Triple-negative Breast Cancer Cancer Stem Cells Research Articles

Abstract 5435: CUEDC2 involved in the inhibitory effect of dandelion on triple-negative breast cancer stem cells

Abstract Background: Cancer stem cells (CSCs) exert a substantial influence on the initiation, progression, and metastasis of triple-negative breast cancer (TNBC). Dandelion has exhibited encouraging outcomes in impeding the proliferation of TNBC cells. Nevertheless, the extent of investigation regarding the efficacy of dandelion in targeting CSCs remains restricted. Purpose: The aim of this study was to investigate the impact of dandelion extract (DE) on CSCs properties. Methods: In vitro experiments were conducted using TNBC cell lines. Mice bearing TNBC tumors were established for in vivo investigations. Biochemical analysis and cell biological assay were carried to evaluate the effect of dandelion and the underlying mechanisms. Results: DE treatment significantly decreased the proportion of ALDH+ cells, mammosphere formation ability, and CSCs-related markers. CUEDC2 is highly expressed in ALDH+ TNBC cells, and its overexpression increased the expression of markers related to CSCs. Conversely, the tumor growth was impeded in CUEDC2 KO mice and the markers related to CSCs also were also reduced. CUEDC2 overexpression reversed the inhibitory effect of DE on TNBC CSCs properties. Mechanistically, CUEDC2 related pathway involved in the inhibitory effect of DE on TNBC CSCs properties. Conclusion: CUEDC2 played a crucial role in maintaining the stem cell properties of TNBC. DE inhibit the self-renewal of TNBC stem cells by regulating the CUEDC2 related signaling pathway. Keywords: Dandelion; CUEDC2; Triple-negative breast cancer; Cancer stem cell. Citation Format: Xinxin Deng, Yanna Jiao, Huifeng Hao, Zhengwang Guo, Dong Xue, Shuyan Han. CUEDC2 involved in the inhibitory effect of dandelion on triple-negative breast cancer stem cells [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 5435.

Epidermal growth factor receptor inhibition potentiates chemotherapeutics-mediated sensitization of metastatic breast cancer stem cells.

Metastasis has been a cause of the poor prognosis and cancer relapse of triple-negative breast cancer (TNBC) patients. The metastatic nature of TNBC is contributed by the breast cancer stem cells (CSCs) which have been implicated in tumorigenesis. Higher expression of epidermal growth factor receptor (EGFR) in breast CSCs has been used as a molecular target for breast cancer therapeutics. Thus, it necessitates the design and generation of efficacious EGFR inhibitors to target the downstream signaling associated with the cellular proliferation and tumorigenesis of breast cancer. To generate efficacious EGFR inhibitors that can potentiate the chemotherapeutic-mediated mitigation of breast cancer tumorigenesis. We identified small molecule EGFR inhibitors using molecular docking studies. In-vitro screening of the compounds was undertaken to identify the cytotoxicity profile of the small-molecule EGFR inhibitors followed by evaluation of the non-cytotoxic compounds in modulating the doxorubicin-induced migration, in-vitro tumorigenesis potential, and their effect on the pro-apoptotic genes' and protein markers' expression in TNBC cells. Compound 1e potentiated the doxorubicin-mediated inhibitory effect on proliferation, migration, in-vitro tumorigenesis capacity, and induction of apoptosis in MDA-MB-231 cells, and in the sorted CD24+-breast cancer cells and CD24-/CD44+-breast CSC populations. Orthotopic xenotransplantation of the breast CSCs-induced tumors in C57BL/6J mice was significantly inhibited by the low dose of Doxorubicin in the presence of compound 1e as depicted by molecular and immunohistochemical analysis. Thus, the study suggests that EGFR inhibition-mediated sensitization of the aggressive and metastatic breast CSCs in TNBCs toward chemotherapeutics may reduce the relapse of the disease.

Inhibitory effect of the novel tyrosine kinase inhibitor DCC-2036 on triple-negative breast cancer stem cells through AXL-KLF5 positive feedback loop

Triple-negative breast cancer (TNBC), an aggressive histological subtype of breast cancer, exhibits a high risk of early recurrence rate and a poor prognosis, and it is primarily associated with the abundance of cancer stem cells (CSCs). At present, the strategies for effectively eradicating or inhibiting TNBC CSCs are still limited, which makes the development of novel drugs with anti-CSCs function be of great value for the treatment of TNBC, especially the refractory TNBC. In this study, we found that the small-molecule tyrosine kinase inhibitor DCC-2036 suppressed TNBC stem cells by inhibiting the tyrosine kinase AXL and the transcription factor KLF5. DCC-2036 downregulated the expression of KLF5 by decreasing the protein stability of KLF5 via the AXL-Akt-GSK3β signal axis, and in turn, the downregulation of KLF5 further reduced the expression of AXL via binding to its promotor (−171 to −162 bp). In addition, p-AXL/AXL levels were positively correlated with KLF5 expression in human TNBC specimens. These findings indicated that DCC-2036 is able to suppress the CSCs in TNBC by targeting the AXL-KLF5 positive feedback loop. Moreover, our findings indicated that DCC-2036 increased the sensitivity of TNBC chemotherapy. Therefore, this study proposes a potential drug candidate and several targets for the treatment of refractory TNBC.

Addiction of Cancer Stem Cells to MUC1-C in Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited treatment options. TNBC progression is associated with expansion of cancer stem cells (CSCs). Few insights are available regarding druggable targets that drive the TNBC CSC state. This review summarizes the literature on TNBC CSCs and the compelling evidence that they are addicted to the MUC1-C transmembrane protein. In normal epithelia, MUC1-C is activated by loss of homeostasis and induces reversible wound-healing responses of inflammation and repair. However, in settings of chronic inflammation, MUC1-C promotes carcinogenesis. MUC1-C induces EMT, epigenetic reprogramming and chromatin remodeling in TNBC CSCs, which are dependent on MUC1-C for self-renewal and tumorigenicity. MUC1-C-induced lineage plasticity in TNBC CSCs confers DNA damage resistance and immune evasion by chronic activation of inflammatory pathways and global changes in chromatin architecture. Of therapeutic significance, an antibody generated against the MUC1-C extracellular domain has been advanced in a clinical trial of anti-MUC1-C CAR T cells and in IND-enabling studies for development as an antibody–drug conjugate (ADC). Agents targeting the MUC1-C cytoplasmic domain have also entered the clinic and are undergoing further development as candidates for advancing TNBC treatment. Eliminating TNBC CSCs will be necessary for curing this recalcitrant cancer and MUC1-C represents a promising druggable target for achieving that goal.

Abstract 5826: Autophagy regulates cancer stem cell properties in triple negative breast cancer via miR-181a-mediated regulation of ATG5/ATG2B

Abstract Autophagy has a dual role in the maintenance of cancer stem cells (CSCs), but the precise relationship between autophagy and cancer stemness requires further investigation. In this study, it was found that luminal and triple-negative breast cancer require distinct therapeutic approaches because of their different expressions of autophagy flux. We identified that autophagy flux was inhibited in triple-negative breast cancer (TNBC) CSCs. Moreover, miRNA-181a (miR-181a) is upregulated both in TNBC CSCs and patients. ATG5 and ATG2B participate in the early formation of autophagosomes and were revealed as targets of miR-181a. Inhibition of miR-181a expression led to attenuation of TNBC cancer stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. In summary, autophagy inhibits cancer stemness through the miR-181a-regulated mechanism in TNBC. Promoting tumor-suppressive autophagy using curcumin may be a potential method for the treatment of TNBC. Citation Format: Jong Hoon Park, Jee Won Park, Yesol Kim, Jaehee Jun, Yejin Ahn. Autophagy regulates cancer stem cell properties in triple negative breast cancer via miR-181a-mediated regulation of ATG5/ATG2B [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 5826.

Abstract 893: GD2 is a potential biomarker for glioblastoma cancer stem cells

Abstract Introduction: Treatment resistance and tumor recurrence of Glioblastoma (GBM) have been associated with cancer stem cells (CSCs), a subpopulation of cancer cells defined by capabilities of sphere-formation in 3D culture, self-renewal, migration, and survival in serum-deprived conditions. Therefore, therapies specifically targeting CSCs are urgently needed. Previous works have identified Ganglioside GD2 as a biomarker of triple negative breast cancer CSCs and suggested that GD2 is highly expressed in GBM cells. Here, we hypothesize that GD2 is also a potential biomarker of GBM CSCs and is involved with cancer stemness. Methods: GBM cells U-87 MG and U-118 MG were sorted as GD2- and GD2+ cells via FACS. Cells were cultured in 2D and 3D. 3D neurosphere size was measured and GD2 in 2D and 3D cultures was quantified via flow cytometry. Transwell migration assay was used to examine the migration capability of GD2- and GD2+ cells. Unsorted cells were cultured in either complete (10% serum) or deprived (1% serum) media and their GD2 was quantified via flow cytometry. Results: To test our hypothesis, we determined whether GD2+ cells express CSCs capabilities of sphere-formation in 3D culture, migration, and survival in serum-deprived conditions. First, we examined the sphere-forming capability by culturing GD2-, GD2+, and unsorted cells in 3D and found that they all formed neurospheres. However, GD2+ neurosphere size was significantly larger compared with unsorted and GD2- neurospheres for day 1 and 2. Interestingly, the significant difference was no longer seen on day 6. Analysis for GD2 expression in the spheres revealed that GD2- spheres contained GD2+ cells, suggesting that GD2- cells could spontaneously dedifferentiate to generate CSCs. To test this, we sorted GD2- and GD2+ cells into 2D conditions and quantified daily GD2 expression via flow cytometry. We found that GD2- seeded cells spontaneously expressed GD2 over six days, explaining the presence of GD2 in GD2- neurospheres. GD2+ cells also demonstrated the enhanced capability to migrate of CSCs with a significantly higher number of migrated cells compared with GD2- cells in the transwell migration assay. To examine the ability to survive in serum-deprived conditions, we cultured unsorted cells in either complete or deprived media. We found that these cells proliferated in the serum-deprived condition at approximately the same level as complete media. GD2 analysis by flow cytometry also showed a remarkably higher percentage of GD2+ cells in deprived media, suggesting that GD2 is involved in driving cell survival and maintaining cancer stemness in serum-deprived conditions. Conclusion: Our data suggests that GD2+ cells demonstrate characteristics of CSCs and GD2 is a potential biomarker of CSCs in GBM. Therefore, GD2 could be utilized as a differential therapeutic target for GBM. Further mechanistic studies are necessary to elucidate how GD2 is synthesized and maintains the stemness of GBM CSCs. Citation Format: Khoa Nguyen, Minh Tran, Thomas Cheng, Kurtis Willingham, Reza Izadpanah, Matthew Burow. GD2 is a potential biomarker for glioblastoma cancer stem cells [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 893.

Targeted liposomal doxorubicin/ceramides combinations: The importance to assess the nature of drug interaction beyond bulk tumor cells

One of the major assets of anticancer nanomedicine is the ability to co-deliver drug combinations, as it enables targeting of different cellular populations and/or signaling pathways implicated in tumorigenesis and thus tackling tumor heterogeneity. Moreover, drug resistance can be circumvented, for example, upon co-encapsulation and delivery of doxorubicin and sphingolipids, as ceramides. Herein, the impact of short (C6) and long (C18) alkyl chain length ceramides on the nature of drug interaction, within the scope of combination with doxorubicin, was performed in bulk triple-negative breast cancer (TNBC) cells, as well as on the density of putative cancer stem cells and phenotype, including live single-cell tracking.C6- or C18-ceramide enabled a synergistic drug interaction in all conditions and (bulk) cell lines tested. However, differentiation among these two ceramides was reflected on the migratory potential of cancer cells, particularly significant against the highly motile MDA-MB-231 cells. This effect was supported by the downregulation of the PI3K/Akt pathway enabled by C6-ceramide and in contrast with C18-ceramide. The decrease of the migratory potential enabled by the targeted liposomal combinations is of high relevance in the context of TNBC, due to the underlying metastatic potential.Surprisingly, the nature of the drug interaction assessed at the level of bulk cancer cells revealed to be insufficient to predict whether a drug combination enables a decrease in the percentage of the master regulators of tumor relapse as ALDH+/high putative TNBC cancer stem cells, suggesting, for the first time, that it should be extended further down to this level.

Autophagy inhibits cancer stemness in triple-negative breast cancer via miR-181a-mediated regulation of ATG5 and/or ATG2B.

Autophagy has a dual role in the maintenance of cancer stem cells (CSCs), but the precise relationship between autophagy and cancer stemness requires further investigation. In this study, it was found that luminal and triple‐negative breast cancers require distinct therapeutic approaches because of their different amounts of autophagy flux. We identified that autophagy flux was inhibited in triple‐negative breast cancer (TNBC) CSCs. Moreover, miRNA‐181a (miR‐181a) expression is upregulated in both TNBC CSCs and patient tissues. Autophagy‐related 5 (ATG5) and autophagy‐related 2B (ATG2B) participate in the early formation of autophagosomes and were revealed as targets of miR‐181a. Inhibition of miR‐181a expression led to attenuation of TNBC stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. In summary, autophagy inhibits cancer stemness through the miR‐181a‐regulated mechanism in TNBC. Promoting tumor‐suppressive autophagy using curcumin may be a potential method for the treatment of TNBC.

MUC1-C Dictates JUN and BAF-Mediated Chromatin Remodeling at Enhancer Signatures in Cancer Stem Cells.

These findings show that MUC1-C, which is necessary for the CRPC and TNBC CSC state, activates a novel pathway involving JUN/AP-1 and ARID1A/BAF that regulates chromatin accessibility of stemness-associated gene enhancers.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Clinically Translatable Approaches of Inhibiting TGF-β to Target Cancer Stem Cells in TNBC.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that disproportionally accounts for the majority of breast cancer-related deaths due to the lack of specific targets for effective treatments. In this review, we highlight the complexity of the transforming growth factor-beta family (TGF-β) pathway and discuss how the dysregulation of the TGF-β pathway promotes oncogenic attributes in TNBC, which negatively affects patient prognosis. Moreover, we discuss recent findings highlighting TGF-β inhibition as a potent method to target mesenchymal (CD44+/CD24−) and epithelial (ALDHhigh) cancer stem cell (CSC) populations. CSCs are associated with tumorigenesis, metastasis, relapse, resistance, and diminished patient prognosis; however, due to differential signal pathway enrichment and plasticity, these populations remain difficult to target and persist as a major barrier barring successful therapy. This review highlights the importance of TGF-β as a driver of chemoresistance, radioresistance and reduced patient prognosis in breast cancer and highlights novel treatment strategies which modulate TGF-β, impede cancer progression and reduce the rate of resistance generation via targeting the CSC populations in TNBC and thus reducing tumorigenicity. Potential TGF-β inhibitors targeting based on clinical trials are summarized for further investigation, which may lead to the development of novel therapies to improve TNBC patient prognosis.

Lovastatin Inhibits EMT and Metastasis of Triple-Negative Breast Cancer Stem Cells Through Dysregulation of Cytoskeleton-Associated Proteins.

Triple-negative breast cancer (TNBC) is more aggressive and has poorer prognosis compared to other subtypes of breast cancer. Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells transform into mesenchymal-like cells capable of migration, invasion, and metastasis. Recently, we have demonstrated that lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor and a lipid-lowering drug, could inhibit stemness properties of cancer stem cells (CSCs) derived from TNBC cell in vitro and in vivo. This study is aimed at investigating whether lovastatin inhibits TNBC CSCs by inhibiting EMT and suppressing metastasis and the mechanism involved. In the present study, we found that lovastatin dysregulated lysine succinylation of cytoskeleton-associated proteins in CSCs derived from TNBC MDA-MB-231 cell. Lovastatin inhibited EMT as demonstrated by down-regulation of the protein levels of Vimentin and Twist in MDA-MB-231 CSCs in vitro and vivo and by reversal of TGF-β1-induced morphological change in MCF10A cells. Lovastatin also inhibited the migration of MDA-MB-231 CSCs. The disruption of cytoskeleton in TNBC CSCs by lovastatin was demonstrated by the reduction of the number of pseudopodia and the relocation of F-actin cytoskeleton. Combination of lovastatin with doxorubicin synergistically inhibited liver metastasis of MDA-MB-231 CSCs. Bioinformatics analysis revealed that higher expression levels of cytoskeleton-associated genes were characteristic of TNBC and predicted survival outcomes in breast cancer patients. These data suggested that lovastatin could inhibit the EMT and metastasis of TNBC CSCs in vitro and in vivo through dysregulation of cytoskeleton-associated proteins.

TrkA Interacts with and Phosphorylates STAT3 to Enhance Gene Transcription and Promote Breast Cancer Stem Cells in Triple-Negative and HER2-Enriched Breast Cancers.

Simple SummaryBreast cancer is the leading cancer in American women. Due to the inherent aggressiveness of triple-negative and HER2-enriched breast cancers, it is imperative to identify novel molecular targets for therapeutic intervention. Due to their abnormal activities in metastatic breast cancers, JAK2–STAT3 and TrkA pathways have been individually implicated in aggressive breast tumors. However, their co-activation and signaling interactions have not been thoroughly investigated. Therefore, our study aimed to elucidate the extent of crosstalk between JAK2–STAT3 and TrkA signaling pathways and its impact on breast cancer. Our data revealed a novel interaction between TrkA and STAT3, and that this interaction results in STAT3 phosphorylation and activation by TrkA, leading to enhanced stemness gene expression and stem cell renewal. We further found that the co-activation of JAK2–STAT3 and TrkA pathways is correlated with shorter time to develop overall and organ-specific metastasis, suggesting that this signaling crosstalk underlies the aggressiveness of triple-negative and HER2-enriched breast cancers.JAK2–STAT3 and TrkA signaling pathways have been separately implicated in aggressive breast cancers; however, whether they are co-activated or undergo functional interaction has not been thoroughly investigated. Herein we report, for the first time that STAT3 and TrkA are significantly co-overexpressed and co-activated in triple-negative breast cancer (TNBC) and HER2-enriched breast cancer, as shown by immunohistochemical staining and data mining. Through immunofluorescence staining–confocal microscopy and immunoprecipitation–Western blotting, we found that TrkA and STAT3 co-localize and physically interact in the cytoplasm, and the interaction is dependent on STAT3-Y705 phosphorylation. TrkA–STAT3 interaction leads to STAT3 phosphorylation at Y705 by TrkA in breast cancer cells and cell-free kinase assays, indicating that STAT3 is a novel substrate of TrkA. β-NGF-mediated TrkA activation induces TrkA–STAT3 interaction, STAT3 nuclear transport and transcriptional activity, and the expression of STAT3 target genes, SOX2 and MYC. The co-activation of both pathways promotes breast cancer stem cells. Finally, we found that TNBC and HER2-enriched breast cancer with JAK2–STAT3 and TrkA co-activation are positively associated with poor overall metastasis-free and organ-specific metastasis-free survival. Collectively, our study uncovered that TrkA is a novel activating kinase of STAT3, and their co-activation enhances gene transcription and promotes breast cancer stem cells in TNBC and HER2-enriched breast cancer.

Nrf2 inhibition sensitizes breast cancer stem cells to ionizing radiation via suppressing DNA repair

Radiation is widely used for cancer treatment but the radioresistance properties of cancer stem cells (CSCs) pose a significant challenge to the success of cancer therapy. Nuclear factor erythroid-2-related factor 2 (Nrf2) has emerged as a prominent regulator of cellular antioxidant responses and its over-activation is associated with drug resistant in cancer cells. However, the role of Nrf2 signaling in regulating the response of CSCs to irradiation has yet to be defined. Here, we show that exposure of triple-negative breast cancer (TNBC) cells to ionizing radiation (IR) upregulates Nrf2 expression and promotes its nuclear translocation in a reactive oxygen species (ROS)-dependent manner. Ectopic overexpression of Nrf2 attenuates, whereas knockdown of Nrf2 potentiates IR-induced killing of TNBC CSCs. Mechanistically, we found that Nrf2 knockdown increases IR-induced ROS production and impedes DNA repair at least in part via inhibition of DNA-PK. Furthermore, activation of Nrf2 by sulforaphane diminishes, whereas inhibition of Nrf2 by ML385 enhances IR-induced killing of TNBC CSCs. Collectively, these results demonstrate that IR-induced ROS production can activate Nrf2 signaling, which in turn counteracts the killing effect of irradiation. Therefore, pharmacological inhibition of IR-induced Nrf2 activation by ML385 could be a new therapeutic approach to sensitize therapy-resistant CSCs to radiotherapy.

Autophagy Regulates Cancer Stem Cell Properties in Triple Negative Breast Cancer Via miR-181a-Mediated Regulation of ATG5/ATG2B

Background: Cancer stemness, miRNA, and autophagy flux are known to contribute to the maintenance of cancers; however, determination of the precise interactions among autophagy, miRNAs, and cancer stemness requires further investigation. Methods: Western blot, immunocytochemistry, immunofluorescence staining, quantitative real-time PCR were performed to detect gene expression. Tumorsphere formation assay, Aldefluor assay, limiting dilution assay were carried out to measure cancer stemness. Acridine orange and Cyto-ID® staining were used to detect autophagy vacuoles. Findings: Autophagy flux was inhibited in triple-negative breast cancer (TNBC) cancer stem cells (CSCs). Moreover, miRNA-181a (miR-181a) is upregulated both in TNBC CSCs and patients. ATG5 and ATG2B participate in the early formation of autophagosomes and were revealed as targets of miR-181a. Inhibition of miR-181a expression led to attenuation of cancer stemness and an increase in autophagy flux. Furthermore, treatment with curcumin led to attenuation of cancer stemness in TNBC CSCs; the expression of ATG5 and ATG2B was enhanced and there was an increase of autophagy flux. Interpretation: It could be suggested that luminal and triple-negative breast cancer require distinct therapeutic approaches because of their different expressions of autophagy flux. These results indicated that ATG5 and ATG2B are involved in the suppression of cancer stemness in TNBC. Autophagy inhibits cancer stemness through the miR-181a-regulated mechanism in TNBC. Promoting tumor-suppressive autophagy using curcumin may be a potential method for the treatment of TNBC. Funding: This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (2019R1A2B5B03069738, NRF- 2016R1A5A1011974). Declaration of Interest: All the authors declared no competing interests. Ethical Approval: This study was approved by the 235 Institutional Review Board of Seoul National University Hospital (IRB number: 1704-236 019-843). A breast cancer tissue array (BC081120f, US Biomax Inc, USA).

Signaling pathways essential for triple-negative breast cancer stem-like cells.

Since the discovery of breast cancer stem cells (CSCs), a significant effort has been made to identify and characterize these cells. It is a generally believe that CSCs play an important role in cancer initiation, therapy resistance, and progression of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with poor prognosis. Thus, therapies targeting these cells would be a valuable addition to standard treatments that primarily target more differentiated, rapidly dividing TNBC cells. Although several cell surface and intracellular proteins have been described as biomarkers for CSCs, none of these are specific to this population of cells. Recent research is moving toward cellular signaling pathways as targets and biomarkers for CSCs. The WNT pathway, the nuclear factor-kappa B (NF-κB) pathway, and the cholesterol biosynthesis pathway have recently been identified to play a key role in proliferation, survival, and differentiation of CSCs, including those of breast cancer. In this review, we assess recent findings related to these three pathways in breast CSC, with particular focus on TNBC CSCs, and discuss how targeting these pathways, in combination with current standard of care, might prove effective and improve the prognosis of TNBC patients.

89Zr-Labeled Multifunctional Liposomes Conjugate Chitosan for PET-Trackable Triple-Negative Breast Cancer Stem Cell Targeted Therapy.

PurposeTherapy for triple-negative breast cancer (TNBC) is a global problem due to lack of specific targets for treatment selection. Cancer stem cells (CSCs) are responsible for tumor formation and recurrence but also offer a promising target for TNBC-targeted therapy. Here, zirconium-89 (89Zr)-labelled multifunctional liposomes (MLPs) surface-decorated with chitosan (CS) were fabricated to specifically target and trace cluster of differentiation 44+ (CD44+) TNBC CSCs specifically.Patients and MethodsThe biological basis of CS targeting CD44 for cancer therapy was investigated by detecting the expression of CD44 in TNBC CSCs and TNBC tissues. Molecular docking and dynamics simulations were performed to investigate the molecular basis of CS targeting CD44 for cancer therapy. Gambogic acid (GA)-loaded, 89Zr@CS-MLPs (89Zr-CS-GA-MLPs) were prepared, and their uptake and biodistribution were observed. The anti-tumor efficacy of 89Zr@CS-GA-MLPs was investigated in vivo.ResultsCD44 is overexpressed in TNBC CSCs and tissues. Molecular docking and dynamics simulations showed that CS could be stably docked into the active site of CD44 in a reasonable conformation. Furthermore, 89Zr@CS-GA-MLPs were able to bind specifically to CD44+ TNBC stem-like cells and accumulated in tumors of xenograft-bearing mice with excellent radiochemical stability. 89Zr@CS-GA-MLPs loaded with GA showed remarkable anti-tumor efficacy in vivo.ConclusionThe GA-loaded, 89Zr-labelled, CS-decorated MLPs developed in this study represent a novel strategy for TNBC imaging and therapy.

DNMT1: A key drug target in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Altered epigenetics regulation including DNA hypermethylation by DNA methyltransferase 1 (DNMT1) has been implicated as one of the causes of TNBC tumorigenesis. In this review, the oncogenic functions rendered by DNMT1 in TNBCs, and DNMT1 inhibitors targeting TNBC cells are presented and discussed. In summary, DNMT1 expression is associated with poor breast cancer survival, and it is overexpressed in TNBC subtype. The oncogenic roles of DNMT1 in TNBCs include: (1) Repression of estrogen receptor (ER) expression; (2) Promotion of epithelial-mesenchymal transition (EMT) required for metastasis; (3) Induces cellular autophagy and; (4) Promotes the growth of cancer stem cells in TNBCs. DNMT1 confers these phenotypes by hypermethylating the promoter regions of ER, multiple tumor suppressor genes, microRNAs and epithelial markers involved in suppressing EMT. DNMT1 inhibitors exert anti-tumorigenic effects against TNBC cells. This includes the hypomethylating agents azacitidine, decitabine and guadecitabine that might sensitize TNBC patients to immune checkpoint blockade therapy. DNMT1 represents an epigenetic target for TNBC cells destruction as well as to derail their metastatic and aggressive phenotypes.

Reversal of HER2 Negativity: An Unexpected Role for Lovastatin in Triple-Negative Breast Cancer Stem Cells.

Effective treatment modality for triple-negative breast cancer (TNBC) is currently lacking due to the absence of defined receptor targets. Recently, we have demonstrated that lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor and a lipid-lowering drug, can selectively inhibit TNBC by targeting cancer stem cells in vivo and in vitro. Interestingly, we found that lovastatin induced the reappearance of human epidermal growth factor receptor 2 (HER2), one of the triple receptors that are missing in TNBC. This prompted us to explore the possibility of regaining sensitivity of TNBC cancer stem cells to receptor tyrosine kinase-targeting drugs. We found that while the combination of lovastatin with a HER2 inhibitor was not sufficient to show synergism, addition of an epidermal growth factor receptor (EGFR/HER1) inhibitor to this combination resulted in significant synergistic inhibitory effect on cell viability. Our findings provide a potential novel strategy of designing a cocktail composed of a lipid-lowering drug and two receptor tyrosine kinase inhibitors for the treatment of TNBC.

Schlafen12 Reduces the Aggressiveness of Triple Negative Breast Cancer through Post-Transcriptional Regulation of ZEB1 That Drives Stem Cell Differentiation.

Schlafen12 (SLFN12) promotes human intestinal and prostatic epithelial differentiation. We sought to determine whether SLFN12 reduces triple-negative breast cancer (TNBC) aggressiveness. We validated bioinformatics analyses of publicly available databases by staining human TNBC. After virally overexpressing or siRNA-reducing SLFN12 in TNBC cell lines, we measured proliferation by CCK-8 assay, invasion into basement-membrane-coated pores, mRNA by q-RT-PCR and protein by Western blotting. Flow cytometry assessed proliferation and stem cell marker expression, and sorted CD44+/CD24- cells. Stemness was also assessed by mammosphere formation, and translation by click-it-AHA chemistry. SLFN12 expression was lower in TNBC tumors and correlated with survival. SLFN12 overexpression reduced TNBC MDA-MB-231, BT549, and Hs578T proliferation. In MDA-MB-231 cells, AdSLFN12 reduced invasion, promoted cell cycle arrest, increased E-cadherin promoter activity, mRNA, and protein, and reduced vimentin expression and protein. SLFN12 knockdown increased vimentin. AdSLFN12 reduced the proportion of MDA-MB-231 CD44+CD24- cells, with parallel differentiation changes. SLFN12 overexpression reduced MDA-MB-231 mammosphere formation. SLFN12 overexpression decreased ZEB1 and Slug protein despite increased ZEB1 and Slug mRNA in all three lines. SLFN12 overexpression accelerated MDA-MB-231 ZEB1 proteasomal degradation and slowed ZEB1 translation. SLFN12 knockdown increased ZEB1 protein. Coexpressing ZEB1 attenuated the SLFN12 effect on E-cadherin mRNA and proliferation in all three lines. SLFN12 may reduce TNBC aggressiveness and improve survival in part by a post-transcriptional decrease in ZEB1 that promotes TNBC cancer stem cell differentiation.