Inhibits Triple-negative Breast Cancer Growth Research Articles

Injectable Autocatalytic Hydrogel Triggers Pyroptosis to Stimulate Anticancer Immune Response for Preventing Postoperative Tumor Recurrence.

Modulating immunosuppression while eliminating residual microscopic tumors is critical for inhibiting the postoperative recurrence of triple-negative breast cancer (TNBC). Although immunotherapy has shown potential in achieving this goal, due to multiple immunosuppression and poor immunogenicity of apoptosis, a satisfactory anti-recurrence effect still faces the challenge. Herein, an injectable hydrogel-encapsulated autocatalytic copper peroxide (CP@Gel) therapeutic platform is designed and combine it with the clinical-grade DNA methyltransferase inhibitor decitabine (DAC) to effectively inhibit TNBC growth and postoperative recurrence via pyroptosis, killing residual cancer cells that bypass apoptosis resistance while also improving immunogenicity and modulating immunosuppression to achieve an intense anti-tumor immune response. Following injection of the CP@Gel, the sustained release of CP leads to the autocatalytic generation of reactive oxygen species, resulting in caspase-3 activation, and the pre-administered DAC inhibits the methylation of Gsdme to elevate the GSDME protein levels, leading to intense pyroptosis and anti-tumor immune responses. The in vivo results show a 67% elimination of local tumor recurrence via treatment with DAC+CP@Gel, suggesting the successful integration of sustained drug release with autocatalysis and epigenetic modification. The results thus suggest great potential for pyroptosis-based and injectable hydrogel-aided strategies for preventing the postoperative recurrence of TNBC.

ASAH1 facilitates TNBC by DUSP5 suppression-driven activation of MAP kinase pathway and represents a therapeutic vulnerability

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is prone to metastasis and therapy resistance. Owing to its aggressive nature and limited availability of targeted therapies, TNBC is associated with higher mortality as compared to other forms of breast cancer. In order to develop new therapeutic options for TNBC, we characterized the factors involved in TNBC growth and progression. Here, we demonstrate that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in TNBC cells and is regulated via p53 and PI3K-AKT signaling pathways. Genetic knockdown or pharmacological inhibition of ASAH1 suppresses TNBC growth and progression. Mechanistically, ASAH1 inhibition stimulates dual-specificity phosphatase 5 (DUSP5) expression, suppressing the mitogen-activated protein kinase (MAPK) pathway. Furthermore, pharmacological cotargeting of the ASAH1 and MAPK pathways inhibits TNBC growth. Collectively, we unmasked a novel role of ASAH1 in driving TNBC and identified dual targeting of the ASAH1 and MAPK pathways as a potential new therapeutic approach for TNBC treatment.

Abstract 5697: MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production

Abstract Despite the progress in endocrine therapy, targeted therapy and immunotherapy, the treatment of triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer, is challenging. This is closely associated with the lack of effective molecular targets in TNBC cells for therapeutic intervention. Here we report that the long noncoding RNA (lncRNA) MILIP drives TNBC cell survival, proliferation and tumorigenicity through complexing with transfer RNAs (tRNAs) to promote protein production, and thus represents a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, yet MILIP silencing diminished TNBC cell viability and retarded TNBC xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its well-established role in repressing p53 in other types of cancers. Mechanistical investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with tRNALeu and tRNASer, type II tRNAs, through their variable loops, thus facilitating the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein production and cell viability, with therapeutic potential revealed where targeting MILIP using Gapmers inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identified MILIP as an RNA translation elongation factor to promote protein production in TNBC cells and demonstrated that experimental therapy with MILIP Gapmers inhibits TNBC growth, indicating MILIP targeting is a potential avenue for developing improved TNBC treatment through blocking protein synthesis. Citation Format: Yuchen Feng, Simin Zheng, Xiaohong Zhao, Yuanyuan Zhang, Ran Xu, Liang Xu, Thomas Preiss, Lei Jin, Jinnan Gao, Xu Dong Zhang. MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5697.

The Streptococcus virulence protein PepO triggers anti-tumor immune responses by reprograming tumor-associated macrophages in a mouse triple negative breast cancer model

BackgroundThe efficacy of current surgery and chemotherapy for triple negative breast cancer (TNBC) is limited due to heterogenous and immunosuppressive tumor microenvironment (TME). Tumor associated macrophages (TAMs), which are regarded as an M2 tumor-promoting phenotype, are crucial in the development of the immunosuppressive TME. Targeting TAM reprograming is a promising strategy in anti-tumor therapy since reprogramming techniques provide the opportunity to actively enhance the antitumor immunological activity of TAM in addition to eliminating their tumor-supportive roles, which is rarely applied in TNBC clinically. However, how to drive M2 macrophages reprogramming into M1 with high potency remains a challenge and the molecular mechanisms how M2 macrophages polarized into M1 are poorly understood. Here, we identified a new immunoregulatory molecular PepO that was served as an immunoregulatory molecule governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells and represented an effective anti-tumor property.ResultsAt the present study, we identified a new immunoregulatory molecular PepO, as a harmless immunoregulatory molecule, governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells efficiently. PepO-primed M2 macrophages decreased the expression of tumor-supportive molecules like Arg-1, Tgfb, Vegfa and IL-10, and increased the expression of iNOS, Cxcl9, Cxcl10, TNF-α and IL-6 to inhibit TNBC growth. Moreover, PepO enhanced the functions of macrophages related to cell killing, phagocytosis and nitric oxide biosynthetic process, thereby inhibiting the development of tumors in vivo and in vitro. Mechanistically, PepO reprogramed TAMs toward M1 by activating PI3K-AKT-mTOR pathway via TLR4 and suppressed the function of M2 by inhibiting JAK2-STAT3 pathway via TLR2. The PI3K inhibitor LY294002 abrogated the role of PepO in switching M2 macrophages into M1 and in inhibiting TNBC growth in vivo. And PepO failed to govern the M2 macrophages to reprogram into M1 macrophages and inhibit TNBC when TLR2 or TLR4 was deficient. Moreover, PepO enhanced the antitumor activity of doxorubicin and the combination exerted a synergistic effect on TNBC suppression.ConclusionsOur research identified a possible macrophage-based TNBC immunotherapeutic approach and suggested a novel anticancer immunoregulatory molecular called PepO.

MicroRNA-Mediated Mitochondrial Dysfunction Is Involved in the Anti-triple-Negative Breast Cancer Cell Activity of Phytosesquiterpene Lactones.

Aims: Emerging evidence suggests that modulating redox homeostasis through targeting mitochondrial functions may be a useful strategy for suppressing triple-negative breast cancer (TNBC) activities. However, whether there are specific microRNAs (miRNAs) involved in regulating oxidative stress-associated mitochondrial functions that can act as therapeutic targets to suppress TNBC activities remains unclear. Here, we aimed to identify the role of redox-associated miRNAs in TNBC and investigated their potential as therapeutic targets. Results: We identified oxidative stress-responsive differentially expressed miRNAs (DEMs) regulated by phytosesquiterpene lactone deoxyelephantopin (DET) and its novel derivative DETD-35, which are known to inhibit TNBC growth and metastasis in vitro and in vivo, using comparative miRNA microarray analysis and reactive oxygen species (ROS) scavenging approaches. Mitochondrial dysfunction was identified as a major biological function regulated by a few specific DEMs. In particular, miR-4284 was identified to play a role in DET- and DETD-35-mediated ROS production, mitochondrial basal proton leak, and antiproliferation activity in TNBC cells. Moreover, DET- and DETD-35-induced mitochondrial DNA damage was observed in TNBC cells and xenograft tumors. miR-4284 was also identified to play a role in oxidative DNA damage in TNBC tumors. Innovation: We identified a novel role for miR-4284 in regulating mitochondrial basal proton leak in TNBC cells, and highlighted its significance in TNBC tumor oxidative DNA damage, and its direct correlation with TNBC patient survival. Conclusion: We used DET and DETD-35 as proof of concept to demonstrate that activities of anticancer agents can involve regulation of multiple miRNAs playing different roles in cancer progression. Antioxid. Redox Signal. 38, 198-214.

Novel potent azetidine-based compounds irreversibly inhibit Stat3 activation and induce antitumor response against human breast tumor growth in vivo

Signal transducer and activator of transcription (Stat)3 is a valid anticancer therapeutic target. We have discovered a highly potent chemotype that amplifies the Stat3-inhibitory activity of lead compounds to levels previously unseen. The azetidine-based compounds, including H172 (9f) and H182, irreversibly bind to Stat3 and selectively inhibit Stat3 activity (IC50 0.38–0.98 μM) over Stat1 or Stat5 (IC50 > 15.8 μM) in vitro. Mass spectrometry detected the Stat3 cysteine peptides covalently bound to the azetidine compounds, and the key residues, Cys426 and Cys468, essential for the high potency inhibition, were confirmed by site-directed mutagenesis. In triple-negative breast cancer (TNBC) models, treatment with the azetidine compounds inhibited constitutive and ligand-induced Stat3 signaling, and induced loss of viable cells and tumor cell death, compared to no effect on the induction of Janus kinase (JAK)2, Src, epidermal growth factor receptor (EGFR), and other proteins, or weak effects on cells that do not harbor aberrantly-active Stat3. H120 (8e) and H182 as a single agent inhibited growth of TNBC xenografts, and H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse TNBC growth and improved survival in syngeneic models. We identify potent azetidine-based, selective, irreversible Stat3 inhibitors that inhibit TNBC growth in vivo.

D-mannose facilitates immunotherapy and radiotherapy of triple-negative breast cancer via degradation of PD-L1

Breast cancer is the most frequent malignancy in women worldwide, and triple-negative breast cancer (TNBC) patients have the worst prognosis and highest risk of recurrence. The therapeutic strategies for TNBC are limited. It is urgent to develop new methods to enhance the efficacy of TNBC treatment. Previous studies demonstrated that D-mannose, a hexose, can enhance chemotherapy in cancer and suppress the immunopathology of autoimmune diseases. Here, we show that D-mannose can significantly facilitate TNBC treatment via degradation of PD-L1. Specifically, D-mannose can activate AMP-activated protein kinase (AMPK) to phosphorylate PD-L1 at S195, which leads to abnormal glycosylation and proteasomal degradation of PD-L1. D-mannose-mediated PD-L1 degradation promotes T cell activation and T cell killing of tumor cells. The combination of D-mannose and PD-1 blockade therapy dramatically inhibits TNBC growth and extends the lifespan of tumor-bearing mice. Moreover, D-mannose-induced PD-L1 degradation also results in messenger RNA destabilization of DNA damage repair-related genes, thereby sensitizing breast cancer cells to ionizing radiation (IR) treatment and facilitating radiotherapy of TNBC in mice. Of note, the effective level of D-mannose can be easily achieved by oral administration in mice. Our study unveils a mechanism by which D-mannose targets PD-L1 for degradation and provides methods to facilitate immunotherapy and radiotherapy in TNBC. This function of D-mannose may be useful for clinical treatment of TNBC.

Macrophage migration inhibitory factor inhibition as a novel therapeutic approach against triple-negative breast cancer

Triple-negative breast cancer (TNBC), defined as loss of estrogen, progesterone, and Her2 receptors, is a subtype of highly aggressive breast cancer with worse prognosis and poor survival rate. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine aberrantly expressed in many solid tumors and known to promote tumor progression and metastasis. However, its role in TNBC progression and metastasis is unexplored. Here we have shown that in TNBC patients, MIF expression was significantly enriched in the tumor compared to adjacent normal tissue. Using publically available patient datasets, we showed that MIF overexpression correlates with worse survival in TNBC compared to other hormonal status. Orthotopic implantation of TNBC cells into MIF knockout mice showed reduced tumor growth compared to wild-type mice. In addition, we have shown that MIF downregulation inhibits TNBC growth and progression in a syngeneic mouse model. We further showed that CPSI-1306, a small-molecule MIF inhibitor, inhibits the growth of TNBC cells in vitro. Mechanistic studies revealed that CPSI-1306 induces intrinsic apoptosis by alteration in mitochondrial membrane potential, cytochrome c (Cyt c) release, and activation of different caspases. In addition, CPSI-1306 inhibits the activation of cell survival and proliferation-related molecules. CPSI-1306 treatment also reduced the tumor growth and metastasis in orthotopic mouse models of mammary carcinoma. CPSI-1306 treatment of tumor-bearing mice significantly inhibited TNBC growth and pulmonary metastasis in a dose-dependent manner. Histological analysis of xenograft tumors revealed a higher number of apoptotic cells in CPSI-1306-treated tumors compared to vehicle controls. Our studies, for the first time, show that MIF overexpression in TNBC enhances growth and metastasis. Taken together, our results indicate that using small molecular weight MIF inhibitors could be a promising strategy to inhibit TNBC progression and metastasis.

Abstract P3-03-10: Targeting Wnt/β-catenin pathway by antidepressant imipramine for triple negative breast cancer treatment

Abstract Aberrant activation of Wnt signaling has been implicated in various human cancers including breast cancer. Several intra-cellular signal transduction pathways are induced by Wnt, notably by Wnt/β-catenin dependent pathway. In breast cancer, triple negative breast cancer (TNBC) subtypes have increased Wnt/β-catenin signaling associated with high grade tumors, metastasis, poor prognosis and tumor regression. Importantly, significant upregulation of Wnt/β-catenin pathway and overexpression of Wnt transcriptional targets have emerged in breast cancer stem cells (BCSCs) than normal mammary epithelial stem cells. Blockade of Wnt/β-catenin signaling has been reported to suppress breast cancer growth, invasion, migration and induced apoptosis and “stemness”. WNT pathway inhibition preferentially reduces stem cell like activity in patient-derived metastatic breast cancer compared to normal cells. Collectively, these studies suggest that Wnt/β-catenin pathway could be the potential target in inhibiting TNBC growth, metastatic progression and “stemness”. We recently identified that imipramine, an FDA approved antidepressant used to treat chronic depression, inhibits TNBC growth and progression. Imipramine treatment significantly reduced cell viability, migration and invasion of breast cancer cells in general and TNBC cells in particular. Our preliminary findings demonstrated that systemic delivery of imipramine suppressed TNBC growth in orthotopic mouse xenograft model. Our studies further show that imipramine inhibits several Wnt/β-catenin catenin pathway proteins including WNT1, FZD1, β-catenin, cyclin D1 and c-myc, which are reported to support breast cancer “stemness” and metastasis. Importantly, our preliminary studies revealed that imipramine inhibits TNBC stem cells as evidenced by reduced CD44+ population. Our results suggest the therapeutic efficacy of imipramine as a single/or neo-adjuvant agent for treating TNBC patients. Citation Format: Subapriya Rajamanickam, Santosh Timilsina, Ismoil Jatoi, Virginia Kaklamani, Ratna K Vadlamudi, Manjeet K Rao. Targeting Wnt/β-catenin pathway by antidepressant imipramine for triple negative breast cancer treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-10.

Abstract 2018: Cannabinoid receptor 2 agonist JWH-015 inhibits growth and metastasis of triple negative breast cancers through regulation of autophagy mechanism

Abstract Metastasis to distant organs is the major cause of mortality associated with breast cancer. Triple-negative breast cancer (TNBC) subtype has been shown to be associated with worst patient survival due to high metastasis rate and lack of targeted therapies. Therefore, there is an utmost need to develop novel therapeutic strategies against TNBC growth and metastasis. Cannabinoids are known to possess anti-cancer activity. However, the associated psychotropic activity limits the therapeutic use of Cannabinoids in breast cancer patients. Here, we used synthetic cannabinoid JWH-015 which activates cannabinoid receptor 2 (CB2) and is devoid of psychotropic activity. In vitro molecular analysis revealed that JWH-015 induces TNBC cell death by activating autophagy associated apoptosis. In addition, we have further shown using in vivo tumor models, that JWH-015 potentially inhibits TNBC growth and metastasis through activation of autophagy mechanisms. Further analysis showed that JWH-015 treated tumors recruited the reduced number of macrophages compared to vehicle control treated tumors. In addition, there were more anti-tumor M1-type macrophages present in JWH-015 treated tumors. Conversely, vehicle control treated tumors possessed a higher number of pro-tumor M2-type macrophages. These observations have established the anti-metastatic potential of JWH-015 in different TNBC pre-clinical mouse models. The mechanistic studies have elaborated the role of JWH-015-induced autophagy in promoting tumor cell death to inhibit TNBC growth and metastasis. Citation Format: Nabanita Chatterjee, Subhadip Das, Dinesh K. Ahirwar, Sanjay Mishra, Ramesh Ganju. Cannabinoid receptor 2 agonist JWH-015 inhibits growth and metastasis of triple negative breast cancers through regulation of autophagy mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2018.

Macrophage migration inhibitory factor (MIF):A novel therapeutic target against aggressive breast cancer

Triple negative breast cancer (TNBC), defined as loss of estrogen, progesterone and Her2 receptors, is a subtype of breast cancer with worse prognosis and poor survival rate. Increased inflammation further reduces the TNBC patient survival. Macrophage migration inhibitory factor (MIF) is a pro‐inflammatory molecule known to enhance breast cancer progression and metastasis. However, not much is known about the role of MIF in TNBC progression and metastasis. MIF interacts with various oncogenic molecules and signaling pathways. Therefore, targeting MIF might be a promising strategy to inhibit TNBC progression and metastasis. Here we report that CPSI‐1306, a small molecule inhibitor specific to MIF, possesses the ability to target TNBC cell in vitro by inducing apoptosis. The mechanistic studies revealed that CPSI‐1306 induces apoptosis by enhancing reactive oxygen species (ROS), mitochondria membrane potential alteration, cytochrome C release and activation of Caspases. In addition, CPSI‐1306 also inhibits cell survival and apoptosis signaling molecules, including AKT, PDK and RAF expression. Next, we analyzed the clinical utility of CPSI‐1306 using mouse MVT‐1 cells derived orthotopic and human MDA‐MB‐231 cells derived xenograft TNBC mouse models. The oral treatment of tumor bearing mice with CPSI‐1306 significantly inhibited TNBC growth and metastasis to the lungs in a dose dependent manner. The histological analysis of tumors showed higher number of apoptotic cells in CPSI‐1306 treated tumors compared to vehicle controls. We also observed an association of higher MIF mRNA expression with poor patient survival, including TNBC, by using publically available datasets. These results suggest that CPSI‐1306 induces cell death in TNBC cells via activating ROS and mitochondria‐mediated apoptosis and significantly inhibits TNBC growth and metastasis in pre‐clinical TNBC mouse models.Support or Funding InformationDOD, Breakthrough AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study of diffusion-weighted magnetic resonance imaging in the evaluation of the response to AAV2-VEGF-Trap neoadjuvant treatment in a triple-negative breast cancer animal model.

ObjectiveEvaluation of the efficacy of adeno‐associated virus 2 mediated gene transfer of vascular endothelial growth factor Trap (AAV2‐VEGF‐Trap) alone or combination with paclitaxel in a mouse model of triple‐negative breast cancer (TNBC) using diffusion‐weighted magnetic resonance imaging (DW‐MRI) and in vivo fluorescence imaging.Materials and MethodsXenografted TNBC tumors were established by subcutaneous injection of MDA‐MB‐231 cells into nude mice. Tumors were treated with AAV2‐VEGF‐Trap, paclitaxel, AAV2‐VEGF‐Trap combined with paclitaxel and control. A 7.0‐Tesla magnetic resonance (MR) was used to obtain the apparent diffusion coefficient (ADC) values and ΔADC values. In vivo fluorescence imaging coupled with the optical imaging probe AngioSense680 EX was acquired to obtain average luminous intensity values. Immunohistochemical staining of tumor Ki‐67 and vascular endothelial cell marker antigen (CD31) were used to evaluate the effects on tumor proliferation and angiogenesis.ResultsThe combination of AAV2‐VEGF‐Trap with paclitaxel exhibited greater tumor growth inhibition compared with the other groups. The ADC values in the paclitaxel group and the AAV2‐VEGF‐Trap in combination with paclitaxel group were significant greater compared with the control group, and the ΔADC values of all treatment groups were significantly increased compared with the control group on the 14th day after administration. Decreased microvessel density and luminous intensity in the treatment groups that contain AAV2‐VEGF‐Trap were observed. Reduced proliferation activity was noted in groups that contained paclitaxel.ConclusionAAV2‐VEGF‐Trap inhibits TNBC growth though inhibiting tumor neovascularization with a single intravenous injection, and AAV2‐VEGF‐Trap exhibits a synergistic effect when used in combination with paclitaxel for TNBC neoadjuvant therapy. In vivo fluorescence imaging can detect the anti‐angiogenesis effect of AAV2‐VEGF‐Trap early and noninvasively. DW‐MRI can longitudinally monitor the neoadjuvant efficacy of TNBC.

Abstract P2-06-24: Cannabinoid receptors agonist induces lysosome-mediated cell death in TNBC

Abstract Breast cancer is one of the leading causes of death among women in the United States. Among all the breast cancer subtypes, triple-negative breast cancer (TNBC) is denoted as having the highest rate of mortality due to inefficacious therapy, drug resistance, and high recurrences rate. Despite many therapeutic advances over the past decades, novel therapeutic approaches are required to treat TNBC. Although lysosomes have recently been identified as novel targets in a variety of cancers, not much known about their role in breast cancer especially in TNBC. Due to the altered features of lysosomes in cancerous cells, its functional ability affects cell proliferation, metabolism, adaptation, and autophagy in tumor cells. Thus, the induction of lysosomal membrane permeabilization (LMP) may be used as a novel approach in controlling the aberrant cellular functions in TNBC. Cannabinoid receptor 2 (CB2) is expressed in breast cancer and its agonist JWH-015 has been shown to inhibit TNBC growth and metastasis. However, not much is known about the mechanisms by which JWH015 inhibit growth and metastasis. To investigate JWH015-mediated therapeutic mechanisms, we examined the lysosome-mediated cell death in TNBC cells. We found that JWH015 induces cell death in TNBC cells through lysosomal disruption mechanism. We also showed that JWH-015 treatment increases releases of lysosomal proteins, cathepsin D/B, and autophagy marker LC3B and that leads to activation of apoptotic cell death signaling in TNBC. These alterations result in the formation of autolysosomes to promote LMP. Our findings elucidate the mechanism by which JWH-015 promotes LMP induced autophagy and regulates TNBC cell proliferation. These studies elucidate novel lysosomal-mediated cell death mechanisms through activation of CB2 receptors in TNBC. Citation Format: Chatterjee N, Das S, Ahiwar D, Mishra S, Kaul K, Ganju R. Cannabinoid receptors agonist induces lysosome-mediated cell death in TNBC [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 P2-06-24.

Transcriptional Downregulation of miR-4306 serves as a New Therapeutic Target for Triple Negative Breast Cancer.

Rationale: Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor alpha (ER-α), human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR) expression, but the effect of lacking the three factors on TNBC is unclear. Whether loss of the three factors contributes to deregulate genes that participate in the progress of TNBC remains unknown.Methods: We performed microRNA arrays and comprehensive analysis to screen for miRNAs that are transcriptionally regulated by ER-α, HER2 and PR. Functional assays and molecular mechanism studies were used to investigate the role of miR-4306 in TNBC. An orthotopic mouse model of TNBC was used to evaluate the therapeutic potential of a cholesterol-conjugated miR-4306 mimic.Results: We found that miR-4306 is transcriptionally regulated by ER-α, HER2 and PR, and the downregulation of miR-4306 in TNBC is caused by the loss of ER-α, HER2 and PR. Clinically, low miR-4306 expression is strongly associated with lymph node metastasis and poor survival for TNBC. Upregulation of miR-4306 greatly suppresses TNBC cell proliferation, migration and invasion and abrogates angiogenesis and lymphangiogenesis in vitro. According to in vivo models, miR-4306 overexpression considerably inhibits TNBC growth, lung metastasis, angiogenesis and lymph node metastasis. Mechanistic analyses indicate that miR-4306 directly targets SIX1/Cdc42/VEGFA to inactivate the signaling pathways mediated by SIX1/Cdc42/VEGFA. Finally, the orthotopic mouse model of TNBC reveals that miR-4306 mimic can be used for TNBC treatment in combination with cisplatin.Conclusions: Our findings suggest that miR-4306 acts as a tumor suppressor in TNBC and is a potential therapeutic target for TNBC treatment.

Schisandrin B exhibits potent anticancer activity in triple negative breast cancer by inhibiting STAT3

Triple negative breast cancer (TNBC) is an aggressive subgroup of human breast cancer. In this study, we have examined the potential of Schisandrin B (Sch B), a bioactive chemical compound found in Schisandra chinensis, against TNBC. We used MDA-MB-231, BT-549, and MDA-MB-468 TNBC cells and immunodeficient mice to study the effect of Sch B. Our results show that Sch B inhibits TNBC growth by inducing cell cycle arrest and by triggering apoptotic death. Sch B also inhibited the migration and colony formation of tumor cells, and prevented the growth of TNBC cells in mice. We found that these inhibitory activities were mediated through suppression of signal transducer and activator of transcription-3 (STAT3) phosphorylation and nuclear translocation. Taken together, our studies show that Sch B has potent anti-tumor activity against TNBC via a novel mechanism involving STAT3 inactivation.

Abstract 4039: Riluzole synergizes with paclitaxel to induce apoptosis and inhibit cell growth in triple negative breast cancer

Abstract Introduction: Systemic treatment for triple negative breast cancer (TNBC) includes paclitaxel, which works by inhibiting the breakdown of microtubules. We previously observed that riluzole, an FDA-approved drug for the treatment of amyotrophic lateral sclerosis, can inhibit TNBC proliferation, invasion, and colony formation. We now test whether riluzole can act synergistically with paclitaxel to inhibit TNBC growth and induce apoptosis in TNBC. Methods: After treatment of various TNBC cell lines with constant ratio concentrations of riluzole and paclitaxel we conducted cell proliferation transformation assays (MTT) to measure cell inhibition potential. Synergy or additivity was determined by the Chou-Talalay using Compusyn software. PARP cleavage normalized to GAPDH was also measured by Western blot to estimate apoptosis due to the riluzole-paclitaxel combination. In vivo synergy was studied using a xenograft study with MDA-MB-231, a human TNBC cell line sensitive to riluzole. Results: MTT results show that riluzole and paclitaxel work synergistically to inhibit cell proliferation in all TNBC cell lines tested, as determined by isobolograms and CI values <1. The strongest synergistic effect was observed in the cell lines SUM159, SUM149, and SUM229, where synergism occurred at Fa values as low as 0.5 (50% inhibition of cell proliferation). Measuring PARP cleavage, we demonstrated that the riluzole-paclitaxel drug combination induces greater apoptosis than does either drug alone. The significant impact of the drug combination was best demonstrated in cell lines SUM159, SUM149 and SUM229, but was observed in all cell lines tested. In the xenograft study, the highest non-toxic dose of paclitaxel (7.2 mg/kg) combined with riluzole resulted in 8/9 tumor-free mice, with a tumor growth inhibition ratio (T/C) of 0%, where <10% is considered highly therapeutic. This result was better than riluzole monotherapy (0/9 tumor-free and T/C = 99%) or paclitaxel monotherapy (4/8 tumor-free and T/C = 8%). Conclusions: Our results demonstrate that the addition of riluzole to paclitaxel results in at least additive and in many cases synergistic effects against TNBC. Our observations thus suggest that repurposing riluzole, which is an oral drug with an excellent safety profile, to add to paclitaxel to treat TNBC represents a potential strategy to improve the efficacy of adjuvant and neoadjuvant treatments for TNBC and/or reduce chemotherapy doses and toxicity in patients. Our data support the need to proceed to clinical trials to test this approach in patients. Citation Format: Miriam A. Bukhsh, Cecilia L. Speyer, Waris S. Jafry, Rachel E. Sexton, David Thomas, David Gorki. Riluzole synergizes with paclitaxel to induce apoptosis and inhibit cell growth in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4039. doi:10.1158/1538-7445.AM2017-4039

Nuclear receptor NR4A1 is a tumor suppressor down-regulated in triple-negative breast cancer.

The nuclear receptor (NR) superfamily contains hormone-inducible transcription factors that regulate many physiological and pathological processes through regulating gene expression. NR4A1 is an NR family member that still does not have an identified endogenous ligand, and its role in cancer is also currently unclear and controversial. In this study, we aimed to define the expression profiles and specific role of NR4A1 in the highly malignant triple-negative breast cancer (TNBC), which still lacks available targeted therapies. Bioinformatic analysis revealed a decrease of NR4A1 mRNA expression in human TNBC samples. Semi-quantitative analysis of NR4A1 protein expression by immunohistochemistry also identified a progressive NR4A1 reduction during the development of mouse basal-like mammary tumors and a significant NR4A1 downregulation in human TNBC samples. Furthermore, the expression levels of NR4A1 in human TNBC were negatively associated with tumor stage, lymph node metastasis and disease recurrence. Moreover, ectopic expression of NR4A1 in MDA-MB-231, a TNBC cell line with little endogenous NR4A1, inhibited the proliferation, viability, migration and invasion of these cells, and these inhibitions were associated with an attenuated JNK1–AP-1–cyclin D1 pathway. NR4A1 expression also largely suppressed the growth and metastasis of these cell-derived tumors in mice. These results demonstrate that NR4A1 is downregulated in TNBC and restoration of NR4A1 expression inhibits TNBC growth and metastasis, suggesting that NR4A1 is a tumor suppressor in TNBC.

Targeting Metabolic Remodeling in Triple Negative Breast Cancer in a Murine Model.

Background: Chemotherapy is the backbone of systemic treatment for triple negative breast cancer (TNBC), which is one of the most relevant breast cancers molecular types due to the ability of tumor cells to develop drug resistance, highlighting the urgent need to design newer and safer drug combinations for treatment. In this context, to overcome tumor cell drug resistance, we employed a novel combinatorial treatment including Doxorubicin, Metformin, and Sodium Oxamate (DoxMetOx). Such pharmacological combination targets indispensable hallmarks of cancer-related to aerobic glycolysis and DNA synthesis.Materials and Methods: Thirty-five female nude mice were transplanted subcutaneously with MDA-MB-231 triple negative human cancer cell line. Once tumors were visible, mice were treated with doxorubicin, metformin, oxamate or all possible pharmacologic combinations. Treatments were administered daily for 15 days and tumors were measured by calipers every day. MicroPET images were taken in three different occasions, basal state, in the middle of the treatment, and at the end of treatment. Western blot analyses, qRT-PCR, flow cytometry, and cytotoxicity assays were performed to elucidate the mechanism of cell death promoted by the drugs in vitro.Results: In this work we assessed the proof of concept of metabolic correction in solid tumors as an effective drug treatment; hence, mice bearing tumors treated with the DoxMetOx therapy showed a complete inhibition of the tumor mass growing in 15 days of treatment depicted by the micro PET images. In vitro studies displayed that the three drugs together act by inhibiting both, mTOR-phosphorylation and expression of LDH-A gene, promoting apoptosis via dependent on the caspase-3 pathway, accompanied by cleavage of PARP. Moreover, induction of autophagy process was observed by the accumulation of LC3-II, a primordial protein implicated in the conformation and elongation of the autophagolysosome.Conclusions: The lack of effective drugs to inhibit TNBC growth is the main cause of therapy failure and tumor relapse. We have showed that targeting crucial molecular pathways in cancer by the combination of Doxorubicin, Metformin, and Oxamate resulted as an efficient and rapid tumor growth inhibitor in a triple negative xenograft model. Our findings are promising for patients diagnosed with TNBC tumors, for which unfortunately there are no reliable drug therapies.

Molecular profiling and computational network analysis of TAZ-mediated mammary tumorigenesis identifies actionable therapeutic targets.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer (BC) cases and contributes disproportionately to BC mortality. TAZ, a key transducer of the Hippo pathway, has recently been demonstrated to confer breast cancer stem cell (CSC) traits. However, TAZ target genes and the underlying transcriptional regulatory pathways responsible for the CSC phenomenon remain unknown. Here, we demonstrate that the oncogenic activity of TAZ is essential for propagation of the malignant phenotype. We further show that constitutively active TAZ tumor-derived cells exhibit unique tumor-initiating properties, including increased self-renewal and metastatic seeding potential, acquired chemotherapy resistance and the ability to efficiently regenerate tumor formation in vivo. Combined digital RNA expression analysis and computational network approaches identify several signaling pathways that distinguish breast cancer tumor-initiating cells (T-ICs) from bulk tumor cells. We demonstrate the utility of this approach by repositioning the small molecule tyrosine kinase inhibitor, Dasatinib, which selectively targets T-ICs and inhibits TNBC growth in vivo.

CCN5/WISP-2 promotes growth arrest of triple-negative breast cancer cells through accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation.

The matricellular protein CCN5/WISP-2 represents a promising target in triple-negative breast cancer (TNBC) because treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model. Our studies found that the p27(Kip1) tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (upregulation and relocalization of p27(Kip1)) are critical for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27(Kip1) resides mostly in the cytoplasm, which is associated with the aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize the p27(Kip1) protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate the transcriptional regulation of p27(Kip1). The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Moreover, inhibition of α6β1 receptors diminishes CCN5 action on p27(Kip1) in TNBC cells. Collectively, these data suggest that CCN5 effectively inhibits TNBC growth through the accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation, and thus, activation of CCN5 may have the therapeutic potential to kill TNBC.