244502), PRL (rs849872) SNPs using Taqman genotyping assay on an Applied Biosystems 7900HT machine. Results: The median age of Yielders and Non-yielders was 51 and 54, respectively, with the same range of 31-70 for both groups (P<0.0001). There were 209(38%) premenopausal, 257(46%) postmenopausal and 81(15%) perimenopausal of yielders vs. 99(28%) premenopausal, 229(64%) postmenopausal and 26(7%) perimenopausal of non-yielders (P<0.0001). ABCC11 genotype distribution was significantly different between yielders and non-yielders (P = 0.00163). However, genotype distribution was not significantly different for PRLR (rs37364), PRL (rs2244502) and PRL (rs849872). View this table: Conclusion: Our analysis demonstrates a correlation between ABCC11 (rs17822931) SNP and NAF yield status, with the major homozygous CC allele associated with NAF yielder and minor homozygous TT allele associated with non-yielder status. If NAF-based risk biomarkers are to be developed, a better understanding of the genetics of NAF yield is required. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-11.

Abstract P4-11-02: Mammary tumor formation induced by N-methyl-N-nitrosourea (MNU) is accelerated by natural and synthetic progesterone but suppressed by an anti-progesterone CDB4124

Background: CDB4124, anti-progesterone suppresses the development of carcinogen-induced ER+/PR+ mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer. We hypothesize that progesterone (P4) and medroxyprogesterone acetate (MPA) will accelerate mammary carcinogenesis induced by MNU, however CDB4124 will efficiently suppress tumor formation stimulated by progesterone. Methods: ovary intact female Sprague Dawley rats received a single intraperitoneal injection of 50mg/kg MNU at 4-5 weeks of age. 30mg of CDB4124 and 25mg of P4 or MPA (90 release pellets, Innovative research of America, Inc) were implanted in dorsal area at 3 weeks and 4 weeks after MNU injection, respectively. 10-11 rats were used for each treatment group. Tumor incidence, latency, multiplicity, and burden were recorded weekly. 9 weeks after MNU injection all the mice were euthanized and mammary tumors and glands were fixed in 10% (v/v) neutral buffered formalin. Plasma concentrations of CDB4124 and its metabolite CDB4453 were determined by LC-MS/MS. Results: The first tumor appeared in the control group at 5 weeks, and in the P4 and MPA treated groups at 6 weeks after MNU injection. 7 weeks after MNU injection, mammary tumor incidence of MPA and P4 treated groups were 80% and 50%, respectively compared to 30% in the control group. 9 weeks after MNU injection all MPA treated mice, 80% of P4 treated mice, and 60% of control mice developed tumors. Tumor incidence, latency, multiplicity, and tumor weight were summarized as mean ± SD in Table 1. Tumor latency of CDB4124 treated groups was increased; tumor incidence and burden (g) of CDB4124 treated groups were decreased compared to P4 and MPA treated groups. In particular, tumor incidence and burden of CDB4124 + P4 treated group were significantly lower than those of the control group. Plasma CDB4124 and CDB4453 were 11.6 ±5.88 ng/mL and 3.4±1.68 ng/mL, respectively. Histopathology of tumors and mammary glands and immuno-histochemical evaluations of Ki67, activated caspase-3, CD34, ER, and PR are currently underway. Conclusions: Our results indicated that natural progesterone promotes MNU- induced mammary tumor formation similar to synthetic progesterone, MPA in rats. Under this tumor permissive environment, CDB4124 provided excellent prevention efficacy, suggesting good potential as breast cancer prevention agent. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-02.