Spontaneous Mammary Tumors Research Articles

Tenascin-C increases lung metastasis by impacting blood vessel invasions

Metastasis is a major cause of death in cancer patients. The extracellular matrix molecule tenascin-C is a known promoter of metastasis, however the underlying mechanisms are not well understood. To further analyze the impact of tenascin-C on cancer progression we generated MMTV-NeuNT mice that develop spontaneous mammary tumors, on a tenascin-C knockout background. We also developed a syngeneic orthotopic model in which tumor cells derived from a MMTV-NeuNT tumor. Tumor cells were transfected with control shRNA or with shRNA to knockdown tenascin-C expression and, were grafted into the mammary gland of immune competent, wildtype or tenascin-C knockout mice. We show that stromal-derived tenascin-C increases metastasis by reducing apoptosis and inducing the cellular plasticity of cancer cells located in pulmonary blood vessels invasions (BVI), before extravasation. We characterized BVI as organized structures of tightly packed aggregates of proliferating tumor cells with epithelial characteristics, surrounded by Fsp1+ cells, internally located platelets and, a luminal monolayer of endothelial cells. We found extracellular matrix, in particular, tenascin-C, between the stromal cells and the tumor cell cluster. In mice lacking stromal-derived tenascin-C, the organization of pulmonary BVI was significantly affected, revealing novel functions of host-derived tenascin-C in supporting the integrity of the endothelial cell coat, increasing platelet abundance, tumor cell survival, epithelial plasticity, thereby promoting overall lung metastasis. Many effects of tenascin-C observed in BVI including enhancement of cellular plasticity, survival and migration, could be explained by activation of TGF-β signaling. Finally, in several human cancers, we also observed BVI to be surrounded by an endothelial monolayer and to express tenascin-C. Expression of tenascin-C is specific to BVI and is not observed in lymphatic vascular invasions frequent in breast cancer, which lack an endothelial lining. Given that BVI have prognostic significance for many tumor types, such as shorter cancer patient survival, increased metastasis, vessel occlusion, and organ failure, our data revealing a novel mechanism by which stromal tenascin-C promotes metastasis in human cancer, may have potential for diagnosis and therapy.

Abstract 2898: Stochastic variation in gene expression is selected during clonal evolution of spontaneous mouse mammary tumors

Abstract Molecular and cellular diversity in cancer cells enables evolutionary processes that govern tumor progression and contributes to treatment failures, but the mechanisms governing this variability remain incompletely understood. We previously described tumor-specific epigenetic alterations in DNA devoid of canonical CpG methylation sites throughout the genomes of spontaneous breast tumors which arise in genetically uniform, yet heterozygous, [FVB X BALB/c MMTV-NeuT+] F1 mice. Those changes were detected in purified tumor DNA using a template-assisted quantitative ligation assay employing locus-specific and SNP-specific oligonucleotide genotyping probes, and the assay was subsequently shown to be sensitive to epigenetic modifications of the templates. We now describe losses of heterozygosity similarly distributed throughout the transcriptomes of tumors in this same model. We quantified expressed alleles across a population of clonal tumors and tested each expressed variant for the occurrence of allelic ratio outliers. The results revealed variegated patterns of expression in hundreds of genes, highly significantly enriched in pathways typically co-opted by tumors (Molecular Mechanisms of Cancer, P < 10-10, IPA analysis). An astounding 87% (2634 of 3044) of expressed genes that could be measured using this approach were represented in at least one tumor. Furthermore, the frequency of these outliers in any one individual tumor was found to correlate strongly with the transcriptional repression of an additional large set of non-polymorphic genes. These findings reveal underappreciated, latent mechanisms driving sporadic errors in epigenetic programming that promote repression of a multiple cis-linked transcripts. This genome-wide molecular chaos presents as dysregulated cellular homeostasis and is the foundation for heritable diversity as tumors evolve. Citation Format: Sara J. Felts, Xiaojia Tang, Virginia P. Van Keulen, Krishna R. Kalari, Larry R. Pease. Stochastic variation in gene expression is selected during clonal evolution of spontaneous mouse mammary tumors [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 2898.

Abstract 4561: Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response

Abstract Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Sli2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent. Citation Format: Dinesh K. Ahirwar, Nabanita Chatterjee, Sanjay Mishra, Kontestine Shilo, Ramesh Ganju. Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response [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 4561.

Abstract 138: TNBC induces immune suppressive tumor microenvironments through microRNAs

Abstract Metastatic breast cancer is the second leading cause of cancer-related deaths for US women. The triple negative breast cancer subtype (TNBC) is one of the major contributors to breast cancer mortality, among all types of breast cancer. There are no effective treatments available for metastatic tumors. Therefore, identifying and targeting novel tumorigenesis pathways will facilitate developing effective therapeutic approaches that could ultimately lead to effective treatment for breast cancer, particularly metastatic cancer and TNBC. Our results demonstrate that miR-146a promotes tumorigenesis and metastasis of breast cancer. We found that miR-146a was markedly overexpressed in TNBC cell lines. Notably, miR-146a deficiency in a spontaneous mammary tumor model leads to reduced tumor growth and metastasis. Notably, miR-146a shifts the polarization of macrophages into immunosuppressive phenotypes. Our studies suggest that miR-146a may contribute to tumor development through modulation of tumor microenvironments. Note: This abstract was not presented at the meeting. Citation Format: Beichu Guo, Melvin Kantono, Wei Sun. TNBC induces immune suppressive tumor microenvironments through microRNAs [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 138.

Abstract LB-024: Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer

Abstract Purpose : The hormonally active form of vitamin D [1,25(OH)2D3], has several antitumor effects including antiproliferative, prodifferentiative and proapoptotic functions in tissues expressing and binding the vitamin D receptor. 1,25(OH)2D3 is synthesized from its precursor 25-hydroxyvitamin D [25(OH)D] via the catalytic action of the mitochondria cytochrome P450 enzyme 25(OH)D-1α-hydroxylase (CYP27B1). In the current study we investigated the role of CYP27B1 ablation on vitamin D signaling using the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein (MMTV-PyMT) mouse, an oncogene-driven model of highly aggressive spontaneous mammary tumors that closely mimics the estrogen receptor negative breast cancer in human disease. Methods: Tumors from animals were digested and primary cells were obtained from both CYP27B1 ablated AOH93Cre+ cells and wild type MT1107 Cre- control cells. The cells were next grown in complete DMEM medium, transfected with either a GFP-tagged human VDR or RXRα and treated with 1,25(OH)2D3 (10−7 mol/L) or 25(OH)D3 (10−7 mol/L) before data acquisition. Results: Using live and fixed cell fluorescence imaging, in situ proximity ligation assay and immunoblotting techniques, we show that the nuclear localization of both endogenous and exogenously tagged VDR and RXR were impaired in CYP27B1 ablated cells when compared to non-ablated cells. Furthermore, the intranuclear shuttling of VDR and its interaction with RXR were significantly reduced in CYP27B1 ablated cells as shown by both Florescence Recovery After Photobleaching and Fluorescence Resonance Energy Transfer techniques. Our results also show that CYP27B1 ablation disrupts VDR function by significantly impairing its nuclear localization, intranuclear transport, interaction with adaptor proteins, hVDR/hRXR, and this correlated with impaired binding to DNA and chromatin. Lastly, we demonstrate impaired induction of 53BP1, p65 and γ-H2AX DNA double-strand breaks in the CYP27B1 ablated cells compared to non-ablated cells. Conclusion: These results suggest that ablation of CYP27B1 results in dysregulation in vitamin D signaling which may impair its anticancer functions. Citation Format: Sylvester Jusu, John Presley, Bertrand Jean-Claude, Ursula Stochaj, Richard Kremer. Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer [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 LB-024.

Abstract 4683: Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies

Abstract G protein coupled receptors (GPCRs) are the largest family of cell surface receptors with over 800 members. Aberrant activation of GPCRs has been implicated in cancer initiation and development. However, many of GPCRs are overexpressed in cancer cells, making it difficult to target individual GPCRs for cancer treatment. In this study, we have tested a new approach of targeting GPCRs in HER2+ breast cancer by blocking their common pathways downstream of G proteins. By mammary gland-specific expression of the pertussis toxin (PTx) catalytic subunit, a selective inhibitor that uncouples a subgroup of GPCRs from activating Gi/o proteins, we demonstrated that blocking GPCR downstream signaling delayed the onset of HER2-driven spontaneous mammary tumor formation and suppressed lung metastasis. Moreover, we showed that aberrant HER2 signaling upregulated Gi/o-GPCR expression in breast cancer cells, which in turn activated the ErbB/HER family of protein-tyrosine kinases via Src and PI3K pathways to maintain cancer stem cell tumorigenicity. Targeting Gi/o-GPCR signaling by PTx, Src or PI3K inhibitors sensitizes HER2+ breast cancer to HER2-targeted therapies. Together, our data demonstrate that targeting GPCR downstream signal pathways may represent a new approach to ablate CSCs to block tumor progression and augment HER2-targeted therapeutics. Citation Format: Wei Wang, Dharmendra Bhargava, Yuan Chao Ye, Songhai Chen. Targeting GPCR signaling in HER2+ breast cancer suppresses cancer stem cell tumorigenicity and sensitizes HER2-targeted therapies [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 4683.

Acute phase proteins and biomarkers of oxidative status in feline spontaneous malignant mammary tumours.

Acute phase proteins (APP) and biomarkers of oxidative status change in human and canine mammary tumours, however, they have not been studied in feline mammary tumours. The aims of this study were to investigate the APP and antioxidant responses in feline malignant mammary tumours, to evaluate their relation with tumour features, and to assess their prognostic value. Serum amyloid A (SAA), haptoglobin (Hp), albumin, butyrylcholinesterase (BChE), insulin-like growth factor1 (IGF1), paraoxonase1 (PON1), total serum thiols (Thiol), glutathione peroxidase (GPox) and total antioxidant capacity determined by different assays, including trolox equivalent antioxidant capacity assessed by two different methodologies (TEAC1/2), ferric reducing ability of plasma (FRAP), and cupric reducing antioxidant capacity (CUPRAC), were determined in serum of 50 queens with spontaneous mammary carcinomas and of 12 healthy female cats. At diagnosis, diseased queens presented significantly higher SAA and Hp, and lower albumin, BChE, GPox, TEAC1, TEAC2 and CUPRAC than controls. Different tumour features influenced concentrations of APP and antioxidants. Increases in serum Hp, and decreases in albumin, Thiol and FRAP were significantly associated with neoplastic vascular emboli, metastasis in regional lymph nodes and/or in distant organs. Distant metastasis development during the course of the disease was associated with increases in SAA and TEAC1. At diagnosis, decreased albumin was associated with a longer survival, and BChE <1.15 μmoL/mL.minute was associated with a shorter survival time on multivariate analysis. Feline malignant mammary tumours are associated with an APP response and oxidative stress, and different tumour features influence the inflammatory response and the oxidative damage. Furthermore, some of these analytes proved to have prognostic value.

Anti-HER2/Neu antibody therapy can reduce the immunosuppressive activity of MDSCs in breast tumor model.

e14181 Background: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell populations that play a critical role in tumor associated immune suppression. MDSCs consist of immature cells with myeloid lineage markers CD11b and Gr1 in mice and accumulate in large numbers in tumor bearing mice including HER2/neu+ breast cancers. This study was conducted to test whether anti-neu antibody treatment can affect on MDSC populations and immunosuppressive functions. Methods: Female BALB/c mice(5–6weeks of age) were used. The TUBO mammary carcinoma cell line was cloned from a spontaneous mammary tumor in a BALB Neu Tg mouse, as cell lines and reagents. Tumor-infiltrating lymphocyte (TIL) populations were evaluated by flow cytometry. The RT2 Profiler PCR array was used for cytokine expression analysis according to the manufacturer’s instructions. Results: Total number of MDSC in tumor and spleen were decreased within 3 days after anti-neu antibody treatment, but percentage of MDSC in tumor (day2) was reduced more quickly than percentage of MDSC in spleen (day3). The relative populations of MDSC in CD45.2+ cells were significantly decreased such as MO-MDSC. Immunosuppressive activity of MDSC was also reduced by anti-neu antibody treatment. MDSC reduced by anti-neu antibody was due to inhibition of the immunosuppressive factors Arg1 and IDO. In addition, combination therapy with 5-FU may enhance MDSC reduction. Conclusions: MDSC can be a potential therapeutic target in anti-neu antibody treated therapeutic model and the effect of anti-neu antibody could be further improved using other therapeutic treatment to target MDSC.

Pro-Apoptotic and Anti-Angiogenesis Effects of Olive Leaf Extract on Spontaneous Mouse Mammary Tumor Model by Balancing Vascular Endothelial Growth Factor and Endostatin Levels

It has been proven that olive associated products such as olive leaf extract (OLE) causes significant reduction in cancer cells viability and proliferation. Female BALB/c adult mice were divided into four groups. Three days prior to oral treatments, tumors were transplanted. First group were treated with distilled water and other three groups were received, respectively, 75, 150, and 225 mg/kg/day of OLE for three weeks. For assessment of anti-angiogenesis and pro-apoptotic effect of OLE on tumor tissue, tumor volume, cell mitosis and apoptosis, and also vascular endothelial growth factor (VEGF) and endostatin levels were assessed. OLE treatment with 150 and 225 mg/kg/day lead to significant reduction in tumor volume and cell mitosis compared with the control group, while the same doses significantly increase tumor cell apoptosis. OLE treatment with 150 mg/kg/day increase endostatin levels, while the same dose did not significantly decrease VEGF levels. The VEGF level is significantly reduced by the treatment with OLE 225 mg/kg/day for three weeks. Although, further studies are needed to clarify anti-angiogenesis and anti-apoptotic mechanism of OLE, consumption of OLE polyphenols after tumor transplantation reduced spontaneous mouse mammary tumor growth.

SAT-331 Elucidating the Role of Breast Cancer Specific GATA3 Mutation in Estrogen Receptor Positive Breast Cancer

Approximately 266,000 women will be newly diagnosed with invasive breast cancer in the US this year, making it the most prevalent cancer in females, resulting in a 1 in 8 lifetime risk. Many of these tumors are of the hormone receptor positive luminal subtype, with 60-80% expressing the estrogen and/or progesterone receptor (ER+/PR+). The transcription factor GATA3 is expressed in the luminal epithelial cells of the mammary gland and is required for mammary development and luminal cell differentiation. Its deletion from the gland results in defects affecting the tightly regulated processes of terminal end bud formation, ductal elongation, and lactation. GATA3 is involved in a positive cross-regulatory loop with ER-alpha expression, and as such, GATA3 is highly expressed in most ER+ breast cancers and is required for breast cancer cell line responses to estradiol. Mutations in GATA3 are observed in approximately 15% of ER+ breast tumors, behind only PIK3CA and TP53 mutation in frequency, and the median tumor onset is eight years earlier than in ER+ tumors without GATA3 mutation. Analysis of TCGA breast tumors illustrated that most mutations cluster to the C-terminal end of GATA3, suggesting a selective advantage for expressing a significant portion of the wild-type protein. Previous studies have relied on xenografts in immunocompromised mice to characterize the effects of GATA3 mutations on tumor biology. We developed a transgenic mouse expressing a hotspot frameshift mutation at GATA3 codon 335 (GATA3335fs) in the mammary gland. These mice did not develop spontaneous mammary tumors; however, they do exhibit hyperproliferative phenotypes in the mammary epithelium that are recapitulated in breast cancer cell lines and xenografts also expressing the GATA3335fs protein, findings consistent with an active role for mutant GATA3 proteins in regulating mammary cell proliferation. We have used integrated gene expression and ChIP-Seq profiling to demonstrate that these zinc-finger deleted proteins retain the ability to associate with the genome by tethering to complexes associated with FOXA1 and AP-2gamma recognition motifs, where they modulate the expression of adjacent genes. Continuous exposure to a medroxyprogesterone acetate (MPA) depot induces ER+/PR+ mammary tumors at an 80% incidence in wild-type mice with a median latency of one-year. This study follows a cohort of our recently described MMTV-GATA3335fs transgenic mouse in a long-term MPA driven mammary tumorigenesis protocol. Given the requirement for GATA3 in ER-alpha expression and the estradiol response, this experimental model will provide insight into the biology of ER driven tumorigenesis and whether and how GATA3 mutation plays a role in this process. This study was supported by the Gundersen Medical Foundation and a Peter T. Rowley Breast Cancer Research Grant from the New York State Dept. of Health.

Expression of the Short Form of RON/STK in Feline Mammary Carcinoma.

RON is a tyrosine kinase receptor activated by the macrophage-stimulating protein (MSP) ligand that is overexpressed in human breast cancer. In humans, RON protein can be present in different isoforms, and the most studied isoform is represented by the short form of RON ( sf-RON), which is generated by an alternative promoter located in intron 10 of the RON complementary DNA (cDNA). It plays an important role in breast cancer progression. Considering the many similarities between feline mammary carcinoma (FMC) and human breast cancer, the aim of this study was to investigate the expression of both RON and MSP in FMCs and to identify the presence of the sf-RON transcript. Tissue samples of spontaneous mammary tumors were collected from 60 queens (10 benign lesions, 50 carcinomas). All of the samples were tested for RON and MSP expression by immunohistochemistry; moreover, RNA was extracted from paraffin-embedded tissue samples, and the cDNA was tested by reverse transcription-polymerase chain reaction (RT-PCR) to identify the presence of sf-RON. Immunohistochemistry detected the expression of RON and MSP in 34 of 50 (68%) and 29 of 50 (58%) FMCs, respectively. RT-PCR revealed the presence of the short-form in 18 of 47 (38%) FMCs. This form originates, as in humans, from an alternative promoter (P2), and it codes for the proper feline short form ( sf-RON). sf-RON expression was associated with poorly differentiated tumors and with a shorter disease-free ( P < .05; hazard ratio [HR], 2.2) period and a shorter survival ( P < .05; HR, 2.2). These results support FMC as a suitable model in comparative oncology and identify sf-RON expression as potential predictor of outcomes for this disease.

IFNγ-induced Chemokines Are Required for CXCR3-mediated T-Cell Recruitment and Antitumor Efficacy of Anti-HER2/CD3 Bispecific Antibody.

The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.

The SuprMam1 breast cancer susceptibility locus disrupts the vitamin D/ calcium/ parathyroid hormone pathway and alters bone structure in congenic mice

Breast cancer is a complex disease, and approximately 30% of cases are considered to be hereditary or familial, with a large fraction of this being polygenic. However, it is difficult to demonstrate the functional importance of genes of small effect in population studies, and these genes are not always easily targeted for prevention. The SuprMam (suppressor of mammary tumour) breast cancer susceptibility alleles were previously identified as contributors to spontaneous mammary tumour development in Trp53+/− mice. In this study, we have generated and characterised congenic mice that contain the BALB/c SuprMam1 (susceptibility) locus on a C57BL/6 (resistant) background and discovered a subtle impairment in the vitamin D/ calcium/ parathyroid hormone (PTH) pathway. This was evident as altered gene expression in the mammary glands of key players in this pathway. Further functional analysis of the mice revealed elevated PTH levels, reduced Cyp27b1 expression in kidneys, and reduced trabecular bone volume/ tissue volume percentage. Plasma 25(OH)D and serum calcium were unchanged. This impairment was a result of genetic differences and occurred only in females, but the elevated PTH levels could be overcome with either calcium or vitamin D dietary supplementation. Either low levels of active vitamin D (1,25(OH)2D) or chronically elevated PTH levels may contribute to increased breast cancer susceptibility. These indicators are not easily measured in human population studies, but either mechanism may be preventable with dietary calcium or vitamin D supplements. Therefore, SuprMam congenic mice could serve as a valuable model for studying the role of gene-hormone-environment interactions of the vitamin D/ calcium/ PTH pathway in cancer and other diseases and for testing preventive interventions.

Abstract B03: RNAi-mediated β-catenin inhibition sensitizes noninflamed tumors to immune checkpoint blockade

Abstract Anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies are immunotherapeutics which confer benefit to several subpopulations of cancer patients, yet the majority of tumors remain insensitive. Wnt/β-catenin signaling has been strongly implicated in mediating cancer immune evasion and resistance to immune checkpoint therapy. The mechanism by which Wnt/β-catenin signaling causes checkpoint resistance is believed to involve blocking of specific cytokines which trigger immune cell recruitment to the tumor, resulting in the phenomenon of T-cell exclusion and rendering the tumor to a non-inflamed state. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines. DCR-BCAT is an advanced experimental therapeutic containing a chemically-optimized RNA interference (RNAi) trigger targeting CTNNB1, the gene that encodes β-catenin, formulated in a tumor-selective lipid nanoparticle. To understand the role of β-catenin in immunotherapy, we investigated DCR-BCAT in combination with checkpoint inhibitors in syngeneic murine tumor allograft models. β-catenin inhibition promoted T-cell infiltration and significantly improved sensitivity to anti-PD-1 and anti-CTLA-4 therapy. The combination treatment yielded significant tumor growth inhibition compared to monotherapy in B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma and Renca renal adenocarcinoma. T-cell mediated cytotoxicity was identified of the primary mechanism of efficacy, as shown by granzyme B and perforin staining. Interestingly, antitumor efficacy in these combination studies was not correlated with Wnt activation status in syngeneic models. Finally and strikingly, when DCR-BCAT was combined with immuotherapy in mice which develop spontaneous Wnt-driven mammary tumors, checkpoint therapy potentiation yielded complete tumor regressions. These data offer proof-of-concept for conversion of non-inflamed tumors to inflamed tumors by β-catenin inhibition, and support clinical evaluation of this combination approach using a first-in-class RNAi-based agent. Citation Format: Shanthi Ganesh, Serena Shui, Weimin Wang, Bob D. Brown, Marc Abrams. RNAi-mediated β-catenin inhibition sensitizes noninflamed tumors to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B03.

Caloric restriction inhibits mammary tumorigenesis in MMTV-ErbB2 transgenic mice through the suppression of ER and ErbB2 pathways and inhibition of epithelial cell stemness in premalignant mammary tissues.

Caloric intake influences the onset of many diseases, including cancer. In particular, caloric restriction (CR) has been reported to suppress mammary tumorigenesis in various models. However, the underlying cancer preventive mechanisms have not been fully explored. To this end, we aimed to characterize the anticancer mechanisms of CR using MMTV-ErbB2 transgenic mice, a well-established spontaneous ErbB2-overexpressing mammary tumor model, by focusing on cellular and molecular changes in premalignant tissues. In MMTV-ErbB2 mice with 30% CR beginning at 8 weeks of age, mammary tumor development was dramatically inhibited, as exhibited by reduced tumor incidence and increased tumor latency. Morphogenic mammary gland analyses in 15- and 20-week-old mice indicated that CR significantly decreased mammary epithelial cell (MEC) density and proliferative index. To understand the underlying mechanisms, we analyzed the effects of CR on mammary stem/progenitor cells. Results from fluorescence-activated cell sorting analyses showed that CR modified mammary tissue hierarchy dynamics, as evidenced by decreased luminal cells (CD24highCD49flow), putative mammary reconstituting unit subpopulation (CD24highCD49fhigh) and luminal progenitor cells (CD61highCD49fhigh). Mammosphere and colony-forming cell assays demonstrated that CR significantly inhibited mammary stem cell self-renewal and progenitor cell numbers. Molecular analyses indicated that CR concurrently inhibited estrogen receptor (ER) and ErbB2 signaling. These molecular changes were accompanied by decreased mRNA levels of ER-targeted genes and epidermal growth factor receptor/ErbB2 family members and ligands, suggesting ER-ErbB2 signaling cross-talk. Collectively, our data demonstrate that CR significantly impacts ER and ErbB2 signaling, which induces profound changes in MEC reprogramming, and mammary stem/progenitor cell inhibition is a critical mechanism of CR-mediated breast cancer prevention.

PO-220 Defining the role of heparanase in breast cancer progression using the PyMT-MMTV mouse model

IntroductionHeparanase (HPSE) is a beta-d-endoglucuronidase and the only mammalian enzyme that cleaves heparan sulphate (HS), a major structural and regulatory component of the extracellular matrix (ECM) and the vascular basement membrane (BM). The expression of HPSE is tightly controlled and under physiological conditions is limited to immune cells, endothelial cells, placental trophoblasts and keratinocytes. However, during pathological conditions such as cancer, HPSE expression is dysregulated and high expression often correlates with poor patient survival. The cleavage of HS by HPSE expressing tumours degrades the ECM/BM and releases HS bound growth factors and cytokines, promoting cellular signalling in a positive feed-back mechanism. This in turn leads to enhanced primary tumour growth, metastasis, angiogenesis and inflammation. HPSE is therefore a critical promoter of the hallmarks of cancer and has generated significant interest as an anti-cancer drug target. However, despite decades of research, the precise mechanistic role of HPSE in the tumour microenvironment remains poorly defined. Breast cancer is the most prevalent malignancy in women worldwide. Clinical data reveals that the expression of HPSE in mammary tumours results in poor patient survival.Material and methodsWe recently generated a HPSE-deficient C57Bl/6 mouse strain (C57Bl/6xHPSE-/-) that were crossed with spontaneous mammary tumour developing PyMT-MMTV mice to generate the PyMT-MMTVxHPSE-/- strain, providing us with a valuable in vivo model to characterise the role of HPSE in early mammary tumour development, tumour progression and metastasis.Results and discussionsOur data indicate that although HPSE promoted tumour angiogenesis, overall tumour development and metastasis between PyMT-MMTV and PyMT-MMTVxHPSE-/- mice remained comparable. By examination of the tumour microenvironment, we also demonstrate that the tumour stroma positively contributes to mammary tumour HPSE activity, promoting tumour angiogenesis. In contrast, HPSE expressed in the tumour-bearing host appeared to play no significant role in tumour growth and metastasis. These data suggest that in the PyMT-MMTV model and indeed, in certain cancer settings such as breast cancer, HPSE may not play as significant a role to what has been proposed over the last two decades.ConclusionThese findings may have important implications for the ongoing development and application of HPSE inhibitors in the treatment of cancer.

Abstract 1368: Variable penetrance of mammary tumors in mouse models of Li-Fraumeni syndrome is linked to replication-associated repair

Abstract Background: The p53 tumor suppressor gene plays a key role in sporadic and inherited breast cancers. Spontaneous mammary tumors develop in ~55% of female BALB/c-Trp53+/- mice providing a model for breast cancer in Li-Fraumeni Syndrome. In contrast, C57BL/6J-Trp53+/- females are devoid of mammary tumors. This strain difference offers a dramatic example of variable penetrance and tools to define genes contributing to the risk of mammary tumors. Methods: Genome-wide linkage analysis of the mammary tumor incidence was performed in F1 and N2 backcross mice. The C57BL/6J alleles of SM1 locus were introgressed into the BALB/c genetic background through 10 backcrosses to generate SM1-Trp53+/- mice. Radiation sensitivity, DNA double-strand break repair, processitivity of DNA replication and expression of DNA repair genes in SM1-Trp53+/- mice were compared with BALB/c-Trp53+/- and C57BL/6J-Trp53+/-. Results: Genome-wide linkage analysis identified a major modifier locus on chromosome 7 (designated SM1) and another on chromosome 2 with an overall LOD score of 6.1 using a multigenic model of inheritance. The SM1 locus does not undergo loss of heterozygosity in mammary tumors consistent with dominant-acting risk alleles from BALB/c mice. Mammary epithelial cells (MMEC) of SM1 mice, having C57BL/6 alleles of SM1 in BALB/c background, were sensitive to radiation as were BALB/c MMEC. Functional assays of DNA double-strand break repair (1) demonstrate error-prone repair through the single strand annealing (SSA) pathway level in SM1 embryo fibroblasts (MEF) was similar to that in C57BL/6 MEF, which was 2.8-fold higher in BALB/c-Trp53+/- MEF and 2.5-fold higher in [BALB.B6]F1-Trp53+/-. Similarly, DNA fiber assays showed that processivity of DNA replication was significantly decreased in BALB/c and F1 MEF with median fork length (MFL) 6.4µM but increased in C57BL/6 and complemented in SM1 MEF (MFL ~8.2µM). In addition, gene expression profile of at least 5 DNA repair proteins (Ercc5, Rev1, Rev3l, Trex2, Rdm1) that are differentially expressed in C57BL/6 and BALB/c were complemented by SM1 locus. Conclusions: Gene(s) within the 20Mb SM1 locus impair movement of replications forks. Stalling of replication may precipitate fork collapse and DNA double strand breaks resulting in the loss of heterozygosity for Trp53 through recombination observed in 90% of mammary tumors.

PO-386 Dissecting the role of regulatory T cells in metastatic breast cancer

IntroductionBreast cancer is the most frequent malignancy among women worldwide. More than 90% of breast cancer-related deaths are due to metastatic disease. Despite these facts, breast cancer metastasis remains incurable. A key player in metastasis is the immune system. Cancer-induced immunosuppression contributes to a tumour’s ability to evade immune destruction. A major cell type in this process is the regulatory CD4+ T cell (Treg). It has been reported that Tregs are found in the microenvironment of primary tumours and metastases, and that their depletion reduces metastatic burden; however, the underlying mechanisms remain poorly understood. Using state-of-the-art breast cancer mouse models that closely recapitulate primary human breast cancer and metastatic disease, our goal is to elucidate the impact of Tregs on breast cancer metastasis, focusing on differences between (pre-)metastatic tissues and different steps in the metastatic cascade.Material and methodsTo study primary spontaneous mammary tumours and the pre-metastatic niche, we primarily use the FVB K14cre;Cdh1F/F;Trp53F/F (KEP) conditional mouse model for invasive lobular carcinoma. For metastatic disease, KEP tumour fragments are orthotopically transplanted into in wild-type syngeneic FVB mice. Following tumour outgrowth and mastectomy, widespread metastatic disease is present in lungs, lymph nodes and other distant organs, providing an accurate representation of the different steps of the metastatic cascade.Results and discussionsWe observed systemically elevated levels of Tregs prior to metastatic disease. These tumor-educated Tregs display a distinct phenotype and specifically accumulate in the lung and lymph node metastases that arise in our metastasis models, perhaps indicating their possible importance for metastasis formation and progression.In addition to these systemic changes, within primary tumours and metastases a large population of PD1high Tregs is found. Preliminary data suggest that tumor-associated myeloid cells influence this population.ConclusionWe are currently setting out to dissect the mechanism behind this interplay of intra-tumoral immune cells and the role of distinct Tregs found prior to metastatic disease.

PO-386 Dissecting the role of regulatory T cells in metastatic breast cancer

IntroductionBreast cancer is the most frequent malignancy among women worldwide. More than 90% of breast cancer-related deaths are due to metastatic disease. Despite these facts, breast cancer metastasis remains incurable. A key player in metastasis is the immune system. Cancer-induced immunosuppression contributes to a tumour’s ability to evade immune destruction. A major cell type in this process is the regulatory CD4+ T cell (Treg). It has been reported that Tregs are found in the microenvironment of primary tumours and metastases, and that their depletion reduces metastatic burden; however, the underlying mechanisms remain poorly understood. Using state-of-the-art breast cancer mouse models that closely recapitulate primary human breast cancer and metastatic disease, our goal is to elucidate the impact of Tregs on breast cancer metastasis, focusing on differences between (pre-)metastatic tissues and different steps in the metastatic cascade.Material and methodsTo study primary spontaneous mammary tumours and the pre-metastatic niche, we primarily use the FVB K14cre;Cdh1F/F;Trp53F/F (KEP) conditional mouse model for invasive lobular carcinoma. For metastatic disease, KEP tumour fragments are orthotopically transplanted into in wild-type syngeneic FVB mice. Following tumour outgrowth and mastectomy, widespread metastatic disease is present in lungs, lymph nodes and other distant organs, providing an accurate representation of the different steps of the metastatic cascade.Results and discussionsWe observed systemically elevated levels of Tregs prior to metastatic disease. These tumor-educated Tregs display a distinct phenotype and specifically accumulate in the lung and lymph node metastases that arise in our metastasis models, perhaps indicating their possible importance for metastasis formation and progression.In addition to these systemic changes, within primary tumours and metastases a large population of PD1high Tregs is found. Preliminary data suggest that tumor-associated myeloid cells influence this population.ConclusionWe are currently setting out to dissect the mechanism behind this interplay of intra-tumoral immune cells and the role of distinct Tregs found prior to metastatic disease.

Abstract 5114: The role of amphiregulin in mammary gland development and breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor (EGFR), is enriched in invasive breast cancer, particularly ERα-positive breast tumors. In normal physiology, AREG mediates estrogen-induced ductal morphogenesis during mammary gland development. In the absence of AREG, the ductal tree fails to elongate and remains rudimentary throughout adulthood. The structure of a healthy mammary duct consists of luminal cells surrounded by a layer of myoepithelial cells. Studies of mammary gland transplantations suggest that a marker of myoepithelial cells (keratin 14, K14) is only expressed if AREG is present in the mammary epithelium. Multiple studies have provided evidence that myoepithelial cells are natural tumor suppressors. During tumor growth, the disappearance of myoepithelial cells indicates progression to invasive carcinoma. Therefore we hypothesize that AREG plays an important role in maintenance of the maintenance of myoepithelial cells in the mammary epithelium and loss of AREG promotes tumor progression as a result of an early loss of myoepithelial cells. In the mammary glands of AREG WT and AREG KO mice without the PyMT transgene, we have found that mammary ducts in AREG KO mice have significantly fewer myoepithelial cells. Immunofluorescent staining of K14 has revealed a thinner and discontinuous myoepithelial layer in the AREG KO mice. To investigate the role of AREG in breast cancer development and progression, we have utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors (AREG KO PyMT). The absence of AREG in both the mammary epithelial and stromal compartments accelerates tumor growth. By 12 weeks of age, tumors of AREG KO PyMT mice are significantly larger than that of AREG WT PyMT mice. AREG KO tumors are histologically less necrotic, apoptotic, and progressed than AREG WT tumors. Furthermore, AREG KO tumors show higher staining of PyMT by immunofluorescence. Together, these results suggest that loss of AREG stimulates tumor initiation but inhibits progression due to fewer myoepithelial cells beginning during development. Citation Format: Serena Mao, Minji Park, Ramon Cabrera, John Christin, George Karagiannis, Wenjun Guo, John Condeelis, Jeffrey Segall. The role of amphiregulin in mammary gland development and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5114.