Development Of Mammary Carcinoma Research Articles

Reduction of phosphorylated signal transducer and activator of transcription-5 expression in feline mammary carcinoma.

Signal transducers and activators of transcription (STATs) are a family of transcription factors involved in various normal physiological cellular processes. Moreover, STATs have been recently identified as novel therapeutic targets for various human tumors. STAT3, STAT5a, and STAT6 have been suggested to be involved in tumorigenesis in human breast cancer. Owing to the similarity between feline mammary carcinomas (FMCs) and human breast cancers, these factors may play an important role in FMCs. However, studies on the expression of STATs in animal tumors are limited. Therefore, in this study, we aimed to characterize the expression of total STAT5 (tSTAT5) and phosphorylated STAT5 (pSTAT5) in FMCs, feline mammary adenomas, non-neoplastic proliferative mammary gland lesions, and normal feline mammary glands using immunohistochemistry. High expression of tSTAT5 was observed in the cytoplasm of all the samples assessed in this study. Moreover, high expression of tSTAT5 was observed in the nucleus; however, its levels varied depending on the lesion. The percentage of pSTAT5-nuclear positive cells varied among normal feline mammary glands (40.1 ± 25.1%), and non-neoplastic lesions, including mammary hyperplasia (43.2 ± 28.6%) and fibroadenomatous changes (18.0 ± 13.6%). Moreover, the percentage of pSTAT5-nuclear-positive cells in feline mammary adenomas was 24.5 ± 19.2%, which was significantly reduced in feline mammary carcinomas (2.4 ± 5.6%), regardless of histopathological subtype. This study suggests that decreased STAT5 activity may be involved in the development and malignant progression of feline mammary carcinomas.

Effect of 2-methoxyestradiol treatment on early- and late-stage breast cancer progression in a mouse model.

The prevalence of breast cancer (BC) continues to increase and is the leading cause of cancer deaths in many countries. Numerous in vitro and in vivo studies have demonstrated that 2-methoxyestradiol (2-ME) has antiproliferative and antiangiogenic effects in BC, thereby inhibiting tumour growth and metastasis. We compared the effect of 2-ME in early- and late-stage BC using a transgenic mouse model-FVB/N-Tg(MMTV-PyVT)-of spontaneously development of aggressive mammary carcinoma with lung metastasis. Mice received 100 mg/kg 2-ME treatment immediately when palpable mammary tumours were identified (early-stage BC; Experimental group 1) and 28 days after palpable mammary tumours were detected (late-stage BC; Experimental group 2). 2-ME was administered via oral gavage three times a week for 28 days after initiation of treatment, whereas control mice received the vehicle containing 10% dimethyl sulfoxide and 90% sunflower oil for the same duration as the treatment group. Mammary tumours were measured weekly over the 28 days and at termination, blood, mammary and lung tissue were collected for analysis. Mice with a tumour volume threshold of 4000 mm3 were killed before the treatment regime was completed. 2-ME treatment of early-stage BC led to lower levels of mammary tumour necrosis, whereas tumour mass and volume were increased. Additionally, necrotic lesions and anti-inflammatory CD163-expressing cells were more frequent in pulmonary metastatic tumours in this group. In contrast, 2-ME treatment of late-stage BC inhibited tumour growth over the 28-day period and resulted in increased CD3+ cell number and tumour necrosis. Furthermore, 2-ME treatment slowed down pulmonary metastasis but did not increase survival of late-stage BC mice. Besides late-stage tumour necrosis, none of the other results were statistically significant. This study demonstrates that 2-ME treatment has an antitumour effect on late-stage BC, however, with no increase in survival rate, whereas the treatment failed to demonstrate any benefit in early-stage BC.

Characterization of Mammary Tumors Arising from MMTV-PyVT Transgenic Mice.

Among genetically engineered mouse models of breast cancer, MMTV-PyVT is a mouse strain in which the oncogenic polyoma virus middle T antigen is driven by the mouse mammary tumor virus promoter. The aim of the present study was to perform morphologic and genetic analyses of mammary tumors arising from MMTV-PyVT mice. To this end, mammary tumors were obtained at 6, 9, 12, and 16 weeks of age for histology and whole-mount analyses. We conducted whole-exome sequencing to identify constitutional and tumor-specific mutations, and genetic variants were identified using the GRCm38/mm10 mouse reference genome. Using hematoxylin and eosin analysis and whole-mount carmine alum staining, we demonstrated the progressive proliferation and invasion of mammary tumors. Frameshift insertions/deletions (indels) were noted in the Muc4. Mammary tumors showed small indels and nonsynonymous single-nucleotide variants but no somatic structural alterations or copy number variations. In summary, we validated MMTV-PyVT transgenic mice as a multistage model for mammary carcinoma development and progression. Our characterization may be used as a reference for guidance in future research.

Assessment of antitumor activity of BP-C1, a platinum-based anticancer agent with a lignin-derived polymeric ligand, in autochthonous induced and spontaneous carcinogenesis rodent models

BackgroundA standard approach to study the anticancer activity of novel drugs is their testing in animals with inoculated tumors, which has some limitations. An alternative is the use of spontaneous or carcinogen-induced tumor models as they have better translation potential. The carcinogen-induced and transgenic tumor models were used to assess the antitumor activity of BP-C1, a platinum-containing drug with lignin-derived polymeric ligand. MethodsWe used female Swiss-H-derived mice and Wistar female rats to induce autochthonous tumors via exposure to benzo[a]pyrene and 1,2-dimethylhydrazine, respectively. Additionally, transgenic HER-2/neu FVB/N female mice, prone to the development of spontaneous mammary carcinomas, were used. ResultsAntitumor activity of BP-C1 was observed in soft tissue sarcomas, induced by benzo[a]pyrene. The animals treated with BP-C1 exhibited more stabilizations and therapy responses compared to placebo controls. The efficacy of BP-C1 was somewhat reduced compared to cyclophosphamide; however, their combination resulted in an enhanced antitumor effect. For the 1,2-dimethylhydrazine-induced rat colon cancer model, BP-C1 reduced tumor multiplicity by 21–41 %. For mammary adenocarcinomas in HER-2/neu FVB/N mice, short-termed complete responses were observed in the BP-C1 groups with a frequency of 12–13 %, while complete responses were absent in the placebo group. ConclusionThe results acquired indicated a wide spectrum of antitumor activity of BP-C1.

Abstract P2-05-01: Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas

Abstract Basal-like breast cancers, an aggressive breast cancer subtype that has poor treatment options, are thought to arise from luminal mammary epithelial cells but undergo plasticity to a basal-like state upon transformation. Identifying cellular mechanisms underlying this luminal-basal plasticity will allow an improved understanding of the pathogenesis of basal-like breast cancers. The Hippo signaling pathway, an important cell fate regulator across many tissues, has been implicated in breast cancer development. Central to Hippo signaling are the tumor suppressor kinases LATS1 and LATS2 (LATS1/2), which function to inhibit the transcriptional regulators YAP and TAZ (YAP/TAZ). In this study, we investigated the roles of LATS1/2 in the maintenance of mammary epithelial homeostasis and tumorigenesis in vivo. Using genetic mouse models, we found that conditional co-deletion of LATS1/2 in adult luminal cells promotes rapid proliferation and acquisition of basal cell traits, leading to early development of basal-like ductal carcinoma in situ and an eventual transition to a metastatic state. Further co-deletion experiments demonstrated that these phenotypes are dependent on YAP/TAZ. Transcriptional analyses of LATS1/2-null cells sorted from mammary glands demonstrated that these cells adopt gene expression signatures that correlate with mammary basal cells and human basal-like breast cancers, and revealed an association with Sox9, a transcription factor implicated in luminal progenitor fate and basal-like carcinomas. Collectively, this study demonstrates that LATS1/2 are important mediators of luminal cell fate in mammary epithelial homeostasis and that dysregulation of Hippo signaling in vivo promotes luminal-basal cell plasticity leading to basal-like mammary carcinoma development. Citation Format: Joseph Kern, Andrew Tilston-Lunel, Anthony Federico, Boting Ning, Grace Peppler, Eleni Stampouloglou, Marc Lenburg, Jennifer Beane, Stefano Monti, Xaralabos Varelas. Inactivation of LATS1/2 drives luminal-basal plasticity to initiate basal-like mammary carcinomas [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-05-01.

Expression of LIN28A/B and Let-7 miRNAs in canine mammary carcinomas

ABSTRACT: LIN28 is a RNA-binding protein including two highly conserved homologous, LIN28A and LIN28B. Proto-oncogenes such as LIN28A and LIN28B are generally targeted by the let-7 miRNAs in different types of human cancers. Here, we determined the expression of LIN28A in canine mammary tumor samples and the LIN28/let-7 pathway in canine mammary cell lines. In those cell lines, we identified a functional LIN28/let-7 pathway which exhibited high expression of let-7 members and low expression of its targets, including LIN28A and LIN28B. However, the mammary carcinoma tissue samples showed a frequent expression of LIN28A being expressed mainly in the epithelial cells. No association was observed between LIN28A expression and histopathological classification and grade, TNM and survival time. Our results suggested a possible role of the LIN28A protein in the development of canine mammary carcinomas due to the high frequency observed in the tumor samples (28 of 32). The in vitro experiments suggested that the LIN28/let-7 pathway is active in the tumor cells evaluated. However, more studies are necessary to elucidate the exact role of LIN28/let-7 pathway in canine mammary carcinomas.

Attenuation of Tumor Development in Mammary Carcinoma Rats by Theacrine, an Antagonist of Adenosine 2A Receptor.

Caffeine has been reported to induce anti-tumor immunity for attenuating breast cancer by blocking the adenosine 2A receptor. Molecular modeling showed that theacrine, a purine alkaloid structurally similar to caffeine, might be an antagonist of the adenosine 2A receptor equivalent to or more effective than caffeine. Theacrine was further demonstrated to be an effective antagonist of the adenosine 2A receptor as its concurrent supplementation significantly reduced the elevation of AMPK phosphorylation level in MCF-7 human breast cells induced by CGS21680, an agonist of adenosine 2A receptors. In an animal model, the development of mammary carcinoma induced by 7,12-Dimethylbenz[a]anthracene in Sprague–Dawley rats could be attenuated by daily supplement of theacrine of 50 or 100 mg/kg body weight. Both expression levels of cleaved-caspase-3/pro-caspase-3 and granzyme B in tumor tissues were significantly elevated when theacrine was supplemented, indicating the induction of programmed cell death in tumor cells might be involved in the attenuation of mammary carcinoma. Similar to the caffeine, significant elevation of interferon-γ and tumor necrosis factor-α was observed in the serum and tumor tissues of rats after the theacrine supplement of 50 mg/kg body weight. Taken together, theacrine is an effective antagonist of adenosine 2A receptors and possesses great potential to be used to attenuate breast cancer.

Phenotypic Plasticity of Fibroblasts during Mammary Carcinoma Development.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer.

Benzyl isothiocyanate suppresses development and metastasis of murine mammary carcinoma by regulating the Wnt/β‑catenin pathway.

Benzyl isothiocyanate (BITC) has been reported to exhibit antitumor properties in various cancer types; however, the underlying mechanisms of its action remain unclear. In the present study, the efficacy of BITC on murine mammary carcinoma cells was evaluated in vitro and in vivo, revealing a potential mechanism for its action. In vivo bioluminescence imaging indicated dynamic inhibition of murine mammary carcinoma cell growth and metastasis by BITC. A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that BITC also induced apoptosis. BITC further exhibited antitumorigenic activity in 4T1-Luc cells in vitro via the inhibition of cell proliferation, induction of apoptosis and cell cycle arrest, and inhibition of cell migration and invasion. Furthermore, the activity of key molecules of the adenomatous polyposis coli (APC)/β-catenin complex was altered following treatment with BITC, which suggested a potential role for the APC/β-catenin complex in the BITC-mediated induction of apoptosis and inhibition of metastasis in murine mammary carcinoma. BITC upregulated the activity of glycogen synthase kinase-3β and APC proteins, whereas it downregulated β-catenin expression. The inhibition of metastasis was accompanied with the downregulation of vimentin and upregulation of E-cadherin. Conversely, BITC did not exhibit toxicity or side effects in the normal mammary epithelial cell line MCF-10A. The present study indicated that BITC exhibited anticancer properties due to the induction of breast cancer cell apoptosis and inhibition of breast cancer cell metastasis mediated by the Wnt/β-catenin signaling pathway.

Abstract 716: A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma

Abstract Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in about 20% of invasive breast cancers. Anti-HER-2 monoclonal antibody therapy is effective, but its utility is limited by high costs, side effects and development of resistance, thus underlining the need of new therapeutic approaches. A novel anti-HER-2 vaccine made of virus-like particles (VLPs) displaying the extracellular domain (ECD) of the human oncogene/antigen HER-2 induced protective immune responses against transgenic mouse mammary carcinomas expressing human HER-2. We have developed a versatile antigen display platform that, unlike existing technologies, effectively facilitates directional covalent attachment of large antigens at high density on the surface of VLPs (J. Nanobiotechnology 14: 30, 2016). The system uses a tag/catcher conjugation system that was developed by splitting the CnaB2 domain from the fibronectin-binding protein FbaB of Streptococcus pyogenes into a highly reactive peptide (SpyTag) and a protein (SpyCatcher) binding partner. Interaction between SpyTag and SpyCatcher results in the spontaneous formation of an isopeptide bond, occurring at high efficiency in a wide variety of protein contexts and buffer conditions. Here, we genetically fused with SpyCatcher the full extracellular domain (subdomains I-IV) of human HER-2, and bound the fusion antigen (SpyCatcher-HER2) to the surface of VLPs (derived from the AP205 phage), each presenting 360 SpyTag peptides. The vaccine, referred to as HER2-VLP, effectively overcame immune tolerance and induced Th1 cytokines and high-titer, high affinity, therapeutically potent anti-HER-2 antibodies which inhibited tumor growth in wild-type FVB mice implanted with transgenic mammary carcinomas expressing human HER-2. Furthermore, vaccination with HER2-VLP prevented spontaneous development of human HER2-positive mammary carcinomas in tolerant transgenic mice. Vaccination with a control preparation of untagged VLP and HER-2 ECD did not induce protective immune responses. Polyclonal IgG antibodies elicited by HER2-VLP vaccination had an affinity for human HER-2 comparable to trastuzumab and inhibited the 3D growth in vitro of both trastuzumab-sensitive and trastuzumab-resistant BT-474 human breast cancer cells. In conclusion, the HER2-VLP vaccine has the potential to become a tool in the fight against HER-2-positive human cancer. The results also provide strong proof-of-concept for the use of the versatile VLP platform to develop a variety of vaccines against other tumor antigens. Supported by grants from the Italian Association for Cancer Research (AIRC), the University of Bologna, the Danish Research Council, the Eurostars program and the European Research Council (ERC). Citation Format: Arianna Palladini, Susan Thrane, Christoph M. Janitzek, Jessica Pihl, Stine B. Clemmensen, Wilhelm A. de Jongh, Thomas M. Clausen, Giordano Nicoletti, Lorena Landuzzi, Manuel L. Penichet, Tania Balboni, Marianna L. Ianzano, Veronica Giusti, Thor H. Theander, Morten A. Nielsen, Ali Salanti, Pier-Luigi Lollini, Patrizia Nanni, Adam F. Sander. A novel virus-like particle vaccine presenting HER-2 extracellular domain elicits strong immune responses against mammary carcinoma [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 716.

Virus-like particle display of HER2 induces potent anti-cancer responses

ABSTRACTOverexpression of human epidermal growth factor receptor-2 (HER2) occurs in 20–30% of invasive breast cancers. Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active vaccination may represent a safer, more effective and cheaper alternative, although the induction of strong and durable autoantibody responses is hampered by immune-tolerogenic mechanisms. Using a novel virus-like particle (VLP) based vaccine platform we show that directional, high-density display of human HER2 on the surface of VLPs, allows induction of therapeutically potent anti-HER2 autoantibody responses. Prophylactic vaccination reduced spontaneous development of mammary carcinomas by 50%-100% in human HER2 transgenic mice and inhibited the growth of HER2-positive tumors implanted in wild-type mice. The HER2-VLP vaccine shows promise as a new cost-effective modality for prevention and treatment of HER2-positive cancer. The VLP platform may represent an effective tool for development of vaccines against other non-communicable diseases.

LFM-A13, a potent inhibitor of polo-like kinase, inhibits breast carcinogenesis by suppressing proliferation activity and inducing apoptosis in breast tumors of mice.

The goals of the present study were to define the anticancer activity of LFM-A13 (α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)-propenamide), a potent inhibitor of Polo-like kinase (PLK), in a mouse mammary cancer model induced by 7,12-dimethylbenz(a)anthracene (DMBA) in vivo and explore its anticancer mechanism(s). We also examined whether the inhibition of PLK by LFM-A13 would improve the efficiency of paclitaxel in breast cancer growth in vivo. To do this, female BALB/c mice received 1mg of DMBA once a week for 6weeks with oral gavage. LFM-A13 (50mg/kg body weight) was administered intraperitoneally with DMBA administration and continued for 25weeks. We found that LFM-A13, paclitaxel, and their combination have a significant effect on the DMBA-induced breast tumor incidence, mean tumor numbers, average tumor weight, and size. At the molecular level, the administration of LFM-A13 hindered mammary gland carcinoma development by regulating the expression of PLK1, cell cycle-regulating proteins cyclin D1, cyclin dependent kinase-4 (CDK-4), and the CDK inhibitor, p21. Moreover, LFM-A13 treatment upregulated the levels of IκB, the pro-apoptotic proteins Bax, and caspase-3, and down-regulated p53 and the antiapoptotic protein Bcl-2 in mammary tumors. The combination of LFM-A13 with paclitaxel was found to be more effective compared with either agent alone. Collectively, these results suggest that LFM-A13 has an anti-proliferative activity against breast cancer in vivo and that LFM-A13 and paclitaxel combination could be a strategy for the treatment of breast cancer.

Abstract 1835: Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1)

Abstract Amplification within the 8p11-12 locus occurs in approximately 15% of breast cancer (BC) and is associated with poor survival and distant recurrence. Overexpression of many of the genes within the 8p11-12 region can confer a pathological gain of function to breast cells. We and others have previously demonstrated that NSD3 (WHSC1L1), a lysine methyltransferase, can act as a potent transforming oncogene. Predictions by the GISTIC algorithm of the Human Cancer Genome Atlas data suggest that NSD3 is a driving gene within 8p11-12 in multiple tumor types, including BC. We previously showed that NSD3 can regulate the expression of influential genes, such as estrogen receptor alpha (ESR1/ERα). To further explore the role of NSD3 in promoting BC, we generated FVB/N transgenic mice with targeted overexpression of NSD3 in the mammary gland and compared them to matched non-transgenic wild-type (WT) females. We observed that pups nursed by transgenic females were underdeveloped, regardless of genotype. Underdeveloped pups displayed delayed hair growth and eye opening, and were half the weight of pups nursed by WT females. To investigate this phenotype, we characterized thoracic and inguinal mammary glands from virgin, mid-pregnancy, lactating, and post-lactating mice by whole mount and histological analysis. Mammary glands from virgin transgenic females displayed increased branching and terminal bud formation. Alveolar buds from mid-pregnant transgenic glands were more numerous and densely packed. Glands from lactating transgenic females showed large areas in which the alveoli failed to undergo functional differentiation, resulting in a lactation defect. Mammary glands taken post-lactation exhibited areas of ductal and alveolar hyperplasia. At 40 weeks of age, multiple transgenic mice had palpable tumors which were excised at 1 cm3 size. Whole mount and histological analysis of glands from tumor bearing mice commonly demonstrated hyperplasia, dysplasia, and carcinoma in situ, contrary to age-matched WT glands. The areas of ductal dysplasia and carcinoma in situ closely resembled several patterns commonly observed in human breast cancer, including micropapillary, cribriform, and high grade areas of ductal carcinoma in situ. Mammary tumors were analogous to infiltrating ductal carcinomas, many of which were high grade tumors. In summary, overexpression of NSD3 in the mouse mammary gland elicited drastic deformation, inhibited functional differentiation, and caused tumor formation. Continued characterization of this mouse model, the oncogenic role of NSD3, and other mechanistic studies are essential to improve patient outcome in 8p11-12 altered cancers. Citation Format: Alex C. Rutkovsky, Brittany Turner-Ivey, Ericka L. Smith, Laura S. Spruill, Jamie N. Mills, Stephen P. Ethier. Development of mammary hyperplasia, dysplasia, and invasive ductal carcinoma in transgenic mice expressing the 8p11 amplicon oncogene NSD3 (WHSC1L1) [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 1835. doi:10.1158/1538-7445.AM2017-1835

Immunosurveillance and immunoediting in MMTV-PyMT-induced mammary oncogenesis

ABSTRACTEvidence of cancer immunosurveillance and immunoediting processes has been primarily demonstrated in mouse models of chemically induced oncogenesis. Although these models are very tractable, they are characterized by high mutational loads that represent a minority of human cancers. In this study, we sought to determine whether cancer immunosurveillance and immunoediting could be demonstrated in a more clinically relevant oncogene-induced model of carcinogenesis, the MMTV-PyMT (PyMT) mammary carcinoma model. This model system in the FVB/NJ strain background was previously used to demonstrate that adaptive immunity had no role in limiting primary cancer formation and in fact promoted metastasis, thus calling into question whether cancer immunosurveillance operated in preventing the development of breast cancer. Our current study in the C57BL/6 strain backgrounds provides a different conclusion, as we report here the existence of an adaptive immunosurveillance of PyMT mammary carcinomas using two independent models of immune deficiency. PyMT mice bred onto a Rag1−/− background or immune suppressed by chronic tacrolimus therapy both demonstrated accelerated development of mammary carcinomas. By generating a bank of cell lines from these animals, we further show that a subset of PyMT cell lines had delayed growth after transplantation into wild-type (WT) syngeneic, but not immune-deficient hosts. This reduced growth rate in immunocompetent animals was characterized by an increase in immune cell infiltration and tissue differentiation. Furthermore, loss of the immune cell infiltration that characterized immunoediting of slow growing cell lines, changed them into fast growing variants capable of progressing in the immunocompetent model. In conclusion, our study provides evidence that immunosurveillance and immunoediting of PyMT-derived cell lines modulate tumor progression in this oncogene-induced model of cancer.

CCL2/CCR2 Regulates the Tumor Microenvironment in HER-2/neu-Driven Mammary Carcinomas in Mice.

Chronic inflammation is a hallmark of cancer. Inflammatory chemokines, such as C-C chemokine ligand 2 (CCL2), are often present in tumors but their roles in cancer initiation and maintenance are not clear. Here we report that CCL2 promotes mammary carcinoma development in a clinically relevant murine model of breast cancer. Targeted disruption of Ccl2 slowed the growth of activated Her2/neu-driven mammary tumors and prolonged host survival. Disruption of Ccl2 was associated with a decrease in the development and mobilization of endothelial precursor cells (EPCs) which can contribute to tumor neovascularization. In contrast, disruption of Ccr2, which encodes CCL2’s sole signaling receptor, accelerated tumor development, shortened host survival, and mobilized EPCs. However, pharmacological inhibition of CCR2 phenocopied Ccl2 disruption rather than Ccr2 disruption, suggesting that the Ccr2-/- phenotype is a consequence of unanticipated alterations not linked to intact CCL2/CCR2 signaling. Consistent with this explanation, Ccr2-/- monocytes are more divergent from wild type monocytes than Ccl2-/- monocytes in their expression of genes involved in key developmental and functional pathways. Taken together, our data suggest a tumor-promoting role for CCL2 acting through CCR2 on the tumor microenvironment and support the targeting of this chemokine/receptor pair in breast cancer.

Abstract 841: Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS)

Abstract Soy isoflavones such as daidzein and its bioactive metabolite equol converted from daidzein by human intestinal bacterial flora, have been proposed to reduce the risk of breast cancer incidence in Asian women probably due to higher number of the inhabitants capable of producing equol in these countries. Although several in vitro studies shows the anti-cancer activity of equol against human breast cancer cells, there have been few reports that have directly showed the preventive role of equol in the experimental animal model of mammary carcinoma. We investigated here the effects of equol on the development of breast cancer using the EMS chemically induced rat model of hormone-dependent mammary carcinoma. Female WKA rats (n = 30) were equally divided into 2 groups, subsequently given EMS orally for 12 weeks and fed the CE2 diets with or without 50 mg equol/g diet throughout the experiments. All EMS-treated rats fed either diet developed ER- and/or PgR- positive hormone-dependent mammary carcinoma by 24 weeks. Compared with the equol-free diet, the diet supplemented with equol could not significantly delay the development of mammary carcinoma, showing any preventive activity on the development of EMS-induced hormone-dependent mammary carcinoma. The body weight, total EMS uptakes, and urine estradiol concentrations were not significantly different between these 2 groups. These data indicate that high dose administration of equol alone does not exert clear preventive effects on hormone-dependent EMS-induced mammary carcinogenesis, and may require other soy isoflavone components such as genistein that has previously been shown to exhibit a great synergy when combined with equol. The in vivo combination studies using genistein and equol are underway. Citation Format: MISAKI ONO, Takako Higuchi, Mikako Takeshima, Shuji Nakano. Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS). [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 841.

Abstract 1200: HER-2 isoform interaction in mammary carcinoma onset and progression

Abstract Human breast cancer cells express full-length HER-2 along with proteins resulting from mutation, alternative splicing, alternative initiation of translation and post-translational modification. The Delta16 splice variant, lacking exon 16, has the properties of an activated oncogene, but it could also play beneficial roles in the response to targeted therapeutic agents. To study mammary carcinogenesis in a mouse model that mimics human coexpression of full-length HER-2 and Delta16 isoforms, we produced hybrid mice bearing heterozygous copies of both human transgenes (F1 mice), and we compared them to parental mice (Delta16 and HER-2 transgenic mice, respectively). Tumor onset in F1 and Delta16 mice was much faster than in HER-2 mice (30 vs >80 weeks), but the growth of established tumors and metastatic spread were not enhanced. Each mammary carcinoma of F1 mice expressed both isoforms at variable ratios. Most (∼80%) expressed high levels of Delta16 and low levels of full length HER-2, a few (∼5%) expressed full-length HER-2 and little, if any, Delta 16, and the remainder (∼15%) coexpressed at high level both isoforms. The study of tumor vascularization showed that full-length HER-2 tumors mainly contained few large vessels or vascular lacunae, whereas Delta16 tumors were perfused by numerous endothelium-lined small vessels. F1 tumors with high full-length HER-2 expression made few large vessels, whereas tumors with low full-length HER-2 and high Delta16 contained numerous small vessels and expressed high levels of both VEGF and VEGFR2. Administration of trastuzumab to young F1 mice effectively prevented mammary carcinoma onset in ∼85% of mice at 1 year of age. The preventive effect of trastuzumab was stronger in F1 mice than in both parental strain. To analyze the intrinsic sensitivity of F1 mammary carcinoma cells to targeted drugs in 3D, we established cell lines expressing different HER-2 isoform ratios. High Delta16 expression caused high sensitivity to HER-2 inhibitors (trastuzumab, neratinib, lapatinib), whereas high full-length HER-2 was associated with a lower sensitivity. Interestingly, high levels of both isoforms was associated with resistance to trastuzumab, but sensitivity to small molecule inhibitors. In conclusion, the coexpression of full-length HER-2 and Delta16 controls several determinants of mammary carcinoma development, progression and sensitivity to targeted agents, as revealed by the study of F1 mice. Citation Format: Arianna Palladini, Massimiliano Dall’Ora, Tania Balboni, Giordano Nicoletti, Marianna Ianzano, Roberta Laranga, Lorena Landuzzi, Veronica Giusti, Alessia Lamolinara, Carla De Giovanni, Augusto Amici, Serenella M. Pupa, Manuela Iezzi, Patrizia Nanni, Pier-Luigi Lollini. HER-2 isoform interaction in mammary carcinoma onset and progression. [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 1200.

Dynamic monitoring of GPER-mediated estrogenic effects in breast cancer associated fibroblasts: An alternative role of estrogen in mammary carcinoma development

Cancer associated fibroblasts (CAFs) are crucial contributors to breast cancer development. Estrogen affects mammary stroma in both physiological and pathophysiological conditions. We show here that estrogen (G-protein coupled) receptor (GPER) could be detected by immunohistochemistry in stromal fibroblasts of primary breast cancers. The presence of GPER expression was further confirmed by immunofluorescence and quantitative PCR in CAFs isolated from primary breast cancers. Based on dynamic monitoring by real time cell analyzer (RTCA) system, 17-β-estradiol (E2) as well as GPER specific agonist G1 were observed to trigger transient cell index increasing within an hour in a dosage-dependent manner in breast CAFs. In addition, E2 and G1 stimulated intracellular calcium modulation and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 within seconds and minutes in CAFs, respectively. Moreover, E2 and G1 promoted cell proliferation of breast CAFs measured by RTCA monitoring, cell viability assay and cell cycle analysis, and this promotion could be blocked by a GPER-selective antagonist G15. Interestingly, dynamic RTCA monitoring indicated that E2 increased adhesion of resuspended cells, and microscopy confirmed that E2 stimulated cell spreading. Both the adhesion and spreading were proposed to be mediated by GPER, since G1 also stimulated these effects similar to E2, and G15 reduced them. Moreover, GPER was found to mediate migration that was increased by E2 and G1 but reduced by G15 in RTCA cell migration assay and transwell assay. Accordingly, GPER mediates not only rapid actions but also slow effects including adhesion/spreading, proliferation and migration in breast CAFs. Estrogen is likely to affect tumor associated stroma and contributes to mammary carcinoma development through CAFs.

The Cytokine IL-6 Reactivates Breast Stromal Fibroblasts through Transcription Factor STAT3-dependent Up-regulation of the RNA-binding Protein AUF1

The development and spread of mammary carcinomas require synergetic interplay between tumor cells and their microenvironment through paracrine secretions, which are still not well defined. We have shown here that interleukin-6 (IL-6), either recombinant or secreted from highly invasive breast cancer cells, down-regulates the tumor suppressor proteins p16(INK4A), p21(WAF1), and p53 and activates breast stromal fibroblasts in a paracrine manner. The formation of myofibroblasts requires p16(INK4A) down-regulation and the activation of the JAK2/STAT3 pathway. Indeed, the transcription factor STAT3 positively controls the expression of the three major myofibroblast markers, SDF-1, α-smooth muscle actin (α-SMA), and TGF-β1, and mediates IL-6-related down-regulation of p16(INK4A), p21(WAF1), and p53 as well as the activation of stromal fibroblasts. Importantly, these effects were mediated through STAT3-dependent up-regulation of the mRNA-binding protein AUF1, whose promoter contains three canonical STAT3 binding sites. AUF1 binds the SDF-1, α-SMA, TGF-β1, and IL-6 mRNAs and reduces their turnover. Consequently, specific AUF1 down-regulation inhibits IL-6-dependent activation of breast stromal fibroblasts, whereas AUF1 ectopic expression of p37(AUF1) activated these cells and enhanced their paracrine induction of epithelial-to-mesenchymal transition in breast cancer cells, which shows a non-cell-autonomous oncogenic function of AUF1. Together, these results demonstrate a major role of IL-6 in activating breast stromal fibroblasts through STAT3-dependent AUF1 induction.

RANK expression as a prognostic and predictive marker in breast cancer

RANK ligand (RANKL) is crucial for the development of mouse mammary glands during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mammary epithelium via its receptor RANK and has a role in expansion and regenerative potential of mammary stem cells. Pharmacologic inhibition of RANKL attenuates the development of mammary carcinoma and inhibits metastatic progression in multiple mouse models. Primary breast carcinoma samples from the neoadjuvant GeparTrio study were analyzed to correlate the expression of human RANK and RANKL with pathological complete response (pCR), disease-free (DFS), and overall (OS) survival. Pre-treatment FFPE core biopsies (n=601) were analyzed for percentage and intensity of immunohistochemical RANK and RANKL expression. Antibodies against human RANK (N-1H8; Amgen) and human RANKL (M366; Amgen) were used. RANK protein was expressed in 160 (27%) patients. Increased RANK expression was observed in 14.5% of patients and correlated with high tumor grade (p<0.023) and negative hormone receptor (HR) status (p<0.001). Patients with high RANK expression showed a higher pCR rate (23.0% vs. 12.6%, p=0.010), shorter DFS (p=0.038), and OS (p=0.011). However, prognostic and predictive information was not an independent parameter. Only 6% of samples expressed RANKL, which was not correlated with any clinical features. Higher RANK expression in the primary tumor is associated with a higher sensitivity to chemotherapy, but also a higher risk of relapse and death. Our study provides a basis for further exploration of the antitumor activity of clinical antibodies against RANKL.