Signaling In Triple-negative Breast Cancer Research Articles

PTPN20 promotes metastasis through activating NF-κB signaling in triple-negative breast cancer

BackgroundCancer metastasis remains a major challenge in the clinical management of triple-negative breast cancer (TNBC). The NF-κB signaling pathway has been implicated as a crucial factor in the development of metastases, but the underlying molecular mechanisms remain largely unclear.MethodsPTPN20 expression was evaluated using data from the Sweden Cancerome Analysis Network-Breast and The Cancer Genome Atlas database, as well as by western blotting and immunohistochemistry in 88 TNBC patients. The ability of PTPN20 to activate NF-κB was assessed by luciferase reporter assays. The effects of PTPN20 overexpression and knockdown via short hairpin RNA were examined in TNBC cell lines by wound healing and transwell matrix penetration assays. Additionally, we analyzed the growth and metastasis abilitiy of 4T1 xenograft tumors in nude mice.ResultsPTPN20 levels were elevated in TNBC cell lines and patient samples compared to controls, and higher protein levels correlated with metastasis-free survival. Overexpression of PTPN20 enhanced migration and invasion in vitro, and promoted lung metastasis in vivo. Our finding revealed that PTPN20 activates NF-κB signaling by dephosphorylating p65 at Ser468, preventing its binding to COMMD1, thereby protecting p65 from degradation. Downregulation of PTPN20 effectively inhibit, while p65 S468A mutant restored the migratory and invasive abilities of TNBC cells.ConclusionsCollectively, our results demonstrate that PTPN20 plays a critical role in TNBC metastasis through the activation of NF-κB signaling. We propose that PTPN20 may serve as a novel prognostic marker and potential therapeutic target for the treatment of TNBC.

Significance of LIF/LIFR Signaling in the Progression of Obesity-Driven Triple-Negative Breast Cancer.

American women with obesity have an increased incidence of triple-negative breast cancer (TNBC). The impact of obesity conditions on the tumor microenvironment is suspected to accelerate TNBC progression; however, the specific mechanism(s) remains elusive. This study explores the hypothesis that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and assesses the efficacy of LIFR inhibition with EC359 in blocking TNBC progression. TNBC cell lines were co-cultured with human primary adipocytes, or adipocyte-conditioned medium, and treated with EC359. The effects of adiposity were measured using cell viability, colony formation, and invasion assays. Mechanistic studies utilized RNA-Seq, Western blotting, RT-qPCR, and reporter gene assays. The therapeutic potential of EC359 was tested using xenograft and patient-derived organoid (PDO) models. The results showed that adipose conditions increased TNBC cell proliferation and invasion, and these effects correlated with enhanced LIFR signaling. Accordingly, EC359 treatment reduced cell viability, colony formation, and invasion under adipose conditions and blocked adipose-mediated organoid growth and TNBC xenograft tumor growth. RNA-Seq analysis identified critical pathways modulated by LIF/LIFR signaling in diet-induced obesity mouse models. These findings suggest that adiposity contributes to TNBC progression via the activation of the LIF/LIFR pathway, and LIFR inhibition with EC359 represents a promising therapeutic approach for obesity-associated TNBC.

Impact of microRNA variants on PI3K/AKT signaling in triple-negative breast cancer: comprehensive review.

Breast cancer (BC) is a significant cause of cancer-related mortality, and triple-negative breast cancer (TNBC) is a particularly aggressive subtype associated with high mortality rates, especially among younger females. TNBC poses a considerable clinical challenge due to its aggressive tumor behavior and limited therapeutic options. Aberrations within the PI3K/AKT pathway are prevalent in TNBC and correlate with increased therapeutic intervention resistance and poor outcomes. MicroRNAs (miRs) have emerged as crucial PI3K/AKT pathway regulators influencing various cellular processes involved in TNBC pathogenesis. The levels of miRs, including miR-193, miR-4649-5p, and miR-449a, undergo notable changes in TNBC tumor tissues, emphasizing their significance in cancer biology. This review explored the intricate interplay between miR variants and PI3K/AKT signaling in TNBC. The review focused on the molecular mechanisms underlying miR-mediated dysregulation of this pathway and highlighted specific miRs and their targets. In addition, we explore the clinical implications of miR dysregulation in TNBC, particularly its correlation with TNBC prognosis and therapeutic resistance. Elucidating the roles of miRs in modulating the PI3K/AKT signaling pathway will enhance our understanding of TNBC biology and unveil potential therapeutic targets. This comprehensive review aims to discuss current knowledge and open promising avenues for future research, ultimately facilitating the development of precise and effective treatments for patients with TNBC.

Abstract B021: CFLAR targeting selectively exploits extrinsic apoptosis signaling in triple-negative breast cancer

Abstract The extrinsic apoptotic pathway activates programmed cell death through the binding of extracellular death ligands, such as TNFα, to their cognate receptors. Although some death ligands are often upregulated in the tumor microenvironment, this pathway is subverted in cancer cells to instead favor growth and survival rather than the induction of apoptosis. Reflecting the strict control cancer cells exert over this pathway, functional genomics screens, such as DepMap, have identified several of the extrinsic apoptotic components as essential targets, with selective dependency in a subset of cancer cell lines. We have genetically validated that downregulation of CFLAR, a negative regulator of the extrinsic apoptosis pathway, causes apoptosis and cell growth inhibition alone, but specially in combination with TNFα signaling, across multiple cancer types, with increase prevalence in Triple-negative breast cancer. Furthermore, we have shown that in order to have maximal activity most cell lines require targeting of CFLAR short isoform splicing variant. Importantly, CFLAR downregulation in non-tumoral models presented much less activity than the observed in the sensitive cancer models supporting the selective activity of CFLAR in a subset of cancer cells. Citation Format: Victor Quereda, Shane W. O'Brien, Tony C. Della Pietra, James J. Foley, Andy Fedoriw. CFLAR targeting selectively exploits extrinsic apoptosis signaling in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B021.

Abstract 1549: Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer

Abstract Background: CD146, an adhesion molecule involved in inflammatory regulation, is linked to increased aggressiveness in Triple Negative Breast Cancer (TNBC). Yet, the molecular mechanisms of CD1146-driven TNBC metastasis in an inflammatory context remain elusive. We synthesized CD146 nanobody (anti-CD146 #112-2), which degraded CD146 protein specifically, inhibiting IL-6-mediated JAK/STAT3 activation and suppressing TNBC metastasis. This study aims to elucidate the mechanisms of CD146 degradation by 112-2 and the role of CD146 in mediating inflammatory responses leading to TNBC metastasis. Methods: CD146 knockout and overxpression cell models were created using CRISPR/Cas9 or lentiviral techniques. Lung metastasis was evaluated by injecting luciferase-labeled cells into the tail veins of Balb/c-nu mice, monitored with the IVIS Kinetic system. The impact of CD146 on TNBC metastasis in an inflammatory environment was explored through cell-adhesion assays and trans-endothelial migration assays using CD146 knockout models and HUVEC cells. Transwell assays assessed the influence of IL-6 and 112-2 on TNBC. CD146 mutant vectors with specific truncations were generated to confirm the binding sites between CD146 and 112-2. ELISA and immunofluorescence assays examined CD146 protein internalization. CD146 degradation by 112-2 was confirmed using proteasome inhibitor MG132 and lysosome inhibitor Bafilomycin A1, along with western blotting and immunofluorescence experiments. Results: Stimulation of TNBC cells with IL-6 increased CD146 expression and tumor metastasis. Knocking down CD146 significantly reduced IL-6 secretion by TNBC cells, leading to reduced tumor-endothelial cell adhesion and trans-endothelial migration. CD146 knockout or 112-2 treatment inhibited IL-6-mediated JAK/STAT3 activation and tumor metastasis. Experiments with CD146 mutants and molecular docking identified the specific 112-2 binding site at leucine positions 331-332 of CD146 protein. 112-2 treatment led to CD146 protein internalization and cleavage, resulting in an 80 kDa fragment recognized by both 112-2 and C-terminus-specific CD146 antibodies, ultimately inhibiting the activation of IL-6/JAK/STAT3 signaling. Notably, the inhibition of proteasome or lysosome activity failed to reverse the 112-2-induced reduction in CD146 protein levels. However, inhibiting lysosomal acidification resulted in significant co-localization of CD146 with lysosomes, suggesting a potential role of lysosomes in 112-2-mediated cleavage of CD146. Conclusion: CD146 promotes TNBC metastasis by modulating IL-6/JAK/STAT3 activation. The nanobody anti-CD146 #112-2 specifically binds to CD146 extracellular domain induces the proteolysis of CD146 protein, thereby inhibiting the tumor-promoting effect of IL-6. Citation Format: Kaiyuan Huang, Hexing Sun, Chengyu Wu, Danping Lin, Jinling Chen, Yanhan Chen, Xinqiang Fang, Shuzhao Chen, Yuanke Liang, Haoyu Lin. Molecular mechanism of CD146 nanobody in tumor metastasis inhibition mediating by IL-6/JAK/STAT3 signaling in triple-negative breast cancer [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 1549.

SLC25A17 inhibits autophagy to promote triple-negative breast cancer tumorigenesis by ROS-mediated JAK2/STAT3 signaling pathway

BackgroundSLC25A17, a peroxisomal solute carrier, has been implicated in various physiological and pathological processes. However, its precise roles and underlying mechanisms in triple-negative breast cancer (TNBC) remain incompletely understood.MethodsThe expression and survival data of breast cancer were derived from TCGA and GEO databases. A variety of in vitro assays were conducted, including proliferation, apoptosis, cell cycle, migration, and invasion. Reactive oxygen species (ROS) were measured by immunofluorescence microscopy and flow cytometry. The levels of autophagy were assessed by mRFP-GFP-LC3 confocal microscopy scanning, western blotting, and electron microscopy.ResultsSLC25A17 was highly expressed in breast cancer tissues, which was found to be associated with unfavorable prognosis. Functional assays demonstrated that SLC25A17 knockdown suppressed proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion. Moreover, it prompted apoptosis and autophagy. On the other hand, SLC25A17 knockdown promoted autophagy through triggering ROS accumulation, which was counteracted by N-acetyl-l-cysteine (NAC). Furthermore, the pro-apoptotic effect of SLC25A17 knockdown was reversed when treated with autophagy inhibitor 3-MA in TNBC cells, suggesting that SLC25A17 knockdown-induced autophagic cell death. Mechanistically, SLC25A17 performed its function through regulation JAK2/STAT3 signaling in TNBC. In a nude mice xenograft study, SLC25A17 knockdown markedly decreased breast tumor growth and metastasis.ConclusionSLC25A17 up-regulation may be a critical factor driving TNBC progression by modulating ROS production and autophagy. Consequently, targeting SLC25A17 could be an effective therapeutic strategy against TNBC.

Ectopic expression of tumor suppressive miR-181c-5p downregulates oncogenic Notch signaling in MDA-MB-231 cells

Triple negative breast cancer (TNBC) is a very invasive subtype of breast cancer (BCa), this is accounted for 15–20% of all BCa cases. TNBC patients have very limited therapy option due to lack of effective targets and patients shows the worse survival. Therefore, present study has tried to introduce the target based therapy by studying the tumor suppressive role of miR-181c-5p on oncogenic Notch1 signaling. Transient transfection, bioinformatics, qRT-PCR, Notch1 luciferase assay and western blotting techniques were utilized to study the effect of induced expression of miR-181c-5p on oncogenic Notch1 signaling in MDA-MB-231 cells. Results shows that miR-181c-5p mimic increase the expression of miR-181c-5p by 45.26% and 75.96% in 24 and 48 h incubation, respectively (p < 0.0003) in transfected cells. The miR-181c-5p binds at NOTCH1 3′ UTR target binding site with a minimum free energy of − 26.0 kcal/mol. The AGO protein showed significant interaction with the miR-181c-5p and miR-181c-5p-NOTCH1 complex. Decreased expression of NOTCH1 by 32.88% and 45.87% (p < 0.0001); and HES1 expression by 14.06% and 53.24% (p < 0.0001) was observed in 24 and 48 h transfected cells respectively. Notch1 promoter luciferase activity was reduced by 25.72% and 46.98% in 24 and 48 h miRNA-mimic transfected cells. Western blot analysis also showed significant reduction in NOTCH1 and HES1 proteins expression. In conclusion, present study suggests that the forced expression of tumor suppressive miR-181c-5p negatively regulates oncogenic Notch1 signaling in TNBC. Negative regulation of Notch1 signaling via miR-181c-5p mimic could be a hopeful therapeutic strategy in TNBC patient treatment.

SHC4 orchestrates β-catenin pathway-mediated metastasis in triple-negative breast cancer by promoting Src kinase autophosphorylation

Triple-negative breast cancer (TNBC) is highly aggressive and metastatic, and has the poorest prognosis among all breast cancer subtypes. Activated β-catenin is enriched in TNBC and involved in Wnt signaling-independent metastasis. However, the underlying mechanisms of β-catenin activation in TNBC remain unknown. Here, we found that SHC4 was upregulated in TNBC and high SHC4 expression was significantly correlated with poor outcomes. Overexpression of SHC4 promoted TNBC aggressiveness in vitro and facilitated TNBC metastasis in vivo. Mechanistically, SHC4 interacted with Src and maintained its autophosphorylated activation, which activated β-catenin independent of Wnt signaling, and finally upregulated the transcription and expression of its downstream genes CD44 and MMP7. Furthermore, we determined that the PxPPxPxxxPxxP sequence on CH2 domain of SHC4 was critical for SHC4-Src binding and Src kinase activation. Overall, our results revealed the mechanism of β-catenin activation independent of Wnt signaling in TNBC, which was driven by SHC4-induced Src autophosphorylation, suggesting that SHC4 might be a potential prognostic marker and therapeutic target in TNBC.

Verteporfin induces YAP-dependent cell cycle arrest and caspase-mediated cellular apoptosis in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) subtype endows distinctive biological features, including aberrant proliferation, and deducing the molecular mechanism harmonizing the TNBC characteristics is crucial for a greater understanding and prognosis of the disease. The aim of the present study was to analyze the anti-tumorigenic effects of verteporfin (VP) in TNBC in vitro. We evaluated the tumorigenic properties of TNBC cells on VP treatment to assess the cell viability, apoptosis, cell cycle, cell survival, and protein/mRNA expressions in MDA-MB-231 and SUM-159 breast cancer cells. Transient silencing of yes-associated protein (YAP) was performed to validate the data. TNBC cells exhibited a comparatively higher active YAP downstream signaling, and VP resulted in the nuclear exclusion of YAP to a significant extent. VP inhibits the proliferation of TNBC cells by altering cyclin-dependent kinase inhibitors, thereby rendering cellular arrest at the G0/G1 phase. In a dose-dependent manner, VP induces the apoptotic machinery in TNBC cells. Moreover, transient silencing of YAP in TNBC cells exhibited a similar pattern of antiproliferative effects. Elevated YAP nuclear activity and downstream signaling in TNBC are associated with sustaining the proliferative capacity of the cells by inhibiting cellular apoptosis. VP induces antiproliferative effects on TNBC through cytoplasmic retention of YAP. Consequently, cells rewire the course of the cell cycle and stimulate cellular death. We suggest YAP signaling as a prerequisite for TNBC cell progression, and VP without light activation exerts anti-tumorigenic effects on TNBC cells.

RIPK1-dependent necroptosis promotes vasculogenic mimicry formation via eIF4E in triple-negative breast cancer

Necroptosis is a caspase-independent form of programmed cell death. Receptor interacting protein kinase 1 (RIPK1) is a key molecule in the initiation of necroptosis and the formation of the necrotic complex. Vasculogenic mimicry (VM) provides a blood supply to tumor cells that is not dependent on endothelial cells. However, the relationship between necroptosis and VM in triple-negative breast cancer (TNBC) is not fully understood. In this study, we found that RIPK1-dependent necroptosis promoted VM formation in TNBC. Knockdown of RIPK1 significantly suppressed the number of necroptotic cells and VM formation. Moreover, RIPK1 activated the p-AKT/eIF4E signaling pathway during necroptosis in TNBC. eIF4E was blocked by knockdown of RIPK1 or AKT inhibitors. Furthermore, we found that eIF4E promoted VM formation by promoting epithelial-mesenchymal transition (EMT) and the expression and activity of MMP2. In addition to its critical role in necroptosis-mediated VM, eIF4E was essential for VM formation. Knockdown of eIF4E significantly suppressed VM formation during necroptosis. Finally, through clinical significance, the results found that eIF4E expression in TNBC was positively correlated with the mesenchymal marker vimentin, the VM marker MMP2, and the necroptosis markers MLKL and AKT. In conclusion, RIPK1-dependent necroptosis promotes VM formation in TNBC. Necroptosis promotes VM formation by activating RIPK1/p-AKT/eIF4E signaling in TNBC. eIF4E promotes EMT and MMP2 expression and activity, leading to VM formation. Our study provides a rationale for necroptosis-mediated VM and also providing a potential therapeutic target for TNBC.

PRR15 deficiency facilitates malignant progression by mediating PI3K/Akt signaling and predicts clinical prognosis in triple-negative rather than non-triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast neoplasms with a higher risk of recurrence and metastasis than non-TNBC. Nevertheless, the factors responsible for the differences in the malignant behavior between TNBC and non-TNBC are not fully explored. Proline rich 15 (PRR15) is a protein involved in the progression of several tumor types, but its mechanisms are still controversial. Therefore, this study aimed to investigate the biological role and clinical applications of PRR15 on TNBC. PRR15 gene was differentially expressed between TNBC and non-TNBC patients, previously described as an oncogenic factor in breast cancer. However, our results showed a decreased expression of PRR15 that portended a favorable prognosis in TNBC rather than non-TNBC. PRR15 knockdown facilitated the proliferation, migration, and invasive ability of TNBC cells in vitro and in vivo, which was abolished by PRR15 restoration, without remarkable effects on non-TNBC. High-throughput drug sensitivity revealed that PI3K/Akt signaling was involved in the aggressive properties of PRR15 silencing, which was confirmed by the PI3K/Akt signaling activation in the tumors of PRR15Low patients, and PI3K inhibitor reversed the metastatic capacity of TNBC in mice. The reduced PRR15 expression in TNBC patients was positively correlated with more aggressive clinicopathological characteristics, enhanced metastasis, and poor disease-free survival. Collectively, PRR15 down-regulation promotes malignant progression through the PI3K/Akt signaling in TNBC rather than in non-TNBC, affects the response of TNBC cells to antitumor agents, and is a promising indicator of disease outcomes in TNBC.

Therapeutic Potential of Tumor Metabolic Reprogramming in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with clinical features of high metastatic potential, susceptibility to relapse, and poor prognosis. TNBC lacks the expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It is characterized by genomic and transcriptional heterogeneity and a tumor microenvironment (TME) with the presence of high levels of stromal tumor-infiltrating lymphocytes (TILs), immunogenicity, and an important immunosuppressive landscape. Recent evidence suggests that metabolic changes in the TME play a key role in molding tumor development by impacting the stromal and immune cell fractions, TME composition, and activation. Hence, a complex inter-talk between metabolic and TME signaling in TNBC exists, highlighting the possibility of uncovering and investigating novel therapeutic targets. A better understanding of the interaction between the TME and tumor cells, and the underlying molecular mechanisms of cell-cell communication signaling, may uncover additional targets for better therapeutic strategies in TNBC treatment. In this review, we aim to discuss the mechanisms in tumor metabolic reprogramming, linking these changes to potential targetable molecular mechanisms to generate new, physical science-inspired clinical translational insights for the cure of TNBC.

Abstract 3408: The role of obesity in promoting the LIF/LIFR signaling in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) that accounts for a disproportionate amount of BC mortality. The relationship(s) between obesity and the TNBC is crucial because the obesity prevalence in the USA is on the rise. Obese patients are generally diagnosed with large primary tumors, more lymph node metastases, and obesity effects on tumor microenvironment (TME) are suspected to accelerate the development of TNBC, but the exact mechanism(s) by which this occurs remain unknown. The goal of this study is to investigate the hypothesis that obesity enhances leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and to test the utility of LIFR inhibitor in blocking obesity driven progression of TNBC. Methods: Established TNBC cell lines were co-cultured with human primary adipocytes, incubated with adipocyte conditioned media, or exposed to high glucose (HG), then treated with the LIFR inhibitor EC359. Cell viability, colony formation, and invasion assays were used to analyze the impact of obesity on TNBC cells and to test utility of EC359. RT-qPCR, Western blotting, reporter gene assays, and RNA-seq analyses were used in the mechanistic studies. Xenografts and patient-derived organoid (PDO) models were used to evaluate the effectiveness of the EC359. Results: The cell proliferation and invasion of TNBC cells were accelerated by adipocyte conditioned media or when exposed to HG. RNA-seq and RT-qPCR analysis revealed a correlation between elevated LIFR expression and downstream LIFR signaling, including STAT3, as well as the subsequent activation of STAT3 target genes. The cell viability, colony formation, and invasion of TNBC cells under HG and adipose conditions were all markedly decreased after treatment with LIFR inhibitor EC359. Results from Western blotting demonstrated that co-culture with adipocytes or incubation with HG dramatically increased LIFR downstream signaling in TNBC model cells, and that this signaling is effectively suppressed by EC359 therapy. In addition, administration of EC359 prevented organoid proliferation that was mediated by the adipose conditioned media. Importantly, co-implantation of adipocytes greatly increased the growth of the TNBC xenograft tumor; however, therapy with EC359 significantly reduced the growth of TNBC caused by adipocyte co-implantation. Conclusions: Collectively, these findings suggest that obesity conditions promote the activation of LIF/LIFR pathway, which in turn enhances TNBC cell proliferation. The LIFR inhibitor EC359 may be employed as a new therapeutic drug to treat obesity driven TNBC and LIF/LIFR axis represents a potential therapeutic target for obesity driven TNBC. Citation Format: Lois Randolph, Alondra Rodriguez Sanchez, Logan Blankenship, Uday P. Pratap, Xue Yang, Durga Meenakshi Panneerdoss, Swapna Konda, Bindu Santhamma, Gangadhara R. Sareddy, Manjeet K. Rao, Edward R. Kost, Rajeshwar R. Tekmal, Hareesh B. Nair, Ratna K. Vadlamudi, Suryavathi Viswanadhapalli. The role of obesity in promoting the LIF/LIFR signaling in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3408.

YAP transduction drives triple-negative breast cancer aggressiveness through modulating the EGFR‒AKT axis in patient-derived xenograft cells.

Aside from the high prevalence of incidents of breast cancer, the high grade of heterogeneity and the dearth of standard treatment guidelines make triple-negative breast cancer (TNBC) the most refractory subtype. Though still in its infancy, the Hippo pathway has been known to play a critical role in tumorigenesis. However, the molecular mechanics through which the pathway exploits the breast cancer (BC) cell vulnerability are largely unexplored. In this study, we observed a relatively higher expression of the Hippo effector, yes-associated protein (YAP), in TNBC patients compared to non-TNBC patients. Thus, we sought to investigate the contribution of Hippo signaling in TNBC by focusing particularly on transducers of the pathway. Impeding YAP transactivation by means of RNA interference or pharmacological inhibition was carried out, followed by evaluation of the subsequent biological changes at the molecular level. We successfully translated the observed data into a TNBC patient-derived xenograft cell line (PDXC). We discovered that nuclear translocation of YAP was associated with TNBC aggressive characteristics and activated the EGFR-AKT axis. Here, we explored the putative role of the Hippo transducer in enhancing cancer hostility and observed that YAP transduction drives proliferation, migration, and survival of TNBC by preventing cellular apoptosis through mediating EGFR activation. These observations suggest that YAP represents a major vulnerability in TNBC cells that may be exploited therapeutically.

The WAVE3/β-catenin oncogenic signaling regulates chemoresistance in triple negative breast cancer

BackgroundMetastatic breast cancer is responsible for the death of the majority of breast cancer patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the USA and worldwide. Triple negative breast cancer (TNBC), which lacks expression of hormone receptors (ER-α and PR) and ErbB2/HER2, is especially lethal due to its highly metastatic behavior, propensity to recur rapidly, and for its resistance to standard of care therapies, through mechanisms that remain incompletely understood. WAVE3 has been established as a promoter of TNBC development and metastatic progression. In this study, we investigated the molecular mechanisms whereby WAVE3 promotes therapy-resistance and cancer stemness in TNBC, through the regulation of β-catenin stabilization.MethodsThe Cancer Genome Atlas dataset was used to assess the expression of WAVE3 and β-catenin in breast cancer tumors. Kaplan–Meier Plotter analysis was used to correlate expression of WAVE3 and β-catenin with breast cancer patients’ survival probability. MTT assay was used to quantify cell survival. CRISPR/Cas9-mediated gene editing, 2D and 3D tumorsphere growth and invasion assays, Immunofluorescence, Western blotting, Semi-quantitative and real-time quantitative PCR analyses were applied to study the WAVE3/β-catenin oncogenic signaling in TNBC. Tumor xenograft assays were used to study the role of WAVE3 in mediating chemotherapy resistance of TNBC tumors.ResultsGenetic inactivation of WAVE3 in combination of chemotherapy resulted in inhibition of 2D growth and 3D tumorsphere formation and invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 did not. Further studies revealed that dual blocking of WAVE3 expression or phosphorylation in combination with chemotherapy treatment inhibited the activity and expression and stabilization of β-catenin. Most importantly, the combination of WAVE3-deficiency or WAVE3-phospho-deficiency and chemotherapy suppressed the oncogenic behavior of chemoresistant TNBC cells, both in vitro and in vivo.ConclusionWe identified a novel WAVE3/β-catenin oncogenic signaling axis that modulates chemoresistance of TNBC. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors.

Abstract P4-08-23: ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease in women, while the oncogenic mechanisms which contribute to TNBC development, progression, and metastasis are poorly investigated and therefore warrant the critical need to identify novel oncogenic signalings and develop new therapeutic targets. Results: We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α protein level and activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggesting that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles in controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC. Conclusion: ZHX2 has been highlighted as one critical hypoxia-related factors regulator contributing to triple-negative breast cancer in this work, which has enriched the upstream regulatory network of HIF-1α signaling. Moreover, identification of key residuals determining biological function of ZHX2 provides novel approaches for treating TNBC via targeting the hypoxia pathway. Citation Format: Chengheng Liao, Wentong Fang, Rachel Shi, Jeremy Simon, Travis Ptacek, Giada Zurlo, Youqiong Ye, Leng Han, Cheng Fan, Lei Bao, Christopher Llynard Ortiz, Hong-Rui Lin, Ujjawal Manocha, Weibo Luo, Yan Peng, William Y Kim, Lee-Wei Yang, Qing Zhang. ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-23.

Asiaticoside hampers epithelial-mesenchymal transition by promoting PPARG expression and suppressing P2RX7-mediated TGF-β/Smad signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes because of its high propensity to develop metastases. Here, the anticancer effects of asiaticoside (AC) against TNBC and the possible underlying mechanism were examined. We found that AC inhibited the TGF-β1 expression and the SMAD2/3 phosphorylation in TNBC cells, thereby impairing the TGF-β/SMAD signaling. AC inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells by suppressing the TGF-β/SMAD signaling. Meanwhile, AC inhibited the lung metastasis of TNBC cells in vivo and the expression of p-SMAD2/3 and vimentin, and increased the expression of E-cadherin and ZO-1 in the lung. Peroxisome proliferator activated receptor gamma (PPARG) was identified as a potential target of AC. AC increased PPARG expression, while PPARG knockdown attenuated the therapeutic effect of AC. AC-mediated PPARG overexpression suppressed the transcription of P2X purinoceptor 7 (P2RX7). The restoration of P2RX7 reversed the therapeutic effect of AC. These results suggested that AC blocked P2RX7-mediated TGF-β/SMAD signaling by increasing PPARG expression, thereby suppressing EMT in TNBC.

Histone H3 proline 16 hydroxylation regulates mammalian gene expression.

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression.

Abstract 967: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT)

Abstract Triple-negative breast cancers (TNBC) are a subset of breast cancer that is highly aggressive and has a poor prognosis. The TNBC subtype has no effective molecular targeted therapies. Gene expression studies have identified a subset of TNBC that is enriched with androgen receptors (AR) and androgen receptor signaling. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Interference with androgen signaling in TNBC with AR-inhibiting drugs have shown a reduction in epithelial-to-mesenchymal transition (EMT), which is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal-like stem cells. Approximately one-third of TNBC expressed AR, and evaluation of AR-positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. Previous studies have shown that AR inhibition or AR knockdown significantly reduces migration and invasion and EMT in different TNBC cell lines. γ-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer effects and have little or no effect on normal cell viability. Studies show that treatment with 0-7 µM γ-tocotrienol reduced AR levels, proliferation, migration and invasion in a dose-responsive manner in TNBC MDA-MB-231 and MDA MB-453 cell lines. Results also show that treatment with 5 µM (MDA-MB-231) and 7 µM (MDA-MB-453) γ-tocotrienol induced a reversal in EMT cell biomarkers, as well as reversal in EMT morphological and behavioral characteristics in these TNBC cell lines. Western blot analysis show that similar treatment with γ-tocotrienol significantly decreased DHT-induced N-cadherin (mesenchymal cell biomarker) and increased expression in cytokeratin 18 and E-cadherin (epithelial cell biomarkers) in these cells. AR signaling is known to play an important role in stimulating DHT-induced EMT by stimulating the STAT3/Snail signaling pathway in MDA-MB231. However, results in the present study shows that γ-tocotrienol treatment significantly inhibited activation of STAT3 and Snail proteins. Additional immunocytochemistry experiments showed that γ-tocotrienol treatment significantly inhibited DHT-induced cytoskeleton changes in TNBC MDA-MB-231 and MDA MB-453 cell lines. In conclusion, these results demonstrated that γ-tocotrienol treatment inhibits AR expression and DHT-dependent EMT and cytoskeleton changes in TNBC cells. These findings suggest that AR may be a potential therapeutic target for treating both LAR and non-LAR TNBC subtypes. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Nayef Aldabaan, Tasmin A. Sultana, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 967.

Abstract 3998: Discovery of a small molecule inhibitor for triple-negative breast cancer (TNBC)

Abstract Introduction: Little progress has been made towards developing alternative therapeutic strategies for triple-negative breast cancer (TNBC). Aberrant Notch1 activation, particularly in breast cancer stem cells (BCSCs) in TNBC tumors, enhances this cancer's aggressivity and resistance and consequently contributes to the poor overall survival of patients with TNBC. In contrast, inhibition of Notch1 has been shown to suppress TNBC growth. Thus, Notch1 presents as an attractive target for inhibiting TNBC growth. This study aims to overcome the limitations of current Notch1 inhibitors and identify a more specific inhibitor. Methods: Small molecule screening was performed using structure-activity relationship (SAR) studies, and molecular docking and isothermal calorimetric (ITC) assays were used to determine the target sites of our rationally designed Notch1 inhibitor on the Notch1 receptor. The therapeutic potential of the identified small molecule Notch1 inhibitor was determined on BCSC and TNBC (ALDH+, BCSC, ALDH-, MDA-MB-231) cell lines using molecular studies and in vivo assays. Results: We identified ASR490, a small molecule that binds to the NRR (the activation switch of the Notch receptor) of Notch1. Molecular docking studies indicated a strong interaction between ASR490 and the NRR domain with binding energy of -52.55 kcal/mol. Residue-wise interaction analysis estimated three hydrogen-bond interactions between ASR490 and NRR residues Asn-1483, Glu-1673, and Gly-1664 mediated by water molecules. The finding was further confirmed using biophysical assays (thermal shift assay and isothermal calorimetric analysis) using recombinant Notch1 protein. In vitro results demonstrated that ASR490 significantly inhibited TNBC growth (p &amp;lt;0.001) at nM concentrations, with an IC50 of 760 nM at 24h. Further, the inhibitory effect was confirmed by TNBC’s colony-forming abilities. Western blot analysis confirmed that ASR490 downregulated the expressions of NICD (the active form of Notch1) and its downstream effectors Hey-1 and HES1 by ubiquitinating NRR of Notch1. ASR490 was found to be non-toxic to healthy cells. Moreover, ASR490 exhibited a maximum tolerated dose of more than 100 mg/kg body weight in mice and abrogated TNBC tumor growth in vivo xenograft models. Conclusion: Our results demonstrate ASR490 as a potential therapeutic agent that abolishes Notch1 signaling in TNBC and BCSCs by specifically targeting Notch1 NRR. Further investigation is required for the clinical translational potential of this agent. Citation Format: Uttara Saran, Balaji Chandrasekaran, Ashish Tyagi, Amandeep Singh, Murali K. Ankem, Arun K. Sharma, Chendil Damodaran. Discovery of a small molecule inhibitor for triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3998.