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Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

Introduction: Triple negative breast cancer (TNBC) is a highly aggressive form of breast cancer prevalent in African-American (AA) women defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative. Because ER- and HER2-targeted therapies are ineffective in TNBC, systemic chemotherapy is the standard of care and there is a tremendous need for new effective therapies with less toxicity. Steroid hormone receptors are highly druggable targets, and orphan nuclear receptors, members of the nuclear receptor superfamily, are emerging as targets for cancer therapy. In fact, we have previously shown that treatment of TNBC cells with a small molecule agonist ligand (DY131) for estrogen related receptor beta (ERRβ), has growth inhibitory and anti-mitotic activity. We have also shown that increased mRNA expression of ERRβ, correlates with better recurrence- and distant metastasis-free survival in TNBC/basal-like breast cancer. The goal of our current work is to comprehensively characterize ERRβ copy number and mRNA status in TNBC and determine its association with patients' prognosis. Methods: ESRRB copy number was determined in 106 primary breast tumors (TNBC n=56, nonTNBC n=50) by array-CGH, using the Agilent SurePrint G3 Human CGH platform. ESRRB mRNA data and its association with overall survival was determined in systemically untreated patients from METABRIC using Illumina gene expression array data (probe ID ILMN_1707398). Results: Copy number alterations (CNAs). Copy number losses at the ESRRB locus (14q24.3) were observed in 10/56 (17.8%) of TNBC vs. 10/50 (20%) of nonTNBC, while copy number gains were detected in 43/56 (76.8%) of TNBC vs. 29/50 (58%) of nonTNBC (c2 *p=0.036). Interestingly, in both TNBC and non-TNBC, ESRRB loss was seen with markedly higher frequency in AA patients when compared to Caucasian (CA) patients (c2 *p=0.012 for TNBC, p=0.052 for non-TNBC). mRNA expression. Among patients not treated with systemic chemotherapy in the METABRIC dataset, low ESRRB mRNA was significantly associated with shorter overall survival in TNBC, but not ER+ or HER2+ patients (TNBC hazard ratio 0.24, 95% confidence interval 0.07-0.85, *p=0.016). Low ESRRB also correlated with reduced overall survival in TP53 mutant (but not wild type) tumors (hazard ratio 0.28, 95% confidence interval 0.1-0.82, *p=0.013). Conclusions: ESRRB presents significantly high levels of copy number losses in TNBC when compared to non-TNBC tumors. In breast tumors from AA women, both the TNBC and non-TNBC subtypes are significantly more likely to have reduced ESRRB copy number vs. CA women. Low ESRRB mRNA expression predicts for poor overall survival in TNBC and TP53 mutant tumors. These data advocate that ERRβ expression has prognostic value in breast cancer, particularly TNBC. Future goals include immunohistochemistry staining, and analysis, of a tissue microarray consisting of 150 primary breast tumors (50 TNBC, 50 ER+, 50 HER2+); as well as ERRβ overexpression and knock-down studies in TNBC cell lines to define the role it plays in TNBC. Citation Format: Fernandez AI, Graham G, Győrffy B, Cavalli L, Mahajan A, Riggins RB. ERRβ copy number and expression in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-09.

MiRNA dysregulation has been linked to cancer initiation and progression, through their control in multiple cancer associated signaling pathways. MiRNA low specificity of target interaction confer them the ability to cooperatively regulate a single biological process. Epithelial mesenchymal transition (EMT) plays pivotal roles in cancer progression and metastasis, through the regulation of transcription factors and proteins, such as E-cadherin, Slug, Snail, Twist and Zeb1. This process is also controlled post-transcriptionally by a complex and regulated network of miRNAs. We have shown that miR-150-5p and miR-661, up-regulated in triple negative breast cancer (TNBC) when compared to normal tissue and other breast cancer subtypes, exert an oncogenic action and modulate cell proliferation, clonogenicity, migration and cytotoxic response in in vitro TNBC models. The main objective of this study was to determine whether the modulation of these phenotypes were due to the regulation of their corresponding gene targets associated with EMT. By conducting an integrated computational and experimental analysis, in which miR-150-5p and miR-661 were suppressed or ectopically expressed in transfected MDA-MB-231 cells, we showed that they present common regulatory roles in cell adhesion and motility signaling networks, including those involved in EMT. Gene expression analysis in a targeted panel of 770 genes with primary functional annotations in EMT, cell adhesion and motility and metastasis, revealed that: i. the suppression of miR-150-5p and miR-661, affected the expression of 160 and 123 genes, respectively; 18% of these genes were commonly affected by both miRNAs, including ACVR1C (ALK-7), CHAD, GIMAP6, SFRCL1, SPARCL1 and TBX1; ii. the ectopic expression of miR-150-5p and miR-661, affected the expression of 283 and 267 genes, respectively; 27% of which commonly affected by both miRNAs, including AKT1, DCL1, MAPK2, MMP1, SFRP1 and TPM2. iii. 26 genes were inversely affected by the suppression and ectopic expression of miR-150-5p and miR-661 (consistent with the general post-transcriptional regulation of miRNA/mRNA targets); 73% of these genes were predicted to be miRs150-5p and 661 direct or indirect targets. In summary, based on the computational and experimental data presented, we provide evidences that strongly suggest that miRNA-150-5p and miR-661 modulate the aggressive tumor phenotype in TNBC cells by regulating common gene targets associated with the EMT process. Further functional studies in TNBC experimental models will allow us to dissect the mechanisms by which they contribute to the TNBC aggressive phenotypes by synergistically regulating EMT. Funding: Partially funded by Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), CNPq, Capes-Brazil (scholarship to PMMO, BMS, ASF). Citation Format: Patricia MM Ozawa, Bruna M. Sugita, Aline S. Fonseca, Yara Rodriguez, Yuriy Gusev, Paolo Fadda, Deepak Kumar, Luciane R. Cavalli. The synergistic role of miR-150-5p and miR-661 in regulating epithelial mesenchymal transition in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 496.

Introduction: Ductal Carcinoma In Situ (DCIS) has become the most diagnosed breast tumor type in North America. Amplified In Breast Cancer 1 (AIB1) is amplified in 5 to 10% of primary breast tumors. We have recently demonstrated that AIB1 causes development of DCIS lesions in the mammary gland of 30% of the AIB1 transgenic mice. Our laboratory has also shown that deletion of one allele of AIB1 prevents HER2-induced mammary tumor development in mice, suggesting a pivotal role of AIB1 during earlier stage of breast cancer. We hypothesize that AIB1-mediated pathways promote the transformation of the mammary epithelial cells and the alterations of the functional integrity of the mammary gland. By contrast, decrease in AIB1 functionality may result in prevented DCIS initiation and development. Results: We show by immunohistochemistry that AIB1 is expressed at low levels in normal breast but is highly expressed in DCIS lesions. We investigated the impact of decreasing AIB1 expression on the early transformed MCFDCIS cell line. MCFDCIS cells generate multiacinar disorganized structures with filling of the lumen, loss of polarization and escape from the proliferative suppression. Reduction of AIB1 in human MCFDCIS cells restored a more normal mammary acinar structure with basement membrane in 3D growth in Matrigel. Then, we studied the effect of regulating AIB1 on the histopathology development of the DCIS lesions both prior to DCIS development or in existing MCFDCIS lesions in a xenograft mouse model by subcutaneously injecting MCFDCIS cells deficient or not in AIB1 into nude mice. The MCFDCIS xenografted tumors obtained reproduced the human breast cancer progression and displayed multiple comedo DCIS-like lesions. Decreased levels of AIB1 in MCFDCIS cells, inhibited tumor growth and led to smaller, necrotic lesions. To investigate how AIB1 affects initiation and progression of DCIS, we compared global gene expression changes in MCFDCIS cells +/-AIB1 shRNA in vitro using cDNA array analysis. Among the most significant expression changes observed were genes that are differentially expressed in Breast Cancer Initiating Cells (BCIC) including CD24 and members of the NOTCH signaling pathway (DLL1 and DLL3). We demonstrated by flow cytometric and immunohistochemical analysis that AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24-/CD44+ BCIC population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. Loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, HER2 and HER3 in vivo. Conclusions: These data indicate that AIB1 plays a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through maintenance of BCIC, facilitates formation of DCIS, a necessary step prior to development of invasive disease. Thus, targeting AIB1 may represent a new therapeutic paradigm especially for women with AIB1-positive primary tumor. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-03.

Introduction: Breast cancer is the most common cancer among South African women, where as in many other Sub-Saharan countries, is the leading cause of female death. Although several risk factors can be attributed to their observed high mortality rate, the biology of their tumors are not extensively investigated; consequently the molecular profiles associated with their disease is poorly characterized. Main goal: In this study our main goal was to characterize the wide-genomic copy number profile of a group of patients with breast cancer diagnosed at the Groote Schuur Hospital, Cape Town, South Africa and associate with their corresponding clinical-pathological data. Material and Methods: Twenty-eight specimens of formalin fixed paraffin embedded (FFPE) material from twenty-eight patients with invasive breast cancer were acquired prior to any treatment and with complete clinical annotation from the archives of the Division of Anatomical Pathology, National Health Laboratory Service-Groote Schuur Hospital. The mean age and tumor size of the patients at diagnosis was 48.2±12.9 years and 38.2±14.0 mm, respectively. Most of the tumors were of grade 2 and 3 (39.2% and 37.7%, respectively) and stage III (67.8%). ER and ERBB2 positivity was observed in 57.1% and 53.8% of the patients, respectively. PR status was available for only 21.4% of the patients, out of which 33% (2/6) were positive. Three patients were negative for ER, PR and ERBB2 receptors. Race information was self-reported; 61.4% of the patients were from the Colored group, followed by 19.2% from the White and Black group, which are the three most predominant ethnic groups in South Africa. Array-CGH analysis was performed in microdissected tumor areas using the Agilent 8x60K oligo nucleotide array platform. Results: The average number of copy number alterations (CNAs) observed in these patients was 8.3±1.2 which was significantly associated with patients' tumor grade (P Conclusions: Our results indicated that CNAs occur in South African breast cancer patients, in association with advanced tumor grades, but irrespectively of their ethnic descendance and other factors at diagnosis that are known to influence prognosis. Additional molecular studies, such as ours, that aim to determine the biologic contributions to breast-cancer risk in women in South Africa and other African countries are imperative. The understanding of the genetic determinants that are associated with the etiology of their tumors is a direction towards the reduction of their observed high mortality rates. Citation Format: Kamil Lupicki, Selene Elifio-Esposito, Aline S. Fonseca, Akanksha Mahajan, Silma R. Pereira, B Langa, Dhirendra Govender, Eugenio Panieri, Donavon Hiss, S Abdul-Rasool, Luciane R. Cavalli. Copy number profiling in South African breast cancer patients [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A14.

One-third of all estrogen receptor (ER)–positive breast tumors treated with endocrine therapy fail to respond, and the remainder is likely to relapse in the future. Almost all data on endocrine resistance has been obtained in models of invasive ductal carcinoma (IDC). However, invasive lobular carcinomas (ILC) comprise up to 15% of newly diagnosed invasive breast cancers each year and, whereas the incidence of IDC has remained relatively constant during the last 20 years, the prevalence of ILC continues to increase among postmenopausal women. We report a new model of Tamoxifen (TAM)-resistant invasive lobular breast carcinoma cells that provides novel insights into the molecular mechanisms of endocrine resistance. SUM44 cells express ER and are sensitive to the growth inhibitory effects of antiestrogens. Selection for resistance to 4-hydroxytamoxifen led to the development of the SUM44/LCCTam cell line, which exhibits decreased expression of ERα and increased expression of the estrogen-related receptor γ (ERRγ). Knockdown of ERRγ in SUM44/LCCTam cells by siRNA restores TAM sensitivity, and overexpression of ERRγ blocks the growth-inhibitory effects of TAM in SUM44 and MDA-MB-134 VI lobular breast cancer cells. ERRγ-driven transcription is also increased in SUM44/LCCTam, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity. These data support a role for ERRγ/AP1 signaling in the development of TAM resistance and suggest that expression of ERRγ may be a marker of poor TAM response. [Cancer Res 2008;68(21):8908–17]

NKX3.1 is a prostate-specific homeoprotein and tumor suppressor that is affected by the loss of 8p21 in prostate cancer. In mice, Nkx3.1 haploinsufficiency results in prostatic dysplasia and complements cancer formation induced by loss of other suppressor genes. However, NKX3.1 expression can be immunohistochemically detected in most primary prostate cancers. We examined the relationship between suppressor gene haploinsufficiency, methylation, and quantitative NKX3.1 expression levels in primary prostate cancer. NKX3.1 gene copy number was assessed by microsatellite analysis, fluorescence in situ hybridization, and quantitative PCR. NKX3.1 gene methylation was determined in prostate cancer cell lines and we thereby identified potential CpG methylation sites for methylation-specific PCR analysis in tissues. We validated and then applied an internally controlled fluorescence immunomicroscopic assay for NKX3.1 protein expression in 48 primary prostate cancer specimens from radical prostatectomies. NKX3.1 loss of heterozygosity was found in 27 of 43 tissues tested. Classic CpG island methylation of the NKX3.1 gene was not found in either prostate cancer cell lines or tissues. However, in 33 of 40 samples tested, CpG sites at −921, −903, and −47 were methylated to a greater degree in malignant than in adjacent normal cells. In 43 of 48 samples, NKX3.1 protein expression was reduced from 0.34 to 0.90 compared with adjacent normal luminal epithelium (mean of all samples, 0.68; 95% confidence interval, 0.05). In 12 cases that also had high-grade prostatic intraepithelial neoplasia, NKX3.1 expression levels were similar in preinvasive and invasive cancer cells and significantly lower than adjacent normal cells. Even in the presence of allelic loss, NKX3.1 expression is reduced over a wide range in prostate cancer at the time of prostatectomy, suggesting that diverse factors influence expression. Samples with protein expression below the median level in cancer cells had both NKX3.1 deletion and selective CpG methylation.

Prostate cancer is the second most commonly diagnosed neoplasia in men. Currently, there is no available non-invasive tool to assist in the identification of the aggressive form of this disease. Cell-free nucleic acid has been tested as a potential new molecular tool that can be useful in the diagnosis and prognosis of prostate cancer. MicroRNAs are a class of non-coding RNAs, that are shown to impact the development and progression of prostate cancer and their detection in plasma have been indicated as a promising non-invasive tool for disease screening and prognosis assessment. Among the several deregulated miRNAs that are observed in prostate cancer, the miRNA-141-3p was observed with upregulated expression levels in prostate cancer, particularly in the metastatic lesions. Therefore, the main aim of this study was to verify the diagnostic potential of miR-141 as a circulating tumor marker for prostate cancer and determine its functional role in modulating tumorigenesis in prostate cancer metastatic cell lines. MiR-141 expression analysis was performed in plasma samples of 102 prostate cancer patients without treatment and of 50 health controls by RT-qPCR. The experimental assays were performed by transfecting the PC3 cell line using miRNA-141 mimic/ inhibitor systems and directly accessing their effect in classical tumor phenotypes. Expression of the miR-141 were found significantly upregulated in the metastatic patients’ plasma specimens (FC=9.11, p=0.04; AUC=0.66) when compared to the patients without metastasis, showing its screening potential for prostate cancer metastasis. The in vitro ectopic expression of miR-141 in PC3 cell line showed a significant increased in cell proliferation, associated with changes in cell cycle (decreased number of cells in the G1 phase). Conversely, the inhibition of miR-141 showed an increase in the migratory ability, cell adhesion and in the Docetaxel cytotoxicity of the PC3 cells. Western-blot analysis showed that the inhibition of the miR-141 levels reduced the expression of the EMT inducers markers E-CADHERIN and CLAUDIN, and up-regulated the levels of ZEB-1, pAKT and VIMENTIN, consistent with their repression roles in the EMT process. These findings showed that miR-141 presents a direct function in controlling some of the in vitro metastatic phenotypes in prostate cancer, through the regulation of the epithelial-mesenchymal transition expression proteins. In conclusion, miR-141 presents a potential use as a minimally invasive molecular marker for prostate cancer diagnosis and can be used as a druggable target for therapy, particularly in the metastatic clinical setting. Citation Format: Marilesia Ferreira de Souza, Ilce Mara de Syllos Cólus, Aline Simoneti Fonseca, Hellen Kuasne, Paulo Emilio Fuganti, Deepak Kumar, Silvia Regina Rogatto, Luciane Regina Cavalli. Cell-free miR-141 as a molecular marker for prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1476. doi:10.1158/1538-7445.AM2017-1476

Neuroblastoma (NB) is a tumor derived from neural crest cells, primitive progenitors of sympathetic ganglia. It accounts for 6-10% of all pediatric cancers with approximately 700 new cases per year throughout the US. Although there are highly effective therapies for patients with low-risk and intermediate-risk disease who have local relapses, recurrent disease in patients with high-risk NB is mostly refractory to therapy. Neuropeptide Y (NPY) is a sympathetic neurotransmitter highly expressed in NB. Its elevated release from tumor tissue is associated with unfavorable clinical outcome. NPY, acting via its Y5 receptor (Y5R), stimulates NB cell survival and chemoresistance, however the mechanisms underlying NPY/Y5R axis activation in these tumors remain unclear. The aim of this work was to investigate the correlation between the expression of NPY and NPY5R proteins and the copy number status of the NPY and NPY5R genes in pre- and post-chemotherapy NB. Eighty-five tissue samples, including specimens from the primary tumors, distant metastases and local relapses, pre- and post-chemotherapy, were collected from the Hospital Pequeno Principe, Parana, Brazil. Protein expression was investigated by immunohistochemistry. Copy number alterations (CNAs) for NPY and NPY5R genes were determined using TaqMan copy number assay. Additionally, FISH analysis was performed to assess MYCN amplification status, a genetic marker of high risk NB. Our results show that elevated extracellular NPY staining, which reflects peptide release, correlates with patients’ age above 18 months, relapse and poor clinical outcome. Moreover, the intensity of NPY staining was increased in chemotherapy-treated NBs, as compared to tumors at diagnosis. However, the differences in NPY CNAs between these samples were not statistically significant. Thus, chemotherapy-induced increase in NPY levels observed in post-treatment NB is driven by its elevated expression rather than genomic changes. For NPY5R, significantly higher level of CNAs was observed in the post-chemotherapy samples (P Citation Format: Selene Elifio-Esposito, Akanksha Mahajan, Aline S. Fonseca, Susana Galli, Lucia Noronha, Lisiane C. Poncio, Bonald C. Figueiredo, Joanna B. Kitlinska, Luciane R. Cavalli. Increase in protein expression and copy number drives the activation of NPY/Y5R pro-survival loop in chemotherapy-treated neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5822. doi:10.1158/1538-7445.AM2017-5822

Triple negative breast cancer (TNBC) disproportionately afflicts young African American (AA) women when compared to Non-Hispanic Whites (NHW), being usually diagnosed in advanced stage, likely to experience metastasis and often unresponsive to treatment. An increased number of studies have characterized the differences in the tumor biology between AA and NHW patients and have shown that breast tumors from AA patients present increased cell proliferation, elevated expression of angiogenesis markers and higher migration and invasive properties, the fundamental hallmarks of cancer. MiRNAs are short sequences of non-coding RNAs that act on gene expression regulation. In this study, our main objective was to determine the expression levels of miRNA-661 in the TNBC of AA when compared to the NHWs and to verify it is directly role in affecting the aggressive TNBC phenotype. MiRNA-661 expression analysis was conducted by RT-qPCR in the tumor tissue of 31 AA-TNBC patients and 17 NHW-TNBC patients. AA and NHW samples of 40 and 20 non-TNBC subtype, respectively, and 49 adjacent normal tissue (ANTs) were used as control for the subtype and tumor specificity, respectively. Our results showed a significant down-regulation of miR-661 expression in TNBC of AA in relation to the NWH (P Citation Format: Aline S. Fonseca, Selene Elifio-Esposito, Marilesia F. Souza, Akanksha Mahajan, Yara R. Zabala, Bruna M. Sugita, Luciane R. Cavalli. Regulatory role of miRNA-661 in triple-negative breast cancer of African American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1465. doi:10.1158/1538-7445.AM2017-1465

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that confers disease recurrence, treatment resistance and high mortality rates. MicroRNAs are a class of noncoding RNAs, that when dysregulated, impact tumorigenesis through the control of the expression of multiple mRNA targets involved in critical cancer signaling pathways. MiR-150-5p has been shown to control the expression of several driver oncogenes and/or tumor suppressor genes involved in these critical pathways. Its pattern of expression varies according to the cancer type; it has been observed mostly downregulated in hematological diseases and GI cancers, and upregulated in hormonal dependent cancers, such as prostate and breast cancer. The main objective of this study was to assess the patterns of expression of miR-150-5p in TNBC and determine its functional role in affecting the tumor phenotype. Archived paraffin samples of 113 patients with ductal breast carcinoma (56 of the TNBC and 57 of the non-TNBC subtype) and 49 adjacent normal tissue (ANT), obtained from the the pathology tumor bank of Lombardi Comprehensive Cancer Center, Washington DC, were profiled for miRNA using the wide-genome Nanostring platform and a Taqman specific miRNA-150-5p assay. Significant overexpression levels of miRNA-150-5p were observed in the tumor tissues when compared to the ANT and in the TNBC cases when compared to the non-TNBC cases, demonstrating its tumor and TNBC subtype specificity, respectively. Overexpressed levels of miRNA-150-5p were also preferentially observed in the TNBC cases from patients that presented with LN metastasis and breast cancer recurrence, indicating its association with poor prognosis. Interestingly, the TNBC of African-American patients, which is the ethnic group mostly affected by this cancer subtype, presented overexpression levels of this miRNA when compared to the Non-Hispanic White patients. Functional analysis performed in the TNBC cell lines, MDA-MB-231 and HCC1806, showed after transfection with miR-150-5p inhibitor, reduced levels on cell proliferation, clonogenicity, migration, drug resistance and expression of the EMT promoter markers, SLUG and SNAIL. These findings, indicate an oncogenic type of action of miRNA-150-5p in TNBC. In summary, miRNA-150-5p is upregulated in TNBC clinical cases in association with poor prognostic parameters and its functional inhibition, directly confers to the cells a reduction of their tumorigenic phenotype. Funding: This project was supported by the Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration to promote regulatory science through innovative research and education. This research does not necessarily reflect the views of the FDA. Scholarship to B.S. was provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Citation Format: Bruna M. Sugita, Yara Zabala, Aline Fonseca, Rodrigo Almeida, Yuriy Gusev, Simina Boca, Iglenir J. Cavalli, Enilze M. Ribeiro, Luciane R. Cavalli. The oncogenic role of miR-150-5p in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3431. doi:10.1158/1538-7445.AM2017-3431

Triple negative breast cancer (TNBC) - defined as estrogen receptor- (ER), progesterone receptor- (PR), and human epidermal growth factor receptor 2- (HER2) negative - is a highly aggressive form of breast cancer prevalent in African-American (AA) women. Because ER- and HER2-targeted therapies are ineffective in TNBC, current treatment strategies rely on cytotoxic chemotherapy. There is a tremendous need for new approaches that can identify effective therapies with less toxicity for this devastating form of breast cancer. Nuclear receptors are highly druggable targets. Classical examples include ligand-regulated receptors like ER, but orphan nuclear receptors are also emerging as targets for cancer therapy. We recently published that increased mRNA expression of estrogen-related receptor beta (ERRβ, gene symbol ESRRB) correlates with better recurrence- and distant metastasis-free survival in women with TNBC, and that a small molecule agonist ligand for ERRβ (DY131, DY) has growth inhibitory and anti-mitotic activity in TNBC cell lines of multiple molecular subtypes. The goal of our current work is to validate ERRβ as a novel therapeutic target for TNBC in two distinct ways. First, we sought to determine whether ERRβ is correlated with survival outcome specifically in African-American (AA) women. Analysis of The Cancer Genome Atlas (TCGA) Breast Cancer RNAseq data shows that low ERRβ mRNA expression is specifically associated with poor overall survival in AA women with TNBC. This association is not observed in AA women with ER- or HER2-positive disease. We further find that while ERRβ mRNA expression is decreased in tumor vs. normal breast tissue regardless of race, this decrease is more pronounced in AA women. Ongoing studies in an independent cohort of AA and white women will determine whether this is due, in part, to copy number changes at the ERRβ locus (chromosome 14q24.3). Second, we sought to determine whether ERRβ expression could predict response to current treatment strategies commonly used in breast cancer, particularly TNBC. Using multiple combined datasets, we find that high ERRβ expression is associated with improved recurrence-free survival (RFS) in women with TNBC who received adjuvant or neoadjuvant therapy with anthracycline (e.g. doxorubicin, Adriamycin). No association between ERRβ and RFS is observed in patients treated with taxanes. Ongoing studies in TNBC cell lines seek to determine if activating ERRβ with its agonist ligand DY can enhance doxorubicin-mediated growth inhibition and cell death. Citation Format: Aileen I. Fernandez, Garrett Graham, Balázs Győrffy, Luciane Cavalli, Rebecca B. Riggins. ERRβ: Validating a novel target for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4131. doi:10.1158/1538-7445.AM2017-4131

Ewing sarcoma (ES) is a tumor driven by EWS-ETS fusion proteins. Yet, the same fusions are present in localized and metastatic tumors that carry strikingly different prognoses. Despite low levels of genomic instability in primary ES tumors, the presence of complex karyotypes is one of a few adverse prognostic factors, implicating an acquired chromosomal instability (CIN) in ES progression. As transcriptional targets of EWS-ETS, neuropeptide Y (NPY) and its Y5 receptor (Y5R) are highly expressed in ES and further activated by hypoxia. We have found that overexpression of Y5R leads to defects in cytokinesis, followed by formation of polyploid cells, chromosome loss and CIN. Thus, the goal of our study was to determine whether CIN that is driven by hypoxia-induced activation of NPY/Y5R axis promotes ES metastases. ES cells were injected into gastrocnemius muscles of SCID/beige mice. Hypoxia in the resulting primary tumors was created by 72h ligation of the femoral artery. Then, the tumors were excised and mice were monitored for metastases. Tissues and cells derived from primary tumors and metastases were subjected to cytogenetic analyses. ES metastasis was associated with progressive genomic changes in tumor cells. Cells derived from primary tumors exhibited increases in nuclear sizes and ploidy, as compared to the original cells. Tumor hypoxia exacerbated this effect. This initial increase in ploidy was followed by a decrease in nuclear size, increase in mitotic errors and reduced chromosome numbers in cells from metastatic tissues, suggesting that ES progression associates with increased CIN and is triggered by cell polyploidization. This notion was confirmed by increased DNA copy number alterations in tissues from ES metastases observed in xenografts derived from 2 different cell lines and a clinical case of matched primary tumor and metastasis tissue (array-CGH). In SK-ES1 xenografts, these alterations involved gains in the locus of Y5R. Consequently, FISH identified an SK-ES1 clone with 3 copies of the Y5R gene. The percent of cells with Y5R gene amplification increased with the degree of SK-ES1 progression, with 16-24% cells in the original SK-ES1 cell line, 40-60% in primary tumors and 86-100% in metastases. This was associated with an increase in Y5R expression in metastatic tissues. Thus, the metastasis in SK-ES1 xenografts associated with a selection of the clone with amplified Y5R. SK-ES1 cells subjected to hypoxia in vitro presented with similar increases in nuclear sizes and enrichment in the clone with amplified Y5R (48%), as was observed in primary tumors. Y5R activation in normoxic SK-ES1 cells mimicked this effect. Our findings support the role for acquired CIN in ES progression and metastasis and implicate the hypoxia-induced activation of the NPY/Y5R axis as its potential trigger. Thus, Y5R antagonist may serve as an adjuvant treatment to prevent ES CIN and progression. Citation Format: Akanksha Mahajan, Sung-Hyeok Hong, Jason U. Tilan, Susana Galli, Congyi Lu, Jasmine Rodgers, Anju Duttargi, Rachel Acree, Luciane R. Cavalli, Joanna B. Kitlinska. Ewing sarcoma progression associates with increasing chromosomal instability: A role for neuropeptide Y and its Y5 receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2443.

Triple negative breast cancer (TNBC) is a clinically and molecularly heterogeneous disease, which incidence and outcome vary among the different ethnic and racial groups. These tumors are more commonly seen in younger women of African and Latinas/Hispanic descents, usually diagnosed at more advanced stages, with non-localized disease. Molecular studies have shown differences in the biology of these tumors in Latinas as key contributors for high mortality. The main objective of our study was to identify a miRNA signature associated with TNBC of Latina patients by integrating miRNA and array-CGH data. Archived paraffin samples of 32 TNBC and 25 non-TNBC cases from Latina patients, obtained from the Clinical Hospital (UFPR) in Brazil, were profiled for miRNA and array-CGH using the Nanostring and the Agilent SurePrint G3 Human array-CGH platforms, respectively. The miRNA and array-CGH data, obtained in the same samples, was directly integrated and combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems were performed to identify the most relevant miRNAs and their corresponding gene targets. Eight-nine miRNAs were observed differentially expressed between the TNBC and non-TNBC lesions. Using miRBase and MiRDB target prediction databases 3,378 miRNAs targets were identified. After integration with array-CGH, a number of 15 miRNAs presented concomitant DNA copy number and miRNA alterations, reducing the number of targets to 1,242. Ingenuity pathway analysis identified the most affected canonical pathways, including IL-8, TGF-beta, PI3K-AKT and HER2 signaling pathways. Using our integration approach we were able to identify a robust miRNA signature associated with TNBC of Latina patients and to identify the potential molecular mechanism (s) that underline the observed miRNA deregulation in these patients. This signature revealed miRNAs and corresponding targets biologically relevant, involved in critical cancer signaling pathways and gene networks. Once this signature is functionally validated, its direct role in the aggressive clinical phenotype of these tumors will be determined. In summary, the findings of our study contributes to the identification of race/ethnic specific molecular targets that can set the basis for new TNBC treatments and for the future design of the most appropriate clinical trials and cancer control interventions for Latina women. Funding:This project was supported by the Georgetown University Center of Excellence in Regulatory Science and Innovation (CERSI; U01FD004319), a collaborative effort between the university and the U.S. Food and Drug Administration to promote regulatory science through innovative research and education. This research does not necessarily reflect the views of the FDA. Citation Format: Bruna M. Sugita, Mandeep Gill, Silma R. Pereira, Yara R. Zabala, Aline S. Fonseca, Selene E. Esposito, Catalin Marian, Iglenir J. Cavalli, Enilze MF Ribeiro, Yuriy Gusev, Luciane R. Cavalli. Integrated analysis of copy number and miRNA profiling in triple negative breast cancer of Latina women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1944.

In the African-American (AA) population, an increased incidence and shorter survival rate of triple negative breast cancer (TNBC) has been reported when compared to White women, with their cancer usually diagnosed in more advanced stages and with non-localized disease. The main objective of our study is to identify molecular markers in TNBC in AA patients that may be associated with their observed disparity in incidence and mortality rates. Formalin-fixed paraffin-embedded tumor sections were obtained from 26 and 28 cases of TNBC from AA and Caucasian breast cancer patients, respectively, and analyzed for DNA copy number (Agilent-SurePrint G3 Human CGH Microarray 8×60K) and miRNA expression (Nanostring System) alterations. The cases were obtained with clinically annotated data from the Histopathology Shared Resources at the Lombardi Comprehensive Cancer Center. The miRNA data was directly integrated with the array-CGH data from the same cases. Combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems were performed to identify predicted target functions. A distinct DNA copy number profile was observed between TNBC of AA and Caucasian cases: the cytobands differentially involved in the AA-TNBC cases were 7p22.3-22.1, 12p13.33-p11.1, 17q25.3 and 17p13.3-p11.2. Common cytobands in both groups were 1q21.1-q44, 8q22.1-q24.3, 16p13.33-p11.1 and 20q11.2-q13.12. An average of 30.5 and 16.8 DNA copy alterations were observed between the AA and Caucasian cases respectively and the miRNA profiling revealed 209 miRNAs differentially expressed between the groups. Unsupervised hierarchical clustering analysis separated the cases by ethnicity (with the exception of 6 cases). Among the miRNAs observed with higher and lower expression levels in the AA cases were miR150-5p, miR4286, miR29b3p and miR1283, miR1253, miR378e, respectively. Ingenuity pathway analysis identified molecular and cellular functions mostly related to cellular development, growth, proliferation and movement, and cell death and survival. Clinical pathological data analysis revealed higher tumor grades and stages in the TNBC of AA women when compared to Caucasians; association of these parameters with the genomic and expression miRNA data is being performed. In conclusion, a distinct DNA copy number and miRNA profile was observed in the AA-TNBC cases when compared to the Caucasians cases of our study. These initial findings will provide the basis for the functional analysis of the identified molecular markers and their potential prognostic and therapeutic impact for this group. In addition to providing a better understanding of the biology of their tumors, these analyses can reveal molecular targets for tailored and ethnic-based clinical trials, which can ultimately lead to the reduction of the observed cancer disparity. Support: FDA U01 FD004319, NIH-NIGMS GM101997 and Susan Komen Foundation. Citation Format: Kenny Regis, Michelle Jaldin, Akanksha Mahajan, Anju Duttargi, Laura Sheahan, Yuriy Gusev, Subha Madhavan, Luciane R. Cavalli. Array-CGH and miRNA expression profiling of triple negative breast cancer in African-American women. [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 4818. doi:10.1158/1538-7445.AM2015-4818

Liver cancer is one of the major human tumors in the world. Basic and epidemiological studies have proposed that the major risk factors for liver cancer include alcohol and diet as well as infection with hepatitis B and C viruses. However, the mechanistic clues for the development of this type of cancer is largely unknown. Poly(ADP-ribose) polymerase (PARP-1) and a component of nonhomologous end-joining (NHEJ) machinery, Ku80, are two major DNA end-binding molecules that play a multifunctional role in DNA damage signaling and repair, recombination as well as the maintenance of genomic stability. Here we show that the interaction of PARP-1 and Ku80 is essential for development because PARP-1/Ku80 double null mice died at embryonic day (E) 9.5. Interestingly, haplo-insufficiency of Ku80 in PARP-1-/- mice promotes the development of hepatocellular adenoma and hepatocellular carcinoma (HCC). These tumors exhibited a multistage tumor progression associated with the loss of E-cadherin expression and the mutation of beta-catenin. Cytogenetic analysis revealed that Ku80 heterozygosity elevated chromosomal instability in PARP-1-/- cells and that these liver tumors harbored a high degree of chromosomal aberrations including fragmentations, end-to-end fusions, and recurrent nonreciprocal translocations (NRT). These features are reminiscent of human HCC. Taken together, these data implicate a synergistic function of Ku80 and PARP-1 in minimized chromosome aberrations and cancer development and suggest that defects in DNA end-processing molecules may be etiological factors in human HCC formation.

Background: Triple negative breast cancers (TNBC) lack the expression of ER, PR and HER2 receptors, making targeted treatments against these receptors ineffective. We are studying BETA PROTEIN 1 (BP1), a member of homeobox gene family, which we cloned and showed is activated in 80% of invasive breast cancers; its expression correlates with breast cancer progression and invasion. The BP1 gene is located on 17q21, a region that we showed is altered in 30% of breast tumors. BP1 protein has been shown to regulate genes including BCL2, BRCA1, VEGF and c-MYC. Also BP1 mRNA and protein are highly expressed in ER and PR negative and clinically aggressive tumors. Thus we hypothesize that BP1 may play a role in TNBC. Methods: To delineate pathways in TNBC cells that are dysregulated by BP1 expression, we studied the 5 TNBC parental cell lines, 2 non-TNBC cell lines and a stably overexpressed BP1 TNBC cell line. Microarray analysis was performed in all these cell lines and the data obtained was verified using qPCR and ELISA. A BP1 shRNA plasmid was stably transfected into MDA-MB-231 cell lines to generate sh-MDA-MB-231 cell lines with about 80% knockdown of BP1 protein. ELISA was performed to verify the effects of BP1 expression on target genes. Formalin-Fixed, Paraffin-Embedded (FFPE) tissues of TNBC were analyzed by array-CGH and confirmed by Taqman copy number (using a BP1-TAM specific probe) and FISH (using our constructed BP1-BAC probe) assays. Results: All of the TNBC cell lines had statistically more BP1 protein (pBP1) than non-TNBC cell lines, suggesting that TNBC tumors may have higher levels of pBP1 than non-TNBC tumors. Gene expression microarrays in the TNBC cell lines revealed up/down regulation of several genes in comparison to the normal breast cell lines, including the IL-6 and IL-8 genes, which were significantly upregulated in TNBC cells overexpressing BP1. ELISA assays confirmed a direct correlation between elevated levels of IL-6 in conditioned media and overexpression of BP1. Knockdown of BP1 led to a decrease in secreted IL-6. Studies are underway to establish the mechanism by which these pathways are regulated by BP1. Increased BP1 copy number was observed in 37% (25/67) of the clinical cases and all (8/8) of TNBC cell lines by array-CGH. Confirmation of these changes by FISH and TaqMan copy number assays revealed increased copy number in 22% (18/82) and 23% (9/38) of the clinical cases, respectively. Protein expression analysis by IHC is currently being conducted to determine if BP1 increased DNA copy number may be one of the mechanisms leading to pBP1 overexpression. Conclusions: Both BP1 copy number and protein expression are dysregulated in TNBC. Microarray analysis in TNBC cell lines have revealed important cancer related genes and oncogenic pathways that may be upregulated by BP1, suggesting that BP1 could be a valuable molecular marker and therapeutic target in this aggressive form of breast cancer. Citation Format: Saurabh Kirolikar, Mandeep Gill, Silma Pereira, Svetlana Ghinbouschi, Luciane Cavalli, Patricia Berg. The BP1 homeobox gene is dysregulated in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1409. doi:10.1158/1538-7445.AM2014-1409

Introduction: Triple negative breast cancers (TNBC) are clinically aggressive tumors that lack the expression of ER, PR and HER2 receptors, and therefore do not respond effectively to the available target therapies. The study of the molecular alterations that are present in these tumors can lead to the identification of potential therapeutic targets that can improve patient's outcome. MicroRNAs (miRNAs) are short non-coding sequences that play a role in breast tumorigenesis regulating the expression of cancer-associated genes. In the present study, we investigated miRNAs expression profile in TNBC and non-TNBC tumors and integrated the data with DNA copy number changes (array-CGH) obtained from the same samples, to obtain the most relevant miRNA targets that may be involved in the pathogenesis of TNBC. Methods: Formalin-fixed paraffin-embedded samples from 43 TNBC and 16 non-TNBC cases were profiled for miRNA using the Nanostring system. Significant differentially expressed miRNAs (with at least 2 fold differences and p≤0.05) between the lesions were calculated by the comparative Ct method (ΔΔCt). DNA copy number analysis from the same samples was performed using an Agilent array-CGH platform. The miRNA data was directly integrated with the array-CGH data from the same cases. Combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems (Ingenuity Pathway, Pathway Studio) were performed to identify predicted target functions. Results: The miRNA profiling revealed 89 miRNAs significant differentially expressed between the TNBC and non-TNBC lesions. Using miRBase and MiRDB target prediction databases we identified 3,378 target genes of upregulated miRNAs and 823 for downregulated miRNAs. A number of 15 miRNAs (out of the 89) presented concomintant genomic gains or losses and miRNA increase or decrease expression, respectively. This direct integration of the data reduced the number of miRNA targets to 1,242. Ingenuity pathway analysis on these predicted targets function, identified canonical pathways, including p53, IL-8 and Rac signaling. Gene expression profiling analysis is underway to confirm the regulatory effect of the selected miRNAs on the targets identified. Conclusions: We have observed significant differentially expressed miRNAs in the analysis of TNBC cases in comparison with non-TNBC cases. The integration analysis with DNA copy number data let us to select the miRNAs with concomitant changes in copy number and gene expression and predicted target algorithms identified key pathways that may be related to the TNBC phenotype. The functional validation of these targets, by in vitro and in vivo experimental models, through the assessment of miRNA-mRNA interaction and/or modulation of miRNA expression will allow us to identify the most relevant miRNAs that are mechanistically involved in the pathogenesis of TNBC. Note: This abstract was not presented at the meeting. Citation Format: Mandeep Gill, Bruna Sugita, Silma R. Pereira, Catalin Marian, Xi Li, Yuriy Gusev, Enilze MSF Ribeiro, Iglenir J. Cavalli, Luciane R. Cavalli. Identification of microRNA targets in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1545. doi:10.1158/1538-7445.AM2014-1545

Introduction: MicroRNAs (miRNAs) are a class of non-coding endogenous RNA molecules that play a role in the invasion and metastatic breast cancer process. Correlation of miRNA alterations and lymph node (LN) breast cancer metastasis status are described, however most of the data are in advanced metastatic cancer cell lines and/or mouse mammary models. There are limited miRNA analysis that are conducted directly in axillary LN specimens and to our knowledge, there are no analysis performed specifically in sentinel lymph nodes (SLN), the first axillary metastatic site. Therefore our main objective is to identify the miRNA profile and its corresponding miRNA targets in paired samples of primary breast tumors (PBT) and SLN metastatic lesions. We hypothesize that these changes will represent the ones that occur in early stages of the axillary breast cancer metastatic process, before distant metastasis occurs. Methods: RNA was isolated from microdissected 50μm tumor sections of formalin-fixed paraffin-embedded from 30 paired samples of PBT and SLN metastasis. MiRNA profiling was performed using the Nanostring miRNA system and significantly differentially expressed miRNAs (with at least 2 fold difference and p≤0.05) between the lesions were calculated by the comparative Ct method (ΔΔCt). The miRNA data was integrated with previous array-CGH data from the same cases, to select for targets with concomitant copy number and miRNA expression alterations. Combinatorial target predicted algorithms in conjunction with functional and pathway annotation enrichment systems were performed to identify predicted target functions. Results: The miRNA profiling of the paired PBT and SLN metastasis, revealed 25 miRNAs significantly differently expressed between these lesions. Among the significant miRNAs observed, were the miR-24-3p, miR-221-3p and miR-99a-5p, which are involved in epithelial-mesenchymal transition (EMT) of metastatic cells. Using miRBase and MiRDB target prediction databases we identified 4691 target genes of deregulated miRNAs, that were reduced to 41 after integration with DNA copy number data from the same cases. Ingenuity pathway analysis on these predicted targets function, identified 73 canonical pathways, including the cyclin D1 cell cycle regulation, p53 and cAMP-mediated signaling. Conclusions: The miRNA profiling in our set of paired PBT and SLN metastasis revealed significant differently expressed miRNAs between these lesions. The selection of the most relevant miRNA targets in the pathways identified with a role in tumor invasion/progression are underway and will be evaluated for its co-expression with the corresponding miRNAs. Finally, the direct functional validation of these targets, by in vitro and in vivo experimental models, will allow us to identify the most relevant ones that are mechanistically involved in the initial stages of axillary breast cancer metastases. Citation Format: Mandeep Gill, Catalin Marian, Clarissa Torresan, Enilze Ribeiro, Iglenir J. Cavalli, Yuriy Gusev, Xi (James) Li, Luciane R. Cavalli. Identification of miRNA targets in axillary breast cancer metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2013-5333

Background: Triple negative breast cancers (TNBC) lack the expression of ER, PR and HER2 receptors, thus targeted treatments against these receptors are not effective. We are studying BETA PROTEIN 1, a member of homeobox gene family, which we cloned and showed is activated in 80% of invasive breast cancers; its expression correlates with breast cancer progression and invasion. The BP1 gene is located on 17q21, a region that is reported to be altered in 30% of breast tumors. Since BP1 mRNA and protein are highly expressed in ER and PR negative and clinically aggressive tumors, we hypothesize that BP1 may play a relevant role in TNBC. Our main objective was to evaluate the gene copy number and mRNA/protein expression status of BP1 in TNBC, in both clinical cases and established TNBC cell lines. Methods: We have characterized seven cell lines (5 TNBC and 2 normal breast cell lines) for the expression of BP1 using qPCR and Western blot analysis. To delineate pathways in TNBC cells that are dysregulated by BP1 expression, we studied both the seven parental cell lines and a stably overexpressed BP1 TNBC cell line, Hs578T-OE. Microarray analysis was performed in all these cell lines and the data obtained was verified using qPCR and ELISA. Formalin-Fixed, Paraffin-Embedded tissues of TNBC were analyzed by array-CGH and confirmed by Taqman copy number (using a BP1-TAM specific probe) and FISH (using our constructed BP1-BAC probe) assays. Results: All of the parental TNBC cells had at least 2.8-fold more pBP1 than the normal breast cell lines suggesting that TNBC tumors have higher levels of pBP1 than non-TNBC tumors. Gene expression microarrays in the TNBC cell lines revealed up/down regulation of several genes in comparison to the normal breast cell lines, including the EGFR. Interestingly, the IL-6 and IL-8 genes were significantly upregulated in TNBC cells overexpressing BP1. ELISA assays confirmed a correlation between elevated levels of IL-6 and IL-8 production and BP1 in the conditioned media from Hs578T cells overexpressing BP1 compared with the empty vector. Studies are underway to establish the mechanism by which these pathways are regulated by BP1. BP1 increase copy number was observed in 37% (25/67) of the cases by array-CGH. Confirmation of these changes by FISH and TaqMan copy number assays revealed increased copy number in 20% (13/66) and 23% (9/38) of the cases, respectively. Protein expression analysis by IHC is currently been conducted in these cases to determine if BP1 increase DNA copy number can be one of the mechanisms that leads to pBP1overexpression. Conclusions: Both BP1 copy number and protein expression are dysregulated in TNBC. Microarray analysis in TNBC cell lines have revealed important cancer related genes and oncogenic pathways that may be upregulate by BP1, suggesting that BP1 could be a valuable molecular marker and therapeutic target in this aggressive form of breast cancer. Citation Format: Mandeep Gill, Saurab Kirolikar, Svetlana Ghimbovschi, Luciane R. Cavalli, Patricia E. Berg. The Role of the BP1 homeobox gene in triple negative breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2013-1812

Triple negative breast cancer (TNBC) are defined by the lack of expression of ER, PR and HER2 receptors and characterized by an aggressive clinical history and poor outcome. Considering that these tumors do not present a good response to the available adjuvant target therapies (hormonal and HER-2 based therapies), there is a critical need to identify new markers to improve treatment options for these patients. The use of genome-wide methodologies is a progress towards this direction, through the identification of biomarkers that can be translated into novel target-based therapies. We report the genomic profiling performed by array-CGH (Agilent 4x44K) in 33 cases of TNBC obtained from Hospital N.S. das Gracas and Hospital das Clinicas (UFPR), Curitiba, South of Brazil. Copy number alterations in potentially therapeutic target genes for TNBC (involved in the PI3K-AKT pathway) were evaluated by TaqMan copy number assay (Applied Biosystems). The data was correlated with pathological parameters from the patients. Copy number changes were observed by array-CGH in 87% (25/30) of the TNBC cases analyzed, with a median of 9.97+12.37 alterations per case. Based on this value, the samples were divided into two categories (>10 and 0.05) or the presence of lymph node (LN) metastasis (≥2=1.8;P>0.10) and tumor grade (≥2=1.18;P>0.20). Although a high degree of molecular heterogeneity was observed, amplification on 8q was observed in 50% (15/30) of the cases, mostly involving 8q24.3. Tumor size was not significantly different among the cases with or without 8q amplification (t=1,88; P>0.20). These cases were also equally distributed in relation to LN metastasis (≥2=2.67;P>0.10) and tumor grade (≥2=0.5;P>0.50). Finally, alterations in at least one gene involved in the PIK3/AKT pathway were observed in 88% (29/33) of the cases and in more than three genes in 45% (13/29) of the cases. PIK3CA and PTEN were the most frequently altered genes: gained and lost in 42% and 40% of the cases respectively. AKT2, AKT1, and FGFR2 genes were observed gained in 24%, 21% and 21% of the cases respectively. Alterations in those genes were also not significantly correlated with tumor size (t=0.95; P>0.30), LN metastasis (≥2=0.16; P>0.50) and tumor grade (≥2=3.23; P>0.05). Our results indicate a high level of DNA copy number changes in TNBC cases, involving mostly chromosome 8q. Alterations on genes involved in the PIK3/AKT pathway were also common; immunohistochemistry analyses are in progress to evaluate those alterations at the expression level. No significant correlations were observed among the genomic alterations and the pathological parameters from the patients. This lack of correlation can be due to the limited sample size or can indicate that in our set of TNBC cases tumor size, LN metastasis and tumor grade are not associated with an elevated genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5100. doi:1538-7445.AM2012-5100

Background: Triple negative breast cancer (TNBC) is associated with poor prognosis and a high mortality rate when compared to the other breast cancer subtypes. These tumors lack the expression of ER, PR and HER2 receptors, and do not respond well to the currently available adjuvant therapies. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions. Alterations of genes in this pathway have been shown to predict responsiveness to cancer targeted therapies. In this study, five genes involved in this signaling pathway, PIK3CA, AKT1, AKT2, PTEN and FGFR, were evaluated for DNA copy number changes in two sets of TNBC specimens. Methods: TNBC samples were obtained from the Hospital Nossa Senhora das Gracas, Curitiba, PR, Brazil. Two sets of TNBC samples were analyzed: the first set was composed of 30 formalin fixed-paraffin embedded tissue specimens and were analyzed for the PIK3CA, AKT1, AKT2, PTEN and FGFR genes by FISH. Locus-specific probes were prepared from BAC contigs for each gene and a centromeric probe for chromosome 3 was used as a control for copy number. The second set was composed of 17 frozen samples and were analyzed by array-CGH using a 44K-Agilent array platform. Real-Time PCR (TaqMan assay), was used to validate the array-CGH data for the above five genes. Results: FISH: PTEN loss and FGFR3 amplification were the most common findings occurring in 56% and 41% of the TNBC samples respectively. PIK3CA, AKT2 and AKT1 were amplified in 31%, 25% and 9.5% of the samples respectively. Array-CGH/Real time PCR: DNA copy number changes were observed by array-CGH in all, but one TNBC sample. The gene with highest frequency of amplification detected by array-CGH was PIK3CA (amplified in 77% of the cases). These findings were confirmed in 70% of the cases. FGFR2, AKT2 and PTEN were amplified in 35%, 24% and 24% of the cases respectively, These findings were confirmed for FGFR2 in 83% of the samples and for PTEN in all the samples. AKT2 amplification was only confirmed in 25% of the samples. Finally AKT1 was observed amplified in by array-CGH in 6% of the cases, which was confirmed by Real-time PCR.Conclusions: Based on our initial results, we conclude that TNBC presents DNA copy number alterations in genes involved in the PIK3 signaling pathway. Upon validation of these alterations in a larger number of cases, with correlation to prognostic parameters, their potential use as therapeutic targets can be considered for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2011-3822

Background: BP1 is a homeobox gene which was reported to be amplified and overexpressed in breast tumors with poor clinical outcome. Its exact role and function in breast tumorigenesis is not known and no studies have investigated its role in familial breast cancer cases. BP1 is located on 17q21, a chromosomal region that is altered in a high percentage of breast tumors; CGH analysis of the 17q region demonstrated gains in 50% and 85% of BRCA1 and BRCA2 mutated familial breast tumors, respectively. BP1 has been found to be overexpressed in breast cancer cells defective in double-strand break repair by homologous recombination (BRCA defective) and has been shown to directly regulate BRCA1 expression. The above information formed our rationale to evaluate the amplification of BP1 in familial breast cancer patients with known BRCA1/2 mutation status. We hypothesized that BP1 would be preferentially amplified in BRCA1/2 mutations carriers and correlated with poor clinical prognostic factors. Methods: Three tissue microarrays, containing tumor tissues of a total of 67 familial breast cancer patients, with known BRCA1/2, ER and PR status and full clinical data, were obtained from the Familial Cancer Registry from Georgetown University. FISH analysis was performed using a specific BP1 FISH probe developed in our lab. Results: FISH scoring in 100 cells was performed in 87% of the patients. Amplification of BP1 was observed in 26% of the cases and was significantly correlated with the presence of mutations in the BRCA1/2 genes, as opposed to patients with full negative (FN), Jewish panel negative (JPN) and variant with unknown significance (VUS). BP1 amplification was also significantly correlated with large tumor size (>1.5cm) and poorly differentiated tumors. Upon stratification of the samples by histological type, a total of 173 lesions (from cores containing different lesions from the same patients) were analyzed. Similar frequencies of BP1 amplification were observed in invasive (24%) and ductal carcinoma in situ (23.6%) lesions. Conclusions: Our findings demonstrate that the BP1 homeobox gene is amplified in familial breast cancer patients and its amplification is correlated with the presence of mutations in the BRCA1/2 genes as well as poor clinical prognostic factors, such as large tumor size and poorly differentiated tumors. Studies evaluating the expression of BRCA1 and BP1 proteins in these samples are in progress, in order to identify possible mechanism of interactions and related pathways between BP1 positive and BRCA1 mutant tumors in familial breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3056. doi:10.1158/1538-7445.AM2011-3056

The sentinel lymph node (SLN) is the first node in the axilla to harbor malignant cells in breast tumors with metastasis, and its positivity is an indication for axillary lymph node dissection. Despite significant improvement in the methods for detection of tumor cells at this site, there are a still significant number of false negative cases and the inability to detect malignant cells in lower abundance, such as in the case of micrometastasis. We propose to use methods based on the genomic alterations, which present higher sensitivity and accuracy, and can be used to augment the current methods to identify the epithelial malignant cells at the SLN. In this study, we performed CGH and array-CGH analysis in microdissected tumors cells from the SLN metastatic lesions to identify DNA copy number changes. We compared the changes observed in the SLN lesion to the ones present in the corresponding primary tumors (PT) from the same patients. Here we present the results from the initial analysis of 10 FFPE (formalin-fixed paraffin-embedded) breast cancer samples (5 pairs of SLN and PT). DNA copy number changes were detected by both methods in all the cases analyzed. A remarkable similarity in the DNA profiles between the SLN metastatic lesions and the corresponding PT were observed, despite the high level of heterogeneity observed among the cases. CGH analysis revealed most frequently gains of chromosomal regions (at both lesions) involving 1p, 1q, 6q, 9q, 11p, 11q, 13q, 16p, 17q, 19p, 19q and Xq. Array-CGH analysis detected both amplifications and deletions, affecting mostly the chromosome regions 1p (genes affected KLHL17, NOC2L, PPIAL4), 9q (ANKRD20A, COL5A), 11p (PSMD13, SIRT3), 15q (MAN2C1), 19p (MUC16, AZU1) and 20q regions (PSMA7). In addition, several genes of the HIST, FLJ, SLC and TRIM family as well as the homeobox families HOX and NK were involved in the alterations. FISH analysis is being conducted to confirm the array-CGH findings as well as the analysis of additional matching samples. The assessment of copy number changes in the SLN metastasis (and in its correspondent PT) using genomic profiling is a very sensitive method and will lead to the identification of genomic alterations that can reliably characterize breast tumor cells at the SLN lesions. Once these alterations are validated in larger series of patients, they can be used as molecular markers that can improve the predictive value of the current established methods of SLN evaluation in the identification of patients at higher risk of developing axillary lymph node metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 327.

The two main histological types of infiltrating breast cancer, lobular (ILC) and the more common ductal (IDC) carcinoma are morphologically and clinically distinct revealing different patterns of chromosomal aberrations and gene expression. Loss of heterozygosity (LOH) at chromosome 3p is frequently observed in breast tumors and a whole genome study utilizing a SNP array assay found a significantly higher frequency of LOH on 3p in ductal than lobular tumors. In order to investigate the molecular alterations at 3p associated with these breast cancer subtypes we analyzed LOH in 118 IDC and 18 ILC samples using seven microsatellite markers. Genomic DNA was obtained from frozen tumor tissue and peripheral blood was used as a normal reference. All samples were genotyped on a MegaBace® system and analyzed using Fragment Profiler® Version 2.1 software. Significance was determined using Chi-square test (X2). A significant higher frequency of LOH was observed in IDC (62.7%) when compared with ILC (38.9%) (X2=4.60 P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2158.