Gland Tumors In Mice Research Articles

Abstract P2-20-11: Progesterone impacts the growth and immune cell infiltration of murine mammary gland tumors

Abstract Clinical studies have linked usage of progestins (synthetic progesterone) to breast cancer risk. However, little is understood regarding the role of native progesterone (P4), signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we demonstrated that P4 treatment or PR overexpression can drive changes in immune cell populations in the murine mammary gland and that PR overexpression leads to increased development of mammary gland tumors in mice. Given these findings, we sought to investigate whether P4 impacts tumor growth and immune cell infiltration of mammary gland tumors. We utilized syngeneic mammary gland tumor models to evaluate the effect of P4 on PR+ mammary gland tumor growth, which revealed that P4 promoted tumor growth and impacted immune cell infiltration of PR+ mammary gland tumors. Of note, numbers of tumor-infiltrating dendritic cells were decreased and exhausted T cells and regulatory T cells were increased with P4 treatment in PR+ tumors. To determine if anti-progestin therapies could reverse the growth-promoting effect of P4 on mammary gland tumors, mice bearing PR+ mammary gland tumors were treated with the anti-progestin onapristone. Onapristone treatment led to significantly decreased tumor volumes in two syngeneic mammary gland tumor models and reversed the effect that P4 had on tumor-infiltrating regulatory T cells. To determine if inhibition of tumor growth by onapristone was immunologically mediated, SCID mice bearing PR+ mammary gland tumors were treated with onapristone. Results revealed a decreased ability of onapristone to inhibit tumor growth in SCID mice compared to immunocompetent mice, suggesting that inhibition of tumor growth is, in part, immunologically mediated. These findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of anti-progestin therapies to promote immune-mediated elimination of mammary gland tumors. Citation Format: Lauryn R Werner, Dominika E. Helm, Julio Tinoco, Eilidh I Chowanec, Sean M Holloran, Richard C Hastings, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Mary A Markiewicz, Prabhakar Chalise, Justin Balko, Zachary C Hartman, Christy R Hagan. Progesterone impacts the growth and immune cell infiltration of murine mammary gland tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-11.

Inhibition of the invasion and metastasis of mammary carcinoma cells by NBD peptide targeting S100A4 via the suppression of the Sp1/MMP‑14 axis.

A synthetic peptide that blocks the interaction between the metastasis-enhancing calcium-binding protein, S100A4, and its effector protein, methionine aminopeptidase 2 (MetAP2) (the NBD peptide), was previously demonstrated to inhibit the angiogenesis of endothelial cells, leading to the regression of human prostate cancer in a xenograft model. However, the effects of the NBD peptide on the malignant properties of cancer cells that express S100A4 remain to be elucidated. The present study demonstrates that the NBD peptide inhibits the invasiveness and metastasis of highly metastatic human mammary carcinoma cells. The introduction of the peptide into MDA-MB-231 variant cells resulted in the suppression of matrix degradation in a gelatin invadopodia assay and invasiveness in a Matrigel invasion assay. In line with these results, the peptide significantly downregulated the expression of matrix metalloproteinase (MMP)-14 (MT1-MMP). Mechanistic analysis of the downregulation of MMP-14 revealed the suppression of the expression of the transcription factor, specificity protein 1 (Sp1), but not that of nuclear factor (NF)-κB, early growth response 1 (EGR1) or ELK3, all of which were reported to be involved in transcriptional regulation of the MMP-14 gene. At the same time, evidence suggested that the NBD peptide also suppressed Sp1 and MMP-14 expression levels in MDA-MB-468 cells. Importantly, the intravenous administration of the NBD peptide encapsulated in liposomes inhibited pulmonary metastasis from mammary gland tumors in mice with xeno-graft tumors. These results indicate that the NBD peptide can suppress malignant tumor growth through the suppression of the Sp1/MMP-14 axis. Taken together, these results reveal that the NBD peptide acts on not only endothelial cells, but also on tumor cells in an integrated manner, suggesting that the peptide may prove to be a promising cancer therapeutic peptide drug.

Prevention of DMBA-induced mammary gland tumors in mice by a dual-function inhibitor of JAK3 and EGF receptor tyrosine kinases

ABSTRACTObjectives: We tested the chemopreventive effect of WHI-P131 in side by side evaluation with the standard anti-breast cancer drug paclitaxel in the well-established 7,12‐dimethylbenz(a)anthracene (DMBA)-induced breast cancer model.Methods: One hundred BALB/cmice were divided into five groups. (i) Control (ii) DMBA (iii) DMBA+ Paclitaxel (10 mg/kg) (iv) DMBA+WHI-P131 (Janex1, 50 mg/kg of BW, i.p, three times per week) (“J”) (v) DMBA+P+J. The duration of study was 25 weeks.Results: Our findings demonstrate that WHI-P131 impedes DMBA-induced carcinogenesis, reduces size, weight, and load of tumors (P < 0.001) in DMBA-challenged mice and improves their survival outcome (P < 0.01). The tumors developing despite WHI-P131 chemoprevention displayedattenuated levels of JAK3, STAT3, and NF-κB as well as increased I-κB expression (P < 0.001). Notably, these tumors exhibited significantly decreased levels of phosphorylated AKT-PI3-Kinase pathway signaling proteins p-mTOR, p-p70S6K1, and p-4E-BP1 (P < 0.001). Our findings are consistent with a model in which DMBA-induced malignant clones with low-level expression of the six signature proteins JAK3/STAT3/NF-κB/p-mTOR, p-p70S6K1/p-4E-BP1, albeit not as aggressive as their JAK3/STAT3/NF-κB overexpressing counterparts are capable of escaping chemo-preventive effects of WHI-P131.Conclusion: These insights may provide the foundation for new chemo-preventive strategies in which WHI-P131 is applied to prevent the development of aggressive forms of breast cancer.

Abstract GS6-06: Identifying metastatic drivers in patient-derived xenograft models of triple negative breast cancer

Abstract Metastases are responsible for the vast majority of deaths due to breast cancer. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor prognosis. We are employing patient derived xenograft (PDX) models of TNBC to identify drivers of metastasis. Tumor samples are obtained from the breast tumors of patients with TNBC and engrafted immediately into the humanized mammary fat pads of immune compromised mice. Lentiviral transduction was employed to express bioluminescent and fluorescent markers in two independent PDX models of TNBC. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain, and lymph nodes. Dynamic and reversible epithelial to mesenchymal transition (EMT) was observed as tumors metastasized to lung and were re-passaged to recipient mouse mammary glands. Lung metastases were isolated using bioluminescence imaging and lung metastasis gene expression signatures were generated. Metastasis signatures from two independent PDX models were compared to identify genes that were commonly de-regulated in lung metastases relative to corresponding mammary tumors. Comprehensive gain-of-function screens were then conducted in vivo to identify functional drivers of TNBC metastasis. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver in this screen. CEACAM5 mRNA and protein levels were elevated in lung metastases relative to corresponding mammary gland tumors in mice. In addition, we demonstrated that CEACAM5 expression was upregulated in the lung metastases of breast cancer patients, and its expression inversely correlated with patient survival. Our data indicate that the metastatic function of CEACAM5 is to promote growth of breast tumors in the lung by inducing MET (mesenchymal to epithelial transition). Citation Format: Powell E, Shao J, Picon HM, Ge Z, Echeverria GV, Peoples M, Bristow C, Cai S, Tu Y, McCoy AM, Piwnica-Worms D, Draetta G, Edwards JR, Moulder SL, Symmans WF, Heffernan TP, Liang H, Piwnica-Worms H. Identifying metastatic drivers in patient-derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS6-06.

Dietary exposure of acrylamide from the fifth Chinese Total Diet Study

The levels of acrylamide in 240 food composite samples from the 5th Chinese Total Diet Study (TDS) were measured using an LC-MS/MS method and the exposure estimates for the general population were evaluated. The samples were collected from 20 provinces in China, covering about two thirds of the Chinese population. Acrylamide was detected in 40.0% of composite samples with the concentrations ranged from 0.8 to 211.8 μg/kg. The average dietary intakes from the 5th Chinese TDS were 0.319 μg kg−1 bw day−1 and an increase with about 70% was observed from the 3rd Chinese TDS in 2000 to the 5th Chinese TDS between 2009 and 2012. The main food group contributors to acrylamide exposure were vegetables (35.2%), cereals (34.3%) and potatoes (15.7%). Based on the benchmark dose lower confidence limit at 10% risk (BMDL10) of 0.31 mg kg−1 bw day−1 for the induction of mammary tumors in rats and 0.18 mg kg−1 bw day−1 for Harderian gland tumors in mice, the margins of exposure (MOEs) were 973 and 565 for Chinese general population, respectively. These MOEs indicate a human health concern.

Combination of Ononis hirta and Bifidobacterium longum decreases syngeneic mouse mammary tumor burden and enhances immune response

The resistance of solid tumors to conventional therapies has prompted the need for alternative therapies. To evaluate in vitro and in vivo effect of extracts from Ononis hirta against resistant mouse mammary gland cell line (66 cl-4-GFP) and to use a combination of Ononis hirta extract with Bifidobacterium longum to target resistant solid tumors in mice. Different solvent extracts of Ononis hirta were prepared and their in vitro antiproliferative activity was tested against 66 cl-4-GFP cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used to identify the active extracts. Balb/C mice were transplanted with 66 cl-4-GFP cell line and in vivo antitumor activity was assessed for the plant extract, Bifidobacterium longum, and a combination of plant extract and Bifidobacterium longum. Histological examination of tumors was performed using standard hematoxylin/eosin staining protocol while gram stain was used to detect the presence of anaerobic bacteria in these sections. A combination of Ononis hirta methanol extract and Bifidobacterium longum showed high ability in targeting solid mammary gland tumors in mice. It also induced extensive necrosis in these tumors. Thirty percent of mice treated with such combination were cured of their cancers. The mechanism underlying this anticancer activity involves immune system activation exemplified by the observed rejection of reinoculated tumors by cured mice. Chemical TLC analysis of the active methanol extract showed the presence of flavonoids, terpenoids, and alkaloids. HPLC analysis confirmed the presence of flavonoids and alkaloids in Ononis hirta methanol extract. The complete regression of the tumor is encouraging and shows that plant extracts in combination with Bifidobacterium longum is an inviting option to treat solid tumors.

Non-targeted effects and the dose response for heavy ion tumor induction

Non-targeted effects (NTE), including bystander effects in neighbor cells of cells directly hit by radiation tracks and genomic instability in the progeny of irradiated cells, challenge traditional radiation protection paradigms on Earth. It is thus of interest to understand how NTE could impact our understanding of cancer risks from galactic cosmic rays (GCR), which are comprised of high-energy protons and heavy ions. The most comprehensive data set for tumor induction by heavy ions is the induction of Harderian gland tumors in mice by high-energy protons, helium, neon, iron and niobium ions after doses of 0.05 to several Gy. We report on an analysis of these data that compares a dose response model motivated by the conventional targeted effects (TE) model to one which includes a dose response term descriptive of non-targeted effects (NTE) in cell culture. Results show that a NTE model provides an improved fit to the Harderian gland data over the TE model. Relative biological effectiveness (RBE) factors are shown to have much larger values at low doses based on a NTE model than the maximum RBE estimates based on estimates of the ratio of initial linear slopes of heavy ions compared to γ-rays in the TE model. Our analysis provides important in vivo support for the deviation from linear dose responses at low doses for high LET radiation, which are best explained by a NTE model.

Essential Roles of mTOR/Akt Pathway in Aurora-A Cell Transformation

We have recently demonstrated that Aurora-A kinase is a potential oncogene to develop mammary gland tumors in mice, when expressed under MMTV promoter. These tumors contain phosphorylated forms of Akt and mTOR, suggesting that Akt-mTOR pathway is involved in transformed phenotype induced by Aurora-A. In the present studies, we discovered that stable cell lines expressing Aurora-A contain phosphorylation of Akt Ser473 after prolonged passages of cell culture, not in cells of the early period of cell culture. Levels of PTEN tumor suppressor are significantly reduced in these late passage cells at least in part due to increased poly ubiquitination of the protein. Akt-activated Aurora-A cells formed larger colonies in soft agar and are resistant to UV-induced apoptosis. Aurora-A inhibitor, VX-680, can cause cell death of Aurora-A cells in which Akt is not activated. siRNA-mediated depletion of mTOR in those cells resulted in decreased phosphorylation of Akt Ser473, suggesting that TORC2 complex phosphorylates Akt in Aurora-A cells. Treatment of late-passage Aurora-A cells with mTOR inhibitor reduced colony formation in soft agar. These results strongly suggest that commitment of cell transformation by Aurora-A is determined by at least co-activation of Akt/mTOR pathway.

Tissue-specific tumor suppressor activity of retinoblastoma gene homologs p107 and p130.

The retinoblastoma gene family consists of three genes: RB, p107, and p130. While loss of pRB causes retinoblastoma in humans and pituitary gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107 and p130. To test this hypothesis, we have generated chimeric mice from embryonic stem cells carrying compound loss-of-function mutations in the Rb gene family. We found that Rb/p107- and Rb/p130-deficient mice were highly cancer prone. We conclude that in a variety of tissues tumor development by loss of pRB is suppressed by its homologs p107 and p130. The redundancy of the retinoblastoma proteins in vivo is reflected by the behavior of Rb-family-defective mouse embryonic fibroblasts in vitro.

Mammary tumors in mice conditionally mutant for Brca1 exhibit gross genomic instability and centrosome amplification yet display a recurring distribution of genomic imbalances that is similar to human breast cancer.

BRCA1 mutation carriers have an increased susceptibility to breast and ovarian cancer. Excision of exon 11 of Brca1 in the mouse, using a conditional knockout (Cre-loxP) approach, results in mammary tumor formation after long latency. To characterize the genomic instability observed in these tumors, to establish a comparative map of chromosomal imbalances and to contribute to the validation of this mouse model of breast cancer, we have characterized chromosomal imbalances and aberrations using comparative genomic hybridization (CGH), and spectral karyotyping (SKY). We found that all tumors exhibit chromosome instability as evidenced by structural chromosomal aberrations and aneuploidy, yet they display a pattern of chromosomal gain and loss that is similar to the pattern in human breast carcinomas. Of note, nine of 15 tumors exhibited a gain of distal chromosome 11, a region that is orthologous to human chromosome 17q11-qter, the mapping position of Erbb2. However, our analysis suggests that genes distal to Erbb2 are the main targets of amplification. Four of the tumors also exhibited a copy number loss of proximal chromosome 11 (11A-B), a region orthologous to human 17p. In eight of the tumors we observed whole or partial gain of chromosome 15 centering on 15D2-D3 (orthologous to human chromosome 8q24), the map location of the c-Myc gene, and six of the tumors exhibited copy number loss of whole or partial chromosome 14, including 14D3, the map location of Rb1. We conclude that despite the tremendous shuffling of chromosomes during the course of mammalian evolution, the pattern of genomic imbalances is conserved between BRCA1-associated mammary gland tumors in mice and humans. Western blot analysis showed that while p53 is absent or mutated in some tumors, at least two tumors revealed wild-type protein, suggesting that other genetic events may lead to tumorigenesis. Similar to BRCA1-deficient mouse embryonic fibroblasts, the tumor cells contained supernumerary functional centrosomes with intact centrioles whose presence results in multipolar mitoses and aneuploidy.

High-LET radiation carcinogenesis

Recent results for neutron radiation-induced tumors are presented to illustrate the complexities of the dose-response curves for high-LET radiation. It is suggested that in order to derive an appropriate model for dose-response curves for the induction of tumors by high-LET radiation it is necessary to take into account dose distribution, cell killing and the susceptibility of the tissue under study. Preliminary results for the induction of Harderian gland tumors in mice exposed to various heavy ion beams are presented. The results suggest that the effectiveness of the heavy ion beams increases with increasing LET. The slopes of the dose-response curves for the different high-LET radiations decrease between 20 and 40 rads and therefore comparisons of the relative effectiveness should be made from data obtained at doses below about 20–30 rads.

Localization of Gene Action in the Causation of Lung and Mammary Gland Tumors in Mice&lt;xref ref-type="fn" rid="fn1"&gt;1&lt;/xref&gt;

Localization of Gene Action in the Causation of Lung and Mammary Gland Tumors in Mice&lt;xref ref-type="fn" rid="fn1"&gt;1&lt;/xref&gt;