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Introduction: We have reported that mouse and human adipocytes take up and metabolize the anthracycline, daunorubicin (DNR), reducing its concentration in the adipocyte microenvironments. This may contribute to anthracycline resistance for cancers, which reside in adipocyte rich environments such as omentum and bone marrow. Adipocytes express several carbonyl- and aldo-keto reductases (CBRs and AKRs) which metabolize and inactivate anthracyclines, and it is unclear which of these might be important targets to improve treatment outcome. Experimental Procedures: We knocked out AKR1C3 in the human preadipocyte cell line Chub S7 using CRISPR/Cas9 gene editing technology. We chose AKR1C3 first as it is one of the overexpressed enzymes in adipocytes with the highest anthracycline-metabolizing activity. We delivered ribonucleoprotein complexes of CRISPR-Cas9 enzyme plus guide RNAs by nucleofection. Then we established single-cell derived clones and tested for successful KO by Western blot. Finally, we quantified adipocyte lysate AKR activity using a colorimetric assay based on NADPH-dependent reduction of phenanthrenequinone. Data Summary: We chose three Chub S7 preadipocyte clones that demonstrated successful AKR1C3 knockout based on almost undetectable protein expression by western blot. AKR activity was significantly reduced in all three clones; control preadipocytes had 44±4.7, while clones had activity of 28±7.3, 33±3.4, and 35±5.7 pmol/min/µg (p Conclusions: These findings demonstrate that AKR1C3 knockout can be successfully done in human preadipocytes using a CRISPR-Cas9 system. Knockout of AKR1C3 significantly reduces overall aldoketoreductase activity in preadipocyte cells. This implies that a substantial portion of preadipocyte AKR activity is dependent on the isoenzyme, AKR1C3. Future testing is needed to determine whether AKR1C3 KO will reduce the clearance of anthracyclines from the cancer microenvironment, and may represent a treatment target to enhance anthracycline cytotoxicity. Citation Format: Vladislava Paharkova, Etan Orgel, Michael Neely, Stan. Louie, Steven Mittelman. Targeting the anthracycline metabolizing enzyme AKR1C3 in adipocytes to improve cytotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6059.

Despite the exciting progresses in the treatment of breast cancer, the effectiveness of the current therapeutic modalities is still restricted by drug toxicity, resistance, and lack of predictive and prognostic biomarkers. Breast cancer continues to be the second leading cause of cancer death among women in the U.S. Therefore, the development of new therapeutic targets and further understanding of the tumor microenvironment is extremely critical for accelerating the progress against breast cancer. Human AC133+/CD34+ stem cells are a highly promising and novel therapeutic option for targeting tumor angiogenesis. We and others have described the incorporation of bone marrow derived AC133+/CD34+/KDR+ cells in the neovasculature around implanted tumors supporting their growth and metastasis. Many mediators have been involved in the cross talk between AC133+/CD34+ cells, endothelial cells, and the tumor cells, but most of these have insufficient clinical benefits as reported by several trials. In this study, we evaluated the secretome of the AC133+/CD34+ stem cells that were isolated by positive selection from human umbilical cord blood and their role in breast cancer progression. Using flow cytometry, we show that the high proliferative AC133+/CD34+ stem cells maintain their capacity to differentiate in to AC133+/CD34+/KDR+ endothelial progenitor cells even after long period of in vitro expansion. In order to evaluate the effect of AC133+/CD34+ stem cells on breast cancer cells, a proliferation (XTT) assay was performed using conditioned medium (CM) from AC133+/CD34+ stem cells and examined on MCF-7 and MDA-MB-231 proliferation. As anticipated, CM significantly induced breast cancer cells proliferation. This effect was in part due to the high expression of a large range of proinflammatory and proangiogenic cytokines in the CM of the AC133+/CD34+ cells. In particular, angiogenin, GRO, IL-8, MCP, and TIMP2. Next, we examined if exosomes, a component of paracrine secretion are involved in the paracrine effect of the AC133+/CD34+ stem cells. Surprisingly, exosomes from AC133+/CD34+ stem cells significantly increased MCF-7 and MDA-MB-231 proliferation at a comparable level as the CM. Further analysis of the exosomes using miRNA array screen reveals that exosomes of AC133+/CD34 cells are highly enriched with oncogenic miRNAs including miR-21-5p, miR-142-3p, and miR-223-3p. These miRNAs are up-regulated in breast cancer. Several studies have confirmed their role in mediating breast cancer cells invasiveness. However, miR-142-3p and miR-223-3p are exclusively expressed in hematopoietic cells. Therefore, we propose that shuttling of the exosomes between AC133+/CD34+cells and breast cancer cells induces breast cancer invasiveness. The analysis of the paracrine interactive mediators between breast cancer cells and AC133+/CD34+ cells is likely to yield viable novel clinically translatable therapeutic targets. Citation Format: Ghada Ben Rahoma, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Anitha Srinivasan, Abraham Mittelman, Jan Geliebter, Raj K. Tiwari. Exosomes secreted by AC133+/CD34+cells harbor invasion potentiating miRNAs [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 3562.

BACKGROUND Metabolic syndrome (MetS) and obesity are associated with increased risk of cardiovascular disease, type 2 diabetes, and cancer recurrence, and are higher in breast cancer survivors than age-matched postmenopausal women. We previously reported that exercise improves MetS and reduces pro-inflammatory biomarkers in obese breast cancer survivors. Whether these exercise-induced changes impact cancer outcomes is currently unknown. In this pilot study, we apply a robust mathematical analysis to identify biomarkers of MetS and obesity associated with clinical outcomes in obese breast cancer survivors following participation in a 16-week exercise intervention. EXPERIMENTAL DESIGN Eleven obese postmenopausal breast cancer survivors were randomized to either the exercise or control group. The exercise group participated in 16 weeks of supervised aerobic and resistance exercise sessions 3 times/week. Fasting blood and adipose tissue samples were analyzed for cytokine secretion, macrophage phenotype, and MetS. Prognostic outcomes included disease-free survival (DFS), overall survival (OS), distant DFS (DDFS), and recurrence-free interval (RFI). Partial least squares regression (PLSR) was used to quantify the importance of specific patient measurements in predicting the clinical response. PLSR is a multivariate regression analysis that quantifies the relationships between the participant characteristics and tissue and plasma measurements (inputs) and the clinical response (outputs) and can be used to identify which patient measurements most significantly associate with specific clinical outcomes. RESULTS MetS, macrophage phenotype, and inflammatory biomarkers were significantly improved in the exercise group compared to the control group (p CONCLUSIONS Exercise improves biomarkers related to MetS, obesity, and inflammation. The PLSR model quantifies patient characteristics and measurements associated with clinical outcomes. Our analysis provides quantitative insight into potential biomarkers to predict response to exercise and prompts the need for an in depth longitudinal study to determine the effects of exercise on long-term survival in obese breast cancer patients. Citation Format: Christina M. Dieli-Conwright, Jean-Hughes Parmentier, Steven D. Mittelman, Nathalie Sami, Kyuwan Lee, Stacey Finley. A mathematical model to predict prognosis in breast cancer survivors following an exercise intervention [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 2627.

Exciting advances in the treatment of breast cancer have occurred over the past decade. However, the efficacy of the current therapeutic modalities is still limited by drug toxicity, resistance, and lack of predictive and prognostic biomarkers. Breast cancer remains the second leading cause of cancer death among women in the U.S. Thus, the development of new therapeutic targets and further understanding of the tumor microenvironment is extremely crucial for accelerating the progress against breast cancer. Human AC133+/CD34+ stem cells serve as a highly promising and novel therapeutic option for targeting tumor angiogenesis. We and others have demonstrated the incorporation of bone marrow-derived AC133+/CD34+/KDR+ endothelial progenitor cells in the neovasculature around implanted tumors supporting their growth and spread. Many mediators have been implicated in the crosstalk between AC133+/CD34+/KDR+ endothelial progenitor cells, endothelial cells, and the tumor cells, but most of these have limited clinical benefits as reported by several trials. In this study, we analyzed the secretome of the AC133+/CD34+ stem cells that were isolated by positive selection from human umbilical cord blood and evaluated their role in breast cancer progression. Our results show that AC133+/CD34+ stem cells exhibited significant growth potential that was manifested as seventy-five fold increase in cell number after 10 days in culture. Flow cytometry demonstrated that AC133+/CD34+ stem cells preserve their capacity to differentiate into AC133+/CD34+/KDR+ endothelial progenitor cells even after long term in vitro expansion. In order to evaluate the effect of AC133+/CD34+ stem cells on breast cancer cells, we performed a simple proliferation (XTT) assay using conditioned medium (CM) from AC133+/CD34+ stem cells and tested on MCF-7 and MDA-MB-231 proliferation. As expected, CM significantly induced proliferation of breast cancer cells. This effect was in part due to the high expression of a repertoire of proinflammatory and proangiogenic cytokines in the CM of the AC133+/CD34+ stem cells. In particular, angiogenin, GRO, IL-8, MCP, PDGF.BB, TIMP2. Further, we examined if exosomes, a component of paracrine secretion are involved in the paracrine effect of the AC133+/CD34+ stem cells. Interestingly, exosomes from AC133+/CD34+ stem cells significantly enhanced MCF-7 and MDA-MB-231 proliferation at a comparable level as the CM. Further analysis of the exosomes reveals that the pro-proliferative miR-141-3p, miR-182-5p, miR-200b-3p, and miR-203a are highly expressed in AC133+/CD34+ exosomes. The analysis of the paracrine interactive mediators between breast cancer cells, AC133+/CD34+ stem cells, and AC133+/CD34+/KDR+ endothelial progenitor cells is likely to yield viable novel clinically translatable therapeutic targets. Citation Format: Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Jan Gelibter, Raj K. Tiwari. Exosomes from AC133+/CD34+ stem cells mediate a paracrine effect in breast cancer cells [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 2053.

Breast cancer affects one in eight women in the USA. Considerable progress in the identification of genetic lesions and their modulation has resulted in newer therapies making breast cancer a manageable disease. However, triple negative breast cancer is still difficult to treat and warrants a search for newer targets. To this end, we focused our attention towards the modulation of the breast cancer epithelium by other cell types such as the endothelial cells and the macrophages. The migratory macrophages and the estrogen sensitive migratory endothelial progenitor cells (EPCs) constitute the cellular milieu within the tumor microenvironment which continuously modulates breast cancer epithelium. We analyzed the interactions of the breast cancer cell lines (MCF-7 and MDA-MB-231) with the highly proliferative human umbilical cord derived CD133+/CD34+/VEGFR-2+ EPCs and M1 polarized macrophages (activated THP-1 cell line) in two separate in vitro studies. The readouts were cell proliferation, changes in epithelial to mesenchymal transition (EMT), and cellular differentiation. We observed morphological and cellular growth changes in the EPCs on treatment with conditioned medium (CM) generated from breast cancer cells, consistent with vasculogenesis and in vitro tubulogenesis. Both, MDA-MB-231 and MCF-7 CM, treatments resulted in enhanced EPCs proliferation and differentiation. However, the differentiation patterns were distinct, with MCF-7 CM increasing the number of cell clusters, whereas MDA-MB-231 CM increasing the number of adherent spindle shaped cells. The paracrine interaction was also assessed with M1 polarized macrophages. We observed decreased cell viability in MCF-7 and MDA-MB-231 cells following activated THP-1 CM and exosome treatments. Analysis of exosomes from activated THP-1 indicated an upregulation of 13 miRNAs compared to unactivated THP-1. The miRNA hsa-miR-146a-5p had the highest upregulation (44 fold increase). This specific miRNA has been observed in senescent cell and it inhibits cell proliferation, suggesting a possible mechanism for exosome-associated growth inhibition. The analysis of the paracrine interactive mediators between breast cancer cells, EPCs, and M1 polarized macrophages is likely to yield viable novel clinically translatable therapeutic targets. Citation Format: Tiwari RK, Ben Rahoma G, Tuli N, Bednarczyk R, Maniyar RR, Chakraborty S, Singh S, Mittelman A, Geliebter J. Novel targets of breast cancer associated with inflammatory tumor microenvironment [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 P5-03-06.

Obesity is associated with increased cancer incidence and mortality. We have previously found that obesity in children is associated with a 50% increased recurrence of acute lymphoblastic leukemia (ALL) in high-risk patients. We have therefore developed novel in vivo and in vitro preclinical models to study the mechanism(s) of this association. Obesity increased relapse after monotherapy with vincristine (P = 0.03) in obese mice injected with syngeneic ALL cells. This occurred although the drug was dosed proportionally to body weight, equalizing blood and tissue drug levels. In coculture, 3T3-L1 adipocytes significantly impaired the antileukemia efficacy of vincristine, as well as three other chemotherapies (P < 0.05). Interestingly, this protection was independent of cell-cell contact, and it extended to human leukemia cell lines as well. Adipocytes prevented chemotherapy-induced apoptosis, and this was associated with increased expression of the two prosurvival signals Bcl-2 and Pim-2. These findings highlight the role of the adipocyte in fostering leukemia chemotherapy resistance, and may help explain the increased leukemia relapse rate in obese children and adults. Given the growing prevalence of obesity worldwide, these effects are likely to have increasing importance to cancer treatment. [Cancer Res 2009;69(19):7867–74]

Heat shock protein gp96 induces a tumor-specific protective immunity in a variety of experimental tumor models. Because the primary sequences of the glycoprotein, gp96 are identical between tumor and normal tissues, the peptides associated with gp96 and/or the posttranslational modifications of gp96, determine its immunogenicity. Gp96-associated peptides constitute the antigenic repertoire of the source tissue; thus, purified gp96-peptide complexes have clinical significance as autologous cancer vaccines. However, the role of altered glycosylation and its contribution in the biological as well as immunologic activity of gp96 still remains uncharacterized. We examined the cancer-specific glycosylation patterns of gp96. To this end, monosaccharide compositions of gp96 were compared between normal rat prostate and two cancerous rat prostate tissues, nonmetastatic/androgen-dependent Dunning G and metastatic/androgen-independent MAT-LyLu, as well as two human nonmetastatic prostate cancer cell lines, androgen-dependent LnCaP and androgen-independent DU145. Marked differences were observed between the gp96 monosaccharide compositions of the normal and cancerous tissues. Furthermore, gp96 molecules from more aggressive cellular transformations were found to carry decreasing quantities of several monosaccharides as well as sum total content of neutral and amino sugars. We believe that the unique glycosylation patterns contribute to cellular phenotype and that the posttranslational modifications of gp96 may affect its functional attributes.

Breast cancer affects one in eight women in the USA. Early diagnosis and newer treatment modalities have rendered breast cancer manageable. However, triple negative breast cancer is still difficult to treat and warrantes a search for newer targets. One strategy that has emerged in cancer research involves targeting of tumor associated blood vessels which provide growing tumors with oxygenated blood and growth factors necessary for maintenance and metastasis. Antiangiogenic drug therapy is transient and has not been able to gain mainstream therapeutic modality. We discovered that endothelial progenitor cells (EPCs) are mobilized from the bone marrow to the tumor site and contribute to the development of breast tumor vessel formation in an estrogen dependent manner. Therefore, characterization of tumor associated endothelial progenitor cells in breast cancer may provide a more specific antivascular therapy. Using the highly proliferative human umbilical cord blood derived EPCs, having the phenotype (CD133+, CD34+, VEGFR-2+), the effect of growth factor and chemokine rich EPCs conditioned medium (CM) was assessed in luminal (MCF-7), and post-EMT (MDA-MB-231) breast carcinoma cell lines. We observed an initial halt in cellular proliferation in MCF-7 followed by a significant increase in proliferation after forty eight hours of treatment. On the other hand, MDA-MB-231 showed decreased proliferation even after forty eight hours of treatment. Treating the EPCs with breast cancer conditioned medium resulted in morphological and cellular growth changes in the EPCs. MDA-MB-231 CM resulted in an increase of the EPCs proliferation and differentiation by increasing the number of spindle shaped attaching cells, and MCF-7 CM resulted only in an increase in the differentiation rate by increasing the number of cell clusters. This increase in EPCs proliferation and differentiation associated with MDA-MB-231 CM treatment might explain the invasiveness of this breast cancer cells through the increase in the tumor associated neovascularization. The analysis of the paracrine interaction between breast cancer cells and EPCs along with the associated cellular changes will facilitate identification of the interactive mediators and subsequent development of effective antivascular therapy. Citation Format: Ghada Ben Rahoma, Neha Tuli, Rachana Maniyar, Sanjukta Chakraborty, Sarnath Singh, Abraham Mittelman, Raj K. Tiwari. Human endothelial progenitor cells: A new target for anti-vascular therapy [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 793. doi:10.1158/1538-7445.AM2017-793

Background and Purpose: Obesity is a leading modifiable contributor to breast cancer mortality due to its association with increased recurrence and decreased overall survival rate. There are over 2.5 million breast cancer survivors, 64% of whom are overweight/obese (BMI >25 kg/m2). A central mechanism by which obesity stimulates cancer progression is through chronic, low-grade inflammation in white adipose tissue, leading to accumulation of adipose tissue macrophages (ATMs), in particular the pro-inflammatory M1 phenotype macrophage. Exercise has been shown to reduce M1 ATMs, and increase the more anti-inflammatory M2 ATMs in obese adults. The purpose of this study was to determine whether a 16-week exercise intervention would positively alter adipose tissue inflammation by changing ATM phenotype and cytokine secretion in obese postmenopausal breast cancer survivors. Experimental Design: Twenty obese postmenopausal breast cancer survivors were recruited from USC and randomized to either the exercise (EX) or control (CON) group. The EX group participated in 16 weeks of supervised exercise sessions 3 times/week. Sessions included total-body resistance training consisting of 8 exercises with a rest period of 45 seconds between each set of resistance exercise followed by 30 minutes of moderate-vigorous intensity (65-80% HRmax) aerobic exercise. The CON group was asked to maintain their current activity levels. Superficial subcutaneous abdominal adipose tissue biopsies were performed at baseline and following the 16-week study period. Adipose tissue samples were analyzed using fluorescence-activated cell sorting (FACS) to characterize ATM characterization (M1 vs M2). Portions (~100 mg) of each biopsy were incubated in media overnight to measure cytokine secretion. A 2x2 (group x time) repeated measures ANOVA was used to evaluate changes in adipose tissue and systemic inflammation. Summary of Results: At baseline, there were no group differences (p>0.05) in age (55.1±5.2 yrs), BMI (34.4±7.5 kg/m2), percent body fat (36.2±4.9%), or ATM M1 (25.4±6.7%) and M2 (4.2±0.9%) levels. EX was associated with a significant decrease in ATM M1 (-18.8±7.3%) and increase in ATM M2 (9.6±1.6%; p0.01). Conclusions: A 16-week aerobic and resistance exercise intervention attenuates adipose tissue inflammation in obese postmenopausal breast cancer survivors. Future large randomized controlled trials are warranted to investigate the impact of exercise-induced reductions in adipose tissue inflammation and breast cancer recurrence. Citation Format: Christina M. Dieli-Conwright, Jean Hughes-Parmentier, Kyuwan Lee, Darcy Spicer, Wendy Mack, Fred Sattler, Steven D. Mittelman. Adipose tissue inflammation in breast cancer survivors: Effects of a 16-week aerobic and resistance exercise intervention [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 985. doi:10.1158/1538-7445.AM2017-985

Obesity is associated with poorer outcome from many cancers, including childhood acute lymphoblastic leukemia (ALL). We have previously shown that adipocytes protect ALL cells from the anthracycline, daunorubicin (DNR). We therefore investigated whether adipocytes sequester and/or metabolize DNR in the ALL microenvironment. Using fluorescence and LC/MS measures, we demonstrated that adipocytes absorb DNR, reducing the intracellular DNR concentration in co-cultured BV173 ALL cells (after 48 hours, median fluorescent intensity of ALL cultured with adipocytes was 1.7±1.0 vs. 5.0±1.7 of those cultured alone, p Citation Format: Xia Sheng, Jean-Hugues Parmentier, Jonathan Tucci, Hua Pei, Omar Cortez-Toledo, Christina Dieli-Conwright, Matthew Oberley, Michael Neely, Etan Orgel, Stan Louie, Steven D. Mittelman. Adipocytes sequester and metabolize daunorubicin [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 2960. doi:10.1158/1538-7445.AM2017-2960

Background: Adipocytes protect acute lymphoblastic leukemia (ALL) cells from several chemotherapeutic drugs. This protection is associated with an oxidative stress response in adipocytes. Since adipocytes are known to release reactive oxygen species, and anthracyclines are sensitive to inactivation mediated by hydrogen peroxide (H2O2), we investigated whether adipocyte release of H2O2 into the micro-environment might contribute to drug resistance of ALL cells. Methods: Pre-B lymphoblast/myeloblast BV173 cells in complete media (CM) were co-cultured for 3 days in transwells over 3T3-L1 fibroblasts or adipocytes. Viable cells were determined by Trypan blue exclusion for BV173 cells. Leukemia Conditioned Media (LCM) was prepared from BV173 cultured for 48 hrs. Total antioxidant capacity was measured by ABTS-based assay. H2O2 released in the extracellular compartment of adipocytes was monitored with Amplex Red and ROS-Glo. Results: BV173 cells were protected from DNR in presence of 1 mM H2O2 (9.34±2.15x104 vs. 3.33±1.01x104 viable cells with and without H2O2, p=0.03). Interestingly, this protection was only observed when pyruvate was present in the culture media at equimolar concentrations with H2O2 (DNR: 4.14±2.14x104; DNR+pyruvate: 4.17±1.53x104; DNR+H2O2: 3.77±2.74x103; DNR+pyruvate+H2O2: 3.21±0.59x105, p=0.02). Adipocytes released H2O2 into the media measured with Amplex Red fluorescence in a time-dependent manner significantly more than fibroblasts (1.96-fold, p=0.001). LCM potentiated adipocyte H2O2 released (1.5-fold over CM, p=0.05). Human adipose tissue biopsies from breast cancer survivors also released H2O2 (4.37x103 RFU for biopsies vs. 0.65x103 RFU for HBSS, 90 min, n=5). Co-culture of adipocytes and BV173 cells increases the survival of BV173 to DNR treatment (1.55±0.34x105 vs. 6.88±2.91x103 viable cells with and without adipocytes, p=0.006). Adipocyte-mediated protection of BV173 cells was reversed in a dose-dependent manner by adding the H2O2-inactivating enzyme catalase to the extracellular media (7.28±1.44x104 vs. 15.58±3.48x104 viable cells with and without 500 U/ml catalase, p=0.01). Conclusion: These data show that adipocytes release H2O2, which in the presence of pyruvate, protects ALL cells from DNR. Further work is needed to determine whether adipocytes secrete significant amounts of H2O2 in the bone marrow micro-environment to contribute to anthracycline resistance. Citation Format: Jean-Hugues Parmentier, Christina M. Dieli-Conwright, Steven D. Mittelman. Adipocyte-derived hydrogen peroxide promotes chemoresistance to daunorubicin in leukemia cells [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 3185. doi:10.1158/1538-7445.AM2017-3185

Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC’s) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC’s, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3’-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 μM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 μM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect. Citation Format: Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer. [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 3263.

Obesity is associated with the development and progression of many cancers, including acute lymphoblastic leukemia (ALL). We have shown that ALL cells induce adipocyte lipolysis and take up adipocyte-derived free-fatty acids (FFAs). Here, we use targeted lipidomics with carbon-13 labeling to 1) determine which adipocyte-derived FFAs are taken up by ALL cells, and 2) identify the specific alterations in the ALL cell lipidomic profile caused by co-culture with adipocytes. Mouse pre-adipocytes (3T3-L1) were differentiated into adipocytes in the presence of U13C-glucose to allow 13C incorporation into adipocyte lipids. ALL cells were then cultured alone or co-cultured over 13C-labeled or non-labeled adipocytes for 72 hours. Following co-culture, ALL cells were harvested and analyzed by nanospray desorption electrospray ionization mass spectrometry (nanoDESI-MS). Lipidomic spectra were analyzed to characterize uptake of adipocyte-derived FFAs identifying 13C-enrichment in ALL lipid moieties. The relative intensities of selected lipid peaks were compared between conditions, after normalization, to evaluate the effects of the presence of adipocytes on ALL lipidome. Adipocytes differentiated in the presence of U13C-glucose incorporated 13C into numerous triglyceride moieties. In ALL cells co-cultured with labeled adipocytes, 13C enrichment was identified in FFA and phospholipids. Labeling of oleic acid primarily occurred in groups of two, suggesting incorporation of U13C-glucose into adipocyte FFAs through acetyl-CoA-mediated lipogenesis. Similarly, 13C enrichment of ALL cell phospholipids occurred in groups of two and three, mirroring adipocyte incorporation of U13C-glucose into phospholipids through both acetyl-CoA and glycerol. Moreover, ALL cell unsaturated FFAs had a greater 13C enrichment than saturated FFAs (Unsaturated FFAs: 14.1±5.5 vs. Saturated FFAs: 6.6±2.3, p = 0.02; n = 5). When co-cultured over adipocytes, ALL cells contained significantly more FFAs than ALL cells alone (Oleic Acid: 63.0±49.2 vs. 24.1±7.4, p = 0.02; Stearic acid: 20.9±17.5 vs. 8.3±2.2, p = 0.03; Palmitoleic Acid: 11.5±9.8 vs. 3.3±1.0, p = 0.01; Palmitic Acid: 15.1±11.7 vs. 5.5±1.4, p = 0.01; n = 5). Using stable-isotope lipidomics, we can effectively identify and quantify which ALL cell lipids are derived from nearby adipocytes. This methodology provides comprehensive insight into the cancer cell lipidome and can be extended to understand how genetics, the microenvironment, and treatment affect cancer lipid metabolism. Citation Format: Jonathan Tucci, Katy Margulis, Cheng-Chih Hsu, Wesley Dixon, Richard N. Zare, Steven D. Mittelman. Using stable isotope lipidomics to identify adipocyte-induced alterations in the acute lymphoblastic leukemia lipidome. [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 5.

Obesity promotes the pathogenesis of many cancers, including acute lymphoblastic leukemia (ALL), the most common childhood malignancy. Clinical studies have shown that children who are obese at the time of ALL diagnosis have a greater risk of relapse than normal weight children; however, this risk may be attenuated if their weight normalizes over the majority of treatment. One possible explanation is that nutrient-deficiency may induce host, but not ALL, cell senescence, thereby augmenting chemotherapy specificity and efficacy. Here, we hypothesize that a calorie- and fat-restricted diet during chemotherapy differentially alters host and ALL cell metabolism, improving chemotherapy efficacy and overall survival of high-fat diet-induced obese mice with ALL. To test the effect of dietary restriction on survival, C57BL/6J mice were placed on a high-fat diet (60% calories from fat) upon weaning. At 20 weeks of age, obese and control mice were implanted with syngeneic BCR/ABL+ ALL cells. After a 7-day latency, half of the obese mice were switched onto a low-fat (10% calories from fat) diet. Concurrently, vincristine chemotherapy (0.5mg/kg/wk x 4wks) was initiated and mice were tracked for survival. Separate groups of implanted mice were sacrificed 1 day before, 1 day and 1 week after dietary restriction for bone marrow and spleen collection. BrdU incorporation and p-AKT(Ser 473), p-eIF2A(Ser51) and p-S6K(Thr412) levels in marrow and spleen ALL and host cells were analyzed via flow cytometry for changes in cell cycle and metabolism. Finally, we also evaluated the effect of dietary restriction on human ALL outcome, using an obese xenograft ALL model treated with vincristine, dexamethasone and L-asparaginase. Dieted obese C57BL/6J leukemic mice exhibited a greater percentage of weight loss over the first two weeks of chemotherapy compared to non-dieted obese mice (Dieted vs. non-dieted obese: -25.2±10.6% vs. -0.3±4.5%, p Based on these findings, a calorie and fat restricted diet initiated at the onset chemotherapy improves overall survival in obese mice with syngeneic ALL, though this phenomenon was not observed in a human xenograft model. Further work is needed to explore the possibility of a dietary intervention as an effective potential adjuvant during chemotherapy in obese cancer patients. Citation Format: Jonathan Tucci, Waseem Alhushki, Xia Sheng, Steven D. Mittelman. Calorie and fat restriction during chemotherapy improves survival in obese mice with syngeneic acute lymphoblastic leukemia. [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 1633. doi:10.1158/1538-7445.AM2015-1633

Multiple cell types interact within the tumor microenvironment (TME) through crosstalk events that are mediated by many biomolecules including cytokines, mRNA, and miRNA-containing exosomes that can influence cancer progression as well as suppression. Inflammation within the TME is a major determinant in tumor progression. Since tumor associated macrophages (TAMs) are a major cellular factor in tumor inflammation, we hypothesized that crosstalk events initiated by macrophage secreted factors such as cytokines and exosomes will modulate breast cancer phenotype. We evaluated the cytokine profile of a human monocytic cell line, THP-1, following stimulation with a phorbol ester TPA (12-O-Tetradecanoylphorbol-13-acetate) in vitro. Activated THP-1 cells exhibited an M1 macrophage phenotype. In addition, we assessed the morphological and cellular growth changes of luminal (T47D, MCF-7) and basal-like (MDA-MB 231) breast cancer cell lines in vitro following treatment with activated THP-1 conditioned medium (CM). Each cell line displayed mesenchymal-like features and decreased cellular growth following activated THP-1 CM treatment. MCF-7 and MDA-MB 231 displayed increased MEK/ERK signaling, while all three cell lines showed increased p-AKT expression. Furthermore, epithelial-like T47D and MCF-7 cells exhibited an increase in EMT transcription factor expression. Moreover, although T47D and MCF-7 did not show drastic changes in E-cadherin expression via western blot analysis, immunofluorescence examination showed a decrease in E-cadherin following activated THP-1 CM treatments emphasizing a possible partial EMT event. Lastly, activated THP-1 exosomes were isolated and used to treat MDA-MB 231 to determine the effects on breast cancer cell proliferation and senescence. MDA-MB 231 exhibited significantly decreased cellular growth and increased senescence following incubation with activated THP-1 exosomes. The variety of secretory factors within the TME as a result of inflammation may contribute to tumor suppression as well as tumor progression via a secretion of cytokines and exosomes. Citation Format: Robert Bronislaw Bednarczyk, Yuki Kitadai, Neha Y. Tuli, Elyse K. Hanly, Ghada Benrahoma, Abraham Mittelman, Raj K. Tiwari. Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches. [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 5087. doi:10.1158/1538-7445.AM2015-5087

The regulation of oxidative stress is fundamental to cell survival and function. Oxidative stress is a result of accumulation of reactive oxygen species (ROS), which are produced by cell metabolism and in response to certain stressors. Cancer cells have higher than normal ROS levels and elevated antioxidant defense mechanisms. Daunorubicin (DNR), a widely used chemotherapy, is a topoisomerase II inhibitor that induces cell death through DNA double strand breaks and increased ROS. We have previously shown that adipocytes protect acute lymphoblastic leukemia (ALL) cells against DNR in vitro and that media cultured by both adipocytes and ALL cells together (ALCM) can protect ALL cells from DNR. We hypothesize that this protection is in part mediated by adipocytes alleviating oxidative stress in ALL cells. Murine pre-B ALL cells isolated from a BCR/ABL transgenic mouse (8093) and human leukemia cell lines (BV173, Nalm6, REH, SEM, RS4;11, HL60, and Molt4) were used. The ALL cells were co-cultured with 3T3-L1 adipocytes using a transwell system. Cells were collected for gene expression analysis using qPCR. Intracellular ROS was measured by flow cytometry with 2′-7′-dichlorodihydrofluorescein (DCFH-DA). Four of the six human leukemia cell lines tested were protected by adipocytes against DNR treatment. Western blots showed that DNR induced a significant increase in the cleaved to procaspase-3 ratio, which was reversed by ALCM (No drug: 0.24±0.2, DNR: 0.83±0.14, ALCM+DNR: 0.24±0.19; p = 0.004 for DNR vs. ALCM+DNR, n = 4). Expression of the oxidative stress response genes gclc, gclm and p21 was upregulated in 8093 cells upon DNR treatment (fold change over baseline: 1.82±0.37, 2.35±0.73, 5.55±1.50; p These findings demonstrate that adipocytes contribute to drug resistance by alleviating oxidative stress in ALL cells. Further research is required to identify the soluble factors responsible for these protective effects. Citation Format: Xia Sheng, Jonathan Tucci, Steven D. Mittelman. Reduction of oxidative stress promotes daunorubicin resistance in acute lymphoblastic leukemia cells. [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 1247. doi:10.1158/1538-7445.AM2015-1247

Background: The epidemic of childhood obesity poses substantial treatment challenges for the care of children with acute lymphoblastic leukemia (ALL). Obesity at time of diagnosis of ALL is associated with worse survival as well as treatment-related morbidity. Inability to achieve a deep initial remission following Induction as evidenced by persistent MRD in the bone marrow is associated with poor survival and has thus become the predominant modern prognostic indicator. We therefore hypothesized that obesity at time of diagnosis of ALL is associated with MRD positivity at the end-of-Induction disease evaluation. Methods: In a cohort of children 1 - 20 years old treated for B-precursor ALL (BP-ALL) at our institution from 2008 to 2013 on Children's Oncology Group (COG) treatment regimens, we examined MRD at end-of-Induction as determined by multidimensional flow cytometry. A threshold of 0.01% was used to determine MRD positive versus negative as per COG regimens. Overweight and obese categories were defined as 85-95th and ≥95th percentile for body mass index (BMI) according to the CDC population norms. Logistic regression analyses were performed controlling for age, gender, ethnicity/race, presence/absence of Down Syndrome (due to the high prevalence of obesity), National Cancer Institute (NCI) risk category, cytogenetic classification (standard, favorable, unfavorable, unknown), and CNS status at diagnosis. BMI was evaluated as both a continuous variable and also as per normal, overweight, obese categories. Results: We identified 198 patients with BP-ALL and evaluable MRD, 119 with NCI SR-ALL and 79 with NCI HR-ALL. Of these, 55 (27.8%) had positive MRD at end-of-Induction [27 SR-ALL (22.7%), 28 HR-ALL (35.4%). In the obese group, 45.2% (19/42) were MRD+, compared to only 22.8% (29/127) of normal weight and 24.1% (7/29) of overweight patients. After controlling for above covariates, obese patients had a statistically significantly increased risk for MRD positivity at end-of-Induction [Odds Ratio (OR) 2.64, 95 percent Confidence Interval (95%CI) 1.13-6.19, p=0.025]. Similarly, each percentile increase in BMI significantly corresponded to an increased risk for MRD positivity (OR 1.08, 95%CI 1.00-1.17, p=0.046). Conclusion: Obese children at diagnosis of BP-ALL are at significantly increased risk for persistent MRD in the bone marrow at end-of-Induction. With the established strong adverse association of persistent MRD with poorer survival, this implies that obesity adversely influences survival as early as this initial phase of treatment. Our results support the need for further research into mechanisms underlying this phenomenon and potential up-front strategies to improve efficiency of remission induction in this population. Citation Format: Etan Orgel, Jonathan Tucci, Wassem Alhushki, David R. Freyer, Hisham Abdel-Azim, Steven D. Mittelman. Childhood obesity is associated with persistent minimal residual disease (MRD) following induction therapy for pediatric acute lymphoblastic leukemia. [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 2185. doi:10.1158/1538-7445.AM2014-2185

Obesity promotes the pathogenesis of many cancers, including acute lymphoblastic leukemia (ALL), the most common childhood malignancy. We have shown that ALL cells interact with adipocytes in vivo and in vitro, and that adipocytes protect ALL cells from chemotherapies. Since cancer cells require free fatty acids (FFA) for energy and as molecular building blocks, we hypothesize that ALL cells gain a survival advantage by stimulating adipocyte lipolysis and using adipocyte-derived FFAs. To test whether ALL cells stimulate adipocyte lipolysis, 3T3-L1 adipocytes were exposed to ALL-conditioned media for 72 hours, and glycerol release and adipocyte lipid content were measured. To determine whether ALL cells take up adipocyte-derived FFA, adipocytes were pre-labeled with fluorescent FFA (BODIPY), and then co-cultured with murine (8093) and human (BV173, RS4;11, Molt4, Nalm6, SEM, SupB15) ALL cells in a Transwell system. ALL uptake of BODIPY-FFAs was analyzed via flow cytometry. To determine the fate of FFA in ALL cells, ALL cells were incubated with BODIPY-FFA and evaluated by confocal and live cell microscopy, and lipid classes resolved through thin layer chromatography (TLC). Real-time PCR was employed to analyze adipocyte-induced alterations in the transcription of key enzymes in the ALL de novo lipogenesis pathway. Finally, TOFA, an acetyl-CoA carboxylase 1 (ACC1) and steroyl-CoA desaturase 1 (SCD1) inhibitor, was used to block de novo lipogenesis in ALL cells in vitro, and evaluate the ability of ALL cells to use adipocyte-derived FFAs for proliferation. ALL-conditioned media stimulated 3T3-L1 lipolysis (191.0 vs. 165.5 mg/L glycerol, n=1), reducing adipocyte lipid content as quantified with Oil Red O by 16.8 ± 7.1% (p = 0.047). When murine or human ALL cells were co-cultured with BODIPY-labeled adipocytes, they accumulated the fluorescent label within 48 hours (97-98% of cells labeled positive). TLC, live cell imaging and confocal microscopy demonstrated the majority of this label accumulated within ALL cell phospholipid membranes and triglyceride-laden lipid droplets. Adipocyte co-culture reduced expression of lipogenic enzymes such as fatty acid synthase (FAS), ACC1 and SCD1 in ALL cells. Finally, while TOFA inhibited ALL cell proliferation (6.1 ± 1.1x104 vs. 41.9 ± 4.7x104 cells, p=0.004, n=3), this proliferation was partially rescued by adipocyte conditioned media (ACM, 29.8 ± 9.2 x104, p=0.045 vs. TOFA alone, n=3). Therefore, we have demonstrated that ALL cells stimulate adipocyte lipolysis and use adipocyte-derived FFAs to supplement de novo lipogenesis and proliferation. This may contribute to the effect of obesity on ALL development and relapse. Citation Format: Jonathan Tucci, Xia Sheng, Steven D. Mittelman. Acute lymphoblastic leukemia cells stimulate adipocyte lipolysis and utilize adipocyte-derived free-fatty acids for proliferation. [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 4339. doi:10.1158/1538-7445.AM2014-4339

Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been shown to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have demonstrated that murine ALL cells actively migrate into adipose tissue in mice, and that adipocytes protect ALL cells from daunorubicin (DNR) in vitro. We have further shown that media cultured by both adipocytes and ALL cells together (ALCM) can protect ALL cells from DNR. We hypothesize that adipocytes protect ALL cells from DNR by decreasing ALL cell intracellular drug accumulation. Human pre B ALL cells (BV173) were co-cultured with 3T3-L1 adipocytes using a transwell system. Intracellular DNR was visualized using con-focal fluorescence microscopy at various time points in leukemia cells during DNR treatment. Drug uptake in leukemia cells treated with DNR and co-cultured with adipocytes was measured by flow cytometry. By preloading cells with DNR, drug efflux assays were also performed with ALL cells. Gene expression of ABCB1a and cell surface expression of MDR1 were quantified using qPCR and flow cytometry, respectively, in ALL cells co-cultured with adipocytes. After 72 hours of DNR treatment, there were significantly more viable BV173 cells in the transwells placed over adipocytes than those over fibroblasts (290,000± 60,500 vs. 108,000± 13,000, p=0.008, n=4). Co-culture with adipocytes was associated with a lower intracellular DNR in BV173 cells. After 4 hours, DNR was 30% lower, and by 24 hours intracellular DNR was 80% less (n=1). However, co-culturing ALL cells with adipocytes was not associated with a significant increase in efflux rate of DNR from preloaded BV173 (half-life = 36±13 vs. 31±9 min., p=0.23, n=3). The mRNA expression of ABCB1a, the gene encoding MDR1, was increased in BV173 cells (130% increase, n=1) co-cultured with adipocytes for 24 hours. However, we did not detect an increase in surface MDR1 expression in BV713 co-cultured with adipocytes. We then tested whether adipocyte-secreted factors would cause efflux of DNR from ALL cells. There was a tendency for DNR efflux rate to be higher in BV173 cells cultured in ALCM (half-life = 36±13 vs. 27±10 min., p=0.08, n=3). Further investigation is needed to confirm this effect. These findings demonstrate that adipocytes may protect ALL cells from daunorubicin treatment in part by decreasing intracellular drug accumulation. However, it is not clear whether this effect is due to altered uptake, efflux, or intracellular drug action. Citation Format: Xia Sheng, Jonathan Tucci, James Behan, Steven D. Mittelman. Adipocytes decrease daunorubicin concentration in acute lymphoblastic leukemia cells. [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 172. doi:10.1158/1538-7445.AM2014-172

Cancer growth and progression is influenced by tumor cells, stromal cells, as well as various secreted molecules found within the tumor microenvironment (TME). These secreted molecules participate in various crosstalk events that contribute to cancer development and progression. One such secreted molecule found within the tumor microenvironment is the proinflammatory cytokine interleukin-8 (IL-8). IL-8 influences cancer development by binding to its G-protein coupled receptors CXCR1 and CXCR2 in turn leading to activation of various signal transduction pathways that promotes cell proliferation, survival, angiogenesis, and invasion. We hypothesized that infiltrating macrophages and tumor cells participate in cellular crosstalk through the release of inflammatory cytokines which function to modulate the breast cancer phenotype. Using a cell culture model, conditioned media (CM) generated from activated THP-1 monocytes as well as recombinant IL-8 (rIL-8) were individually tested on the breast cancer cell lines T47D (ER +) and MDA-MB 231 (ER -) in order to evaluate cell proliferation, cell signaling pathways, invasion, and migration. Increased proliferation was observed for both T47D and MDA-MB 231 following treatment with rIL-8 as opposed to an inhibition in proliferation, which was observed following culturing of T47D and MDA-MB 231 with THP-1 CM. It is presumed that THP-1 CM contains both inhibitory and stimulatory factors, which include IL-8. Activation of the MAPK signaling pathway, as observed by phosphorylation of MEK and ERK, was modulated in response to activated THP-1 CM, which may explain the enhanced migration and invasion observed in the breast cancer cells upon treatment with rIL-8. In order to further characterize the crosstalk between THP-1 and breast cancer cells, the inverse experiments were performed by incubating non-activated THP-1 monocytes with CM generated from MDA-MB 231 cells. Interestingly, MDA-MB 231 CM was capable of activating THP-1 monocytes comparable to that of the classical activating agent phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA). Characterization of the MDA-MB 231 CM by cytokine array analysis revealed an abundance of the proinflammatory cytokines IL-6 and IL-8 and although this needs further characterization, we believe that secretion of IL-6 and IL-8 is essential to the crosstalk between breast cancer cells and THP-1 monocytes, which modulates the breast cancer cell phenotype. Citation Format: Robert Bednarczyk, Neha Tuli, Ghada Benrahoma, Rui Kitadai, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. Proinflammatory chemokine interleukin-8 in the tumor microenvironment modulates breast cancer phenotype. [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 1171. doi:10.1158/1538-7445.AM2014-1171

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine (Asn) and glutamine (Gln). We and others have shown that its cytotoxicity depends more on Gln than Asn depletion. Novel ASNases with different activities have been developed in an attempt to decrease side effects and allergic reaction. To evaluate the importance of glutaminase activity, we developed a novel form of Helicobacter pylori ASNase (dm HpA) using site-directed mutagenesis, with amino acid substitutions M121C/T169M. This mutant form was designed to have the same asparaginase activity as wild type (wt), but lack glutaminase activity. WT and dm HpA were compared with the clinically used ASNases from E. coli (EcA-II) and Erwinia chrysanthemi (ErA). Asparaginase and glutaminase activities of each enzyme were measured with Nessler's assay at pH 8.6. The ratio of glutaminase/asparaginase activity was 1.4% for dm HPase, 7.5% for wt HPase, 41% for EcA-II, and 99% for ErA (at 0.6 IU), respectively. L-Asparaginases (0.01 to 3 IU/ml over 72 hrs) were tested on 8 human leukemia cell lines: pre B ALL (BV173, Nalm-6, RCH-ACV, RS4;11, SEM, SupB15), T cell ALL (Molt-4), and acute promyelocytic leukemia (HL-60). Viable cells were determined by trypan blue exclusion. Two cell lines which we had previously shown to be Asn-dependent (RS4;11 and SupB15) were equally sensitive to the asparaginase isoforms (>90% kill at 0.03 IU/mL). The other 6 lines were more sensitive to isoforms with higher glutaminase activities (Table). ErA was the most effective on all three cell lines on which it was tested. These data show that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full efficacy. These sensitivities to ASNases match previous data on Asn- and Gln-dependent cell proliferation (Ehsanipour et al., Cancer Research, 2013). % cell growth inhibition at 3 IU/ml ASNase (n=1-2 for each cell line; nd: not done)Cell linedm HpAwt HpAEcA-IIErARS4;11919291ndSupB15919193ndRCH-ACV13362866SEM22484165BV17324326ndNalm-61621574HL-60204033ndMolt4325963nd Citation Format: Jean-Hugues Parmentier, Maristella Maggi, Erika Tarasco, Claudia Scotti, Vassilios Avramis, Steven D. Mittelman. Glutaminase activity determines cytotoxicity of L-asparaginases on leukemia cell lines. [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 3378. doi:10.1158/1538-7445.AM2014-3378

Breast cancer stem cells (BCSCs), often characterized by cell surface marker expression as being CD44+/CD24- or CD44+/CD24low phenotypes, constitute a subpopulation of tumor cells that are highly tumorigenic. With respect to CD44, it has been reported that its expression is influenced by tumor microenvironment (TME) and that modulation of CD44 is detrimental to breast cancer stem cell self-renewal and differentiation. Interestingly, the factors that mitigate this effect are still unknown, which has led us to hypothesize that factors within the TME, comprising of differentiating agents, modulate the BCSC phenotype by regulating cell surface markers. MDA-MB 231 showed presence of BCSCs as CD44 positive cells were detected using immunofluorescence and Western blot analyses. The expression of CD44 on MDA-MB 231 was not responsive to the proinflammatory molecule IL-8 or estrogen. Both MDA-MB 231 and T47D were responsive to IL-8 and estrogen with respect to migration and invasion as examined in vitro using a Boyden Chamber assay suggesting that migration and invasion was independent of CD44 expression. Alternatively IL-8 responsiveness may be on a select clonal population of CD44- cells. The presence of differentiating agents within the TME has been explored. To examine the effect of differentiating agents, we used the classical all-trans-retinoic acid (ATRA) previously shown to promote tumor regression. CD44 expression was highly responsive to ATRA as it was down regulated following treatment. ATRA treatments also resulted in decreased migration and invasion of breast cancer cells. Our results led us to conclude that specific differentiating agents can affect breast cancer stem cell activity by directly down regulating key adhesion cell surface markers. Citation Format: Rui Kitadai, Robert Bednarczyk, Neha Tuli, Elyse Hanly, Ghada Benrahoma, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. Modulation of breast cancer stem cell marker CD44 by all-trans-retinoic acid (ATRA). [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 3889. doi:10.1158/1538-7445.AM2014-3889

The tumor microenvironment (TME) is a heterogeneous niche consisting of numerous stromal cells, structural components, and secreted molecules from both the malignant and surrounding cells that contribute to tumor growth and progression. Importantly, these diverse types of secreted molecules can initiate crosstalk events between the tumor cells and TME. One such molecule, CXCL8/Interleukin-8 (IL-8), is a proinflammatory chemokine, primarily involved in neutrophil mobilization, which activates multiple cellular signaling pathways via the membrane associated G protein-coupled CXCL8 cognate receptors, CXCR1 and CXCR2. We hypothesized that IL-8, a ubiquitous inflammatory molecule that is secreted by macrophages, activated fibroblasts and mesenchymal cells, can alter breast cancer phenotype. To this end, using a cell culture model we analyzed both estrogen receptor (ER) positive and ER negative breast cancer cell lines T47D and MDA-MB-231 respectively that were found to express the CXCL8 receptors CXCR1 and CXCR2. The cellular source of IL-8 was from conditioned media (CM) from in vitro cultures of primary stromal cells including primary mesenchymal and fibroblast cells, as well as the established monocytic cell lines HL-60 and THP-1. Each cell line secreted adequate amounts of IL-8 as measured by ELISA, however primary mesenchymal cells produced four times that of the fibroblasts and macrophages suggesting that activated mesenchymal cells may be a very potent source of chronic inflammation in the TME. Interestingly, treatment with IL-8 or CM from the phorbol ester treated macrophages THP-1 led to increased proliferation in T47D in vitro however only IL-8 treatment led to proliferation of MDA-MB-231. Recombinant IL-8 as well as CM of THP-1 and mesenchymal cells enhanced migration and invasion of both T47D and MDA-MB-231 cells as evaluated by a modified Boyden chamber assay. Supplementation of the triple negative MDA-MB-231 line with THP-1 CM enhanced its invasive propensity nearly two fold. THP-1 CM treatment also led to the activation of the PI3K/p-Akt and Mek/Erk pathways in the ER− MDA-MB-231 cells but not the ER+ T47D cells suggesting a link between CXCL8 mediated inflammation and constitutive signal transduction pathways that is translatable into enhanced metastatic propensity. Our studies are directed towards the analysis of both chronic and acute inflammation of the TME and we believe IL-8 and its associated intracellular molecular markers may be important preventive and therapeutic targets in triple negative breast cancer. Citation Format: Andrea L. George, Robert Bednarczyk, Shilpi Rajoria, Elyse Hanly, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. CXCL8 is a secretory inflammatory stimulus of the activated TME that modulates breast cancer phenotype. [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 1432. doi:10.1158/1538-7445.AM2013-1432

The cellular composition of the tumor microenvironment (TME) and soluble effector molecules mediating intercellular cross-talk are important therapeutic and preventive targets for breast cancer. Chemokines released within the TME work in a synergistic and reciprocal manner to induce malignant progression of tumor cells. CXCL8, a proinflammatory CXC chemokine shown to be released within the TME, can act as a chemoattractant, angiogenic factor, and inflammatory factor via cellular receptors CXCR1 and CXCR2. However, the specific role of CXCL8 in the signaling cascades within the tumor microenvironment has yet to be fully classified and warrants further investigation. Treatment of human breast cancer cell lines MCF-7, MDA-MB-231, and T47D with 50nM exogenous CXCL8 enhanced the expression of CXCR2. The acquisition of chemokine receptors by tumor cells, in this case CXCR2, can mediate phenotypic changes that enhance migratory and invasive potential. Treatment of these human breast cancer cell lines with 50nM exogenous CXCL8 resulted in phenotypic morphology changes characteristic of an epithelial-to-mesenchymal transition (EMT), a phenomenon commonly exhibited by metastatic tumor cells. We validated this phenomenon with 50nM exogenous CXCL8 treatment, which led to enhanced expression of both Slug (Snail2) and Twist in all three human breast cancer cell lines. Furthermore, we observed that treatment with 50nM exogenous CXCL8 promoted the migration and invasion of these breast cancer cell lines in an in vitro modified Boyden chamber assay. These results suggest that CXCL2 secreted from cells within the TME act to induce characteristic changes in human breast cancer cells, propagating the transition to more aggressive tumors. Tumor-associated macrophages (TAMs) are an important component of the heterogeneous population of cells that make up the TME. Breast cancer cells exposed to conditioned media from human monocytic cell lines THP-1 and U937, previously differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA), exhibited distinct morphology changes characteristic of EMT and enhanced migratory and invasive ability, similar to exogenous CXCL8 treatment. Together, our results suggest that CXCL8 can have profound implications in determining the fate of tumor cells by engaging in extensive cross-talk within the TME, thus providing a potential target for the treatment and prevention of breast cancer tumor progression and metastasis. 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 304. doi:1538-7445.AM2012-304

Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been demonstrated to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have shown that obesity impairs the survival of mice transplanted with ALL and treated with the front-line chemotherapy, L-Asparaginase (ASNase), which acts through the deamination of asparagine (ASN) and glutamine (GLN). We have also shown that adipocytes protect ALL cells from ASNase in vitro. We hypothesized that adipocytes protect ALL cells from ASNase via production of GLN. Fat pads were excised from control and diet-induced obese mice. Glutamine synthetase, the rate-limiting enzyme for GLN synthesis, was analyzed by western blot. Amino acids secreted by fat pads were measured using HPLC. ALL cell sensitivity to ASN and/or GLN depletion, and ASNase, was determined by trypan blue exclusion in culture alone and in transwells over 3T3-L1 adipocytes. Mouse fat pads expressed high levels of glutamine synthetase protein, and this was not affected by obesity or 5 days of L-asparaginase treatment (3,000 IU/kg/day). Fat pad explants secreted GLN (251±50 nmoles/100 mg tissue), but not ASN, over 72 hours. Confirming that GLN secretion could protect ALL from ASNase, twice daily addition of 500 nmoles of GLN to murine ALL cells completely reversed ASNase cytotoxicity (64.0x10^4 vs. 7.9x10^4 viable cells after 72 hours of 1 IU/mL ASNase, p 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 5245. doi:1538-7445.AM2012-5245

Glioblastoma is characterized by highly proliferative tumor cells, increased cellularity, necrosis, and proliferation of capillary endothelial cells. The incidence rate of glioma in adults is 50% greater in men than in women suggesting that there may be a male hormonal component that modulates glioblastoma progression. Our preclinical developmental program identified Capridine as a chemotherapeutic agent that had specificity towards prostate cancer with minimal bone marrow toxicity. Since the DNA intercalating activity of Capridine does not completely correlate with its cytotoxic activity, we examined other alternative cellular targets. Our investigation identified the androgen receptor (AR) and its dependent signal transduction mechanism as a possible cellular mediator of its cytotoxic activity. In this study, our analysis revealed that glioblastoma cell lines T98G and MO59K expressed AR and their growth was responsive to the AR-mediated signal transduction. Capridine inhibited the growth of glioblastoma cells by down regulating the AR and inhibiting the phosphorylation of Focal adhesion kinase (FAK). Since FAK is associated with integrin and neuropeptide mediated signaling, critical for cell migration and invasion, we examined the activity of Capridine on cell invasion and migration using a Boyden Chamber assay. Capridine inhibited the migration and invasion of the glioblastoma cell lines T98G and MO59K. Our data not only validate AR as a target of Capridine as with prostate cancer but also implicate a novel target, FAK in glioblastoma. Combination approaches using Capridine and anti-hormonal therapy may prove to be viable chemotherapeutic regimen for primary glioblastoma as well as an inhibitor of metastatic propensity. These preclinical studies are being extended in an in vivo animal model with combination treatment approaches with a view to develop novel chemotherapy based treatment for glioblastoma. 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 3803. doi:1538-7445.AM2012-3803

The tumor microenvironment (TME) of breast cancer is a heterogeneous population of tumor cells, infiltrating endothelial cells, immunomodulatory cells, and underlying stroma. This cellular cross-talk is key to tumor progression; however the signaling pathways involved remain unclear. Rapid expansion of breast cancer cells requires sufficient supply of oxygenated blood supported by tumor induced neovasculature. Our previous studies demonstrated that estrogen mediates neovascularization via recruitment of bone marrow derived endothelial progenitor cells (BM-EPCs). However, hypoxia, a major cellular characteristic of cancer cell growth, mimics estrogen induced vascularization as evidenced by HIF-1α translocation and secretion of vascular endothelial growth factor (VEGF). These studies were carried out in the TG1-1 murine mammary cancer cell line and used as a model. Conditioned media from TG1-1 cells enhanced endothelial cell migration and tube formation in vitro. Treatment of tumor cells with the anti-estrogen Fulvestrant as well as the HIF-1 inhibitor YC-1 abrogated the effects of estrogen. Further, use of the PI3K inhibitor wortmannin as well as the protein inhibitor cyclohexamide, elucidated the necessity of a functional AKT signaling pathway in this estrogen induced neovasculogenesis. While the cell-cell interaction studies demonstrate a considerable modulation of the tumor microenvironment, the co-culture experiments using immunomodulatory cells and epithelial cells induced epithelial to mesenchymal transition (EMT) in TG1-1 cells. We demonstrated that the murine macrophage cell line RAW264.7 secretes factors that modulate tumor cell EMT via upregulation of TWIST and Slug and an increase in tumor cell migration and invasion. To elucidate the modulatory secreted factors, we performed western blot analysis of the TGF-β signaling pathway and observed differences in SMAD levels. However, neutralizing antibody studies indicate that TGF-β is not the sole modulator of RAW264.7 induced EMT in Tg1-1 cells. Taken together, cytokines as extrinisic factors and tumor hypoxic conditions generating intrinsic factors, act in concert to alter breast cancer progression. Our data demonstrate a need to target multiple TME interacting pathways as a future breast cancer treatment strategy. 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 1494. doi:1538-7445.AM2012-1494

Active specific immunotherapy has been implemented in patients with advanced malignant melanoma, utilizing the mouse antiidiotypic (anti-id) monoclonal antibody (mAb) MK2-23 which bears the internal image of high molecular weight-melanoma associated antigen (HMW-MAA). In a previous study, development of anti-HMW-MAA immunity in patients with advanced malignant melanoma immunized with anti-id mAb MK2-23 was found to be associated with a statistically significant survival prolongation. Since no information is available about the relationship between development of immunity and clinical response in patients immunized with anti-id mAb, the present study has characterized the kinetics of the immune response in three patients with advanced malignant melanoma who experienced regression of metastatic lesions following immunization with the anti-id mAb MK2-23. The three patients developed anti-mouse IgG antibodies, anti-anti-id antibodies and anti-HMW-MAA antibodies. The anti-HMW-MAA antibodies are mainly IgG, suggesting that the immune response elicited by anti-id mAb MK2-23 is T-cell dependent. The development of anti-HMW-MAA immunity preceded the reduction in the size of metastatic lesions. This temporal relationship suggests but does not prove that the anti-HMW-MAA immunity elicited by anti-id mAb MK2-23 has a beneficial effect on the clinical course of the disease in patients with malignant melanoma. This finding in conjunction with minor side effects associated with repeated administrations of mouse anti-id mAb MK2-23 suggest that active specific immunotherapy with anti-id mAb which bear the internal image of melanoma-associated antigen represents a viable therapeutic approach to malignant melanoma.