MMTV-PyMT Mice Research Articles

Myeloid Cell–Derived TGFβ Signaling Regulates ECM Deposition in Mammary Carcinoma via Adenosine-Dependent Mechanisms

TGFβ plays a crucial role in the tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGFβ signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a protumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response, and angiogenesis. Here, using an MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGFβ signaling on myeloid cells (PyMT/TGFβRIILysM) affects extracellular matrix (ECM) formation in tumor tissue, specifically increasing collagen and decreasing fibronectin deposition. These changes were associated with mitigated tumor growth and reduced metastases. Reduced TGFβ signaling on fibroblasts was associated with their proximity to CD73+ myeloid cells in tumor tissue. Consistent with these findings, adenosine significantly downregulated TGFβ signaling on fibroblasts, an effect regulated by A2A and A2B adenosine receptors. METABRIC dataset analysis revealed that patients with triple-negative breast cancer and basal type harbored a similar signature of adenosine and ECM profiles; high expression of A2B adenosine receptors correlated with decreased expression of Col1 and was associated with poor outcome. Taken together, our studies reveal a new role for TGFβ signaling on myeloid cells in tumorigenesis. This discovered cross-talk between TGFβ/CD73 on myeloid cells and TGFβ signaling on fibroblasts can contribute to ECM remodeling and protumorigenic actions of cancer-associated fibroblasts. SIGNIFICANCE: TGFβ signaling on fibroblasts is decreased in breast cancer, correlates with poor prognosis, and appears to be driven by adenosine that accelerates tumor progression and metastasis via ECM remodeling.

Infiltrating Mast Cell-Mediated Stimulation of Estrogen Receptor Activity in Breast Cancer Cells Promotes the Luminal Phenotype.

Tumor growth and development is determined by both cancer cell-autonomous and microenvironmental mechanisms, including the contribution of infiltrating immune cells. Because the role of mast cells (MC) in this process is poorly characterized and even controversial, we investigated their part in breast cancer. Crossing C57BL/6 MMTV-PyMT mice, which spontaneously develop mammary carcinomas, with MC-deficient C57BL/6-KitW-sh/W-sh (Wsh) mice, showed that MCs promote tumor growth and prevent the development of basal CK5-positive areas in favor of a luminal gene program. When cocultured with breast cancer cells in vitro, MCs hindered activation of cMET, a master regulator of the basal program, and simultaneously promoted expression and activation of estrogen receptor (ESR1/ER) and its target genes (PGR, KRT8/CK8, BCL2), which are all luminal markers. Moreover, MCs reduced ERBB2/HER2 levels, whose inhibition further increased ESR1 expression. In vivo and in silico analysis of patients with breast cancer revealed a direct correlation between MC density and ESR1 expression. In mice engrafted with HER2-positive breast cancer tumors, coinjection of MCs increased tumor engraftment and outgrowth, supporting the link between MCs and increased risk of relapse in patients with breast cancer. Together, our findings support the notion that MCs influence the phenotype of breast cancer cells by stimulating a luminal phenotype and ultimately modifying the outcome of the disease. SIGNIFICANCE: Mast cells impact breast cancer outcome by directly affecting the phenotype of tumor cells through stimulation of the estrogen receptor pathway.

Abstract IA30: Intrinsic and extrinsic contributions of mitochondrial DNA to metastatic efficiency: A genetic explanation for disparities in metastasis efficiency?

Abstract Single-nucleotide polymorphisms (SNP) in nuclear and mitochondrial DNA are used to define clades (or races) in people, as well as different strains in mice (PMID: 27383787). Previous studies showed that nuclear SNP determine metastasis efficiency; we hypothesized that mtDNA SNP could also play roles in tumorigenicity and metastasis. Mitochondrial Nuclear Exchange (MNX) mice, created by transferring an oocyte nucleus from strainx into an enucleated oocyte from strainy (PMID: 27840835), show that mammary tumor formation and metastasis are regulated by inherited mitochondrial polymorphisms when MNX mice were bred to MMTV-PyMT or MMTV-HER2 mice (PMID: 26471915; 29070615). Since stromal compartments also possess changed mitochondria in MNX mice, we hypothesized that mitochondrial SNP in noncancer compartments could exert effects on tumor formation or metastasis in addition to genetic (cell autonomous) changes. Syngeneic tumor cells were injected into MNX mice with the same nuclear (and, therefore, same histocompatibility) background. Experimental metastasis was compared between wild-type and MNX mice (i.e., with same or different mtDNA backgrounds, respectively). E0771 mammary carcinoma and B16-F10 melanoma cells (both syngeneic to C57BL/6J), formed significantly (P<0.01) more lung metastases in C57BL/6J-mtMNX(C3H/HeN). K1735-M2 melanoma cells (syngeneic to C3H/HeN) formed significantly (P<0.05) fewer lung metastases in C3H/HeNmtMNX(C57BL/6J) mice. These results have been replicated ≥3 times using >10 mice per experiment. C57BL/6J mitochondria confer resistance to metastasis in both cell autonomous and non-cell autonomous experiments. Basal metabolic and ROS differences comparing mouse embryonic fibroblasts isolated from wild-type and MNX mice exist and may be responsible for the stromal effects. Likewise, significant and statistically significant differences in MNX mice for nuclear DNA methylation (PMID: 28663334) and epigenetic marks (J. McGuire & D.R. Welch, in preparation), immune (T.C. Beadnell & D.R. Welch, in preparation) and microbota (S.J. Manley & D.R. Welch, in preparation) profiles are observed. Together, our findings highlight the striking influences that mitochondrial haplotypes can exert on tumorigenicity and metastasis via both intrinsic and extrinsic mechanisms. These findings also suggest that mitochondrial SNP could serve, in part, as a genetic basis to explain racial disparities with regard to cancer aggressiveness. Support: Susan G. Komen for the Cure (SAC11037), Natl. Fndn. Cancer Res., CA168524, W81XWH-18-1-0450, GM103418. Citation Format: Danny R. Welch. Intrinsic and extrinsic contributions of mitochondrial DNA to metastatic efficiency: A genetic explanation for disparities in metastasis efficiency? [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr IA30.

Oroxylin A suppresses ACTN1 expression to inactivate cancer-associated fibroblasts and restrain breast cancer metastasis

Tumor initiation and progression are not only ascribed to the behavior of cancer cells, but also profoundly influenced by the tumor microenvironment. Inside, cancer-associated fibroblasts (CAFs) have become key factors to accelerate growth and metastasis for the abundance in most solid tumors. Our group previously reported that Oroxylin A (OA), a flavone from Scutellaria Baicalensis Georgi, possess the ability to suppress growth and invasion of several tumor cells. However, the regulatory effect of OA on stromal microenvironment is poorly understood. In this study, breast cancer-induced fibroblasts and primary breast CAFs from MMTV-PyMT mice were used to evaluate the influence of OA on the activation of fibroblasts. Results showed that OA could decrease the expression of α-SMA, fibronectin, vimentin and matrix metalloproteinases (MMPs). Thus, OA-deactivated CAFs did not further promote the proliferation and invasion in breast cancer cells. In vivo experiments, OA could also impede tumor metastasis through exhausting progressive CAFs. Mechanically, OA could specifically bind ACTN1 and significantly inhibit its expression to prevent CAF activation. As a consequence, OA could decrease the phosphorylation of FAK and STAT3, and reduce the secretion of CCL2 in CAFs. Altogether, OA could remodel stromal microenvironment and it is a potential therapeutic agent in breast cancer.

The study on the effect of depression on breast cancer incidence through IDO/kynurenine pathway.

e13569 Background: Psychosocial factors have been considered as the most common causes for women with breast cancer. However, it remains unclear whether mental stress can influence cancer incidence and progression. This study aims to investigate the relationship between them and the potential molecular mechanism. Methods: Depression model was established by restricting MMTV-PyMT mice in a 50ml-fixator for 4 h per day for 21 consecutive days. Behavioral tests including light-dark box test, open-field test and force swimming test were performed. Then blood, brain and mammary samples were collected at the indicated time points. ELISA was used to detect the concentration of neurotransmitters serotonin, epinephrine and norepinephrine and inflammatory factors IL-6, TNF-a and IL-1β in brain tissue and serum. Real-time quantitative qPCR and western blot were used to detect indoleamine 2, 3-dioxygenase (IDO) expression. The level of tryptophan was measured by high performance liquid chromatography(HPLC). Flow cytometry was used to evaluate the exhaustion of CD8+ T cells. Besides, the correlation between IDO expression detected by immunohistochemistry and the severity of mammary tumors was analyzed in the tumor specimens. Results: We observed that chronic stress caused depression-like behaviors, abnormal secretion of neurotransmitters serotonin, epinephrine and norepinephrine and promoted the development of breast cancer in MMTV-PyMT mice. Depression elevated the secretion of inflammatory factors IL-6, TNF-a and IL-1β in brain tissue and serum, which upregulated the expression of IDO and decreased the level of tryptophan in mice. Furthermore, T cells are sensitive to low tryptophan concentration, so elevated IDO expression suppressed and exhausted CD8+ T cells, ultimately promoting cancer incidence. Administration of xiaoyao pill (a well-known Chinese herbal formula for treating depression) or IDO inhibitor reversed the depression-like behaviors in mice and slowed the development of breast cancer. At the same time, the analysis of clinical samples showed that depression identified by Hamilton Depression Scale(HAMD) and IDO expression in mammary tumor tissues were positively correlated with the severity of mammary tumors. Conclusions: Altogether, our results suggest that depression can promote tumor immune privilege and cancer occurrence through inducing upregulated IDO expression.

SAT-588 Role of Adipose Tissue in Reproduction, Mammary Gland Development, Function and Cancer

To investigate the role of adipose tissue in the function of the mammary gland (MG) and reproductive system, we have examined lipodystrophic (LD) mice. LD mice of both sexes are sterile, but fertility was restored in both sexes with leptin injections. In addition, leptin was only needed for initial stages of pregnancy and not for parturition. A transplant of mouse embryonic fibroblasts (MEFs) led to the formation of an ectopic fat pad which also rescued the fertility in both sexes. However, pups born to rescued LD mothers died shortly after birth. We therefore examined the mammary glands of these mothers. MGs from LD mice were rudimentary and lacked terminal end buds. Leptin-injected and MEF rescued LD mice were able to become pregnant, showed normal pregnancy-associated glandular proliferation despite a smaller glandular area, were able to produce a small amount of milk that had grossly normal content of milk proteins and neutral lipids, but could not sustain pups to weaning. In order to separate the individual requirements for 1) adipokines such as leptin, 2) estradiol, and 3) epithelial-adipocyte interactions, we performed a series of experiments with both LD and ob (leptin-deficient) mice that received either estradiol or preadipocyte transplant. The resulting fat pad did not rescue the defect in MG development in LD mice. The defect also was not rescued with estradiol pellets. Ob/ob mice, like LD mice, lack leptin and estradiol, but retain adipose tissue. Ob mice have defective MG development. However, in striking contrast to LD mice, reconstitution of a WT fat pad in ob mice rescued the defect in MG development. Estradiol treatment did not rescue MG development in ob mice. Therefore direct interaction between mammary gland epithelia and adipocytes is a requirement for full invasion and expansion of the gland during puberty, but is not required for glandular proliferation during pregnancy and milk production.Given that excess adipose tissue is a risk factor for breast cancer we wanted to determine if breast cancer was affected by the absence of adipose tissue. LD mice were bred to MMTV-PyMT mice that develop spontaneous breast cancer. Remarkably, LD PyMT+ mice had accelerated growth of primary tumors compared to WT PyMT+ mice. Using our MEF transplant model future studies will be directed to understanding whether the accelerated breast cancer growth is due to loss of adipokines or altered epithelial-stromal interactions.

Press Pulse Therapy in MMTV‐PyMT Murine Breast Cancer

BackgroundTumors can be viewed as populations of cells which may succumb to eradication principles observed in nature. For example, population reduction or eradication occurs when populations are exposed to simultaneous press and pulse pressures. Press disturbances are pressures which produce chronic stress in populations, while pulse disturbances produce acute stress. We recently proposed a novel Press‐Pulse therapeutic strategy for the metabolic management of cancer that is based on these principles ( https://nutritionandmetabolism.biomedcentral.com/articles/10.1186/s12986‐017‐0178‐2). We hypothesize that specific press‐pulse protocols will be effective in managing even aggressive cancers, while allowing for the use of low dose and low‐ or non‐toxic metabolic‐targeted therapies.MethodsIn this project, we are testing the press disturbances of the ketogenic diet and ketone supplementation, and the pulse disturbances of 2‐deoxyglucose (glycolytic inhibitor), sodium phenylbutyrate (glutamine targeting agent), hyperbaric oxygen therapy, and pharmacological ascorbic acid, in the MMTV‐PyMT mouse model of breast cancer.Preliminary ResultsTo date, we have tested the proposed metabolic treatments as monotherapies and combination therapies. Tumor growth rate and survival time were largely unaltered by the treatments tested. Ketogenic diet and sodium phenylbutyrate were the only treatments to affect survival time as a monotherapy. Ketogenic diet treated mice lived 8.7% longer than standard diet fed mice (p<0.05). Sodium phenylbutyrate treated mice had a decrease in survival of 8.1% from the standard diet fed mice (p<.05). Data analyses pending for the effects of combination therapies.ConclusionsWe are not yet able to determine if Press‐Pulse targeting is effective in this model system as we have only tested and analyzed monotherapies. Ketogenic diet therapy appears to elicit a minor therapeutic effect as a stand‐alone, but the other metabolic‐targeted treatments utilized were not effective as monotherapies. Studies to test combinations of these therapies have been completed, and the analyses of the data is ongoing.Support or Funding InformationDonner Foundation, Florida High Tech Corridor

Functional Blockade of E-Selectin in Tumor-Associated Vessels Enhances Anti-Tumor Effect of Doxorubicin in Breast Cancer.

Chemotherapy is a mainstay of treatment for solid tumors. However, little is known about how therapy-induced immune cell infiltration may affect therapy response. We found substantial CD45+ immune cell density adjacent to E-selectin expressing inflamed vessels in doxorubicin (DOX)-treated residual human breast tumors. While CD45 level was significantly elevated in DOX-treated wildtype mice, it remained unchanged in DOX-treated tumors from E-selectin null mice. Similarly, intravenous administration of anti-E-selectin aptamer (ESTA) resulted in a significant reduction in CD45+ immune cell density in DOX-treated residual tumors, which coincided with a delay in tumor growth and lung metastasis in MMTV-pyMT mice. Additionally, both tumor infiltrating T-lymphocytes and tumor associated-macrophages were skewed towards TH2 in DOX-treated residual breast tumors; however, ESTA suppressed these changes. This study suggests that DOX treatment instigates de novo intratumoral infiltration of immune cells through E-selectin, and functional blockade of E-selectin may reduce residual tumor burden as well as metastasis through suppression of TH2 shift.

The functional activity of E-cadherin controls tumor cell metastasis at multiple steps

E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, many metastases continue to express E-cadherin, and a full EMT is not always necessary for metastasis; also, positive roles for E-cadherin expression in metastasis have been reported. We hypothesize instead that changes in the functional activity of E-cadherin expressed on tumor cells in response to environmental factors is an important determinant of the ability of the tumor cells to metastasize. We find that E-cadherin expression persists in metastatic lung nodules and circulating tumor cells (CTCs) in two mouse models of mammary cancer: genetically modified MMTV-PyMT mice and orthotopically grafted 4T1 tumor cells. Importantly, monoclonal antibodies that bind to and activate E-cadherin at the cell surface reduce lung metastasis from endogenous genetically driven tumors and from tumor cell grafts. E-cadherin activation inhibits metastasis at multiple stages, including the accumulation of CTCs from the primary tumor and the extravasation of tumor cells from the vasculature. These activating mAbs increase cell adhesion and reduce cell invasion and migration in both cell culture and three-dimensional spheroids grown from primary tumors. Moreover, activating mAbs increased the frequency of apoptotic cells without affecting proliferation. Although the growth of the primary tumors was unaffected by activating mAbs, CTCs and tumor cells in metastatic nodules exhibited increased apoptosis. Thus, the functional state of E-cadherin is an important determinant of metastatic potential beyond whether the gene is expressed.

Abstract A81: Identifying cellular immune components that correlate with response to immunotherapy in breast cancer using murine models

Abstract The heterogeneity of the tumor microenvironment may contribute to the lack of durable responses of immunotherapy in breast cancer. To understand factors that contribute to tumor immune cell heterogeneity, we report a detailed analysis and comparison of the immune tumor microenvironment of the autochthonous MMTV-PyMT murine breast cancer model resembling luminal B breast cancer and corresponding syngeneic models. We obtained tumors from MMTV-PyMT mice and used them to generate syngeneic models using either 1E6, 1E5, or 1E4 cells injected into the mammary fat pad of FVB/NJ wild-type mice. When tumors reached 100 mm3, tumors were harvested and quantitative flow-cytometry and NanoString analysis was performed. We have identified that the number of cells inoculated to generate syngeneic tumors significantly influences tumor latency, the tumor immune microenvironment, and the response to immune checkpoint blockade (ICB). Compared to the autochthonous model, the 1E6 and 1E5 model had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and the highest proportion of PD-L1-positive myeloid cells. We found that increased TILs and expression of PD-L1 on myeloid cells were the best predictors of response to PD-L1 or CTLA-4 therapy but that tumor cell expression of PD-L1 and T-cell expression of PD-1 did not correspond to beneficial outcome of treatment. Both the 1E6 and 1E5 models responded to PD-L1 and/or CTLA-4 ICB therapy, whereas the 1E4 and autochthonous models were resistant. These matched sensitive and resistant tumor models represent a unique opportunity to further interrogate the TME in breast cancer. Citation Format: Anita K. Mehta, Jessica A. Castrillion, Alaba Sotayo, Elizabeth A. Mittendorf, Anthony G. Letai, Jennifer L. Guerriero, Emily Cheney. Identifying cellular immune components that correlate with response to immunotherapy in breast cancer using murine models [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 A81.

Abstract P3-01-01: A dual role of natural killer cells in breast cancer metastasis

Abstract BACKGROUND: Metastatic disease drives breast cancer mortality. We recently discovered that leading cells at the invasive edge of mammary tumor organoids retain a conserved basal epithelial program defined by their expression of keratin-14 (K14), establishing K14 as a good marker of invasive breast cancer cells. K14-positive invasive cells also exhibit characteristics that make them targets of immunosurveillance by natural killer (NK) cells. While NK cells are key immune mediators in the control of metastasis, our understanding of the specific mechanisms behind this regulation and its eventual evasion by metastatic cells remains incomplete. METHODS: We have developed a novel preclinical 3D co-culture assay to discover mechanisms behind interactions between K14+ invasive breast cancer cells and NK cells. Combined with in vivo assays of metastasis, we are able to determine how NK cells limit the early stages of metastasis and also how tumor cells can influence key NK cell properties. RESULTS: In ex vivo co-culture assays of NK cells isolated from healthy mouse donors and mammary tumor organoids from MMTV-PyMT and C31T mouse models of breast cancer, we demonstrate that NK cells limit the early stages of metastasis, namely invasion and colony formation. Antibodies to invasive K14+ cells were able to enhance the ability of NK cells to limit colony formation, suggesting antibody-dependent cell mediated cytotoxicity. Surprisingly, when isolated from tumor bearing mice, NK cells did not limit invasion and instead promoted colony formation. The in vivo adoptive transfer of NK cells from healthy donors prevents the progression of early lung metastatic seeds to macrometastases, while the adoptive transfer of cells isolated from tumor bearing donors promotes macrometastatic development. This growth promoting phenotype can be neutralized with antibodies targeting inhibitory cell surface receptors. CONCLUSIONS: Our ex vivo and in vivo data demonstrate that healthy donor NK cells can limit metastasis through the directed cytotoxicity against pioneering K14+ invasive cells. However, prolonged exposure to tumors reprogram NK cells from tumor killing to tumor promoting, specifically in promoting the outgrowth of macrometastases. Further, we can neutralize this effect using NK cell specific inhibitory antibodies. This is the first time inhibitory signaling on NK cells have been linked with a growth promoting phenotype. These data can provide insight into when the use of NK cell directed therapies can be used to treat or prevent clinically relevant metastatic disease. Citation Format: Isaac S. Chan, Hildur Knútsdoóttir, Gayathri Ramakrishnan, Veena Padmanaban, Joel S. Bader, Elizabeth M. Jaffee, Andrew J. Ewald. A dual role of natural killer cells in breast cancer metastasis [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 P3-01-01.

Abstract P1-10-12: Menstrual cycling critically affects the Oncotype DX 21-gene signature: Implications for predictive biomarker assays in premenopausal women

Abstract Introduction: The 21-gene Oncotype DX Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early stage, hormone receptor (HR)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle can impact gene expression in these hormone-responsive cancers. However, the extent to which hormonal fluctuations affect Oncotype DX Recurrence Scores remains unclear. Aim: To determine the extent to which ovarian cycling affects the Oncotype DX 21-gene signature using paired premenopausal breast cancer samples and mouse models. Methods: To investigate menstrual variation in the Oncotype DX 21-gene signature within the same tumour, paired formalin-fixed paraffin-embedded, HR-positive invasive breast cancer samples were collected on different days of the menstrual cycle from women <50 years old (n=18), and compared to women >50 years old (n=11). Samples were collected an average of 18 days apart and were neoadjuvant therapy naive. To determine the effect of progesterone on gene expression, HR-positive T47D breast cancer cells were xenografted into the mammary fat pad of BALB/c nude mice, treated with exogenous estrogen±progesterone (n=12, 11 respectively). Additionally, HR-positive mammary tumours were collected from naturally cycling Mmtv-Pymt mice at the estrus or diestrus phase of the ovarian cycle (n=25, 28 respectively). The 21-gene Oncotype DX signature was assessed through quantitative RT-PCR and an experimental recurrence score (RS) was calculated using the Oncotype DX Recurrence Score algorithm. Results: Increased discordance in RS was observed between paired samples collected from younger women (3.2±2.5; mean±stdev), compared to older women (2.0±1.7; p=0.04). In young women, discordance was primarily driven by variable expression of proliferative genes, compared to older women, where discordances were a result of variable expression of invasive genes. Variable concentrations of progesterone at the time of tissue collection may influence proliferative gene expression and contribute to discordant RS. In support of this, in HR-positive xenograft tumours, expression of proliferative genes Ki67 (p=0.03) and STK15 (p=0.04), and Ki67 protein expression (p=0.02), were reduced following progesterone treatment. Furthermore, in naturally cycling Mmtv-Pymt mice, mammary tumours collected at diestrus, analogous to the luteal phase of the menstrual cycle in humans, show significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Her2, Grb7, Bag1; p≤0.05) and a significant increase in RS (21.1±2.4; mean±SEM) compared to tumours dissected at estrus (15.5±1.9; p=0.03). Clustering analysis revealed a subgroup of Mmtv-Pymt mammary tumours collected at diestrus characterised by increased expression of proliferative (p<0.001) and invasive (p=0.01) genes and a significant increase in RS (p=0.01). These tumours also exhibited higher expression of estrogen regulated genes (p=0.005) suggesting increased sensitivity to hormonal fluctuations during the ovarian cycle, and possibly greater variability in RS. Conclusion: Our results suggest that menstrual cycling affects the expression of genes included in the Oncotype DX 21-gene signature and influences experimental recurrence scores. Oncotype DX may be less effective for guiding chemotherapy treatment decisions for cycling premenopausal women compared to older postmenopausal women. Citation Format: Sarah M Bernhardt, Pallave Dasari, Danielle J Glynn, Lucy Woolford, Wendy Raymond, Lachlan M Moldenhauer, David Walsh, Amanda R Townsend, Timothy J Price, Wendy V Ingman. Menstrual cycling critically affects the Oncotype DX 21-gene signature: Implications for predictive biomarker assays in premenopausal women [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 P1-10-12.

Oncoprotein GT198 vaccination delays tumor growth in MMTV-PyMT mice.

Targeting early lesion in breast cancer is more therapeutically effective. We have previously identified an oncoprotein GT198 (PSMC3IP) in human breast cancer. Here we investigated GT198 in MMTV-PyMT mouse mammary gland tumors and found that GT198 is a shared early lesion in both species. Similar to human breast cancer even before a tumor appears, cytoplasmic GT198 is overexpressed in mouse tumor stroma including pericyte stem cells, descendent adipocytes, fibroblasts, and myoepithelial cells. Using recombinant GT198 protein as an antigen, we vaccinated MMTV-PyMT mice and found that the GT198 vaccine delayed mouse tumor growth and reduced lung metastasis. The antitumor effects were linearly correlated with vaccinated mouse serum titers of GT198 antibody, which recognized cell surface GT198 protein on viable tumor cells confirmed by FACS. Furthermore, GT198+ tumor cells isolated from MMTV-PyMT tumor induced faster tumor growths than GT198- cells when re-implanted into normal FVB/N mice. Together, this first study of GT198 vaccine in mouse showed its effectiveness in antitumor and anti-metastasis. The finding supports GT198 as a potential target in human immunotherapy since GT198 defect is shared in both human and mouse.

Splice switching an oncogenic ratio of SmgGDS isoforms as a strategy to diminish malignancy

The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.

Single-cell analysis reveals immune modulation and metabolic switch in tumor-draining lymph nodes

ABSTRACT Lymph-node metastasis is a prognosis factor for poor clinical outcome of breast cancer patients. Currently, how breast cancer cells establish pre-metastatic niche in the tumor-draining lymph nodes (TDLNs) is still unclear. To address this question, we isolated heterogeneous cells including immune and stromal cells from naive lymph nodes (LNs) of the FVB/NJ mice and TDLNs of the MMTV-PyMT mice. Single-cell RNA sequencing was performed to investigate the transcriptome of the cells and various bioinformatics analyses were used to identify the altered pathways. Our results revealed several significant changes between naïve LNs and TDLNs. First, according to immunologic signature and pathway analysis, CD4+ and CD8 + T cells showed upregulated angiogenesis pathway genes and higher regulatory T (Treg)-associated genes while they demonstrated downregulation of interferon response and inflammatory response gene signatures, concurrently suggesting an immunosuppressive microenvironment in the TDLNs. Second, profiling of B cells showed down-regulation of marginal zone B lymphocytes in the TDLNs, which was validated by flow cytometric analysis. Third, we found the enhancement of oxidative phosphorylation pathway in the fibroblastic reticular cells (FRCs) of the MMTV-PyMT mice and the elevation of related genes including Prdx3, Ndufa4 and Uqcrb, suggesting massive ATP consumption and TCA cycle metabolism in the FRCs. Collectively, our results reveal the reprogramming of TDLNs during breast cancer progression at single-cell level in a spontaneous breast cancer model and suggest the changes in immune modulation and metabolic switch are key alterations in the preparation of pre-metastatic niche by breast cancer cells.

Critical Adjuvant Influences on Preventive Anti-Metastasis Vaccine Using a Structural Epitope Derived from Membrane Type Protease PRSS14.

We tested how adjuvants effect in a cancer vaccine model using an epitope derived from an autoactivation loop of membrane-type protease serine protease 14 (PRSS14; loop metavaccine) in mouse mammary tumor virus (MMTV)-polyoma middle tumor-antigen (PyMT) system and in 2 other orthotopic mouse systems. Earlier, we reported that loop metavaccine effectively prevented progression and metastasis regardless of adjuvant types and TH types of hosts in tail-vein injection systems. However, the loop metavaccine with Freund's complete adjuvant (CFA) reduced cancer progression and metastasis while that with alum, to our surprise, were adversely affected in 3 tumor bearing mouse models. The amounts of loop peptide specific antibodies inversely correlated with tumor burden and metastasis, meanwhile both TH1 and TH2 isotypes were present regardless of host type and adjuvant. Tumor infiltrating myeloid cells such as eosinophil, monocyte, and neutrophil were asymmetrically distributed among 2 adjuvant groups with loop metavaccine. Systemic expression profiling using the lymph nodes of the differentially immunized MMTV-PyMT mouse revealed that adjuvant types, as well as loop metavaccine can change the immune signatures. Specifically, loop metavaccine itself induces TH2 and TH17 responses but reduces TH1 and Treg responses regardless of adjuvant type, whereas CFA but not alum increased follicular TH response. Among the myeloid signatures, eosinophil was most distinct between CFA and alum. Survival analysis of breast cancer patients showed that eosinophil chemokines can be useful prognostic factors in PRSS14 positive patients. Based on these observations, we concluded that multiple immune parameters are to be considered when applying a vaccine strategy to cancer patients.

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

Hepatic thrombopoietin gene silencing reduces platelet count and breast cancer progression in transgenic MMTV-PyMT mice

AbstractIn humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm3 combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

Stromal integrin α11 regulates PDGFR-β signaling and promotes breast cancer progression.

Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRβ+ CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11-deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRβ was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRβ expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using five CAF subpopulations (one murine, four human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, integrin α11 pro-invasive activity relies on its ability to interact with PDGFRβ in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a pro-invasive matricellular protein. Pharmacological inhibition of PDGFRβ and JNK impaired tumor cell invasion induced by integrin α11-positive CAFs. Collectively, our study uncovers an integrin α11-positive subset of pro-tumoral CAFs that exploits PDGFRβ/JNK signalling axis to promote tumor invasiveness in BC.

KCa3.1 Channels Confer Radioresistance to Breast Cancer Cells.

KCa3.1 K+ channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of KCa3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed KCa3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological KCa3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional KCa3.1 for therapy response. The KCa3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. KCa3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca2+ signals and DNA repair pathways. Consistently, KCa3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that KCa3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since KCa3.1 is druggable, KCa3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.