Spontaneous Breast Cancer Research Articles

RGD-functionalised melanin nanoparticles for intraoperative photoacoustic imaging-guided breast cancer surgery

PurposeObtaining tumour-free margins is critical for avoiding re-excision and reducing local recurrence following breast-conserving surgery; however, it remains challenging. Imaging-guided surgery provides precise detection of residual lesions and assists surgical resection. Herein, we described water-soluble melanin nanoparticles (MNPs) conjugated with cyclic Arg-Gly-Asp (cRGD) peptides for breast cancer photoacoustic imaging (PAI) and surgical navigation.MethodsThe cRGD-MNPs were synthesised and characterized for morphology, photoacoustic characteristics and stability. Tumour targeting and toxicity of cRGD-MNPs were determined by using either breast cancer cells, MDA-MB-231 tumour-bearing mice or the FVB/N-Tg (MMTV-PyVT) 634Mul/J mice model. PAI was used to locate the tumour and guide surgical resection in MDA-MB-231 tumour-bearing mice.ResultsThe cRGD-MNPs exhibited excellent in vitro and in vivo tumour targeting with low toxicity. Intravenous administration of cRGD-MNPs to MDA-MB-231 tumour-bearing mice showed an approximately 2.1-fold enhancement in photoacoustic (PA) intensity at 2 h, and the ratio of the PA intensity at the tumour site to that in the surrounding normal tissue was 3.2 ± 0.1, which was higher than that using MNPs (1.7 ± 0.3). Similarly, the PA signal in the spontaneous breast cancer increased ~ 2.5-fold at 2 h post-injection of cRGD-MNPs in MMTV-PyVT transgenic mice. Preoperative PAI assessed tumour volume and offered three-dimensional (3D) reconstruction images for accurate surgical planning. Surgical resection following real-time PAI showed high consistency with histopathological analysis.ConclusionThese results highlight that cRGD-MNP-mediated PAI provide a powerful tool for breast cancer imaging and precise tumour resection. cRGD-MNPs with fine PA properties have great potential for clinical translation.

Type 2 dendritic cells mediate control of cytotoxic T cell resistant tumors.

Type 2 DCs (DC2s) comprise the majority of conventional DCs within most tumors; however, little is known about their ability to initiate and sustain antitumor immunity, as most studies have focused on antigen cross-presenting DC1s. Here, we report that DC2 infiltration identified by analysis of multiple human cancer data sets showed a significant correlation with survival across multiple human cancers, with the benefit being seen in tumors resistant to cytotoxic T cell control. Characterization of DC subtype infiltration into an immunotherapy-resistant model of breast cancer revealed that impairment of DC1s through 2 unique models resulted in enhanced DC2 functionality and improved tumor control. BATF3 deficiency depleted intratumoral DC1s, which led to increased DC2 lymph node migration and CD4+ T cell activation. Enhancing DC2 stimulatory potential by genetic deletion of Hsp90b1 (encoding molecular chaperon GP96) led to a similar enhancement of T cell immunity and improved survival in a spontaneous breast cancer model. These data highlight the therapeutic and prognostic potential of DC2s within checkpoint blockade–resistant tumors.

Peptide-Targeted High-Density Lipoprotein Nanoparticles for Combinatorial Treatment against Metastatic Breast Cancer.

The sonic hedgehog (SHH) signaling pathway exhibits aberrant activation in triple-negative breast cancer (TNBC), wherein it regulates several malignant phenotypes related to tumor metastasis. GANT61, an inhibitor of the SHH signaling pathway, may offer promise when administered in combination with conventional chemotherapy to treat metastatic TNBC. However, poor bioavailability and substantial off-target toxicity limit its clinical application. To address these limitations, we designed a peptide-functionalized dual-targeting delivery system encapsulating paclitaxel and GANT61 in tLyP-1 peptide-modified reconstituted high-density lipoprotein nanoparticle (tLyP-1-rHDL-PTX/GANT61 NP) for metastatic TNBC treatment. The apolipoprotein A-1 and tLyP-1 peptide modified on the surface of nanoparticles enable the delivery system to target tumor cells by binding to the overexpressed scavenger receptor B type I and neuropilin-1 receptor. Moreover, the tLyP-1 peptide also enables the deep tumor penetration of nanoparticles further facilitating paclitaxel and GANT61 delivery. Increased cellular uptake of the nanoparticles was observed in both MDA-MB-231, BT-549 tumor cells, and their 3D tumor spheroids. A series of in vitro experiments reveal that GANT61 was able to suppress key metastasis-related tumor cell activities including angiogenesis, migration, invasion, and stemness. Owing to more effective drug administration, the metastasis suppression efficiency of GANT61 was significantly enhanced by the dual-targeting tLyP-1-rHDL delivery system. Meanwhile, the codelivery of paclitaxel and GANT61 by dual-targeting tLyP-1-rHDL nanoparticles demonstrated superior efficiency of disrupting proliferation and inducing apoptosis in tumor cells compared with drug solutions. In a spontaneous metastasis breast cancer NCG mice model, the tLyP-1-rHDL-PTX/GANT61 nanoparticles exhibited highly tumor-specific distribution and result in significant inhibition of the primary tumor growth and dramatic reduction of lung metastasis without obvious side effects. The present work suggests that a combination of the SHH signaling pathway suppression and chemotherapy assisted by peptide-functionalized targeting tLyP-1-rHDL nanoparticles may provide a promising strategy for metastatic TNBC treatment.

Refractoriness of STING therapy is relieved by AKT inhibitor through effective vascular disruption in tumour

Stimulator of interferon genes (STING) promotes anti-tumour immunity by linking innate and adaptive immunity, but it remains unclear how intratumoural treatment with STING agonists yields anti-tumour effects. Here we demonstrate that intratumoural injection of the STING agonist cGAMP induces strong, rapid, and selective apoptosis of tumour endothelial cells (ECs) in implanted LLC tumour, melanoma and breast tumour, but not in spontaneous breast cancer and melanoma. In both implanted and spontaneous tumours, cGAMP greatly increases TNFα from tumour-associated myeloid cells. However, compared to spontaneous tumour ECs, implanted tumour ECs are more vulnerable to TNFα-TNFR1 signalling-mediated apoptosis, which promotes effective anti-tumour activity. The spontaneous tumour’s refractoriness to cGAMP is abolished by co-treatment with AKT 1/2 inhibitor (AKTi). Combined treatment with cGAMP and AKTi induces extensive tumour EC apoptosis, leading to extensive tumour apoptosis and marked growth suppression of the spontaneous tumour. These findings propose an advanced avenue for treating primary tumours that are refractory to single STING agonist therapy.

JUNB suppresses distant metastasis by influencing the initial metastatic stage

The complex interactions between cells of the tumor microenvironment and cancer cells are considered a major determinant of cancer progression and metastasis. Yet, our understanding of the mechanisms of metastatic disease is not sufficient to successfully treat patients with advanced-stage cancer. JUNB is a member of the AP-1 transcription factor family shown to be frequently deregulated in human cancer and associated with invasion and metastasis. A strikingly high stromal JUNB expression in human breast cancer samples prompted us to functionally investigate the consequences of JUNB loss in cells of the tumor microenvironment on cancer progression and metastasis in mice. To adequately mimic the clinical situation, we applied a syngeneic spontaneous breast cancer metastasis model followed by primary tumor resection and identified stromal JUNB as a potent suppressor of distant metastasis. Comprehensive characterization of the JUNB-deficient tumor microenvironment revealed a strong influx of myeloid cells into primary breast tumors and lungs at early metastatic stage. In these infiltrating neutrophils, BV8 and MMP9, proteins promoting angiogenesis and tissue remodeling, were specifically upregulated in a JUNB-dependent manner. Taken together, we established stromal JUNB as a strong suppressor of distant metastasis. Consequently, therapeutic strategies targeting AP-1 should be carefully designed not to interfere with stromal JUNB expression as this may be detrimental for cancer patients.

JFK Is a Hypoxia-Inducible Gene That Functions to Promote Breast Carcinogenesis

Many carcinomas feature hypoxia, a condition has long been associated with tumor progression and poor prognosis, as well as resistance to chemoradiotherapy. Here, we report that the F-box protein JFK promotes mammary tumor initiation and progression in MMTV-PyMT murine model of spontaneous breast cancer. We find that JFK is inducible under hypoxic conditions, in which hypoxia-inducible factor HIF-1α binds to and transcriptionally activates JFK in breast cancer cells. Consistently, analysis of public clinical datasets reveals that the mRNA level of JFK is positively correlated with that of HIF-1α in breast cancer. We show that JFK deficiency leads to a decrease in HIF-1α-induced glycolysis in breast cancer and sensitizes hypoxic breast cancer cells to ionizing radiation and chemotherapeutic treatment. These results indicate that JFK is an important player in hypoxic response, supporting the pursuit of JFK as a potential therapeutic target for breast cancer intervention.

Breast cancer-derived DAMPs enhance cell invasion and metastasis, while nucleic acid scavengers mitigate these effects

Breast cancer (BC) is the most common malignancy in women. Particular subtypes with aggressive behavior are major contributors to poor outcomes. Triple-negative breast cancer (TNBC) is difficult to treat, pro-inflammatory, and highly metastatic. We demonstrate that TNBC cells express TLR9 and are responsive to TLR9 ligands, and treatment of TNBC cells with chemotherapy increases the release of nucleic-acid-containing damage-associated molecular patterns (NA DAMPs) in cell culture. Such culture-derived and breast cancer patient-derived NA DAMPs increase TLR9 activation and TNBC cell invasion in vitro. Notably, treatment with the polyamidoamine dendrimer generation 3.0 (PAMAM-G3) behaved as a nucleic acid scavenger (NAS) and significantly mitigates such effects. In mice that develop spontaneous BC induced by polyoma middle T oncoprotein (MMTV-PyMT), treatment with PAMAM-G3 significantly reduces lung metastasis. Thus, NAS treatment mitigates cancer-induced inflammation and metastasis and represents a novel therapeutic approach for combating breast cancer.

Abstract 2571: Paternal epigenetic regulation contributes to the prevention of estrogen receptor-negative mammary cancer with combined broccoli sprouts and green tea polyphenols consumption in transgenic mice

Abstract Breast cancer is one of the leading causes of cancer-related death among women in the United States. It is widely appreciated that abnormal epigenetic alterations act as one of the primary contributors to breast cancer initiation and progression. Parental, including maternal and paternal, nutritional interventions have shown considerable impact on fetal development leading to different susceptibility of offspring to various chronic diseases. Moreover, combinatorial dietary administration to parents could be more efficacious in ameliorating epigenetic aberrations and changing cancer risk in an individual's later life. The bioactive dietary broccoli sprouts (BSp) and green tea polyphenol (GTP) are important epigenetic modulators that can prevent various cancers. Our study focusses on investigating the effect of paternal dietary BSp and GTP administration on mammary cancer prevention of their offspring. Two spontaneous breast cancer transgenic mouse models, C3(1)/SV40 and Her2/neu, were employed in this study. Male C3(1)/SV40 or Her2/neu transgenic mice were randomly assigned into four groups and treated with: control AIN-93G diet, 26% BSp (w/w) in food pellets, 0.5% GTPs (w/v) in drinking water or combined BSp and GTPs from 3 wks of age until 10 wks of age. Treated male mice of different groups were then mated with non-treated female mice. Female pups were selected after their weaning and tumor growth was monitored weekly until the termination of the experiment. Tumor- and epigenetic-related protein expression and histone modifications (methylation and acetylation) were measured. Our study indicated that paternal BSp or/and GTPs administration suppressed tumor growth, decreased tumor incidence, and delayed tumor latency compared with the control in both mouse models. Overall, paternal dietary intervention displayed more efficacy for mammary tumor prevention in C3(1)/SV40 mice and the combination group in this model also showed synergistically effects. Subsequent analysis with the C3(1)/SV40 mouse tumors demonstrated that paternal BSp or/and GTPs treatments upregulated the expression of tumor suppressor proteins, such as P16 and P53, and down-regulated tumor promoting proteins, such as MYC and BMI1. Moreover, altered DNA methylation, histone methylation and acetylation levels were also observed. These results suggested that paternal exposure to BSp and GTPs may contribute to ER-negative mammary tumor prevention in their offspring through epigenetic regulations and the combined addition of bioactive botanicals could be a more promising approach for coping with breast cancer initiation and progression in humans. Citation Format: Shizhao Li, Huixin Wu, Trygve Tollefsbol. Paternal epigenetic regulation contributes to the prevention of estrogen receptor-negative mammary cancer with combined broccoli sprouts and green tea polyphenols consumption in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2571.

The Consequences of Soluble Epoxide Hydrolase Deletion on Tumorigenesis and Metastasis in a Mouse Model of Breast Cancer.

Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.

Andrographolide Suppresses the Growth and Metastasis of Luminal-Like Breast Cancer by Inhibiting the NF-κB/miR-21-5p/PDCD4 Signaling Pathway.

Tumor growth and metastasis are responsible for breast cancer-related mortality. Andrographolide (Andro) is a traditional anti-inflammatory drug used in the clinic that inhibits NF-κB activation. Recently, Andro has been found in the treatment of various cancers. Andro inhibits breast cell proliferation and invasion and induces apoptosis via activating various signaling pathways. Therefore, the underlying mechanisms with regard to the antitumor effects of Andro still need to be further confirmed. Herein, a MMTV-PyMT spontaneous luminal-like breast cancer lung metastatic transgenic tumor model was employed to estimate the antitumor effects of Andro on breast cancer in vivo. Andro significantly inhibited tumor growth and metastasis in MMTV-PyMT mice and suppressed the cell proliferation, migration, and invasion of MCF-7 breast cancer cells in vitro. Meanwhile, Andro significantly inhibited the expression of NF-κB, and the downregulated NF-κB reduced miR-21-5p expression. In addition, miR-21-5p dramatically inhibited the target gene expression of programmed cell death protein 4 (PDCD4). In the current study, we demonstrated the potential anticancer effects of Andro on luminal-like breast cancer and indicated that Andro inhibits the expression of miR-21-5p and further promotes PDCD4 via NF-κB suppression. Therefore, Andro could be an antitumor agent for the treatment of luminal-like breast cancer in the clinic.

NF-κB and neutrophil extracellular traps cooperate to promote breast cancer progression and metastasis

Aberrant NF-κB activation and neutrophil extracellular traps (NETs) are associated with breast cancer progression. How NF-κB and NETs modulate each other in breast cancer development remains unclear. Here, we found that NETs induced by phorbol 12-myristate 13-acetate promote breast cancer cell progression. In turn, cancer cells–derived factors, such as IL-8 and granulocyte colony-stimulating factor, stimulate neutrophils to form NETs. Mechanistically, NETs increased the interaction of NF-κB essential modifier (NEMO) with IκB kinase (IKK)α/β and enhanced NF-κB activation. We then employed a cell-permeable peptide corresponding to the NEMO-binding domain (NBD) of IKKα/β, termed NBD peptide, which disrupts NETs-mediated NEMO interaction with IKKα/β and abolished NF-κB activation in vitro. NBD peptide also reduced IL-8 level and NETs formation, and suppressed primary tumor growth and/or lung metastasis in human breast cancer mouse xenograft models and mouse spontaneous breast cancer model. Blockade of NET formation using a peptidylarginine deiminase 4 (PAD4) pharmacologic inhibitor decreased NF-κB activation and tumor metastasis. Collectively, these data suggest that NF-κB associates with NETs to form a positive loop facilitating breast tumor progression and metastasis, and that selective inhibition of NF-κB and PAD4-dependent NETs provides an effective therapeutic approach for treating breast cancer.

Reappraisal of anticancer nanomedicine design criteria in three types of preclinical cancer models for better clinical translation

Anticancer nanomedicines are designed to improve anticancer efficacy by increasing drug accumulation in tumors through enhanced permeability retention (EPR) effect, and to reduce toxicity by decreasing drug accumulation in normal organs through long systemic circulation. However, the inconsistent efficacy/safety of nanomedicines in cancer patients versus preclinical cancer models have provoked debate for nanomedicine design criteria. In this study, we investigate nanomedicine design criteria in three types of preclinical cancer models using five clinically used nanomedicines, which identifies the factors for better clinical translations of their observed clinical efficacy/safety compared to free drug or clinical micelle formulation. When those nanomedicines were compared with drug solution or clinical micelle formulation in breast tumors, long and short-circulating nanomedicines did not enhance tumor accumulation by EPR effect in transgenic spontaneous breast cancer model regardless of their size or composition, although they improved tumor accumulations in subcutaneous and orthotopic breast cancer models. However, when tumors were compared to normal breast tissue, nanomedicines, drug solution and clinical micelle formulation showed enhanced tumor accumulation regardless of the breast cancer models. In addition, long-circulating nanomedicines did not further increase tumor accumulation in transgenic mouse spontaneous breast cancer nor universally decrease drug accumulations in normal organs; they decreased or increased accumulation in different organs, potentially changing the clinical efficacy/safety. In contrast, short-circulating nanomedicines decreased blood concentration and altered drug distribution in normal organs, which are correlated with their clinical efficacy/safety. A reappraisal of current nanomedicine design criteria is needed to ensure consistent clinical translation for improvement of their clinical efficacy/safety in cancer patients.

Nutritional AGEing and RAGEing as a Regulator of the Tumor Microenvironment

Abstract Objectives Advanced glycation end products (AGEs) are reactive metabolites formed endogenously by glyoxidative, oxidative and lipoxidative stresses. Foods associated with modern dietary habits are particularly AGE laden but despite increasing epidemiological evidence for oncogenic potential, cause and effect relationships are lacking. The objective was to provide detailed mechanistic insight and in vivo confirmation that AGEs found in the diet are oncogenic drivers of tumorigenesis. Methods We used the heat driven formation of glyoxidative, oxidative and lipoxidative stresses in experimental mouse chow to reproduce the wide spectrum of the AGEs found in vivo. Syngeneic xenograft and spontaneous prostate and breast cancer mouse models were then fed the AGE specific diets and the effects of chronic AGE consumption on tumor growth assessed. To gain mechanistic insight, human and mouse two compartment co-culture models using primary fibroblasts and matched tumor epithelial cells were then used to assess the effects of AGEs on extracellular crosstalk in the TME. Results A high impact finding from our research is that consumption of AGEs found in our diet promotes prostate tumor growth, aggression and metastasis by functioning as ligand to the transmembrane receptor for AGE (RAGE). Dietary-AGEs promoted neoplastic growth by functioning as ligand to RAGE expressed in the prostate tumor stroma not tumor epithelium. Dietary-AGE activation of stromal RAGE caused a regulatory program of ‘activated fibroblasts’ defined by the increased expression of cancer associated fibroblast markers, NFkB, MYC and pro-tumorigenic paracrine secretion. Fibroblast activation was accompanied by decreased expression of androgen receptor (AR) and the increased expression of neuroendocrine differentiation markers in tumor epithelial cells. AGE exposed primary fibroblasts isolated from patient tissue conferred tumor promoting abilities when cultured with patient matched tumor epithelial cells. Conclusions For the first time these data demonstrate a direct cause and effect relationship between dietary-AGEs and neoplastic growth. This may lay the foundation for strategic self-management strategies aimed at reducing AGE exposure in the diet to reduce cancer incidence and mortality. Funding Sources NIH/NCI; ACS.

PD1 Blockade of Tumor-primed CD4 T Cells Instigates Protective Immunity Against Tumor Rechallenge.

Abstract Blockade of immune checkpoint PD1 in cancer patients results in remarkable therapeutic benefits. Revealing mechanisms underlying this revolutionary therapy is critical to identifying biomarkers for predicting responses to this treatment and developing novel PD1 blockade combined therapeutic regimens for broader clinical efficacy. Rejuvenation of preexisting intratumoral ‘exhausted’ T cells and recruitment of peripheral antitumor T cells into tumors have been suggested to explain how PD1 blockade works, but mechanisms of action of anti-PD1 therapy remain to be fully delineated. In a clinically relevant mouse model of spontaneous metastatic breast cancer 4T1.2-Neu, we show that effective and durable anti-PD1 therapy depends on systemic immunity, including type 1 conventional dendritic cells (cDC1: CD8α+/CD103+DC), B cells, CD4 and CD8 T cells. Increased circulating and/or tumor-infiltrating IL12+CD103+DC and IL2+CD4/CD8 T cells are associated with the response to anti-PD1 therapy. PD1 blockade results in tumor-primed CD44+IL2+CD4T cells in the periphery regardless of cDC1. In the breast cancer model with adoptive cell transfer (ACT), PD1 blockade-driven CD44+CD4+T cells infiltrate into tumors and ignite protective immunity against new tumors in an IL2-dependent and tumor-specific manner. In a distinct ACT model using enforced IL2-expressing melanoma antigen specific TRP1-transgenic (Tg) CD4 T cells, IL2+TRP1-Tg CD4 T cells control the growth of PD1-resistant endogenous BP or B16 melanoma. These data suggest the prominent role of PD1 blockade on the effectiveness of tumor-primed CD4T cells in tumor-specific immunity.

Human Serum Albumin Decorated Indocyanine Green Improves Fluorescence-Guided Resection of Residual Lesions of Breast Cancer in Mice.

ObjectiveAchieving negative resection margin is critical but challenging in breast-conserving surgery. Fluorescence-guided surgery allows the surgeon to visualize the tumor bed in real-time and to facilitate complete resection. We envisioned that intraoperative real-time fluorescence imaging with a human serum albumin decorated indocyanine green probe could enable complete surgical removal of breast cancer in a mouse model.MethodsWe prepared the probe by conjugating indocyanine green (ICG) with human serum albumin (HSA). In vitro uptake of the HSA-ICG probe was compared between human breast cancer cell line MDA-MB-231 and normal breast epithelial cell line MCF 10A. In vivo probe selectivity for tumors was examined in nude mice bearing MDA-MB-231-luc xenografts and the FVB/N-Tg (MMTV-PyMT) 634Mul/J mice model with spontaneous breast cancer. A positive-margin resection mice model bearing MDA-MB-231-luc xenograft was established and the performance of the probe in assisting surgical resection of residual lesions was examined.ResultsA significantly stronger fluorescence intensity was detected in MDA-MB-231 cells than MCF 10A cells incubated with HSA-ICG. In vivo fluorescence imaging showed that HSA-ICG had an obvious accumulation at tumor site at 24 h with tumor-to-normal tissue ratio of 8.19 ± 1.30. The same was true in the transgenic mice model. The fluorescence intensity of cancer tissues was higher than that of non-cancer tissues (58.53 ± 18.15 vs 32.88 ± 11.34). During the surgical scenarios, the residual tumors on the surgical bed were invisible with the naked eye, but were detected and resected with negative margin under HSA-ICG guidance in all the mice (8/8). Recurrence rate among mice that underwent resection with HSA-ICG (0/8) was significantly lower than the rates among mice with ICG (4/8), as well as the control group under white light (7/7).ConclusionsThis study suggests that real-time in vivo visualization of breast cancer with an HSA-ICG fluorescent probe facilitates complete surgical resection of breast cancer in a mouse xenograft model.

Breast Cancer Promotes Cardiac Dysfunction Through Deregulation of Cardiomyocyte Ca2+-Handling Protein Expression That is Not Reversed by Exercise Training.

BackgroundPatients treated for breast cancer have a high incidence of cardiovascular complications. In this study, we evaluated the impact of breast cancer on cardiac function and cardiomyocyte Ca2+‐handling protein expression. We also investigated whether exercise training (ET) would prevent these potential alterations.Methods and ResultsTransgenic mice with spontaneous breast cancer (mouse mammary tumor virus–polyomavirus middle T antigen [MMTV‐PyMT+], n=15) and littermate mice with no cancer (MMTV‐PyMT−, n=14) were studied. For the ET analysis, MMTV‐PyMT+ were divided into sedentary (n=10) and exercise‐trained (n=12) groups. Cardiac function was evaluated by echocardiography with speckle‐tracking imaging. Exercise tolerance test was conducted on a treadmill. Both studies were performed when the tumor became palpable and when it reached 1 cm3. After euthanasia, Ca2+‐handling protein expression (Western blot) was evaluated. Exercise capacity was reduced in MMTV‐PyMT+ compared with MMTV‐PyMT− (P interaction=0.031). Longitudinal strain (P group <0.001) and strain rate (P group=0.030) were impaired. Cardiomyocyte phospholamban was increased (P=0.011), whereas phospho‐phospholamban and sodium/calcium exchanger were decreased (P=0.038 and P=0.017, respectively) in MMTV‐PyMT+. No significant difference in sarcoplasmic or endoplasmic reticulum calcium 2 ATPase (SERCA2a) was found. SERCA2a/phospholamban ratio was reduced (P=0.007). ET was not associated with increased exercise capacity. ET decreased left ventricular end‐systolic diameter (P group=0.038) and end‐diastolic volume (P group=0.026). Other morphological and functional cardiac parameters were not improved by ET in MMTV‐PyMT+. ET did not improve cardiomyocyte Ca2+‐handling protein expression.ConclusionsBreast cancer is associated with decreased exercise capacity and subclinical left ventricular dysfunction in MMTV‐PyMT+, which is at least partly associated with dysregulation of cardiomyocyte Ca2+ handling. ET did not prevent or reverse these changes.

Abstract PS8-13: Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer

Abstract Background: Hereditary breast cancer is common. With enhanced awareness and the recent introduction in affordable multi-gene germ line testing, an estimated 0.5-1 million women will learn that they carry a considerable risk to develop breast cancer. Thus, a rapidly increasing number of women, many of them very young, will be in need of effective strategies for breast cancer prevention. Current options to prevent breast cancer in women at high risk include bilateral mastectomies or systemic anti-estrogen therapy. Both options, while effective, may have a detrimental impact on the physical and emotional well-being of the patient. Localized delivery of an established anti-estrogen to breast tissue only may thus offer an attractive alternative for cancer prevention and may replace systemic therapy for ductal carcinoma in situ and early stage breast cancer with minimal risk for metastases. Methods: As such, we have sought to develop a silastic-silicone device, which when placed under breast tissue, will deliver the anti-estrogen fulvestrant directly to the target tissue. Sustained slow release of fulvestrant from a silastic-silicone device directly into the mammary tissue will provide the risk reducing benefits of systemic hormonal therapy while minimizing systemic exposure and the resulting poor compliance due to adverse effects. Results: Using a combination of in vitro and in vivo studies, we show that fulvestrant can be delivered through a silastic-silicone device. Implanted adjacent to mammary tissue, this drug delivery device provides sustained high levels of fulvestrant to inhibit estrogen receptor signaling and breast cancer cell proliferation. In a MCF-7 breast cancer xenograft model we have shown that silastic-silicone delivers fulvestrant selectively to mouse mammary tissue for more than 1 year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Using the Sprague-Dawley rat DMBA spontaneous breast cancer induction model, we further demonstrate that fulvestrant delivered by silastic-silicone devices implanted adjacent to mammary tissue significantly delays time to first tumor compared to control animals (n=90, HR = 0.42, 95% CI 0.21-0.83) with minimal systemic exposure (plasma average 1.1 ng/mL, SD ±1.5 ng/mL). Initial large animal safety and toxicity studies in female sheep (ewes, n=2) support the surgical strategy to place the device between breast tissue (i.e. udder) and the chest wall (abdominal wall in ewes). Following 1 month of fulvestrant release, gross and histopathological analysis found no adverse effects or implant related toxicity. Bioanalytical analysis of mammary tissues suggested that drug was found highest near the implant (max 341 ng/g, udder average 58.4 ng/g, SD ±17.1 ng/g), with diminishing levels distal from the implant. Systemic exposure was low, with plasma levels 1.2 ng/mL, SD ±0.22 ng/mL, comparing favorably to clinical plasma levels achieved by intramuscular fulvestrant of 500 mg: 28.0 ng/mL, SD ±27.9 ng/mL (FDA package insert). Subsequent large animal studies will focus on the safety and pharmacokinetics of device delivered fulvestrant in longer studies (e.g. 3 to 6 months). Conclusions: The greater awareness and genetic identification of individuals at risk for breast cancer brings about an increased need for novel approaches to breast cancer prevention. The development of a silastic-silicone based device for sustained and localized drug delivery with an approved and effective anti-estrogen should allow rapid transition into clinical testing. This strategy will provide an alternative option to mastectomies and allow breast conservation for women identified to have a more than 40% lifetime chance of developing breast cancer, as well as provide an alternative to systemic hormonal therapy in women with ductal carcinoma in situ or stage I breast cancer. Citation Format: Pamela N Munster, Pujan K Desai, Nela Pawlowska, Elysia Roche, Romain Pacaud, Maxim A Daud, Emily Hsu, David Gingrich, Amelia Deitchman, Fran Aweeka, Scott Thomas. Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-13.

Gain of HIF1 Activity and Loss of miRNA let-7d Promote Breast Cancer Metastasis to the Brain via the PDGF/PDGFR Axis.

Early detection and adjuvant therapies have significantly improved survival of patients with breast cancer over the past three decades. In contrast, management of metastatic disease remains unresolved. Brain metastasis is a late complication frequently observed among patients with metastatic breast cancer, whose poor prognosis calls for novel and more effective therapies. Here, we report that active hypoxia inducible factor-1 (HIF1) signaling and loss of the miRNA let-7d concur to promote brain metastasis in a recently established model of spontaneous breast cancer metastasis from the primary site to the brain (4T1-BM2), and additionally in murine and human experimental models of breast cancer brain metastasis (D2A1-BM2 and MDA231-BrM2). Active HIF1 and let-7d loss upregulated expression of platelet-derived growth factor (PDGF) B/A in murine and human brain metastatic cells, respectively, while either individual silencing of HIF1α and PDGF-A/B or let-7d overexpression suppressed brain metastasis formation in the tested models. Let-7d silencing upregulated HIF1α expression and HIF1 activity, indicating a regulatory hierarchy of the system. The clinical relevance of the identified targets was supported by human gene expression data analyses. Treatment of mice with nilotinib, a kinase inhibitor impinging on PDGF receptor (PDGFR) signaling, prevented formation of spontaneous brain metastases in the 4T1-BM2 model and reduced growth of established brain metastases in mouse and human models. These results identify active HIF1 signaling and let-7d loss as coordinated events promoting breast cancer brain metastasis through increased expression of PDGF-A/B. Moreover, they identify PDGFR inhibition as a potentially actionable therapeutic strategy for patients with brain metastatis. SIGNIFICANCE: These findings show that loss of miRNA let-7d and active HIF1 signaling promotes breast cancer brain metastasis via PDGF and that pharmacologic inhibition of PDGFR suppresses brain metastasis, suggesting novel therapeutic opportunities. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/594/F1.large.jpg.See related article by Thies et al., p. 606.

Breast cancer is marked by specific, Public T-cell receptor CDR3 regions shared by mice and humans.

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.

Epsins 1 and 2 promote NEMO linear ubiquitination via LUBAC to drive breast cancer development.

Estrogen receptor-negative (ER-negative) breast cancer is thought to be more malignant and devastating than ER-positive breast cancer. ER-negative breast cancer exhibits elevated NF-κB activity, but how this abnormally high NF-κB activity is maintained is poorly understood. The importance of linear ubiquitination, which is generated by the linear ubiquitin chain assembly complex (LUBAC), is increasingly appreciated in NF-κB signaling, which regulates cell activation and death. Here, we showed that epsin proteins, a family of ubiquitin-binding endocytic adaptors, interacted with LUBAC via its ubiquitin-interacting motif and bound LUBAC's bona fide substrate NEMO via its N-terminal homolog (ENTH) domain. Furthermore, epsins promoted NF-κB essential modulator (NEMO) linear ubiquitination and served as scaffolds for recruiting other components of the IκB kinase (IKK) complex, resulting in the heightened IKK activation and sustained NF-κB signaling essential for the development of ER-negative breast cancer. Heightened epsin levels in ER-negative human breast cancer are associated with poor relapse-free survival. We showed that transgenic and pharmacological approaches eliminating epsins potently impeded breast cancer development in both spontaneous and patient-derived xenograft breast cancer mouse models. Our findings established the pivotal role epsins played in promoting breast cancer. Thus, targeting epsins may represent a strategy to restrain NF-κB signaling and provide an important perspective into ER-negative breast cancer treatment.