Mammary Tumor Development Research Articles

Effects of Two Types of Calorie Restriction on Methylation Levels of Adiponectin Receptor 1 (AdipoR1) and Leptin Receptor Overlapping Transcript (Leprot) in a MMTV-TGF-α Breast Cancer Mouse Model.

The role of adiponectin and leptin signalling pathways has been suggested to play important roles in the protective effects of energy restriction (ER) on mammary tumour (MT) development. To study the effects of ER on the methylation levels in adiponectin receptor 1 (AdipoR1) and leptin receptor overlapping transcript (Leprot) genes using the pyrosequencing method in mammary fat pad tissue, mouse mammary tumour virus-transforming growth factor-α (MMTV-TGF-α) female mice were randomly assigned to ad libitum (AL), chronic ER (CER, 15 % ER) or intermittent ER (3 weeks AL and 1 week 60 % ER in cyclic periods) groups at 10 weeks of age until 82 weeks of age. The methylation levels of AdipoR1 in the CER group were higher than those in the AL group at week 49/50 (P < 0·05), while the levels of methylation for AdipoR1 and Leprot genes were similar among the other groups. Also, the methylation levels at CpG2 and CpG3 regions of the promoter region of the AdipoR1 gene in the CER group were three times higher (P < 0·05), while CpG1 island of Leprot methylation was significantly lower compared with the other groups (P < 0·05). Adiponectin and leptin gene expression levels were consistent with the methylation levels. We also observed a change with ageing in methylation levels of these genes. These results indicate that different types of ER modify methylation levels of AdipoR1 and Leprot in different ways and CER had a more significant effect on methylation levels of both genes. Epigenetic regulation of these genes may play important roles in the preventive effects of ER against MT development and ageing processes.

Truncated ASPP2 Drives Initiation and Progression of Invasive Lobular Carcinoma via Distinct Mechanisms.

Invasive lobular carcinoma (ILC) accounts for 8%-14% of all breast cancer cases. The main hallmark of ILCs is the functional loss of the cell-cell adhesion protein E-cadherin. Nonetheless, loss of E-cadherin alone does not predispose mice to mammary tumor development, indicating that additional perturbations are required for ILC formation. Previously, we identified an N-terminal truncation variant of ASPP2 (t-ASPP2) as a driver of ILC in mice with mammary-specific loss of E-cadherin. Here we showed that expression of t-ASPP2 induced actomyosin relaxation, enabling adhesion and survival of E-cadherin-deficient murine mammary epithelial cells on stiff matrices like fibrillar collagen. The induction of actomyosin relaxation by t-ASPP2 was dependent on its interaction with protein phosphatase 1, but not on t-ASPP2-induced YAP activation. Truncated ASPP2 collaborated with both E-cadherin loss and PI3K pathway activation via PTEN loss in ILC development. t-ASPP2-induced actomyosin relaxation was required for ILC initiation, but not progression. Conversely, YAP activation induced by t-ASPP2 contributed to tumor growth and progression while being dispensable for tumor initiation. Together, these findings highlight two distinct mechanisms through which t-ASPP2 promotes ILC initiation and progression. SIGNIFICANCE: Truncated ASPP2 cooperates with E-cadherin and PTEN loss to drive breast cancer initiation and progression via two distinct mechanisms. ASPP2-induced actomyosin relaxation drives tumor initiation, while ASPP2-mediated YAP activation enhances tumor progression.

Abstract A88: RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor

Abstract Genetic mutations in cancer cells play key roles in shaping the tumor immune microenvironment. RUNX1 is a transcription factor (TF) essential for estrogen receptor (ER)+ luminal mammary epithelial cells (MECs) and is mutated in a significant portion of human ER+ luminal breast cancers. It is also an important regulator of immune cells, raising an intriguing question as to whether its loss-of-function mutations found in ER+ breast cancers affect both MECs and immune cells. To address this, we established an in vivo mouse model by inducing loss of both RUNX1 and p53 in luminal MECs, upon intraductal injection of a Cre-expressing adenovirus under the control of the Keratin 8 promoter (Ad-K8-Cre) to mammary glands of female mice carrying their conditional knockout alleles. Compared to similarly injected wild-type (WT) control mice or mice with induced loss of p53 alone, induced loss of both led to hyperproliferation of Runx1/Trp53-null MECs and eventually development of mammary tumors after a long latency (a tumor-free period of ~9-13 months after Ad-K8-Cre injection). Characterization of these tumors revealed that they are ER+ mammary tumors with extensive leukocyte infiltration. CD45+ leukocytes in them constituted ~32-84% of total live tumor cells, which is unusual for a mouse cancer model. Among CD45+ cells, ~45% were macrophages and ~15% were CD3+ T cells. Among tumor-infiltrating myeloid cells (CD45+CD11b+), many were myeloid-derived suppressor cells (MDSCs). In addition, significant differential expression of PD-1 on CD3+ T cells (38.5% vs. 7.3%) and PD-L1 on CD45- cells (2.7% vs. 0.3%) was observed in these Runx1/Trp53-null tumors, when compared to WT controls. This immune profile suggests an immunosuppressive microenvironment in these tumors. The tumor immune phenotype induced by RUNX1-loss in our model is supported by genomic data from human breast cancers analyzed by the TIMER online tool (Li et al., Cancer Res 77(21):e108-e110). The TIMER analysis showed that in human breast cancers, levels of most infiltrating immune cell subtypes (e.g., macrophages, T cells) in RUNX1-mutated cases are higher than those of RUNX1-WT cases; in contrast, a similar analysis for GATA3-mutated cases exhibited the opposite. As both RUNX1 and GATA3 are key TFs for luminal MECs and their mutations are both found in luminal breast cancers, the data from both human cases and our mouse model suggest that RUNX1-mutated breast cancers may represent a unique, immune “hot” subtype of ER+ luminal breast tumors. ER+ breast cancers are typically immune “cold” and do not respond to immune checkpoint blockade (ICB) well. Our data suggest that RUNX1-mutated breast cancers may represent a subset of ER+ breast cancers that may respond to ICB-based therapy. Collectively, our data suggest that loss of RUNX1 promotes development of ER+ breast cancer and modulates the immune microenvironment; a better understanding of how these two processes are interconnected may provide novel strategies to treat breast cancer with RUNX1-mutation. Citation Format: Dongxi Xiang, Zhe Li. RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A88.

Abstract P4-08-03: Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer

Abstract Research Overview and Objective Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant challenge. The co-deficiency of tumor suppressors p53 and ARF is a significant genetic signature enriched in TNBC, but it is not yet clear how TNBC is regulated by this genetic alteration. Methods To answer this question, we established p53/ARF-defective murine embryonic fibroblast (MEF) to study the molecular and phenotypic consequences in vitro. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. Results Increased transformation capability was observed in p53/ARF-defective cells, and formation of aggressive mammary tumors was also seen in p53−/−ARF−/− mice. RNA-editing enzyme ADAR1 was identified as a potential mediator for the elevated oncogenic potential. Interestingly, we found that the overexpression of ADAR1 is also prevalent in human TNBC cell lines and patient specimen. Using short hairpin RNA (shRNA) to reduce ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. Different levels of RNA editing of known ADAR1 targets were detected in shRNA-treated human TNBC cell lines, suggesting that ADAR1-mediated RNA editing contributes to TNBC pathogenesis. Implication/Discussion These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, partially through affecting ADAR1-mediated RNA editing. Further understanding of this pathway could shed light on potential vulnerabilities of TNBC and inform the development of personalized therapies based on patients’ genetic signiatures. Citation Format: Che-Pei Kung, Catherine E Kuzmicki, Emily A Bross, Sua Ryu, Eric Freeman, Thwisha Sabloak, Emily R Bramel, Leonard B Maggi Jr, Jason D Weber. Tumor suppressors p53 and ARF control ADAR1-driven tumorigenicity in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-03.

Abstract OI: Novel non-canonical functions of EZH2 in triple negative breast cancer

Abstract Patients with distant metastasis at the time of diagnosis have significantly worse prognosis. Triple negative breast carcinomas (TNBC) are heterogeneous tumors with propensity to metastasize and a guarded short term prognosis. The link between breast cancer and regulators of cell type identity was demonstrated in 2003, when enhancer of zeste homolog 2, EZH2, was reportedly overexpressed in approximately 55% of breast cancer tissue samples and was significantly associated with estrogen receptor negative status and worse disease free and overall survival. As the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), the methyltransferase EZH2 deposits trimethyl marks on histone tails of lysine 27 of histone H3 (H3K27me3) to effect transcriptional repression. However, in a subset (~ 30%) TNBC high levels of EZH2 are associated with low H3K27me3, suggesting the intriguing possibility that in this group of tumors EZH2 may promote progression through other unknown mechanisms. Recent studies have shown that in TNBC EZH2 may indeed function independent of PRC2 and H3K27me3 activity. During breast cancer initiation, EZH2 enhances breast cancer stem cells and mammary tumor development through transcriptional activation of NOTCH1 and upregulation of its signaling pathway. More recent data show that EZH2 protein binds to p38 MAP kinase with resulting EZH2 phosphorylation at threonine 367 and accumulation in the cytoplasm of TNBC cells. In breast cancer tissue samples, cytoplasmic pEZH2 T367 is significantly associated with low H3K27me3 levels and a triple negative phenotype. Mechanistically, pEZH2 T367 protein binds to cytoplasmic regulators of cell migration and invasion, and is required for metastasis. These studies demonstrate a H3K27me3-independent mechanism of EZH2 function in TNBC progression. Future investigations will be required to test the therapeutic potential of inhibiting EZH2 non-canonical mechanisms alone or in combination with pharmacological targeting of EZH2 methyltransferase activity in TNBC. Citation Format: CG Kleer. Novel non-canonical functions of EZH2 in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OI.

Abstract P5-04-20: Progesterone promotes immunosuppression in breast cancer

Abstract Why are some early neoplastic lesions identified through immunosurveillance and subsequently destroyed, whereas other tumors evade this clearance step and progress to clinically-relevant tumors? The cancer immunoediting hypothesis highlights that the innate and adaptive immune responses work together to “flag” early neoplastic lesions for immune-mediated elimination. An early mediator of this “elimination” process is activation of type I interferon signaling. Thus, suppression of type I interferon signaling may help developing tumors evade the critical early steps of immune recognition and clearance. Preliminary data from our lab suggest that progesterone, working through the progesterone receptor (PR), may be a key player in tumor immune evasion through downregulation of interferon signaling. In breast cancer cells, we have shown that presence/activation of PR can attenuate interferon-signaling at multiple steps within the signaling cascade: decreased STAT1 phosphorylation, decreased STAT1/STAT2/IRF9 transcriptional complex formation (called ISGF3, required for interferon-alpha signaling), decreased recruitment of ISGF3 to ISG promoter sequences, and decreased transcriptional activation of interferon-stimulated genes (ISG, end point of interferon signaling). These data suggest a concerted effort aimed at reducing interferon-signaling through activation of PR. These data translate to the immune microenvironment in the murine mammary gland. In mice treated with progesterone for 21 days, we see immunosuppressive changes in the mammary gland: increased T-regulatory cells (immunosuppressive), decreased frequency of antigen-presenting cells (decreased ability to present potential tumor-associated antigens to CD8 T-cells), and a decrease in MHC class I presentation in non-lymphoid cells (decreased ability for epithelial cells to present potential tumor-associated antigens). We observe similar phenotypes in mice with transgenic overexpression of PR. These changes in the immune system suggest that PR can promote immunosuppressive changes in the mammary gland. Moreover, 80% of these mice in a multiparous cohort develop mammary gland tumors. Therefore, early changes in the immune microenvironment in the mammary gland may translate to the development of mammary gland tumors in PR transgenic mice. Cumulatively, these data suggest a mechanism through which PR aids early breast cancer lesions in escaping immune surveillance. These data have significant implications for the use of progesterone-containing hormone replacement therapy, as well as underscore the importance of studying anti-progestins as novel chemo-preventative agents for breast cancer. Citation Format: Christy Hagan, Merit Goodman, Gloria Trinca, Katherine Walter, Katelin Gibson, Prabhakar Chalise, Mary Markiewicz. Progesterone promotes immunosuppression in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-20.

Leptin Signaling in Liver Tissue of a Transgenic Breast Cancer Mouse Model.

Leptin, an adipocytokine, is secreted from various tissues including the liver. The roles of both leptin and leptin receptor (ObR) in numerous pathophysiological conditions including mammary tumor (MT) development have been reported. However, the roles of leptin signaling-related proteins in the liver have not been reported previously in MT development. The objective of this study was to examine the expression levels of leptin and ObR in liver tissue of a transgenic breast cancer mouse model to investigate whether the roles of leptin in MT development are systemic or local. MMTV-TGF-α transgenic female mice were fed ad-libitum from week 10 up to week 74. Protein expression levels of leptin and ObR were measured in liver tissues of 74-week-old MMTV-TGF-α mice with and without MT by western blot. Serum leptin and insulin levels were measured using a enzyme-linked immunosorbent assay. Protein expression levels of leptin and ObR were similar in mice with MT compared to the ones without MT. Serum leptin and insulin levels were also not significantly different between the two groups. These results indicate that the effects of leptin signaling in MT development might be important at a local tissue level, such as mammary fat pad, and not as important at a systemic level.

A Non-canonical Function of BMAL1 Metabolically Limits Obesity-Promoted Triple-Negative Breast Cancer.

SummaryThe epidemiological association between disrupted circadian rhythms and metabolic diseases is implicated in increased risk of human breast cancer and poor therapeutic outcomes. To define a metabolic phenotype and the underlying molecular mechanism, we applied chronic insulin treatment (CIT) to an in vitro model of triple-negative breast cancer to directly address how BMAL1, a key circadian transcription factor, regulates cancer cell respiration and governs tumor progression. At the cellular level, BMAL1 suppresses the flexibility of mitochondrial substrate usage and the pyruvate-dependent mitochondrial respiration induced by CIT. We established an animal model of diet-induced obesity/hyperinsulinemia and observed that BMAL1 functions as a tumor suppressor in obese, but not lean, mice. Downregulation of BMAL1 is associated with higher risk of metastasis in human breast tumors. In summary, loss of BMAL1 in tumors confers advantages to cancer cells in both intrinsic mitochondrial metabolism and extrinsic inflammatory tumor microenvironment during pre-diabetic obesity/hyperinsulinemia.

Abstract B090: Tyrosine 211 phosphorylation of PCNA: A new paradigm of linking cell proliferation to cancer stemness and metastasis

Abstract Cancer metastasis is the fundamental cause of cancer-related death. While 5-year survival rate of breast cancer with local involvement is very optimistic, the number precipitately dropped for patients whose disease has spread to distant organs. Some cancer cells can remain dormant for a long time and evolve to full blown malignancy at a later time. Despite intensive study, the mechanisms underpinning metastasis and tumor dormancy continue to drive cancer morbidity. PCNA forms a homotrimer ring encircling the DNA double helix and acts as a sliding platform indispensable for DNA replication, DNA damage repair, and chromatin remodeling. While a positive role of PCNA in is conceivable, how PCNA functions in tumor progression remains elusive due to the embryonic lethal phenotype in PCNA-deficient mice. We have shown that PCNA is regulated by phosphorylation at tyrosine 211 (Y211) meditated through multiple growth factor-stimulated signaling pathways. Y211 phosphorylation enhances chromatin-bound PCNA to promote cell proliferation and is frequently detected in multiple human cancers including breast cancer. To further dissect its biological functions, we have generated genetic mouse strain in which Y211 is replaced with phenylalanine which is structurally similar to tyrosine but cannot be phosphorylated. Comparing to wild-type mice, 211F mice have significantly reduced mammary tumor formation upon induction by the carcinogen DMBA. To understand its biological role in oncogene-induced mammary tumorigenesis, mice were crossed with the MMTV-PyMT mice in which mammary gland expression of the oncogene polyoma virus middle T antigen (PyMT) is driven by the mouse mammary tumor virus (MMTV) enhancer/promoter. Female 211F/PyMT mice have statistically significant but only moderate decrease in mammary tumor development. However, their lung metastasis is dramatically ablated compared to WT/PyMT mice harboring primary tumors of similar size. These results suggest that pY211-PCNA has an essential function not only in tumorigenesis but more importantly in progression to distant metastasis. Consistently, mammary tumors derived from WT/PyMT mice exhibits more pronounced stromal activity than tumors of 211F/PyMT mice. The suppression of metastasis and stromal activity in 211F/PyMT mice is associated with reduced number of tumor-initiating cells (TICs) as determined by in vitro and in vivo analyses. Further study using syngeneic orthotopic model of transplanting murine cancer cells into the mammary glands of WT and 211F mice unveils an unexpected function of pY211-PCNA in the stromal compartment on tumor development. Thus, our study reveals a novel tumor-promoting function of PCNA in both parenchymal and stromal compartments besides its traditional role in cell proliferation. Numerous FDA-approved drugs are targeting the signaling pathways leading to Y211 phosphorylation, suggesting that pY211-PCNA is a new druggable mechanism to target tumor metastasis. Citation Format: Yuan-Liang Wang, Chuan-Chun Lee, Pei-Le Lin, Yi-Chun Shen, Wan-Rong Wu, Wei-Ya Xia, Mien-Chie Hung, Shao-Chun Wang. Tyrosine 211 phosphorylation of PCNA: A new paradigm of linking cell proliferation to cancer stemness and metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B090. doi:10.1158/1535-7163.TARG-19-B090

Association between DLA-DRB1.2 allelic diversity and development of mammary gland tumors in dogs

BackgroundThe major histocompatibility complex (MHC) is the best-characterized genetic region related to resistance/susceptibility to a wide range of infectious and immune-mediated diseases. Evidences suggest that MHC class II genes may play an important role in developing different types of tumors including breast cancer. Canine mammary gland tumors (CMTs) are the most common neoplasms in female dogs. In the current study, the association of canine MHC class II DLA-DRB1.2 genotypes with development of mammary gland tumor profiles in dogs was investigated. DLA-DRB1.2 allelic diversity was determined in 40 dogs (18 CMT cases and 22 controls) using HRM technique and DNA sequencing. Association of the DLA-DRB1.2 genotypes with CMT profiles was expressed as odds ratio (OR).ResultsBased on the histopathological typing of tumors, CMT cases were categorized into 4 groups: simple carcinoma, complex carcinoma, carcinoma arising in a benign tumor and special types of carcinoma. A total of eight HRM profiles (A to H) were identified in dogs sampled. The association study revealed a significant correlation between DLA-DRB1.2 genotypes with different CMT profiles. The E genotype was significantly associated with increased risk of carcinoma arising in a benign tumor, and the B genotype represented a positive correlation with complex carcinoma. Significant association was also observed between the heterozygosity of DLA-DRB1.2 genotypes and decreased risk of developing tumor in dogs.ConclusionsThese results provide additional support for the association between DLA-DRB1 genes and development of mammary gland tumors in dogs and could potentially be used for early diagnosis of neoplasia and identifying susceptible dogs.

Effects of two types of energy restriction on methylation levels of adiponectin receptor 1 and leptin receptor overlapping transcript in a mouse mammary tumour virus-transforming growth factor-α breast cancer mouse model.

The role of adiponectin and leptin signalling pathways has been suggested to play important roles in the protective effects of energy restriction (ER) on mammary tumour (MT) development. To study the effects of ER on the methylation levels in adiponectin receptor 1 (AdipoR1) and leptin receptor overlapping transcript (Leprot) genes using the pyrosequencing method in mammary fat pad tissue, mouse mammary tumour virus-transforming growth factor-α (MMTV-TGF-α) female mice were randomly assigned to ad libitum (AL), chronic ER (CER, 15 % ER) or intermittent ER (3weeks AL and 1 week 60 % ER in cyclic periods) groups at 10weeks of age until 82weeks of age. The methylation levels of AdipoR1 in the CER group were higher than those in the AL group at week 49/50 (P < 0·05), while the levels of methylation for AdipoR1 and Leprot genes were similar among the other groups. Also, the methylation levels at CpG2 and CpG3 regions of the promoter region of the AdipoR1 gene in the CER group were three times higher (P < 0·05), while CpG1 island of Leprot methylation was significantly lower compared with the other groups (P < 0·05). Adiponectin and leptin gene expression levels were consistent with the methylation levels. We also observed a change with ageing in methylation levels of these genes. These results indicate that different types of ER modify methylation levels of AdipoR1 and Leprot in different ways and CER had a more significant effect on methylation levels of both genes. Epigenetic regulation of these genes may play important roles in the preventive effects of ER against MT development and ageing processes.

Arsenite and cadmium promote the development of mammary tumors.

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.

Estrogen receptors genotypes and canine mammary neoplasia

BackgroundEstrogens are essential for the development and proper function of several hormone-dependent organs. There are, however, several lines of evidence associating estrogens with mammary carcinogenesis. A marked individual genetic variability concerning estrogens biosynthesis, metabolism and mechanism of action was recognized and associated with human breast cancer susceptibility, clinical features and progression. Although some genetic variations in canine ESR1 gene were reported, their influence in clinicopathological features and progression of canine mammary tumors has not been fully evaluated. This study aims to assess the influence of SNPs in ESR1 gene (rs397512133, rs397510462, rs851327560, rs397510612, rs852887655, rs852684753 and rs852398698) in canine mammary tumors characteristics and progression. A group of 155 non-neutered bitches with mammary tumors was included in the study. Follow-up information was assessed 24 months after surgery.ResultsGenetic profiles associated with a later onset of mammary tumors and less aggressive clinicopathological features, namely smaller tumor size (≤ 3 cm) with extensive tubular differentiation and low canine-adapted prognostic index (vet-NPI), were identified in this study.ConclusionsOur data suggest that the ESR1 genetic profile may help on the decision regarding the selection of individual tailored preventive measures against canine mammary tumors development, such as early neutering.

Evaluation of deslorelin implant on subsequent mammary tumors of rats (Rattus norvegicus)

ABSTRACT Background Mammary fibroadenomas are one of the most common tumors of female companion rats (Rattus norvegicus forma domestica). The objectives of this study were to determine if subcutaneous administration of a deslorelin implant following excision of fibroadenomas can prevent or delay development of additional mammary tumors and increase survival in companion rats. Methods Female intact client-owned rats with benign mammary tumors were divided into three groups: no implant (n = 10), placebo implant (n = 10), or 4.7 mg deslorelin implant (n = 10) placed within 2 months of tumor excision. Rats were monitored for subsequent mammary tumors for 10 months following treatment. Expression of estrogen α, progesterone, prolactin, and androgen receptors stained by immunohistochemistry in primary masses of rats included in the deslorelin-treated group was evaluated using the Allred scoring system. Results In the control non-implanted group, four of the 10 rats developed another mammary tumor, including one anaplastic carcinoma. In the control placebo group, five of the 10 rats developed another mammary tumor, including one ductal carcinoma. In the deslorelin-treated group, three of 10 rats developed another mammary tumor, including one adenocarcinoma. Median time between surgery and new mass detection did not differ significantly among groups (55 days in the deslorelin group vs. 77 days in the control placebo group, P = 0.25). Median survival times after surgery did not differ significantly among groups. No correlation was noted between receptor expression and response to treatment with deslorelin. Conclusions and clinical relevance Deslorelin implants placed within 2 months of benign mammary tumor surgical excision were not associated with a decreased risk of developing subsequent mammary tumors, nor with an increased survival in female rats. Further studies are needed to define useful adjunct therapy to surgery.

Ivermectin inhibits canine mammary tumor growth by regulating cell cycle progression and WNT signaling

BackgroundMammary gland tumor is the most common spontaneous tumor in intact female dogs, and its poor prognosis remains a clinical challenge. Ivermectin, a well-known anti-parasitic agent, has been implicated as a potential anticancer agent in various types of human cancer. However, there are no reports evaluating the antitumor effects of ivermectin in canine mammary tumor. Here, we investigated whether ivermectin was able to inhibit canine mammary tumor development and explored the related mechanisms.ResultsIvermectin inhibited the growth of canine mammary tumor cell lines in a dose- and time-dependent manner. The antitumor effects induced by ivermectin were associated with cell cycle arrest at G1 phase via down-regulation of CDK4 and cyclin D1 expression, with no significant induction of apoptosis. Furthermore, significantly reduced β-catenin nuclear translocation was observed after treatment with ivermectin, resulting in the inactivation of WNT signaling. Consistent with the results in vitro, a significant suppression of tumor growth by ivermectin was observed in canine mammary tumor xenografts.ConclusionIvermectin, as a promising anti-cancer agent, inhibits the growth of canine mammary tumor by regulating cell cycle progression and WNT signaling.

Rodent Models Assessing Mammary Tumor Prevention by Soy or Soy Isoflavones.

While epidemiological studies performed in Asian countries generally show that high levels of dietary soy are associated with reduced breast cancer risk, studies in Western countries have typically failed to show this correlation. In an attempt to model the preventative actions of soy on mammary tumor development, rodent models have been employed. Thirty-four studies were identified that evaluated the impact of soy products or purified soy isoflavones on mammary tumor initiation (studies evaluating established mammary tumors or mammary tumor cell lines were not included) and these studies were separated into mammary tumors induced by chemical carcinogens or transgenic expression of oncogenes based on the timing of soy administration. Regardless of when soy-based diets or purified isoflavones were administered, no consistent protective effects were observed in either carcinogen-induced or oncogene-induced mammary tumors. While some studies demonstrated that soy or purified isoflavones could reduce mammary tumor incidence, other studies showed either no effect or tumor promoting effects of soy products or isoflavones. Most importantly, only five studies found a decrease in mammary tumor incidence and six studies observed a decrease in tumor multiplicity, two relevant measures of the tumor preventative effects of soy or isoflavones. The variable outcomes of the studies examined were not completely surprising given that few studies employed the same experimental design. Future studies should be carefully designed to more accurately emulate soy consumption observed in Asian cultures including lifetime exposure to less refined soy products and potentially the incorporation of multigenerational feeding studies.

Co-administering Melatonin With an Estradiol-Progesterone Menopausal Hormone Therapy Represses Mammary Cancer Development in a Mouse Model of HER2-Positive Breast Cancer.

Melatonin has numerous anti-cancer properties reported to influence cancer initiation, promotion, and metastasis. With the need for effective hormone therapies (HT) to treat menopausal symptoms without increasing breast cancer risk, co-administration of nocturnal melatonin with a natural, low-dose HT was evaluated in mice that develop primary and metastatic mammary cancer. Individually, melatonin (MEL) and estradiol-progesterone therapy (EPT) did not significantly affect mammary cancer development through age 14 months, but, when combined, the melatonin-estradiol-progesterone therapy (MEPT) significantly repressed tumor formation. This repression was due to effects on tumor incidence, but not latency. These results demonstrate that melatonin and the HT cooperate to decrease the mammary cancer risk. Melatonin and EPT also cooperate to alter the balance of the progesterone receptor (PR) isoforms by significantly increasing PRA protein expression only in MEPT mammary glands. Melatonin significantly suppressed amphiregulin transcripts in MEL and MEPT mammary glands, suggesting that amphiregulin together with the higher PRA:PRB balance and other factors may contribute to reducing cancer development in MEPT mice. Melatonin supplementation influenced mammary morphology by increasing tertiary branching in the mouse mammary glands and differentiation in human mammary epithelial cell cultures. Uterine weight in the luteal phase was elevated after long-term exposure to EPT, but not to MEPT, indicating that melatonin supplementation may reduce estrogen-induced uterine stimulation. Melatonin supplementation significantly decreased the incidence of grossly-detected lung metastases in MEL mice, suggesting that melatonin delays the formation of metastatic lesions and/or decreases aggressiveness in this model of HER2+ breast cancer. Mammary tumor development was similar in EPT and MEPT mice until age 8.6 months, but after 8.6 months, only MEPT continued to suppress cancer development. These data suggest that melatonin supplementation has a negligible effect in young MEPT mice, but is required in older mice to inhibit tumor formation. Since melatonin binding was significantly decreased in older mammary glands, irrespective of treatment, melatonin supplementation may overcome reduced melatonin responsiveness in the aged MEPT mice. Since melatonin levels are known to decline near menopause, nocturnal melatonin supplementation may also be needed in aging women to cooperate with HT to decrease breast cancer risk.

Abstract 5066: Chemoprevention of breast cancer by targeting glucose metabolism with HJC0152

Abstract Most mammalian cells use glucose as the primary fuel source, which is metabolized via glycolysis to pyruvate and further transferred into mitochondria for generating ATP through the Krebs cycle under normal condition. However, metabolism is characteristically reprogrammed in cancer cells or highly proliferative cells with a preferential ATP production through generating lactate by lactate dehydrogenase (LDH/LDHA), referred to as the Warburg effect or metabolic reprogramming toward anaerobic glycolysis. Efficient control of energy metabolism is the key to maintaining metabolic homeostasis, while disturbance in energy balance provokes diseases such as obesity, diabetes and cancer. However, the mechanisms underlying efficient energy metabolic homeostasis and breast cancer development are poorly understood. The transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) is activated downstream of many cytokines and growth factor receptors. STAT3-targeting genes and their functions vary depending on the cellular context, primarily in correlation with cell survival and proliferation. Recently, reports show that active STAT3 with phosphorylation at Y705 residue plays an important role in regulating energy metabolism via transcriptional induction of its well-recognized transcriptional target HIF-1α. HJC0152, a novel small-molecule glucose metabolism modulator, was proprietarily developed using a combination approach of structure-based drug design strategies and molecular modeling techniques in our attempt to develop orally bioavailable non-peptide STAT3 inhibitors for anticancer use. A panel of mammary epithelial cells and breast cancer cells treated with HJC0152 exhibited suppressed cell growth and induced apoptosis in vitro. Intriguingly, HJC0152 reduces glucose uptake and Glut1 protein level. In addition, HJC0152 treated BC cell line showed decreased protein level of glycolytic enzymes including HK2, PFKL, ALDOA, PDHK, PKM2, LDHA and important metabolism regulator HIF-1α in a time-dependent manner. Furthermore, HJC0152 has significant in vivo efficacy in reducing/preventing mammary tumor development in transgenic mouse models of estrogen receptor (ER)-negative breast cancer. These results provide a rationale to develop HJC0152 as a promising drug candidate and preventive therapy for breast cancer and other cancers with aberrant glucose metabolism. In addition, HJC0152 can serve as a molecular probing tool for elucidating the key factors driving the development of breast cancer in the context of metabolic dysregulation and diseases. This work was supported by grant R01CA226001 from the NIH/NCI. Citation Format: Hyejin Kim, Jiabin Dong, Lili Wang, Jimin Xu, Dan Zhang, Ruping Yan, Hao Zou, Haiying Chen, Xi Liu, Yun Zhu, Yu Xue, Jia Zhou, Qiang Shen. Chemoprevention of breast cancer by targeting glucose metabolism with HJC0152 [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 5066.

Abstract 2361: Progesterone receptor promotes immunosuppressive microenvironment in the mammary gland

Abstract Why are some early neoplastic lesions identified through immunosurveillance and subsequently destroyed, whereas other tumors evade this clearance step and progress to clinically-relevant tumors? The cancer immunoediting hypothesis highlights that the innate and adaptive immune responses work together to “flag” early neoplastic lesions for immune-mediated elimination. An early mediator of this “elimination” process is activation of type I interferon signaling. Thus, suppression of type I interferon signaling may help developing tumors evade the critical early steps of immune recognition and clearance. Preliminary data from our lab suggest that progesterone, working through the progesterone receptor (PR), may be a key player in tumor immune evasion through downregulation of interferon signaling. In breast cancer cells, we have shown that presence/activation of PR can attenuate interferon-signaling at multiple steps within the signaling cascade: decreased STAT1 phosphorylation, decreased STAT1/STAT2/IRF9 transcriptional complex formation (called ISGF3, required for interferon-alpha signaling), decreased recruitment of ISGF3 to ISG promoter sequences, and decreased transcriptional activation of interferon-stimulated genes (ISG, end point of interferon signaling). These data suggest a concerted effort aimed at reducing interferon-signaling through activation of PR. These data translate to the immune microenvironment in the murine mammary gland. In mice treated with progesterone for 21 days, we see immunosuppressive changes in the mammary gland: increased T-regulatory cells (immunosuppressive), decreased frequency of antigen-presenting cells (decreased ability to present potential tumor-associated antigens to CD8 T-cells), and a decrease in MHC class I presentation in non-lymphoid cells (decreased ability for epithelial cells to present potential tumor-associated antigens). We observe similar phenotypes in mice with transgenic overexpression of PR. These changes in the immune system suggest that PR can promote immunosuppressive changes in the mammary gland. Moreover, 80% of these mice in a multiparous cohort develop mammary gland tumors. Therefore, early changes in the immune microenvironment in the mammary gland may translate to the development of mammary gland tumors in PR transgenic mice. Cumulatively, these data suggest a mechanism through which PR aids early breast cancer lesions in escaping immune surveillance. These data have significant implications for the use of progesterone-containing hormone replacement therapy, as well as underscore the importance of studying anti-progestins as novel chemo-preventative agents for breast cancer. Citation Format: Merit Goodman, Gloria Trinca, Katherine Walter, Katelin Gibson, Prabhakar Chalise, Mary Markiewicz, Christy R. Hagan. Progesterone receptor promotes immunosuppressive microenvironment in the mammary gland [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 2361.

Abstract 1922: MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells

Abstract Adenoid Cystic Cancer (ACC) is the most aggressive salivary gland tumor with no effective systemic therapy. MYB/MYBL1 fusion event underlies the etiology of ACC with few recurrent variants focused on NOTCH and chromatin remodeling pathways and low overall mutational tumor burden. No chemotherapy or targeted drugs have been approved for ACC. Therefore, there is an urgent need to develop novel therapeutic strategies. To study MYB biology and test new therapeutic agents we have developed and characterized the phenotype of a new murine conditional MYB-NFIB transgene model and have also generated a mammary tumor cell line expressing the human MYB fusion transgene with ACC-like features. We have also generated new human MYB-NFIB positive ACC tumor cell lines and xenografts from patient samples and have tested these tumor cells with newly developed MYB mimic peptides. We analyzed the phenotype of mice carrying conditional MMTV-CRE MYB-NFIB transgene alone or after crossing to heterozygosity with p16/ARF null alleles. We observed shortened survival in two different mouse transgene strains due to development of B-cell leukemia manifested by elevated WBC, low platelets, and marked splenomegaly. We also noted sporadic solid tumors including the development of a murine mammary tumor which generated a MYB-NFIB positive tumor cell line for testing new therapies. In addition, we have generated patient derived xenografts (PDX) and the successful generation of a human ACC tumor cell line from fresh ACC patient biopsies. Using shRNA reagents we observed reduced tumor cell growth and viability assayed by MTS assay following Notch1 and MYB knockdown. We also tested four candidate MYB inhibitors, celastrol, all-trans retinoic acid, and the peptidomimetic inhibitors, MYBMIM and CRYBMIM, against murine and human ACC tumor samples with immortalized normal cells as controls and will discuss these results. Citation Format: Yue Jiang, Lauren Forbes, Jianping Li, Shelby Freeberg, Chunxia Cao, Maria Zajac-Kaye, Jonathan Licht, Alex Kentsis, Frederic Kaye. MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells [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 1922.