Triple-negative Mammary Tumors Research Articles

MYC Deregulation and PTEN Loss Model Tumor and Stromal Heterogeneity of Aggressive Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response.

Intratumoral Treatment with 5-Androstene-3β, 17α-Diol Reduces Tumor Size and Lung Metastasis in a Triple-Negative Experimental Model of Breast Cancer.

Breast cancer treatment failure is related to low response rates, high costs, and long-term toxicities. Thus, it is necessary to find less toxic, cheaper, and more effective treatments. In situ administration ensures drug delivery to tumor cells and decreases systemic toxic effects. The androstene-3β, 17α-diol (α-AED) reduces breast tumor cell proliferation and is an ideal candidate to treat mammary tumors. This study aims to identify the in vitro and in vivo effects of α-AED on a triple-negative mammary tumor model. An in vitro biphasic steroid effect was observed in mouse and human mammary tumor cells treated with α-AED. In this sense, cells treated with higher doses (100 and 200 μM) showed an antiproliferative effect. The α-AED administrated intratumorally reduced average tumor weight and increased the percentage of natural killer cells (NK), plasmatic, and plasmablast cells in mice tumors. Of note, VEGF levels in all α-AED-treated tumors was lower than in the control and vehicle groups. The tumor in situ increased response was reflected systemically by higher anti-4T1 IgG concentration in serum from α-AED-treated mice, but no other associated systemic changes were detected. The reduction in tumor size for the local injection of α-AED is associated with the anti-proliferative effect of this steroid, and the lower local levels of VEGF may be related to the imperceptible macroscopic metastasis in α-AED-treated mice. The above suggests that α-AED may be used in clinical studies to prove its efficacy as an alternative breast tumor treatment or in conjunction with already established therapies.

Highly metastatic claudin-low mammary cancers can originate from luminal epithelial cells

Claudin-low breast cancer represents an aggressive molecular subtype that is comprised of mostly triple-negative mammary tumor cells that possess stem cell-like and mesenchymal features. Little is known about the cellular origin and oncogenic drivers that promote claudin-low breast cancer. In this study, we show that persistent oncogenic RAS signaling causes highly metastatic triple-negative mammary tumors in mice. More importantly, the activation of endogenous mutant KRAS and expression of exogenous KRAS specifically in luminal epithelial cells in a continuous and differentiation stage-independent manner induces preneoplastic lesions that evolve into basal-like and claudin-low mammary cancers. Further investigations demonstrate that the continuous signaling of oncogenic RAS, as well as regulators of EMT, play a crucial role in the cellular plasticity and maintenance of the mesenchymal and stem cell characteristics of claudin-low mammary cancer cells.

Abstract P2-06-23: New biomarkers for adjuvant bisphosphonate use in triple-negative breast cancer

Abstract We first showed that triple-negative breast cancer (TNBC) patients with low Toll-like receptor 9 (TLR9) expression have significantly worse prognosis than patients with high TLR9 expression. TLR9 is an innate immunity DNA receptor which is also expressed in several cancers, including TNBC. Later, we showed that low-TLR9 cells and tumors are more sensitive to amino-bisphosphonates, especially to zoledronate. Bisphosphonates (BP) inhibit osteoclasts and thereby effectively prevent bone fractures in osteoporosis and in cancer-induced bone disease. BPs also have direct anticancer effects. Interestingly, adjuvant BP use is associated with significantly improved progression-free and overall survival, but only among post-menopausal breast cancer patients. The molecular mechanisms behind this observation is unclear. CD73 is a 70 kDa glycophosphatidylinositol-anchored cell protein, which converts adenosine monophosphate to adenosine and organic phosphate. High CD73 expression has been shown to have cancer cell invasion-promoting properties. On the other hand, CD73 is associated with poor prognosis in TNBC. Interestingly, TLR9 and CD73 have been shown to have inverse relationship in T cells and during colonic inflammation. Currently, we study the relationship of CD73 and TLR9 in vitroand in vivo upon BP treatment. Our preliminary results show that TLR9 and CD73 have an inverse relationship also in TNBC cells. We use both nitrogen-containing (zoledronate and alendronate) and pyrophosphate-like (clodronate) BPs together with doxorubicin to study TNBC cell responses. We have seen that zoledronate and alendronate, but not clodronate, inhibited 4T1 murine triple-negative mammary tumor cell proliferation in a dose-dependent manner. Also, high concentrations of zoledronate and alendronate significantly inhibited 4T1 cell migration. Additionally, CD73 shRNA cells are more sensitive to zoledronate and alendronate, showing morphological changes and disruption in actin organization. Our results suggest that CD73 expression may affect treatment responses to BPs in TNBC. Further, low-CD73 tumors could benefit more of BP therapy, compared with high-CD73 tumors. Citation Format: Sandholm JA, Petruk N, Selander KS, Tuomela JM. New biomarkers for adjuvant bisphosphonate use in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-23.

Deregulation of VEGFR-2 and PDGFR Expression and Microvascular Density in a Triple-Negative Model of Canine Malignant Mammary Tumors with Lymph Node or Lung Metastasis

Canine mammary tumors (CMT) represent the most common cancer in noncastrated female dogs. Interestingly, triple-negative tumors are the most common molecular subtype in female dogs. In this study, we proposed to evaluate the expression of vascular endothelial growth factor receptor 2 (VEGFR-2), Platelet-derived growth factor receptor (PDGFR), and microvascular density (MVD) in a group of metastatic and nonmetastatic triple-negative CMT and compare the expression based on clinical parameters. Twenty-six female dogs with triple-negative mammary tumors were divided into three groups: nonmetastatic tumors (NMT) (n = 11), tumors with lymph node metastasis (LNM) (n = 10), and tumors with lung metastasis (LM) (n = 5). We observed increased VEGFR-2 expression in LNM compared with NMT and a positive correlation between tumor grade and VEGFR-2 expression. A positive correlation was noted between VEGFR-2 and PDGFR expression. Regarding microvascular density (MVD), we identified a higher number of vessels in primary tumors with lymph node metastasis and lung metastasis compared with tumors with no metastasis. The primary tumors with lung metastasis exhibited an increased MVD compared with carcinoma with lymph node metastasis. Overall, our results suggest a deregulation of VEGFR-2 and PDGFR and high MVD in metastatic tumors, indicating a role for angiogenesis in tumor progression.

Leptin Signaling Mediates Obesity-Associated CSC Enrichment and EMT in Preclinical TNBC Models.

Obesity is associated with poor prognosis in triple-negative breast cancer (TNBC). Preclinical models of TNBC were used to test the hypothesis that increased leptin signaling drives obesity-associated TNBC development by promoting cancer stem cell (CSC) enrichment and/or epithelial-to-mesenchymal transition (EMT). MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like, triple-negative mammary tumors, received either a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat) for up to 42 weeks (n = 15/group). Mice were monitored for tumor development and euthanized when tumor diameter reached 1.5 cm. Tumoral gene expression was assessed via RNA sequencing (RNA-seq). DIO mice had greater body weight and percent body fat at termination than controls. DIO mice, versus controls, demonstrated reduced survival, increased systemic metabolic and inflammatory perturbations, upregulated tumoral CSC/EMT gene signature, elevated tumoral aldehyde dehydrogenase activity (a CSC marker), and greater leptin signaling. In cell culture experiments using TNBC cells (murine: E-Wnt and M-Wnt; human: MDA-MB-231), leptin enhanced mammosphere formation, and media supplemented with serum from DIO versus control mice increased cell viability, migration, invasion, and CSC- and EMT-related gene expression, including Foxc2, Twist2, Vim, Akt3, and Sox2 In E-Wnt cells, knockdown of leptin receptor ablated these procancer effects induced by DIO mouse serum. These findings indicate that increased leptin signaling is causally linked to obesity-associated TNBC development by promoting CSC enrichment and EMT.Implications: Leptin-associated signals impacting CSC and EMT may provide new targets and intervention strategies for decreasing TNBC burden in obese women. Mol Cancer Res; 16(5); 869-79. ©2018 AACR.

Melatonin Regulates Angiogenic and Inflammatory Proteins in MDA-MB-231 Cell Line and in Co-culture with Cancer-associated Fibroblasts.

Cancer-associated fibroblast (CAFs) are the most abundant cells in the tumor microenvironment, able to secrete growth factors and act on tumor progression. Melatonin is associated with several mechanisms of action with oncostatics and oncoprotectors effects, and also participate in the reduction of synthesis of surrounding fibroblasts and endothelial cells in breast cancer. The objectives of this study were to determine the effectiveness of melatonin in cell viability and expression of proteins involved in angiogenesis and inflammation in triplenegative mammary tumor cell line (MDA-MB-231) and in co-culture with CAFs. Cell viability was measured by MTT assay and the protein expression was evaluated by Membrane Antibody Array after melatonin treatment. Melatonin treatment (1 mM) for 48 hours reduced the cell viability of MDA-MB-231, CAFs and co-culture (p < 0.05). The semi-quantitative protein analysis showed that when monoculture of tumor cells were compared with co-culture of CAFs, there was a regulation of angiogenic and inflammatory proteins (p < 0.05). Melatonin treatment also leads a differential expression of angiogenic and inflammatory proteins in both monoculture and co-culture of tumor cells and CAFs (p < 0.05). The influence of CAFs under the tumor microenvironment was confirmed, increasing the malignancy of the tumor. In addition, melatonin is effective in both monoculture and co-culture, regulating angiogenic and inflammatory proteins that contribute to tumor progression. This study show an overview of melatonin ability in regulating angiogenic and inflammatory proteins, and opens the way for exploration of each individual protein in further studies.

Abstract 2101: A gene-expression fingerprint predicting sensitivity to all-trans-retinoic acid in breast cancer cells is tumor-context independent

Abstract All-trans retinoic acid (ATRA) and derived natural as well as synthetic retinoids are promising agents in the treatment and chemoprevention of various types of neoplasia, including mammary tumors. ATRA is an important component of the therapeutic schemes used for the treatment of a rare form of Acute Myelogenous Leukemia known as Acute Promyelocytic Leukemia (APL). A rational use of the paradigmatic retinoid, ATRA, in a heterogeneous disease, like breast cancer, requires the definition of the cellular and molecular determinants of sensitivity to the agent. The major aim of the study was the definition of a predictive gene expression fingerprint that can be used for the selection of patients who may benefit from treatment protocols containing ATRA. To this purpose, we selected 53 breast cancer cell lines characterized for the constitutive whole genome gene expression profiles. The sensitivity of 30 cell lines (training set) to the anti-proliferative action of ATRA was defined after challenge with increasing concentrations of the retinoid for 3, 6 and 9 days. This analysis established that Luminal and ER+ cell lines are enriched within the ATRA sensitive group. In contrast, cell lines characterized by a Basal-like phenotype, according to the PAM50 gene expression signature, are generally refractory to the growth inhibitory action of ATRA. The sensitivity of Luminal-A and Luminal-B and the general refractoriness of Basal-like tumors to ATRA was validated in short-term tissue slice cultures of surgical breast cancer specimens. The training set was used to define a gene-expression fingerprint consisting of morethan 100 genes significantly associated with ATRA sensitivity. The fingerprint was generated by reprocessing the RNA sequencing data contained in the CCLE (Cancer Cell line Encyclopedia) of the Broad Institute and it was built from approximately 60,000 coding and non-coding loci. The approach involved the use of general linear models (machine learning algorithm). The identified gene-expression fingerprint was subsequently used to successfully predict ATRA sensitivity in a test set consisting of the remaining 23 cell lines. As a first step towards the use of the fingerprint for the stratification of patients, we evaluated the proportion of predicted ATRA sensitive breast tumors in the TCGA dataset. In accordance with the cell line and primary tumor data, approximately 30% of the Luminal tumors present with a high similarity score to the identified gene expression fingerprint associated with ATRA sensitivity. In contrast, only 5% of the Basal-like or Triple-negative mammary tumors are characterized by the same high similarity score. Interestingly, the ATRA sensitivity signature seems to be tumor context independent, as it correctly identifies the Acute Promyelocytic Leukemia patients present in the Acute Myelogenous Leukemia patients present in two publicly available datasets. Citation Format: Enrico Garattini, Maurizio Gianni’, Marco Bolis, Maddalena Fratelli, Gabriela Paroni, Mineko Terao. A gene-expression fingerprint predicting sensitivity to all-trans-retinoic acid in breast cancer cells is tumor-context independent. [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 2101.

Tumor-Associated Macrophage-Mediated Targeted Therapy of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. TNBC is often infiltrated with a large number of macrophages, which in turn promote tumor growth and metastasis. In this study, tumor-associated macrophages (TAMs) were exploited as a target to deliver doxorubicin (DOX), a chemotherapeutic agent, to TNBC using nanoparticles surface-functionalized by (i) acid-sensitive sheddable PEGylation and (ii) modifying with mannose (i.e., DOX-AS-M-PLGA-NPs). In mice with orthotopic M-Wnt triple-negative mammary tumors, a single intravenous injection of DOX-AS-M-PLGA-NPs significantly reduced macrophage population in tumors within 2 days, and the density of the macrophages recovered slowly. Repeated injections of DOX-AS-M-PLGA-NPs can help maintain the population of the macrophages at a lower level. In M-Wnt tumor-bearing mice that were pretreated with zoledronic acid to nonselectively deplete macrophages, the TAM-targeting DOX-AS-M-PLGA-NPs were not more effective than the DOX-AS-PLGA-NPs that were not surface-modified with mannose and thus do not target TAMs in controlling tumor growth. However, in M-Wnt tumor-bearing mice that were not pretreated with zoledronic acid, the TAM-targeting DOX-AS-M-PLGA-NPs were significantly more effective than the nontargeting DOX-AS-PLGA-NPs in controlling the tumor growth. The AS-M-PLGA-NPs or other nanoparticles surface-functionalized similarly, when loaded with a chemotherapeutic agent commonly used in adjuvant therapy of TNBC, may be developed into targeted therapy for TNBC.

Abstract PD3-08: Modulation of immunity with 2-fluorofucose (2FF) for breast cancer treatment and prevention

Abstract The majority of patients with breast cancer have robust Type II immune responses directed against their tumors with little to no Type I immunity. The dominance of a Type II microenvironment is established early in breast tumorigenesis with a Type II immune signature prevalent even in pre-invasive lesions such as ductal carcinoma in situ. As a result, breast cancer is associated with abundant autoantibodies directed against tumor associated antigens and few infiltrating T-cells in the majority of patients. A recently reported inhibitor of protein and cellular fucosylation, 2-fluorofucose (2FF) has been shown to enhance immune cell function, in part through the generation of fucose-deficient antibodies which result in enhanced antibody dependent cell mediated cytotoxicity, as well as apparent modulation of T-cell dependent activity [Ca Res, Oct 1, 2014 74;2890]. For treatment studies, TgMMTV-neu (MMTVneu; luminal) and C31-Tag (C3T; triple negative) mice were treated orally with vehicle alone and 20mM 2FF when spontaneous tumor volume reached 75-100mm3 until sacrifice (n=20/grp). To assess the ability to prevent breast cancer development, mice were treated orally with vehicle alone, 20 or 50mM 2FF for up to 200 days starting at 6-8 weeks of age (n=20/grp). Tumor kinetics, disease free survival, and overall survival were calculated. Immune responses were evaluated by both DTH and IFN-gamma ELISPOT. To determine immune mechanism of action, NK, B, CD4, or CD8 cells were depleted concurrently with tumor implant. When tumor bearing mice were treated with 2FF, tumor growth was significantly inhibited in both groups over the course of therapy (MMTVneu p&amp;lt;0.0001 and C3T p&amp;lt;0.0001 compared to controls). In addition, survival was significantly prolonged in both models (MMTVneu p&amp;lt;0.0001 and C3T p=0.0014). In the prevention setting, 2FF significantly delayed the average age of tumor onset in both models at both doses with the higher does showing greater efficacy; MMTVneu 20mM, p=0.001, 50mM, p=0.0002. At the time of study termination (300d), 45% of 2FF-treated MMTVneu mice had no evidence of mammary tumors. 2FF also significantly improved the average age of tumor onset in C3T mice when comparing untreated to 20mM (p=0.007), and 50mM (p=0.0007) treated mice. At study termination, 33% of all 2FF-treated C3T animals were disease free. 2FF anti-tumor activity was associated with the induction of tumor antigen specific immunity. After treatment both the MMTVneu (p&amp;lt;0.0001) and C3T (p&amp;lt;0.0001) developed a DTH response to syngeneic tumor lysates. Of note, the DTH response was inversely associated with the rate of tumor growth (p&amp;lt;0.0001). 2FF anti-tumor activity was associated with the induction of tumor antigen specific immunity. Depletion of immune cell subsets using targeting antibodies demonstrated that the anti-tumor response was mediated in large part by CD4 T-cells which are involved in stimulating both humoral and CD8 T-cell responses. 2FF, a novel inhibitor of fucosylation has potent anti-tumor effects in 2 transgenic models of breast cancer; luminal and triple negative mammary tumors. The agent is active in these mouse models in both the treatment and prevention setting and thus may represent a rational therapeutic approach to evaluate in breast cancer patients. Citation Format: Disis ML, Rastetter L, Gad E, Koehnlein M, Senter PD, Gardai S, Okeley NM. Modulation of immunity with 2-fluorofucose (2FF) for breast cancer treatment and prevention. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-08.

Abstract P6-04-09: Cellular and molecular determinants of breast cancer sensitivity to all-trans retinoic acid: Identification of a gene expression fingerprint predicting responsiveness

Abstract All-trans retinoic acid (ATRA) and derived natural as well as synthetic retinoids are promising agents in the treatment and chemoprevention of various types of neoplasia, including mammary tumors. ATRA is an important component of the therapeutic schemes used for the treatment of a rare form of Acute Myelogenous Leukemia known as Acute Promyelocytic Leukemia. A rational use of the paradigmatic retinoid, ATRA, in a heterogeneous disease, like breast cancer, requires the definition of the cellular and molecular determinants of sensitivity to the agent. The major aim of the study was the definition of a predictive gene expression fingerprint that can be used for the selection of patients who may benefit from treatment protocols containing ATRA. To this purpose, we selected 45 breast cancer cell lines characterized for the constitutive whole genome gene expression profiles. The sensitivity of 30 cell lines (training set) to the anti-proliferative action of ATRA was defined after challenge with increasing concentrations of the retinoid for 3, 6 and 9 days. This analysis established that Luminal and ER-positive cell lines are enriched within the ATRA sensitive group. In contrast, cell lines characterized by a Basal-like phenotype, according to the PAM50 gene expression signature, are generally refractory to the growth inhibitory action of ATRA. The sensitivity of Luminal-A and Luminal-B and the general refractoriness of Basal-like tumors to ATRA was validated in short-term tissue slice cultures of surgical breast cancer specimens. The training set was used to define a gene-expression fingerprint consisting of approximately 50 genes significantly associated with ATRA sensitivity. The fingerprint was generated by reprocessing the RNA sequencing data contained in the CCLE (Cancer Cell line Encyclopedia) of the Broad Institute and it was built from approximately 60,000 coding and non-coding loci. The approach involved the use of general linear models (machine learning algorithm). The identified gene-expression fingerprint was subsequently used to successfully predict ATRA sensitivity in a test set consisting of the remaining 15 cell lines. As a first step towards the use of the fingerprint for the stratification of patients, we evaluated the proportion of predicted ATRA sensitive breast tumors in the TCGA dataset. In accordance with the cell line and primary tumor data, approximately 30% of the Luminal tumors present with a high similarity score to the identified gene expression fingerprint associated with ATRA sensitivity. In contrast, only 5% of the Basal-like or Triple-negative mammary tumors are characterized by the same high similarity score. Curiously, the ATRA sensitivity signature seems to be tumor context independent, as it correctly identifies the 20 Acute Promyelocytic Leukemia patients present in the 198 Acute Myelogenous Leukemia patients present in the TCGA dataset. Citation Format: Garattini E, Bolis M, Paroni G, Fratelli M, Zambelli A, Terao M. Cellular and molecular determinants of breast cancer sensitivity to all-trans retinoic acid: Identification of a gene expression fingerprint predicting responsiveness. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-09.

Abstract 3091: Targeting low molecular weight (LMW) cyclin E-Cdk2 pathway for the prevention of breast cancer

Abstract Our laboratory was first to discover that full length cyclin E is proteolytically cleaved into low-molecular weight isoforms (LMW-E) specifically in tumor cells. The LMW-E isoforms are hyperactive and over-expressed in 70% of all triple negative breast cancer (TNBC) compared to only 30% of non-TNBC patients. Overexpression of LMW-E in transgenic mice induces mammary tumors similar to the basal subtype of breast cancer, which accounts for 70% of all human TNBCs. We have reported that Cdk2 deficient mice were completely resistant to LMW-cyclin E mediated mammary tumors. In this study, we have interrogated the ability of roscovitine in preventing the development of triple negative mammary tumors in the MMTV-LMW-E; p53+/- mouse model. Initially, a group of mice were treated from age 3 months with vehicle (DMSO/PBS, 40:60v/v, N = 8) or roscovitine (100 mg/kg, N = 8) once every 4 and 5 days alternatively by intraperitoneal injection and were followed up for mammary tumor development. Mice did not show any signs of toxicity. In this schedule of treatment, we did not find any difference in time to tumor formation or survival between the 2 groups (8.3 vs 7.9 months, P = 0.70, and 8.9 vs 8.4 months, P = 0.98, respectively). However, we found a 2.8-fold increase in tumor doubling time in the roscovitine-treated group indicating that roscovitine treatment decreased tumor growth (12.1+/-4.4 vs 4.4+/-0.9 days, P&amp;lt;0.05). This was due to a decreased proliferation as measured by BrdU immunohistochemistry (IHC) (13.6+/-0.9% vs 30.0 +/- 7.7%, P&amp;lt;0.05) and an increased apoptotic index as measured by cleaved caspase 3 IHC (49.6+/-9.4 CC3+ cells/x40 field vs 25.9 +/- 9.9, P&amp;lt;0.05). A second group of mice were treated at age 3 months with the same schedule but were killed after 2 months of treatment and the mammary glands were collected to investigate the effects of drug treatment on normal and premalignant mammary cells. The BrdU staining showed that 2 months of roscovitine treatment caused a 3-fold decrease in the number of BrdU positive cells (5.4+/-1.5% vs 14.6+/-3.8%, P&amp;lt;0.05) but did not completely block cyclin E-induced proliferation. This result may explain why the roscovitine-treated mice developed tumor with similar kinetics as the control group. Our ongoing work is to use gene expression profiling to determine the RNA changes induced by roscovitine for the discovery of potential biomarkers. The results from the first trial is suggestive that the mice need to be treated on a continued schedule to ensure that proliferation of the cyclin E overexpressing mammary tissue is completely inhibited. To this end, we are now treating the mice twice a day, 7 days on, 21 days off. We predict that the new treatment schedule will be more efficacious in inhibiting tumor formation. These preliminary data could support the development of a clinical trial testing cyclin-dependent kinase inhibitors for the prevention of human triple negative breast cancer especially those with LMW-E overexpression. Citation Format: Said Akli, Dong Yang, Tuyen N. Bui, Khandan Keyomarsi. Targeting low molecular weight (LMW) cyclin E-Cdk2 pathway for the prevention of breast cancer. [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 3091. doi:10.1158/1538-7445.AM2015-3091

A novel role of hematopoietic CCL5 in promoting triple-negative mammary tumor progression by regulating generation of myeloid-derived suppressor cells

CCL5 is a member of the CC chemokine family expressed in a wide array of immune and non-immune cells in response to stress signals. CCL5 expression correlates with advanced human breast cancer. However, its functional significance and mode of action have not been established. Here, we show that CCL5-deficient mice are resistant to highly aggressive, triple-negative mammary tumor growth. Hematopoietic CCL5 is dominant in this phenotype. The absence of hematopoietic CCL5 causes aberrant generation of CD11b(+)/Gr-1(+), myeloid-derived suppressor cells (MDSCs) in the bone marrow in response to tumor growth by accumulating Ly6C(hi) and Ly6G(+) MDSCs with impaired capacity to suppress cytotoxicity of CD8(+) T cells. These properties of CCL5 are observed in both orthotopic and spontaneous mammary tumors. Antibody-mediated systemic blockade of CCL5 inhibits tumor progression and enhances the efficacy of therapeutic vaccination against non-immunogenic tumors. CCL5 also helps maintain the immunosuppressive capacity of human MDSCs. Our study uncovers a novel, chemokine-independent activity of the hematopoietically derived CCL5 that promotes mammary tumor progression via generating MDSCs in the bone marrow in cooperation with tumor-derived colony-stimulating factors. The study sheds considerable light on the interplay between the hematopoietic compartment and tumor niche. Because of the apparent dispensable nature of this molecule in normal physiology, CCL5 may represent an excellent therapeutic target in immunotherapy for breast cancer as well as a broad range of solid tumors that have significant amounts of MDSC infiltration.

Cancer Cell–Associated MT1-MMP Promotes Blood Vessel Invasion and Distant Metastasis in Triple-Negative Mammary Tumors

Functional roles for the cancer cell-associated membrane type I matrix metalloproteinase (MT1-MMP) during early steps of the metastatic cascade in primary tumors remain unresolved. In an effort to determine its significance, we determined the in vivo effects of RNAi-mediated downregulation in mammary cancer cells on the migration, blood and lymphatic vessel invasion (LVI), and lymph node and lung metastasis. We also correlated the expression of cancer cell MT1-MMP with blood vessel invasion (BVI) in 102 breast cancer biopsies. MT1-MMP downregulation in cancer cells decreased lung metastasis without affecting primary tumor growth. The inhibition of lung metastasis correlated with reduced cancer cell migration and BVI. Furthermore, cancer cell-expressed MT1-MMP upregulated the expression of MT1-MMP in vascular endothelial cells, but did not affect MT1-MMP expression in lymphatic endothelial cells, LVI, or lymph node metastasis. Of clinical importance, we observed that elevated MT1-MMP expression correlated with BVI in biopsies from triple-negative breast cancers (TNBC), which have a poor prognosis and high incidence of distant metastasis, relative to other breast cancer subtypes. Together, our findings established that MT1-MMP activity in breast tumors is essential for BVI, but not LVI, and that MT1-MMP should be further explored as a predictor and therapeutic target of hematogenous metastasis in TNBC patients.

Abstract 1812: Effects of vitamin D on obesity-induced increase in ER-positive and ER-negative mammary cancer in mice

Abstract We have previously found that dietary exposure to vitamin D (VD3) reverses the increase in endometrial cancer in obese Pten+/- mice. Since obesity also increases breast cancer risk and recurrence, this study investigated whether VD3 supplementation may inhibit development of mammary cancer in obese mice. As the protective effect of VD3 in the endometrium was not related to the estrogen receptor (ER), we pursued another potential pathway and determined whether VD3 reverses obesity-induced insulin resistance. Methods: Two mouse models were used: DMBA-treated C57BL/6 mice developing ER positive (ER+) mammary tumors, and Pten+/- mice which develop triple negative mammary tumors. C57BL/6 mice were wild type littermates of the Pten+/- mice. Mice in the two models were divided into four groups after weaning, and they were fed (a) AIN93G based control diet, (b) diet supplemented with 20K IU VD3, (c) obesity-inducing diet (OID), or (d) OID supplemented with 25K IU VD3. Results: OID induced insulin resistance and increased mammary tumorigenesis in both C57BL/6 and Pten+/- mice. VD3 significantly inhibited ER+ (p&amp;lt;0.05) and ER- (p&amp;lt;0.04) mammary cancer, but did not reverse the obesity-induced increase in cancer risk. Results obtained in the insulin tolerance test, however, indicated that VD3 prevented the development of insulin resistance in the OID fed obese C57BL/6 and Pten+/- mice. Conclusions: Dietary exposure to VD3 protects against development of ER+ and triple negative mammary cancer, but only in lean mice. VD3 supplementation also prevents insulin resistance in obese mice, but this is not sufficient to reduce mammary cancer risk in either C57BL/6 or Pten+/- mice Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2011-1812