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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.

Ewing Sarcoma (ES) is an aggressive malignancy that arises in children and young adults. Although the survival for patients with localized tumors is relatively high, the metastatic form carries a dismal prognosis, particularly when bone metastases are present. The frequency of metastases and osseous dissemination increases in patients with necrotic ES tumors. In line with this, hypoxia, the major cause of tumor necrosis, has been shown to increase metastatic potential of ES cells. Previous data from our laboratory demonstrated that in an ES orthotopic xenograft model, primary tumor hypoxia specifically promotes bone metastasis, which is associated with accumulation of cells with enlarged nuclei and frequent chromosome gains in bone invasion areas. We have also shown that the progeny of hypoxia-induced polyploid ES cells (ES≈4n cells) preferentially metastasized to bone. Thus, the goal of our study was to determine the mechanisms enabling osseous dissemination of ES≈4n cells. To this end, we used FUCCI Cell Cycle Sensor followed by DNA staining with Hoechst 33342 and cell sorting to isolate tetraploid cells (4n) from hypoxia-exposed SK-ES1 ES cells. Subsequently, we compared the metastatic properties of their progeny (H-SK-ES1≈4n) with a diploid cell population selected from normoxic SK-ES1 cells (N-SK-ES1-2n cells). We have found that H-SK-ES1≈4n cells have increased motility and invasiveness, as compared to the N-SK-ES1-2n cells. H-SK-ES1≈4n cells had also an increased ability to grow in hypoxia in 2D culture and in soft agar. In line with this, H-SK-ES1≈4n cells were highly sensitive to a glycolysis inhibitor, 2D-glucose, but not an inhibitor of oxidative phosphorylation, metformin. Moreover, H-SK-ES1≈4n cells were more sensitive than controls to a growth-inhibitory effect of AICAR, a blocker of anabolic metabolism. In contrary, the tetraploid cell progeny were highly resistant to doxorubicin, particularly under hypoxic conditions, which in contrast sensitized N-SK-ES1-2n and wild type SK-ES1 cells to chemotherapy. Altogether, our data implicate the following mechanisms underlying osseous dissemination of the hypoxia-induced poplyploid cell progeny: 1) increased motility and invasiveness facilitating escape from the primary tumor; 2) ability to survive under low oxygen tension characteristic for bone tissue. Moreover, we have shown that the cells initiating bone dissemination are highly resistant to conventional chemotherapy, while strategies targeting their metabolic dependencies may be more successful in treatment of patients with bone metastases. Citation Format: Shiya Zhu, Akanksha Mahajan, Sung-Hyeok Hong, Susana Galli, Congyi Lu, You-Shin Chen, Sara Misiukiewicz, Stacey Chung, Jason Tilan, Joanna B. Kitlinska. Hypoxia-induced phenotypic and metabolic changes in Ewing sarcoma cells trigger bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3664.

Treating metastatic disease remains a major obstacle in oncology. Neuropeptide Y (NPY) and its Y5 receptor (Y5R) are expressed in a number of malignancies, including pediatric tumors Ewing sarcoma (ES) and neuroblastoma (NB). Our previous clinical and experimental data implicated NPY/Y5R axis in ES and NB metastasis. We have shown that Y5R is highly expressed in angioinvasive NB cells, while elevated release of NPY from NB tumors associates with metastatic disease and poor clinical outcome. Additionally, hypoxia-induced over-activation of NPY/Y5R pathway in ES tumors, which express particularly high levels of Y5R, led to formation of polyploid cells that give rise to chromosomally unstable, metastatic progeny. This phenomenon was reproduced in CHO-K1 cells over-expressing Y5R, which became polyploid due to a cytokinesis defect. Altogether, our data suggested a role of the NPY/Y5R pathway in the regulation of cell migration and cytokinesis, processes essential in tumor growth and dissemination. Thus, the goal of our study was to determine mechanisms of NPY/Y5R actions. We have found that in both CHO-K1/Y5R transfectants and ES cells, Y5R stimulation activated an essential cytoskeleton regulator, RhoA, activity of which is tightly controlled in a spatial and temporal manner during both cell migration and cytokinesis. This data was confirmed by strong co-localization of Y5R and active RhoA in all cell types. In non-dividing cells, expression of Y5R and active RhoA was limited, while their levels were higher in mitotic cells. Notably, in CHO-K1 cells over-expressing Y5R, both Y5R and RhoA accumulated in the narrow zone of the cleavage furrow at the abscission phase of cytokinesis. Such RhoA activation at this stage was previously shown to trigger cytokinesis defects. The concurrent changes in subcellular localization of Y5R and active RhoA were also observed in migratory cells. In single cell migration, both Y5R and active RhoA accumulated on the trailing and leading edges of the cells, while in the cells migrating collectively their expression was elevated in those present at the front of the moving colony. Strong Y5R/active Rho-A co-localization was also observed in membrane blebs of cells in amoebal migration mode. The role of Y5R in stimulation of cell migration was confirmed by transwell assay, in which Y5R antagonist significantly inhibited NPY-induced migration of NB and ES cells. Altogether, our data support the role of NPY/Y5R/RhoA pathway in regulation of cytoskeleton functions. The stimulation of this pathway promotes cell migration, while its over-activation may lead to cytokinesis defects and chromosomal instability, both of which are essential processes in tumor progression. Further studies are required to determine if these functions of the NPY/Y5R/RhoA pathway contribute to metastasis and validate Y5R as a potential therapeutic target. Citation Format: Nouran Abualsaud, Congyi Lu, Akanksha Mahajan, Abrar Bakr, Shiya Zhu, Lindsay Caprio, Sung Hyeok Hong, Joanna Kitlinska. The role of neuropeptide Y and its Y5 receptor in RhoA-mediated regulation of cytokinesis and cell motility [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 4143.

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.

Neuroblastoma (NB) is a pediatric malignancy arising due to defects in sympathetic neuron differentiation. NB is a heterogeneous disease, with phenotypes ranging from spontaneously regressing to highly aggressive, incurable tumors. This clinical variability cannot be explained solely by genetic aberrations. Even in families with hereditary NB the penetrance of the disease is incomplete and the same genetic mutation often results in tumors with phenotypes varying from differentiating ganglioneuromas to undifferentiated, highly aggressive NBs. Thus, other, perhaps non-genetic factors can contribute to the disease development and modify its phenotype. Strikingly, the two factors promoting de-differentiation of NB cells and their malignant phenotype, hypoxia and glucocorticoids, are elevated in the fetus during maternal stress, suggesting a role for prenatal stress in NB tumorigenesis. Previously, using TH-MYCN mice as a model of aggressive NB, we have shown that an increase in maternal corticosterone levels during pregnancy attained by inserting slow release pellets resulted in increased tumor frequency in TH-MYCN offspring. The goal of the current study was to determine the effect of prenatal stress on NB metastasis. To this end, pregnant mice carrying TH-MYCN hemizygous offspring were subjected to chronic stress at embryonic days 10-17, the time of sympathetic neuroblast proliferation and differentiation. Two established stress paradigms were used - chronic unpredictable stress, in which mice were subjected daily to various stressors, and chronic cold stress comprising of daily 30 min exposure to cold. The phenotypes of the disease and its dissemination were compared between offspring of control and stressed mothers. The offspring from both prenatally stressed groups presented with more malignant disease, as manifested by the presence of advanced lung metastases disseminating from small primary tumors (1,000 mm3). Although not common, lung metastases occur preferentially in NB patients with MYCN amplification and are associated with significantly worse prognosis, as compared to patients with metastatic disease, but no pulmonary involvement (14 vs 43% 3-year event-free survival, respectively). Thus, the profound pulmonary dissemination observed in prenatally-stressed TH-MYCN mice mimics one of the most malignant NB phenotypes observed in human disease. Altogether, our data implicate maternal stress during pregnancy as a potential environmental factor modifying the effects of genetic aberrations and promoting malignant phenotype of NB. Citation Format: Sung Hyeok Hong, Larissa Wietlisbach, Susana Galli, Akanksha Mahajan, Shiya Zhu, Jason Tilan, Yichien Lee, Olga Rodriguez, Chris Albanese, Joanna Kitlinska. Prenatal stress increases malignancy of neuroblastoma tumors in TH-MYCN animal model [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 1940. doi:10.1158/1538-7445.AM2017-1940

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

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.

Ewing sarcoma (ES) is a pediatric tumor induced by EWS-ETS fusion proteins, most often EWS-FLI1. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear. Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of nonperfused areas within tumor tissue was associated with poor prognosis. In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes via changes in transcriptional activity of the EWS-FLI1 gene. However, despite this line of evidence, no direct proof for this hypoxia-induced ES progression and spread has been provided. Moreover, the mechanisms by which hypoxia could exert such effects are unknown. To fill this gap, we created an in vivo model of hypoxia in ES and tested its effect on tumor metastasis. SK-ES1 ES cells were injected into gastrocnemius muscles of SCID/beige mice. Once the tumors reached a volume of 250mm3, they were either excised (control) or subjected to femoral artery ligation (FAL) for 72h prior to excision, inducing ischemia of the lower hindlimb, thus creating tumor hypoxia. Then, the mice were monitored for metastases. The extent of the metastatic disease was assessed and compared between experimental groups based on periodic MRI, necropsy and histopathology findings. FAL resulted in profound tumor hypoxia, as evidenced by inhibition of primary tumor growth, severe tissue necrosis and positive staining for a hypoxyprobe, pimonidazole. However, despite the impaired growth of primary tumors, xenografts subjected to FAL were more metastatic. The involvement of hypoxic cells in metastases was evidenced by the accumulation of pimonidazole-positive cells (hypoxic at the time of FAL) in areas of tissue invasion and intravasation. Consequently, mice bearing FAL-treated tumors exhibited a decreased latency of metastases formation and an increase in their number from an average of 0.9 to 2.3 metastases per mouse in control and FAL groups, respectively. We also observed a change in the pattern of metastases, as FAL-treated tumors metastasized more often to distant organs (average of 0.3 organ metastases per mouse in control and 1.3 in FAL group). The hypoxia-induced metastases were most often observed in adrenal gland and spine (50% and 42% of mice in FAL group, respectively), while no such metastases were observed in the control group. Moreover, 100% of FAL-treated mice had signs of bone marrow invasion, while no tumor cells were detectable in bone marrow of control mice. This data provides the first-ever direct evidence for tumor hypoxia as a driver of ES metastases. Moreover, our model of tumor hypoxia in vivo provides an excellent opportunity to identify hypoxia-induced pathways involved in ES metastatic progression that subsequently may become novel therapeutic targets for this disease. Citation Format: Jason U. Tilan, Sung-Hyeok Hong, Susana Galli, Rachel Acree, Katherine Connors, Meredith Horton, Akanksha Mahajan, Larissa Wietlisbach, Yi-Chien Lee, Olga Rodriguez, Christopher Albanese, Joanna Kitlinska. Tumor hypoxia promotes Ewing sarcoma metastases in a mouse xenograft model. [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 2478.

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