Prevention Of Triple-negative Breast Cancer Research Articles

Knockout of KDM3A in MDA-MB-231 breast cancer cells inhibits tumor malignancy and promotes apoptosis.

The histone lysine demethylase 3A (KDM3A) is vital for the regulation of cancer physiology and pathophysiology. The purpose of this study was to investigate the effect of KDM3A expression with triple-negative breast cancer (TNBC) invasion and metastasis. In our results, knockout of KDM3A in TNBC MDA-MB-231 cells promoted apoptosis and inhibited the proliferation, invasion and metastasis of MDA-MB-231 cells. In addition, we found that in vivo experiments indicated that the growth, invasion and metastasis of metastatic neoplasms were significantly inhibited by knockout of KDM3A in a TNBC metastasis model. These findings suggest that KDM3A may be a potential therapeutic target for the treatment and prevention of TNBC, providing a critical theoretical basis for the effective prevention or treatment of breast cancer disease.

Tumor nano-lysate activates dendritic cells to evoke a preventative immune response

A tumor nano-lysate “TNL” vaccine comprised of sonicated 4 T1 cells was developed, characterized and implemented for the prevention of triple-negative breast cancer. This study aims to gain a better understanding of the immune response behind the success of the vaccine in vivo, through use of ex vivo and in vivo assays. Here, we analyze the activation of various immune cells isolated from healthy mouse spleens and find that antigen-presenting cells (APCs) such as dendritic cells (DCs) are being activated following 24 h incubation with 1:10 mg TNL/mg splenocytes. These cells were further explored to determine the pathway by which activation is occurring, and it was observed that TNL are phagocytosed by DCs to activate NF-kB and c-Fos pathways, resulting in enhanced cytokine release after 24 h. An in vivo temporal analysis was performed in mice to understand the immune response at 1, 3, 7 and 10 days after one 100 μL dose of TNL consisting of 105 sonicated 4 T1 cells via cardiac puncture and splenocyte and peripheral blood mononuclear cell (PBMC) analysis. Changes were observed for up to one week. A multiple dose study was performed comparing mice that were vaccinated with one dose of TNL administered every ten days for 3 doses total, as well as a PBS vehicle control. Survival for TNL-vaccinated mice was enhanced compared to the PBS control, and there was an average delay of 10 days in the onset of metastasis. The differences between the groups at the end of the study demonstrate the potential for TNL as a preventative therapeutic.

Adipocyte-derived kynurenine stimulates malignant transformation of mammary epithelial cells through the aryl hydrocarbon receptor

Anti-hormone therapies are not efficacious for reducing the incidence of triple negative breast cancer (TNBC), which lacks both estrogen and progesterone receptors. While the etiology of this aggressive breast cancer subtype is unclear, visceral obesity is a strong risk factor for both pre- and post-menopausal cases. The mechanism by which excessive deposition of visceral adipose tissue (VAT) promotes the malignant transformation of hormone receptor-negative mammary epithelial cells is currently unknown. We developed a novel in vitro system of malignant transformation in which non-tumorigenic human breast epithelial cells (MCF-10A) grow in soft agar when cultured with factors released from VAT. These cells, which acquire the capacity for 3D growth, show elevated aryl hydrocarbon receptor (AhR) protein and AhR target genes, suggesting that AhR activity may drive malignant transformation by VAT. AhR is a ligand-dependent transcription factor that generates biological responses to exogenous carcinogens and to the endogenous tryptophan pathway metabolite, kynurenine. The serum kynurenine to tryptophan ratio has been shown to be elevated in patients with obesity. Herein, we demonstrate that AhR inhibitors or knockdown of AhR in MCF-10A cells prevents VAT-induced malignant transformation. Specifically, VAT-induced transformation is inhibited by Kyn-101, an inhibitor for the endogenous ligand binding site of AhR. Mass spectrometry analysis demonstrates that adipocytes metabolize tryptophan and release kynurenine, which is taken up by MCF-10A cells and activates the AhR to induce CYP1B1 and promote malignant transformation. This novel hormone receptor-independent mechanism of malignant transformation suggests targeting AhR for TNBC prevention in the context of visceral adiposity.

Docking and Molecular Dynamics Simulation Revealed the Potential Inhibitory Activity of Amygdalin in Triple-Negative Breast Cancer Therapeutics Targeting the BRCT Domain of BARD1 Receptor.

Triple-negative breast cancer (TNBC), is diagnosed as the most lethal molecular subtype of breast cancer (BC) preceded by an extremely poor prognosis. For enabling effective TNBC therapy, the identification of novel druggable biomarkers is an earnest need. Multigene paneling and genomewide association studies identify multiple genes with high-to-moderate penetrance in TNBC. Modern computer-aided drug designing techniques, thus aim to design more cost-effective natural small molecule inhibitors for TNBC prevention and diagnosis. Here Amygdalin, a natural glycosidic inhibitor is docked and simulated against three such high-to-moderate penetrance genes identified in TNBC, BARD1, RAD51, and PALB2. The preliminary result of the analysis, reports a highest, intermediate, and least binding energy score of -6.69kcal/mol, -5.09kcal/mol, and -4.89kcal/mol in BARD1, RAD51, and PALB2, respectively. The best-docked protein-ligand complex (BARD1-Amygdalin) was then simulated and compared with an approved drug for TNBC treatment, Olaparib. A comparable binding energy score of -8.53kcal/mol was obtained by docking olaparib with BARD1. A 100ns MD simulation revealed, Amygdalin forms more H-bonds, providing more stable and compact protein-ligand complex with BARD1 than compared to Olaparib. The result was also supported by calculation of solvent accessible surface area and analysis of radius of gyration. Thus, our findings suggest that role of Amygdalin can further be studied in details for TNBC therapeutics, which was found to target the BRCT domain of the BARD1 receptor in stable manner. Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary. Name and affiliations are correctly identified.

Abstract P004: Targeting the RXR pathway for the prevention of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor (ER), progesterone receptor, and Erb-B2 receptor tyrosine kinase 2. TNBC patients exhibit a poor prognosis, even if treated with chemotherapy. Although studies using selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) have shown that breast cancer prevention is feasible, these drugs do not prevent ER-negative or TNBC tumors. It has been shown by our laboratory and others that retinoid X receptor (RXR)-specific ligands (rexinoids) can prevent breast cancers that are both ER-positive and ER-negative in mice. In our previous studies in MMTV-erbB2 mice, IRX-4204, a fourth generation rexinoid, prevented the development of most HER2/erbB2-positive tumors in these mice. For this study, we hypothesized that by targeting RXR pathway, we can prevent the development of triple negative, BRCA1-mutant mammary tumors in mice. To test the hypothesis, we treated MMTV-Cre/BRCA1co/co/p53+/- mice prior to their developing tumors with IRX-4204 to determine whether this RXR agonist effectively prevents triple-negative breast tumors. Methods: We tested the tumor preventative effect of IRX-4204 using the established MMTV-Cre/BRCA1co/co/p53+/- mouse model. These mice were produced by breeding MMTV-Cre/BRCA1co/co/p53+/- males and MMTV-Cre/BRCA1co/co/p53+/+ females, and female pups were PCR genotyped. All mice were separated into 4 groups: 1) sesame oil control, 2) IRX-4204 (10 mg/kg), 3) IRX-4204 (20 mg/kg) and 4) 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage, five days a week from 4 months of age. Mice were observed daily for tumor formation and toxicity. The percentage of tumor free mice were recorded, from which tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the Log-rank test. Results: In MMTV-Cre/BRCA1co/co/p53+/- mice, IRX-4204 reduced tumor incidence and was associated with an increase in median tumor free survival time from 209 days to 336 days at 10 mg/kg dose (p=0.005). At the higher dose (20 mg/kg) IRX-4204 also delayed tumor formation with median tumor free survival from 209 days to 260 days (p=0.039). The rexinoid 9-cis-UAB 30 also significantly delayed tumor formation in MMTV-Cre/BRCA1co/co/p53+/- mice with median survival from 209 days to 270 days (p=0.04). Long term treatment of IRX-4204 was not associated with any toxicity. Conclusion: RXR agonist IRX-4204 delayed ER-negative mammary tumor formation in BRCA1co/co; MMTV-Cre; p53+/- mice. Based on our results, IRX-4204 is an effective cancer preventive drug without observed toxicity. Our results suggest that studies with reduced IRX-4204 dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the IRX-4204 should be considered for the prevention of breast cancer in high-risk patients. (Supported by NCI-PREVENT contract to P. Brown and A. Mazumdar HHSN26100008). Citation Format: Cassandra Moyer, Jamal Hill, Darian Coleman, Shizuko Sei, Altaf Mohammed, Martin Sanders, Powel Brown, Abhijit Mazumdar. Targeting the RXR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P004.

Abstract IA017: Targeting the mTOR pathway for the prevention of triple-negative breast cancer

Abstract Phase III cancer prevention clinical trials have shown that breast cancer prevention is feasible using anti-estrogen drugs. However, these drugs have not been widely accepted because of concerns about toxicity. In addition, anti-estrogen drugs do not prevent estrogen-negative breast cancers, including the most aggressive form of breast cancer, triple-negative breast cancer (TNBC), that does not express the estrogen receptor (ER), progesterone receptor, or the human epidermal growth factor receptor 2 (HER2). Our laboratory is focused on identifying growth-regulatory molecules that are essential for the growth of TNBCs. For this study, we identified mTOR as an essential growth-regulatory molecule that is highly expressed in TNBCs. We then investigated whether the mTOR inhibitor everolimus can prevent mammary tumors in transgenic mouse models including 4 models of TNBC and one model of ER-negative/HER2-positive breast cancer. Everolimus treatment significantly delayed mammary tumor formation but with a varying degree in all five mouse models. Everolimus treatment for up to 1 year was well tolerated with no observable toxicity. These results suggest that mTOR inhibitors may be promising drugs for the prevention of ER-negative and triple-negative breast cancers in women at risk of these aggressive breast cancers. Grant support: This research was supported by an NCI-PREVENT contract to P. Brown and A. Mazumdar (HHSN261201500018I/HHSN26100006). Citation Format: Powel H. Brown, Abhijit Mazumdar, William Tahaney, Jamal Hill, Yun Zhang, Sumankalai Ramachandran, Jitesh Kawedia, Jing Qian, Alejandra Contreras, Michelle Savage, Lana Vornik. Targeting the mTOR pathway for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA017.

Baicalein sensitizes triple negative breast cancer MDA-MB-231 cells to doxorubicin via autophagy-mediated down-regulation of CDK1.

Triple negative breast cancer (TNBC) is a kind of refractory cancer with poor response to conventional chemotherapy. Recently, the combination of baicalein and doxorubicin was reported to exert a synergistic antitumor effect on breast cancer. However, the underlying mechanism how baicalein sensitizes breast cancer cells to doxorubicin remains to be elucidated. Here, it was found that 20μM baicalein increased the autophagy markers including the ratio of LC3B II/I, GFP-LC3 punctate aggregates and down-regulation of p62 expression, and up-regulated mitophagy marker PINK1 and Parkin in TNBC MDA-MB-231 cells as well. In contrast, doxorubicin decreased the levels of autophagy markers, and significantly up-regulated CDK1 in MDA-MB-231 cells. Pretreatment with baicalein markedly inhibited the doxorubicin-induced decrease in autophagy markers and up-regulation of CDK1, which was reversed by the autophagy inhibitor 3-Methyladenine. Moreover, baicalein alleviated the doxorubicin-induced expression and phosphorylation (at Ser616) of mitochondrial fission protein Drp1. Intriguingly, the autophagy inhibitor 3-Methyladenine also significantly weakened the effect of baicalein on doxorubicin-induced viability decrease and apoptosis in MDA-MB-231 cells. Taken together, our data indicate that baicalein improves the chemosensitivity of TNBC cells to doxorubicin through promoting the autophagy-mediated down-regulation of CDK1, also suggest a novel strategy for prevention of TNBC in the future.

Thymoquinone Anticancer Effects Through the Upregulation of NRF2 and the Downregulation of PD-L1 in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a metastatic nature. TNBC lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor receptor 2. The absence of these three receptors limits the therapy options. Meanwhile, conventional medication is not very effective in the treatment and prevention of TNBC. Developing innovative therapeutic agents from natural bioactive compounds is a viable option. In the current study, the natural compound thymoquinone (TQ) was used in MDA-MB-231 TNBC cells. A dose response to TQ (0-50 µM) was obtained following a 24-hour exposure. An Annexin V-FITC apoptosis detection was used to study the apoptotic effect of TQ. With increasing TQ concentrations from 0-20 µM, flow cytometer examination revealed an increase in apoptotic cells. Nearly 80% of the cells studied were in the apoptotic phase at 20 µM. (early and late apoptosis). Meanwhile, at 30 µM, there was a significant drop in apoptotic cells, which may be interpreted as an increase in necrotic cells. Molecular-targeted therapy is a new approach in treating cancer. The effect of TQ on the expression of Nrf2(Nuclear factor erythroid 2- related factor 2) and PD-L1 (Programmed death-ligand 1) was investigated using a specific primary antibody against these proteins. A Western blot analysis confirmed TQ's ability to change the expression of both proteins under investigation. According to normalized data, TQ had the ability to elicit more than 2-fold increase in Nrf2 expression in IFN-γ stimulated MDA-MB-231 cells. In contrast, co-treated cells (IFN-γ + TQ) showed a 65 percent reduction in PD-L1 expression. In conclusion, TQ was suggested as a promising anticancer option for treating TNBC.

Elevated expression of the rhythm gene NFIL3 promotes the progression of TNBC by activating NF-\u03baB signaling through suppression of NFKBIA transcription

BackgroundEpidemiological studies have confirmed that abnormal circadian rhythms are associated with tumorigenesis in breast cancer. However, few studies have investigated the pathological roles of rhythm genes in breast cancer progression. In this study, we aimed to evaluate the aberrant expression of 32 rhythm genes in breast cancer and detect the pathological roles and molecular mechanisms of the altered rhythm gene in regulating the progression of triple negative breast cancer (TNBC).MethodsThe aberrant expression of rhythm genes in breast cancer was screened by searching the GEPIA database and validated by using qRT-PCR and immunohistochemistry staining. Bioinformatics analysis combined with luciferase reporter experiment and chromatinimmunopercitation (ChIP) were used to investigate the molecular mechanism about aberrant expression of identified rhythm gene in breast cancer. The pathological roles of identified rhythm gene in TNBC progression was evaluated by colony formation assay, wound healing experiment, transwell assay, subcutaneous tumor formation and the mouse tail vein injection model through gain-of-function and loss-of-function strategies respectively. mRNA array, bioinformatics analysis, luciferase reporter experiment, ChIP and immunoflurescence assay were employed to investigate the key molecules and signaling pathways by which the identified rhythm gene regulating TNBC progression.ResultsWe identified that nuclear factor interleukin 3 regulated (NFIL3) expression is significantly altered in TNBC compared with both normal breast tissues and other subtypes of breast cancer. We found that NFIL3 inhibits its own transcription, and thus, downregulated NFIL3 mRNA indicates high expression of NFIL3 protein in breast cancer. We demonstrated that NFIL3 promotes the proliferation and metastasis of TNBC cells in vitro and in vivo, and higher expression of NFIL3 is associated with poor prognosis of patients with TNBC. We further demonstrated that NFIL3 enhances the activity of NF-κB signaling. Mechanistically, we revealed that NFIL3 directly suppresses the transcription of NFKBIA, which blocks the activation of NF-κB and inhibits the progression of TNBC cells in vitro and in vivo. Moreover, we showed that enhancing NF-κB activity by repressing NFKBIA largely mimics the oncogenic effect of NFIL3 in TNBC, and anti-inflammatory strategies targeting NF-κB activity block the oncogenic roles of NFIL3 in TNBC.ConclusionNFIL3 promotes the progression of TNBC by suppressing NFKBIA transcription and then enhancing NF-κB signaling-mediated cancer-associated inflammation. This study may provide a new target for TNBC prevention and therapy.Graphical

Propolin G-Suppressed Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells via Glycogen Synthase Kinase 3β-Mediated Snail and HDAC6-Regulated Vimentin Degradation.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with a poor prognosis. The incidence and mortality rate of TNBC are frequently found in younger women. Due to the absence of a good therapeutic strategy, effective remedies for inhibiting TNBC have been developed for improving the cure rate. Epithelial-to-mesenchymal transition (EMT) is a critical mechanism to regulate cancer cell motility and invasion. Furthermore, ectopic expression of EMT molecules correlates with the metastasis and poor prognosis of TNBC. Targeting EMT might be a strategy for the therapy and prevention of TNBC. Propolin G, an active c-prenylflavanone in Taiwanese propolis, has been shown to possess anti-cancer activity in many cancers. However, the anti-metastasis activity of propolin G on TNBC is still unclear. The present study showed that the migration and invasion activities of TNBC cells was suppressed by propolin G. Down-regulated expression of Snail and vimentin and up-regulated expression of E-cadherin were dose- and time-dependently observed in propolin G-treated MDA-MB-231 cells. Propolin G inhibited Snail and vimentin expressions via the signaling pathways associated with post-translational modification. The activation of glycogen synthase kinase 3β (GSK-3β) by propolin G resulted in increasing GSK-3β interaction with Snail. Consequently, the nuclear localization and stability of Snail was disrupted resulting in promoting the degradation. Propolin G-inhibited Snail expression and the activities of migration and invasion were reversed by GSK-3β inhibitor pretreatment. Meanwhile, the outcomes also revealed that histone deacetylase 6 (HDAC6) activity was dose-dependently suppressed by propolin G. Correspondently, the amounts of acetyl-α-tubulin, a down-stream substrate of HDAC6, were increased. Dissociation of HDAC6/Hsp90 with vimentin leading to increased vimentin acetylation and degradation was perceived in the cells with the addition of propolin G. Moreover, up-regulated expression of acetyl-α-tubulin by propolin G was attenuated by HDAC6 overexpression. On the contrary, down-regulated expression of vimentin, cell migration and invasion by propolin G were overturned by HDAC6 overexpression. Conclusively, restraint cell migration and invasion of TNBC by propolin G were activated by the expression of GSK-3β-suppressed Snail and the interruption of HDAC6-mediated vimentin protein stability. Aiming at EMT, propolin G might be a potential candidate for TNBC therapy.

Therapeutic Potential of Thymoquinone in Triple-Negative Breast Cancer Prevention and Progression through the Modulation of the Tumor Microenvironment.

To date, the tumor microenvironment (TME) has gained considerable attention in various areas of cancer research due to its role in driving a loss of immune surveillance and enabling rapid advanced tumor development and progression. The TME plays an integral role in driving advanced aggressive breast cancers, including triple-negative breast cancer (TNBC), a pivotal mediator for tumor cells to communicate with the surrounding cells via lymphatic and circulatory systems. Furthermore, the TME plays a significant role in all steps and stages of carcinogenesis by promoting and stimulating uncontrolled cell proliferation and protecting tumor cells from the immune system. Various cellular components of the TME work together to drive cancer processes, some of which include tumor-associated adipocytes, fibroblasts, macrophages, and neutrophils which sustain perpetual amplification and release of pro-inflammatory molecules such as cytokines. Thymoquinone (TQ), a natural chemical component from black cumin seed, is widely used traditionally and now in clinical trials for the treatment/prevention of multiple types of cancer, showing a potential to mitigate components of TME at various stages by various pathways. In this review, we focus on the role of TME in TNBC cancer progression and the effect of TQ on the TME, emphasizing their anticipated role in the prevention and treatment of TNBC. It was concluded from this review that the multiple components of the TME serve as a critical part of TNBC tumor promotion and stimulation of uncontrolled cell proliferation. Meanwhile, TQ could be a crucial compound in the prevention and progression of TNBC therapy through the modulation of the TME.

Current Advancements of Plant-Derived Agents for Triple-Negative Breast Cancer Therapy through Deregulating Cancer Cell Functions and Reprogramming Tumor Microenvironment.

Triple-negative breast cancer (TNBC) is defined based on the absence of estrogen, progesterone, and human epidermal growth factor receptor 2 receptors. Currently, chemotherapy is the major therapeutic approach for TNBC patients; however, poor prognosis after a standard chemotherapy regimen is still commonplace due to drug resistance. Abnormal tumor metabolism and infiltrated immune or stromal cells in the tumor microenvironment (TME) may orchestrate mammary tumor growth and metastasis or give rise to new subsets of cancer cells resistant to drug treatment. The immunosuppressive mechanisms established in the TME make cancer cell clones invulnerable to immune recognition and killing, and turn immune cells into tumor-supporting cells, hence allowing cancer growth and dissemination. Phytochemicals with the potential to change the tumor metabolism or reprogram the TME may provide opportunities to suppress cancer metastasis and/or overcome chemoresistance. Furthermore, phytochemical intervention that reprograms the TME away from favoring immunoevasion and instead towards immunosurveillance may prevent TNBC metastasis and help improve the efficacy of combination therapies as phyto-adjuvants to combat drug-resistant TNBC. In this review, we summarize current findings on selected bioactive plant-derived natural products in preclinical mouse models and/or clinical trials with focus on their immunomodulatory mechanisms in the TME and their roles in regulating tumor metabolism for TNBC prevention or therapy.

Growth inhibitory efficacy of the nutritional herb Psoralea corylifolia in a model of triple‑negative breast cancer

Triple‑negative breast cancer (TNBC) lacks the expression of estrogen receptor‑α (ER‑α), progesterone receptor (PR) and amplified human epidermal growth factor receptor‑2 (HER‑2). Current treatments involve chemotherapy and molecular targeted therapy. These options lead to dose‑limiting systemic toxicity and acquired tumor resistance. Moreover, no drug is yet available for the secondary prevention of TNBC. Such limitations underscore a need to investigate non‑toxic testable alternatives for the secondary prevention/treatment of TNBC. The Chinese nutritional herb, <em>Psoralea corylifolia</em> (PC), has been shown to be effective against the MCF‑7 cell line, a cellular model for ER‑α‑positive breast cancer. The present study aimed to investigate the growth inhibitory effects of PC against TNBC using the MDA‑MB‑231 cell line. Anchorage‑independent colony formation, cell cycle progression, cellular apoptosis, RB signaling and caspase‑3/7 activity assays were performed to determine the quantitative parameters for the efficacy of PC. Treatment with PC induced a 3.5‑fold increase in the G₁: S + G₂/M ratio, an 11‑fold increase in the apoptotic cell number and a 149‑fold increase in caspase‑3/7 activity. PC treatment also decreased cyclin D1, CDK4/6 and p‑RB expression levels. On the whole, the data of the present study identify mechanistic leads for PC as a naturally occurring testable alternative, and validate a mechanistic approach to prioritize efficacious nontoxic herbal extracts as nutritional supplements for the secondary prevention/therapy of TNBC.

Efficacy of fluvastatin and aspirin for prevention of hormonally insensitive breast cancer

PurposePrimary prevention of hormonally insensitive breast cancers remains an important clinical need and repurposing existing low-toxicity drugs represents a low-cost, efficient strategy for meeting this goal. This study targeted the cholesterol pathway using fluvastatin, a cholesterol-lowering drug, and aspirin, an AMPK activator that acts as a brake in the cholesterol pathway, in a transgenic mouse model of triple-negative breast cancer (TNBC).MethodsUsing SV40C3 TAg mice, the efficacy and mechanism of fluvastatin, aspirin, or both in combination were compared with vehicle alone.ResultsSixteen-weeks of fluvastatin treatment resulted in significant delay in onset of tumors (20 weeks vs. 16.8 weeks in vehicle treatment, p = 0.01) and inhibited tumor incidence and tumor multiplicity by 50% relative to the vehicle control. In animals that developed tumors, fluvastatin treatment inhibited tumor weight by 75% relative to vehicle control. Aspirin alone did not significantly affect tumor latency, tumor incidence or tumor burden compared to vehicle control. Fluvastatin and aspirin in combination delayed the onset of tumors but failed to inhibit tumor incidence and tumor multiplicity. The growth-inhibitory effects of fluvastatin were mediated through increased FAS/FASL mediated apoptotic cell death that was characterized by increased cleaved PARP and driven in part by depletion of an isoprenoid, geranyl geranyl pyrophosphate (GGPP).ConclusionsIn line with NCI’s emphasis to repurpose low-toxicity drugs for prevention of cancer, fluvastatin was effective for prevention of TNBC and warrants further clinical testing. Aspirin did not provide chemopreventive benefit.

Interface between obesity with dysfunctional metabolism and inflammation, and the triple-negative breast cancer in African American women.

Obesity has dramatically increased over the past fifty years. In the last decade, it has been noted that augmented body mass, metabolic abnormalities, and the relevant “obese” tumor microenvironment (TME) are connected with signaling molecular networks, which in turn, may contribute to aggressive tumor biology in some patients with breast malignancies. This article presents the associations between obesity, metabolic derangements, inflammatory processes in the adipose tissue or TME, and aggressive behavior of triple-negative breast cancer (TNBC) in African American (AA) women. It also describes some abnormal molecular signaling patterns in the “obese” TME with relevance to TNBC biology. Ethnic disparities in TNBC can be due to a variety of biological features (e.g., genetic mutations and tumor heterogeneity), comorbidities (e.g., cardio-metabolic diseases, including diabetes mellitus), and reproductive factors (e.g., multiparty or short breastfeeding period). Such a constellation of biological variables potentially leads to the association between obesity, metabolic derangements, inflammatory processes in the adipose tissue or TME, and aggressive behavior of TNBC in AA women. Since the TNBC and its TME can display very aggressive behavior, it is crucial that the afflicted AA women make efforts to maintain healthy body weight, “flexible” metabolism, and a well-functioning immune system. Further studies are merited to explore the multi-disciplinary factors that can affect TNBC prevention, management, and outcomes to optimize treatment strategies and survival among AA women.

Do Olive and Fish Oils of the Mediterranean Diet Have a Role in Triple Negative Breast Cancer Prevention and Therapy? An Exploration of Evidence in Cells and Animal Models.

Breast cancer is the most common malignancy and cause of cancer-related mortality among women worldwide. Triple negative breast cancers (TNBC) are the most aggressive and lethal of the breast cancer molecular subtypes, due in part to a poor understanding of TNBC etiology and lack of targeted therapeutics. Despite advances in the clinical management of TNBC, optimal treatment regimens remain elusive. Thus, identifying interventional approaches that suppress the initiation and progression of TNBC, while minimizing side effects, would be of great interest. Studies have documented an inverse relationship between the incidence of hormone receptor negative breast cancer and adherence to a Mediterranean Diet, particularly higher consumption of fish and olive oil. Here, we performed a review of studies over the last 5 years investigating the effects of fish oil, olive oil and their components in model systems of TNBC. We included studies that focused on the fish oil ω-3 essential fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in addition to olive oil polyphenolic compounds and oleic acid. Both beneficial and deleterious effects on TNBC model systems are reviewed and we highlight how multiple components of these Mediterranean Diet oils target signaling pathways known to be aberrant in TNBC including PI3K/Akt/mTOR, NF-κB/COX2 and Wnt/β-catenin.

Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

The mechanisms through which tumor cells genetically lose antigenicity and evade immune checkpoints remain largely elusive. Here, we report that tissue-specific expression of the human long-noncoding RNA LINK-A in mouse mammary glands initiated metastatic mammary gland tumors, which phenotypically resembled human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein–coupled receptor (GPCR) pathways, attenuating protein kinase A (PKA)-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48–polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A-locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb, and p53, and sensitized mammary gland tumors to immune checkpoint blockers (ICBs). Importantly, PD-1 blockade-resistant TNBC patients exhibited elevated LINK-A levels and downregulated PLC components. Hence, we demonstrated lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which may provide the basis for developing a therapeutic regimen of combinational immunotherapy and effective early prevention for TNBCs.

Abstract P6-12-02: isomiR-140-3p-regulated mevalonic acid pathway as a potiental target for prevention of triple negative breast cancer

Abstract Background: Prevention of triple negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis. Methods: In order to identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represents early stages of TNBC development. We identified direct gene targets of an isomiRNA and using cell based and in vivo model systems we demonstrate the utility of targeting downstream pathways for prevention of TNBC. Results: These analyses showed that 5'isomiRNA of miR-140-3p (miR-140-3p-1) were deregulated in the normal-to- preneoplastic transition. We also identified novel direct gene targets of miR140-3p-1, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, and found that these too are deregulated in the normal to preneoplastic transition, resulting in activation of the cholesterol synthesis pathway. Upregulation in the cholesterol pathway creates a metabolic vulnerability that can be targeted with the statin class of inhibitors. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin inhibits growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin treatment induced HMGCR transcript levels in the explanted xenografts, with higher transcript induction directly correlated with the degree of histological progression of lesions, indicating an adaptive resistance mechanism that circumvents statin targeting of HMGCR. Therefore, we hypothesized that dual targeting of HMGCR and the feedback loops are necessary to overcome this adaptive resistance. To test this hypothesis, we generated MCF10AT1 cells resistant to fluvastatin. We then treated these resistance AT1 cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin effectively abrogated the statin-induced upregulation of HMGCR and sensitized these resistant cells to fluvastatin. Conclusions: Our preclinical data suggests that the statin treatment induced homeostatic upregulation of the cholesterol pathway is a barrier to effective chemoprevention with statins. However, our results also suggest that this adaptive resistance mechanism can be abrogated by combined treatment with statin and AMPK activators such as aspirin. Clinical studies of this combined regimen should be considered in high risk patients. Citation Format: Bhardwaj A, Singh H, Trinidad CM, Albarracin C, Hunt KK, Bedrosian I. isomiR-140-3p-regulated mevalonic acid pathway as a potiental target for prevention of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-12-02.

Germline TP53 and MSH6 mutations implicated in sporadic triple-negative breast cancer (TNBC): a preliminary study

BackgroundGermline BRCA1/2 prevalence is relatively low in sporadic triple-negative breast cancer (TNBC). We hypothesized that non-BRCA genes may also have significant germline contribution to Chinese sporadic TNBC, and the somatic mutational landscape of TNBC may vary between ethnic groups. We therefore conducted this study to investigate germline and somatic mutations in 43 cancer susceptibility genes in Chinese sporadic TNBC.Patients and methodsSixty-six Chinese sporadic TNBC patients were enrolled in this study. Germline and tumor DNA of each patient were subjected to capture-based next-generation sequencing using a 43-gene panel. Standard bioinformatic analysis and variant classification were performed to identify deleterious/likely deleterious germline mutations and somatic mutations. Mutational analysis was conducted to identify significantly mutated genes.ResultsDeleterious/likely deleterious germline mutations were identified in 27 (27/66, 40.9%) patients. Among the 27 patients, 9 (9/66, 13.6%) were TP53 carriers, 5 (5/66, 7.6%) were MSH6 carriers, and 5 (5/66, 7.6%) were BRCA1 carriers. Somatic mutations were identified in 64 (64/66, 97.0%) patients. TP53 somatic mutations occurred in most of the patients (45/66, 68.2%) and with highest mean allele frequency (28.1%), while NF1 and POLE were detected to have the highest mutation counts.ConclusionsOur results supported our hypotheses and suggested great potentials of TP53 and MSH6 as novel candidates for TNBC predisposition genes. The high frequency of somatic NF1 and POLE mutations in this study showed possibilities for clinical benefits from androgen-blockade therapies and immunotherapies in Chinese TNBC patients. Our study indicated necessity of multi-gene testing for TNBC prevention and treatment.

The isomiR-140-3p-regulated mevalonic acid pathway as a potential target for prevention of triple negative breast cancer

BackgroundPrevention of triple-negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis.MethodsTo identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represent early stages of TNBC development. We have identified direct gene targets of isomiRNA-140-3p and by using cell-based and in vivo model systems we have demonstrated the utility of targeting downstream pathways for prevention of TNBC.ResultsThese analyses showed that 5’isomiRNA of miR-140-3p (miR-140-3p-1) and its novel direct gene targets, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, were deregulated in the normal-to-preneoplastic transition. Upregulation in the cholesterol pathway creates metabolic vulnerability that can be targeted. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin to inhibit growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin-induced HMGCR transcript levels to be directly correlated with the degree of histological progression of lesions, indicating that the extent of cholesterol pathway suppression directly correlates with abrogation of the tumorigenic process. To block the HMGCR feedback response to statins, we treated resistant preneoplastic cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin and metformin effectively abrogated the statin-induced aberrant upregulation of HMGCR and sensitized these resistant cells to fluvastatin.ConclusionsThese results suggest the potential use of combined treatment with statin and aspirin for prevention of TNBC.