Mammary Tumor Growth Research Articles

Robust eIF4B levels undermine invasive growth and immune evasion mechanisms in murine triple negative breast cancer models.

Abstract Dysregulated protein synthesis is seen in many aggressive cancers, including metastatic breast cancer. However, the specific contributions of certain translation initiation factors to in vivo disease remain undefined. This is particularly true of eIF4B, an RNA-binding protein and cofactor of the RNA helicase eIF4A and associated eIF4F cap-binding complex. While eIF4A, eIF4G, and eIF4E are known to contribute to the progression of many cancer types, the role played by eIF4B in breast cancer remains relatively unclear. We therefore explored how naturally divergent and experimentally modulated eIF4B levels impact tumor growth and progression in murine triple negative breast cancer (TNBC) models. Surprisingly, we found that higher eIF4B levels in mouse and human breast cancers were associated with less aggressive phenotypes. shRNA-mediated eIF4B knockdown in TNBC lines failed to markedly alter proliferation and global translation in cells grown in vitro and only modestly hindered primary mammary tumor growth in mice. However, eIF4B knockdown significantly enhanced invasive growth in vitro and exacerbated both tumor burden and mortality relative to nontargeting shRNA controls in a model of metastatic disease. Deposited patient data reinforced a link between high eIF4B levels and better outcomes. Interestingly, in vitro and in vivo studies associated low eIF4B expression with more formidable immune evasion. These findings suggest that eIF4B levels impact metastatic disease in part through immunomodulatory mechanisms beyond the factor’s traditional role supporting eIF4/F directed translation. Future studies should explore the impact of these findings for personalizing treatment decisions and developing future therapies.

Abstract 5250: Effect of the β-blocker carvedilol in preventing DMBA-induced mammary gland tumor growth

Abstract According to global cancer statistics, breast cancer has now become the most diagnosed cancer and has become the fifth leading cause of cancer-related deaths worldwide. A promising method of retarding cancer-related mortality is cancer chemoprevention. However, there is no drug available that can be used routinely as a breast cancer-preventative agent. As carvedilol (CAR), an FDA-approved beta-adrenergic receptor blocker, reduces breast cancer mortality, we hypothesized that CAR might act as a breast cancer preventative agent. Three protocols were utilized to evaluate the ability of CAR to prevent 7,12-Dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. In protocol 1, two doses of CAR (2 and 10 mg/kg) were administered in drinking water as a 7-day pretreatment and throughout the study. In protocol 2, 10 mg/kg tamoxifen and 10 mg/kg carvedilol were provided as a 7-day pretreatment, and treatment was halted after six weeks. In protocol 3, CAR and the non-β-blocking enantiomer R-carvedilol (10 mg/kg) were given as a 7-day pretreatment and throughout the study. In all studies, animals were palpated weekly beginning at week 5 to detect the presence and location of mammary tumors. Tumors were measured using a caliper, and the experiments terminated at week 13. In protocol 1, only 10 mg/kg CAR was effective in delaying DMBA-mediated tumor occurrence (p = 0.0002); the first appearance of tumors and median tumor appearance in CAR-treated rats were separated from rats receiving DMBA alone by 2 and 4 weeks, respectively. However, tumors that did form grew at the same rate in all groups. In protocol 2, tamoxifen served as a control to prevent DMBA-mediated tumors; only one tamoxifen-treated rat developed a tumor at the end of the study. The tamoxifen-treated rats tumor formation was not statistically different than negative controls; however, the CAR group was not statistically different from the DMBA group. In protocol 3, CAR and R-CAR treatment showed delayed tumor formation and growth compared to the DMBA-only group, although not statistically significant. Combining all DMBA and 1-week pretreatment CAR data (n = 58 and 40, respectively), CAR effectively prevents DMBA-induced tumor incidence (p = 0.001), with a 3-week differential in the median tumor formation between the two groups. Therefore, continuous CAR treatment is essential to achieve the observed chemopreventive effects. As carvedilol is a clinically used beta-blocker, the results provide significant implications in breast cancer chemoprevention. Citation Format: Pabitra Kumar Sardar, Steven Yeung, Ayaz Shahid, Bradley T. Andresen, Ying Huang. Effect of the β-blocker carvedilol in preventing DMBA-induced mammary gland tumor growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5250.

Abstract 1284: The effect of Semaphorin 7A on breast cancer metastases and chemoresistance in young women

Abstract Our overall goal is to understand drivers of chemoresistance in triple-negative breast cancer (TNBC) and the mechanisms that contribute to metastatic spread in young women. TNBC is more frequently diagnosed in women aged <40 and confers worse overall and distant-metastatic-free survival. Our lab identified a ß1 integrin-ligand, Semaphorin 7A (SEMA7A), that is increased in TNBC and young women's breast cancer (BC). Our central hypothesis is that SEMA7A is increased in TNBC and signals via ß1-integrin to activate epithelial-to-mesenchymal plasticity (EMP) and chemoresistance. We have incorporated genetically manipulated TNBC cell lines and ex vivo tumor samples to define how SEMA7A expression impacts metastasis and chemoresistance. We have also incorporated our Young Women’s BC Cohort to analyze SEMA7A protein expression in human tumors and mammary epithelial tissue. In this cohort, we found that the normal mammary epithelium and tumors exhibit higher SEMA7A expression in women with postpartum BC, defined as BC diagnosed within 10 years postpartum, who are between 30&39-years-old. This sub-population also has increased tumor-associated lymphatic vessel density, as well as increased lymphovascular invasion and LN involvement. Using a 9-month-old mouse model, which is equivalent to a 35-year-old woman, we have shown that mammary tumor growth and metastasis are increased compared to young 8-week-old mice, which can be mimicked by SEMA7A OE tumors in the young mice; these results suggest that the aged microenvironment can replicate the tumor promotional effects of SEMA7A. Similarly, in orthotopic models, SEMA7A OE cells metastasize more frequently to lymph nodes (LNs) and lungs compared to controls. Additionally, via a tail vein injection, we observe that SEMA7A OE cells survive in circulation, seed the lung, and outgrow faster compared to controls. In vitro, SEMA7A promotes migration and invasion via ß1-integrin signaling. Finally, ex vivo metastatic tumor cells, when compared to parental cells, exhibit evidence of EMP. Namely, prior to dissemination, SEMA7A OE cells are mostly mesenchymal-like. Ex vivo, SEMA7A OE cells from LN metastasis express both epithelial/mesenchymal markers, suggesting a hybrid phenotype and maintenance of an invasive phenotype. Whereas those from the lung are more epithelial, which can be more proliferative. EMP is also known to promote chemoresistance and we have found that SEMA7A OE cells are resistant to caspase-mediated cell death in the presence of paclitaxel, a known chemotherapeutic utilized for TNBC, and have increased activation of pAkt, which can mediate pro-survival phenotypes. Overall, our results suggest that the aged mammary gland and SEMA7A expression can promote metastatic spread and chemoresistance and that these patient populations should be considered for direct and indirect targeting of SEMA7A by novel therapeutics. Citation Format: Kelsey Kines, Sarah Tarullo, Virginia Borges, Traci Lyons. The effect of Semaphorin 7A on breast cancer metastases and chemoresistance in young women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1284.

Abstract 622: Progesterone promotes immunosuppression in the mammary gland through regulatory T cell activity

Abstract Estrogen and progesterone signaling together drive hormone-receptor positive breast cancer growth and progression. However, there have been limited studies on the independent role that progesterone and its receptor, the progesterone receptor, play in breast cancer development. Studies in our lab have shown that progesterone signaling promotes mammary tumor growth and changes the immune landscape of the mammary gland towards immunosuppression. Regulatory T-cells (Tregs) are immune cells known to promote immunosuppression in the tumor microenvironment, in part through decreasing the activity of anti-tumor CD8+ T cells. The data from our lab also show that progesterone increases the number of Tregs in the normal mammary gland. To determine if progesterone signaling increases the number of Tregs in the tumor-bearing mammary gland, we used a syngeneic orthotopic murine tumor model, E0771 cells, engineered to express mouse PR or control empty vector. Mice were treated with progesterone or placebo for 7 days before implantation of the modified E0771 cells. Results from this study show that progesterone promoted tumor growth, as well as changed the immune landscape of the tumor. Flow cytometry analysis at tumor endpoint showed that progesterone receptor-positive tumors treated with progesterone harbored more Tregs than placebo treated mice. Additionally, initial studies into activity markers harbored by Tregs in progesterone-treated mice indicate increased Treg activity following progesterone treatment. As progesterone treatment promoted tumor growth and Treg recruitment in progesterone receptor-positive tumors, these data imply that progesterone treatment may promote the release of secreted factors impacting the immune cells in the mammary microenvironment. To determine if progesterone treatment results in cytokine or chemokine changes that may affect Treg recruitment, cytokine arrays and enzyme-linked immunosorbent assays (ELISAs) were performed using cell culture supernatant from murine mammary tumor cells expressing progesterone receptor (E0771-PR) treated with progesterone (0-72 hours). E0771-PR cells treated with progesterone showed higher levels of cytokines and chemokines that have been linked to Treg recruitment and differentiation, such as CCL5. Our findings demonstrate that progesterone signaling promotes an immune suppressed microenvironment in the mammary gland through an increase in Tregs. This evidence provides rationale to investigate the use of anti-progestins as a mechanism to overcome the immune-cold tumor microenvironment seen in progesterone receptor positive breast tumors. Citation Format: Eilidh I. Chowanec, Lauryn R. Werner, Christy Hagan. Progesterone promotes immunosuppression in the mammary gland through regulatory T cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 622.

Abstract 4237: Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis

Abstract Social isolation (SI), are associated with increased mortality from many diseases, including breast cancer. Up to 41% of breast cancer patients are estimated to feel socially isolated when assessed between 6 months and 2 years following their cancer diagnosis. Since socially isolated breast cancer survivors are at a 64% higher risk of breast cancer-specific mortality than socially integrated survivors, the biological mechanisms which mediate the effects of SI need to be identified to prevent increased mortality. In a preclinical model, we found that SI caused recurrence of mammary cancers which had been treated with hormone therapy and thus led cancer cells to emerge from dormancy. We further found that mammary glands of SI rats exhibited upregulation of IL6/JAK/STAT3 signaling pathway and impaired oxidative phosphorylation (OXPHOS), compared with group housed (GH) rats. We investigated if these changes could be mediated through the gut-brain-axis. Stress can affect the gut microbiota, and the gut microbiota is critical for regulating the host immunity. In four separate experiments, the effect of SI on the gut microbiota composition was assessed in C57BL/6 mice that were either group housed or housed singly in SI for 4 weeks. In each study replicate, several differences in the gut microbiota composition were seen, but these differences were mostly unique to each experiment. Beta-diversity was increased in three of the four experiments in SI mice. Nevertheless, at the genus level, SI suppressed the abundance of Akkermansia in all four replicates and increased Acetatifactor in three of the four replicates. These two bacterial changes are expected to disrupt OXPHOS, perhaps by resulting a suppression in short-chain fatty acid production. Further, low Akkermansia and high Acetatifactor are expected to increase inflammation. Thus, it is possible that the changes in the composition of the gut microbiota of SI animals explain an increase in IL6 signaling and a suppression in OXPHOS in their mammary gland. We also have studied immune cells in the spleen in SI and GH mice. SI increased the frequency of pro-inflammatory CD4+RORy+ cells, and the immunosuppressive Treg cells (CD4+Foxp3+) and myeloid derived suppressor cells (PMN-MDSCs). SI also increased PD1 expression in Foxp3+ cells. In an on-going study, we found that fecal microbiota transplant (FMT) from SI mice increased E0771 mammary tumor growth in antibiotic treated GH recipient mice, compared with mice receiving FMT from GH mice. Whether the FMT also altered IL6 and OXPHOS signaling in the mammary glands and tumors of recipient mice is currently being investigated. Our findings suggest that social isolation may increase breast cancer through inducing gut dysbiosis. Citation Format: Fabia de Oliveira Andrade, Maddie McDermott, Lu Jin, Vivek Verma, Karla Andrade de Oliveira, Christopher Staley, Leena Hilakivi-Clarke. Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4237.

Abstract 1721: Dual targeting of JAK2 and TrkA inhibits breast cancer cell stemness, enhances apoptosis, and delays tumor growth of triple-negative and HER2-enriched breast cancers

Abstract Despite the current standard of care, metastatic triple-negative breast cancer (TNBC) and HER2-enriched breast cancer remain difficult to treat due to limited actionable targets, poor response to treatment, and intrinsic/acquired therapeutic resistance. Thus, more effective therapies are needed to reduce the high mortality rates associated with metastatic TNBC and HER2-enriched breast cancer. The Janus Kinase 2 (JAK2)-Signal Transducer and Activator of Transcription 3 (STAT3) pathway is dysregulated in multiple solid tumors, including breast cancer, and is being investigated as a therapeutic target. Similarly, Tropomyosin receptor tyrosine kinase A (TrkA) is an emerging target in metastatic breast cancers. The prevalence of aberrant JAK2 or TrkA signaling led to the development and FDA approval of orally active, selective inhibitors for JAK2 (pacritinib) and TrkA (entrectinib, larotrectinib). However, breast cancer patients receiving either JAK2 or TrkA inhibitors showed suboptimal clinical response, suggesting that oncogenic signaling events are sustained in the absence of either JAK2 or TrkA activity. In support of this paradigm, we previously reported a novel signaling crosstalk between the JAK2-STAT3 and TrkA pathways (Cancers, 10:2340-2360, 2021). We found that JAK2 and TrkA are preferentially co-activated in TNBC and HER2-enriched breast cancer, and both kinases converge to phosphorylate STAT3 to drive gene transcription and enhance breast cancer cell stemness. The observed significant functional crosstalk between JAK2 and TrkA pathways suggest that co-inhibition of these pathways would greatly benefit breast cancer patients with co-activation of these pathways. In this study, we aimed to determine the therapeutic utility of dual targeting JAK2 and TrkA in TNBC and HER2-enriched breast cancer using FDA-approved selective inhibitors. Our data showed that JAK2 and TrkA inhibitors synergize to reduce breast cancer cell viability in vitro. Our study further revealed that co-inhibition of JAK2 and TrkA potently inhibits breast cancer cell stemness since co-inhibition of these kinases significantly reduces the CD44high/CD24low cell population, mammosphere formation, and ALDH activity to a greater extent than vehicle or monotherapies. We also found that co-inhibition of JAK2 and TrkA more effectively induces apoptosis of breast cancer cells, as indicated by Annexin V flow cytometry, Western blot analysis for cleaved PARP, and Caspase 3/7 activity. Importantly, our in vivo studies showed that co-inhibition of JAK2 and TrkA significantly delays mammary tumor growth using cell line-derived and patient-derived xenograft breast cancer mouse models. Taken together, our data illustrates the therapeutic utility of dual targeting JAK2 and TrkA in metastatic TNBC and HER2-enriched breast cancers. Citation Format: Angelina T. Regua, Mariana K. Najjar, Grace L. Wong, Sara G. Manore, Austin Arrigo, Hui-Wen Lo. Dual targeting of JAK2 and TrkA inhibits breast cancer cell stemness, enhances apoptosis, and delays tumor growth of triple-negative and HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1721.

Targeting the NF-κB pathway enhances responsiveness of mammary tumors to JAK inhibitors

Interactions between tumor cells and the tumor microenvironment are critical for tumor growth, progression, and response to therapy. Effective targeting of oncogenic signaling pathways in tumors requires an understanding of how these therapies impact both tumor cells and cells within the tumor microenvironment. One such pathway is the janus kinase (JAK)/signal transducer and activator or transcription (STAT) pathway, which is activated in both breast cancer cells and in tumor associated macrophages. This study demonstrates that exposure of macrophages to JAK inhibitors leads to activation of NF-κB signaling, which results in increased expression of genes known to be associated with therapeutic resistance. Furthermore, inhibition of the NF-κB pathway improves the ability of ruxolitinib to reduce mammary tumor growth in vivo. Thus, the impact of the tumor microenvironment is an important consideration in studying breast cancer and understanding such mechanisms of resistance is critical to development of effective targeted therapies.

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.

Abstract P2-01-05: MBQ-167 chemosensitizes triple-negative breast cancer cells to current chemotherapies and reduces paclitaxel-induced metastasis

Abstract Triple-negative breast cancer (TNBC) is an aggressive and recurrent type of breast cancer that accounts for over 15% of new diagnoses yearly. Patients with TNBC have limited response to hormonal or targeted therapies due to the lack of ER, PR, and HER2. Therefore, the standard treatment for TNBC is anthracycline/taxane-based chemotherapies, paclitaxel (PXL), and doxorubicin (DOX), for which patients often develop chemoresistance. PXL, particularly, induces metastasis in TNBC patients by activating the Toll-Like Receptor 4 (TLR4)/NFkB signaling pathway. Since there are few targeted therapies for TNBC, there is an urgent need to identify new “druggable” molecular targets. The oncogenic GTPases Rac and Cdc42, and their downstream effector, p-21 Activated Kinase (PAK), play critical roles in TNBC development and metastatic cancer progression. Therefore, we developed MBQ-167 as a potent Rac/Cdc42 inhibitor that decreases tumor cell growth and metastasis in TNBC mouse models with no apparent toxicity. To test the hypothesis that inhibiting Rac/Cdc42 with MBQ-167 will chemosensitize TNBC cells to PXL and DOX, we treated TNBC human cell lines MDA-MB-231 and MDA-MB-468 with different concentrations of MBQ-167 (250 nM-500 nM), PXL (5 nM-10 nM), DOX (250 nM-500 nM), and their combinations. The efficacy of single and combined treatments was determined on cell proliferation with the MTT assay, apoptosis with the Caspase-Glo 3/7 assay, and cell migration with the wound healing assay. In addition, we evaluated the effect of single or combined MBQ-167 (per oral 50 mg/kg, 5X a week) and PXL (IP 10mg/kg, 1X a week) on tumor growth and metastasis in an orthotopic Luciferase tagged-MDA-MB-468 TNBC mouse model. Our in vitro results demonstrate that treatments with MBQ-167, PXL, DOX, and their respective combinations, decreased TNBC cell viability and increased apoptosis compared to vehicle controls. Both combination treatments reduced cell viability and increased apoptosis compared to PXL or DOX alone. Moreover, MBQ-167 was more effective than PXL at reducing MDA-MB-231 cell migration. DOX treatment did not affect cell migration, while combination treatment of DOX with MBQ-167 decreased migration. Moreover, in the mouse model, MBQ-167 significantly reduced MDA-MB-468 mammary tumor growth by 85% compared to vehicle treatments. This decrease in tumor growth was comparable to PXL treatment alone and in combination with MBQ-167. As expected, PXL treatment increased metastasis to the lungs and liver, while MBQ-167 prevented lung and liver metastases and reduced PXL-induced metastasis. Therefore, inhibiting Rac/Cdc42 with MBQ-167 improves the responses to current TNBC chemotherapies, PXL and Dox, and reduces PXL-induced metastasis, making MBQ-167 a potential adjuvant treatment against TNBC. This conclusion will be tested since we recently obtained IND approval from the US FDA for MBQ-167 and plan to conduct clinical trials in TNBC patients. Citation Format: Nilmary Grafals-Ruiz, Ailed M. Cruz-Collazo, Anamaris Torres-Sánchez, Surangani Dharmawardhane. MBQ-167 chemosensitizes triple-negative breast cancer cells to current chemotherapies and reduces paclitaxel-induced metastasis [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 P2-01-05.

Abstract B011: Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis

Abstract Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion, modulation of the tumor microenvironment (TME), plasticity, and ability to colonize secondary tissues. Therefore, a viable anti-metastasis strategy needs to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, the purpose of this study was to test the hypothesis that Rac and Cdc42 inhibitors target both migratory cancer cells and immunosuppressive cells in the TME. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. Since the targets of these inhibitors may also affect immune cell behavior, we first tested their potential to target Rac/Cdc42 function in human and rat macrophage cell lines and show that EHop-016 and MBQ-167 inhibit Rac activation (at 5X higher concentration than the IC50 in breast cancer cells), actin cytoskeletal extensions, and migration, without affecting cell viability. EHop-016 and MBQ-167 significantly decrease the proinflammatory cytokine Interleukin-6 (Il-6) from plasma and the TME. Next, we tested the in vivo effect of MBQ-167 on immune cells from breast cancer mouse models. Tumors from immunodeficient mice bearing mammary tumors that were treated with vehicle or 50mg/kg MBQ-167 5x a week for 5 weeks, were analyzed by flow cytometry to quantify different immune cell types. MBQ-167 treated mice demonstrated elevated CD11+F4/80+CD86+ M1 macrophages and reduced MDSCs in mammary tumors. In the immunocompetent 4T-1 breast cancer model, spleens contained a lower frequency of macrophages and neutrophils, accompanied by an increase in activated cytotoxic CD69+, CD8+T cells. Moreover, when metastasis to the lungs was reduced by ~90% following MBQ-167 treatment, granzyme B+ CD8+ T cells were elevated in the lung extracts, without affecting the immunosuppressive CD4+ regulatory T cell counts. In vitro, we also demonstrated that increasing doses of MBQ-167 induced T cell activation and interferon-gamma secretion, a cytokine known to have antitumor properties. Taken together, Rac/Cdc42 inhibitors induce anti-metastatic cancer effects via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells, while promoting the activation of cytotoxic T cells, in the tumor and the metastasis microenvironment. Therefore, MBQ-167 promises to be an effective antimetastatic cancer therapeutic that is poised to enter Phase I clinical trials. Citation Format: Stephanie Dorta-Estremera, Anamaris Torres, Michael Rivera-Robles, Ailed Cruz-Collazo, Luis Borrero-Garcia, Suranganie Dharmawardhane. Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B011.

Antitumor effect of selenium-rich Brazil nuts and selenomethionine dietary supplementation on pre-existing 4T1 mammary tumor growth in mice.

Selenium (Se) is an essential micronutrient known to play an important role in the antioxidant system that can potentially influence tumor growth. We aimed to investigate the effects of dietary Se supplementation after detection of 4T1 mammary tumor growth in BALB/c mice. Thirty female mice received subcutaneous inoculation of 4T1 cells. After five days, all animals presenting palpable tumors were randomly assigned to three groups: a control group (Se-control) receiving a diet with adequate Se (0.15 mg/kg) and two other groups that received Se-supplemented diets (1.4 mg/kg of total Se) with either Brazilian nuts (Se-Nuts) or selenomethionine (SeMet). Data were assessed by either One or Two-way ANOVA followed by Tukey's HSD or Bonferroni's post hoc tests, respectively. Both Se-supplemented diets reduced tumor volume from the thirteenth day of feeding compared with the Se-adequate (control) diet (p < 0.05). The SeMet group presented a higher Se blood concentration (p < 0.05) than the Se-control group, with the Se-Nuts group presenting intermediate values. Selenoprotein P gene expression in the liver was higher in the Se-Nuts group than in the Se-control group (p < 0.05), while the SeMet group presented intermediate expression. Dietary Se supplementation, starting after detection of 4T1 palpable lesions, reduced tumor volume in mice.

Separate and combined effects of advanced age and obesity on mammary adipose inflammation, immunosuppression and tumor progression in mouse models of triple negative breast cancer.

Advanced age and obesity are independent risk and progression factors for triple negative breast cancer (TNBC), which presents significant public health concerns for the aging population and its increasing burden of obesity. Due to parallels between advanced age- and obesityrelated biology, particularly adipose inflammation, we hypothesized that advanced age and obesity each accelerate mammary tumor growth through convergent, and likely interactive, mechanisms. To test this hypothesis, we orthotopically transplanted murine syngeneic TNBC cells into the mammary glands of young normoweight control (7 months), young diet-induced obese (DIO), aged normoweight control (17 months), and aged DIO female C57BL/6J mice. Here we report accelerated tumor growth in aged control and young DIO mice, compared with young controls. Transcriptional analyses revealed, with a few exceptions, overlapping patterns of mammary tumor inflammation and tumor immunosuppression in aged control mice and young DIO mice, relative to young controls. Moreover, aged control and young DIO tumors, compared with young controls, had reduced abundance ofcytotoxic CD8 T cells. Finally, DIO in advanced age exacerbated mammary tumor growth, inflammation and tumor immunosuppression. These findings demonstrate commonalities in the mechanisms driving TNBC in aged and obese mice, relative to young normoweight controls. Moreover, we found that advanced age and DIO interact to accelerate mammary tumor progression. Given the US population is getting older and more obese, age- and obesity-related biological differences will need to be considered when developing mechanism-based strategies for preventing or controlling breast cancer.

Abstract C010: Genes with African ancestry-specific expression in TNBC tumors also show population-specific baseline differences in normal mammary tissue

Abstract Our recent efforts to define ancestry-specific drivers in triple negative breast tumors (TNBC) has been sharply focused on identifying African (AFR) ancestry-associated gene signatures, which we have reported to be distinct from traditional approaches using SRR to categorize patients. In our AFR ancestry enriched TNBC cohort, we have identified a 613 gene signature associated with AFR ancestry, which is enriched in previously implicated canonical pathways, such as RNA post-transcriptional modification and chronic inflammation. We also reported a significant enrichment in disease/function terms associated with immune cell trafficking, where increasing African ancestry was associated with predicted activation of immune cell migration, and inhibition of immune cell activation. While these associations were determined in the TNBC tumor environment, involvement of this 613 AFR ancestry-associated gene signature has yet to be explored in the context of non-diseased breast/mammary tissue. At present, we obtained gene expression (TPM) and ancestry data from the GTEx cohort. Of 459 patients with gene expression data available for breast/mammary tissue, 47 of those were found to be of African ancestry, and 20 identified as female, and were used in subsequent analyses. We found that 17 of the 613 AFR ancestry-associated genes from TNBC tumors were also associated with AFR ancestry in non-diseased breast/mammary tissue. Interestingly we observed that some genes had differencing directionality/correlation between non-diseased and tumor tissue. Of the 17 genes, 7 were positively associated with AFR ancestry in both cohorts, including: AGMAT, CYBA, HEATR3, KCNAB2, PTGES, RAB42, and SHPK. Eight genes were found to be positively associated with AFR in normal, but negatively associated in TNBC (AC068279.3, AP001434.2, C12orf60, FNDC3B, HNRNPA1P50, RP11-214O14.1, SNORA53 and SPATA20P1). The remaining 2 were negatively associated with AFR in normal, and positively associated in tumor (AVPR2 and FAM26D). Tumor agnostic survival analysis using TCGA BRCA cohort data shows some race-specific differences in overall survival of these genes. Specifically, higher expression of AVPR2 and CYBA, and lower expression of AGMAT and SNORA53 show benefit among AA patients, but not EA patients (ns). Pathway analysis of the 17 genes shows significant enrichment with growth of mammary tumor (p = 7.3 E-07) and inflammatory response (p = 2.5 E-07) disease and function terms, specifically related to predicted activation of IFNG, AKT1, and FOS. Ancestry-specific differences in gene expression that are present in non-malignant tissue may implicate baseline differences that could play a significant role in later tumor etiology. Interestingly, 10 of 17 genes showed differing association with African ancestry, potentially revealing malignancy-induced changes to these pathways. Further analyses and curation of robust and diverse datasets of non-diseased tissue is needed to fully contextualize these findings. Citation Format: Rachel Martini, Nicolas Robine, Ishmael Kyei, Ernest Adjei, Mahteme Bekele, Olivier Elemento, Lisa Newman, Melissa Davis. Genes with African ancestry-specific expression in TNBC tumors also show population-specific baseline differences in normal mammary tissue [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C010.

Fusobacterium nucleatum: a novel immune modulator in breast cancer?

Breast cancer was the most commonly diagnosed cancer worldwide in 2020. Greater understanding of the factors which promote tumour progression, metastatic development and therapeutic resistance is needed. In recent years, a distinct microbiome has been detected in the breast, a site previously thought to be sterile. Here, we review the clinical and molecular relevance of the oral anaerobic bacterium Fusobacterium nucleatum in breast cancer. F. nucleatum is enriched in breast tumour tissue compared with matched healthy tissue and has been shown to promote mammary tumour growth and metastatic progression in mouse models. Current literature suggests that F. nucleatum modulates immune escape and inflammation within the tissue microenvironment, two well-defined hallmarks of cancer. Furthermore, the microbiome, and F. nucleatum specifically, has been shown to affect patient response to therapy including immune checkpoint inhibitors. These findings highlight areas of future research needed to better understand the influence of F. nucleatum in the development and treatment of breast cancer.

Prevalence of Iron Deficiency and its Association With Breast Cancer in Premenopausal Compared to Postmenopausal Women in Al Ahsa, Saudi Arabia.

Iron is an essential cofactor needed for normal functions of various enzymes and its depletion lead to increase DNA damage, genomic instability, deteriorate innate, adaptive immunity, and promote tumor development. It is also linked to tumorigenesis of breast cancer cells through enhancing mammary tumor growth and metastasis. There is insufficient data describing this association in Saudi Arabia. This study aims to determine the prevalence of iron deficiency and its association with breast cancer among premenopausal and postmenopausal women referred for breast cancer screening center in Al Ahsa, Eastern Province of Saudi Arabia. Age, hemoglobin level, iron level, history of anemia, or iron deficiency were collected from patients' medical records. The included participants were grouped based on their age into premenopausal (<50 years) or postmenopausal (⩾50 years). The definition of low Hb implemented (Hb below 12 g/dL) and low total serum Iron levels (below 8 μmol/L). Logistic regression test was used to compute the association between having a positive cancer screening test (radiological or histocytological) and participant's lab results. The results are presented as odds ratios and 95% confidence intervals. Thrree hundred fifty-seven women were included, 77% (n = 274) of them were premenopausal. This group cases had more history of iron deficiency (149 [60%] vs 25 (30%), P = .001) compared to those in the postmenopausal group. The risk of having a positive radiological cancer screening test was associated with age (OR = 1.04, 95% CI 1.02-1.06), but negatively was associated with iron level (OR = 0.9, 95% CI 0.86-0.97) among the entire cohort. This study is the first to propose an association between iron deficiency and breast cancer among Saudi young females. This could suggest iron level as a new risk factor that may be used by clinicians to assess breast cancer risk.

Reduction of Staphylococcus epidermidis in the mammary tumor microbiota induces antitumor immunity and decreases breast cancer aggressiveness

The mammary gland hosts a microbiota, which differs between malignant versus normal tissue. We found that aerosolized antibiotics decrease murine mammary tumor growth and strongly limit lung metastasis. Oral absorbable antibiotics also reduced mammary tumors. In ampicillin-treated nodules, the immune microenvironment consisted of an M1 profile and improved T cell/macrophage infiltration. In these tumors, we noted an under-representation of microbial recognition and complement pathways, supported by TLR2/TLR7 protein and C3-fragment deposition reduction. By 16S rRNA gene profiling, we observed increased Staphylococcus levels in untreated tumors, among which we isolated Staphylococcus epidermidis, which had potent inflammatory activity and increased Tregs. Conversely, oral ampicillin lowered Staphylococcus epidermidis in mammary tumors and expanded bacteria promoting an M1 phenotype and reducing MDSCs and tumor growth. Ampicillin/paclitaxel combination improved the chemotherapeutic efficacy. Notably, an Amp-like signature, based on genes differentially expressed in murine tumors, identified breast cancer patients with better prognosis and high immune infiltration that correlated with a bacteria response signature.This study highlights the significant influence of mammary tumor microbiota on local immune status and the relevance of its treatment with antibiotics, in combination with breast cancer therapies.

Lacticaseibacillus rhamnosus Probio-M9-Driven Mouse Mammary Tumor-Inhibitory Effect Is Accompanied by Modulation of Host Gut Microbiota, Immunity, and Serum Metabolome.

Gut microbiome may influence tumor growth and cancer treatment efficacy, so it is a potential target for tumor prevention/treatment. This pilot study investigated the preventive and therapeutic effects of a probiotic strain, Lacticaseibacillus rhamnosus Probio-M9 (Probio-M9), against murine mammary cancer. Thirty-six female mice were randomly divided into three groups (n = 12 per group): control (without tumor transplantation), model (tumor transplantation; no probiotic administration), and probiotic (30-day oral gavage of probiotic, started seven days before tumor transplantation). Changes in tumor size were recorded, and blood, tumor tissue, and stool samples were collected at the end of the trial for analyses. Comparing with the model group, the probiotic group had a significantly smaller tumor volume (p < 0.05), a higher fecal microbiota Shannon diversity index, with significant modifications in the gut microbiota structure (p < 0.05), characterized by more Alistipes sp._2, Porphyromonadaceae bacterium_7, and Bacteroidales bacterium 55_9 (p < 0.05). Additionally, Probio-M9 administration elevated the serum IFN-γ, IL-9, IL-13, and IL-27 levels and several metabolites (e.g., pyridoxal, nicotinic acid, 3-hydroxybutyric acid, glutamine; p < 0.05), while reducing IL-5 (p < 0.05). These changes might be associated with the protective effect of Probio-M9 against mammary tumor growth. Thus, probiotic administration could harness host gut microbiome in anti-cancer responses.

Veterinary Management of Harderian Gland Tumors in Cancer Rainbow (crainbow) HER2-Positive Mice.

A Cancer Rainbow mouse line that expresses 3 fluorescently labeled isoforms of the tumor-driver gene HER2 (HER2BOW) was developed recently for the study of tumorigenesis in the mammary gland. The expression of 1 of the 3 HER2 isoforms in HER2BOW mice is induced through the Cre/lox system. However, in addition to developing palpable mammary tumors, HER2BOW mice developed orbital tumors, specifically of the Harderian gland. Mice were euthanized, and histopathologic examination of the Harderian gland tumors was performed. Tumors were characterized by adenomatous hyperplasia to multinodular adenomas of the Harderian gland. Fluorescent imaging of the Harderian gland tissue confirmed the expression of HER2 in the tumors. Here we discuss monitoring and palliative approaches to allow attainment of humane experimental endpoints of mammary tumor growth in this mouse line. We describe a range of interventions, including close monitoring, topical palliative care, and surgical bilateral enucleation. Based on our data and previous reports in the literature, the overexpression of HER2 in Harderian gland tissue and subsequent tumor formation likely was driven by MMTV-Cre expression in the Harderian gland.

Mammary Tumor Growth and Proliferation Are Dependent on Growth Hormone in Female SV40 C3(1) T-Antigen Mice.

Female SV40 C3(1) T-antigen (C3(1)/TAg) transgenic mice develop mammary tumors that are molecularly similar to human basal-like breast cancers with 100% incidence at 16 weeks of age. To determine the requirement for growth hormone (GH) signaling in these tumors, genetic crosses were used to create cohorts of female mice that were homozygous for a floxed growth hormone receptor (Ghr) gene and carried one copy each of the Rosa-Cre-ERT2 transgene and the C3(1)/TAg transgene (Ghrflox/flox; Rosa-Cre-ERT2; C3(1)/TAg+/0 mice). When the largest mammary tumor reached 200 mm3, mice were treated with tamoxifen to delete Ghr or with vehicle as a control. An additional group of Ghrflox/flox; C3(1)/TAg+/0 mice were also treated with tamoxifen when the largest mammary tumor reached 200 mm3 as a control for the effects of tamoxifen. After 3 weeks, tumors in mice in which Ghr was deleted began to shrink while vehicle and tamoxifen treatment control mouse tumors continued to grow. Pathological analysis of tumors revealed similar growth patterns and varying levels of necrosis throughout all groups. A decrease in cancer cell proliferation in Ghr-/- tumors relative to controls was observed as measured by Ki67 immunohistochemistry labeling index. These data suggest that even established C3(1)/TAg mammary tumors are dependent on the GH/IGF-1 axis.

Self-Splittable Transcytosis Nanoraspberry for NIR-II Photo-Immunometabolic Cancer Therapy in Deep Tumor Tissue.

Cancer photo-immunotherapy (CPIT) as an ideal strategy can rapidly release hostile signals by appropriate dosage of focal laser irradiation to unmask primary tumor immunogenicity and can activate adaptive immunity to control distant metastases. However, many factors, including disordered immunometabolism, poor penetration of photothermal agents and immuno-regulators, inadequate laser penetration into the deep tumor region, restrict the therapeutic outcomes of CPIT. Here, a second near-infrared window (NIR-II) photo-immunometabolic cancer therapy (PICT) by a programmed raspberry-structured nanoadjuvant (PRNMT ) is presented that can potentiates efficient immunogenic cell death (ICD) in deep tumor tissue and alleviates immunometabolic disorder. The PRNMT is architected through self-assembly of indoleamine 2,3-dioxygenase 1 (IDO-1) inhibitor modified small-sized CuS nanoparticles (CuS5 ) and tumor microenvironment (TME) responsive cationized polymeric matrix. The TME can trigger the splitting and surface cationization of PRNMT into small cationized CuS5 that feature high transcytosis potential and TME immunometabolic regulation. Upon NIR-II irradiation, CuS5 induce homogeneous ICD and release immunometabolic regulator in deep tumor tissues, which ameliorates IDO-1 mediated immunometabolic disorder and further suppresses regulatory T cells infiltration. PRNMT mediated PICT effectively delays the primary murine mammary carcinoma 4T1 tumor growth and inhibits the lethal pulmonary metastasis in combination with programmed cell death protein1 (PD1)blockade.