Stemness Of Triple-negative Breast Cancer Cells Research Articles

VEGFA/NRP-1/GAPVD1 axis promotes progression and cancer stemness of triple-negative breast cancer by enhancing tumor cell-macrophage crosstalk.

Triple-negative breast cancer (TNBC) has long been considered a major clinical challenge due to its aggressive behavior and poor prognosis. Cancer stem cells (CSCs) are known as the main cells responsible for tumor origination, progression, recurrence and metastasis. Here, we report that M2-type tumor-associated macrophages (TAMs) contribute to cancer stemness in TNBC cells via the secretion of VEGFA. Reciprocally, elevated VEGFA expression by TAM-educated TNBC cells acts as a regulator of macrophage polarization, therefore constitute a feed-back loop between TNBC cells and TAMs. Mechanistically, VEGFA facilitates the CSC phenotype via the NRP-1 receptor and downstream GAPVD1/Wnt/β-catenin signaling pathway in TNBC cells. Our study underscores the crosstalk between TNBC cells and TAMs mediated by VEGFA and further clarifies the role and underlying mechanisms of the VEGFA/NRP-1/GAPVD1 axis in regulating cancer stemness. We also document an immunosuppressive function of VEGFA in the tumor microenvironment (TME). Therefore, the present study indicates crosstalk between TNBC cells and TAMs induced by VEGFA and provides a potential implication for the combination of immunotherapy and VEGFA-targeted agents in TNBC therapy.

YTHDF1 enhances stemness and chemoresistance in triple-negative breast cancer cells by upregulating SIAH2.

Triple-negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer, and chemoresistance is the major determinant of TNBC treatment failure. This study explores the molecular mechanism of TNBC chemoresistance.The Cancer Genome Atlas, breast cancer integrative platform, and GEPIA databases were used to analyze the expression and correlation of YTHDF1 and seven in absentia homology 2 (SIAH2) in breast cancer. Knockdown of YTHDF1 and SIAH2, or overexpression of SIAH2 in vitro and in vivo, was conducted to evaluate the impact of changes in YTHDF1 and SIAH2 expression on TNBC cell proliferation, apoptosis, stemness, drug resistance, and Hippo pathway gene expression.YTHDF1 and SIAH2 were highly expressed in breast cancer patients and TNBC cells. Knockdown of YTHDF1 and SIAH2 significantly inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells. Mechanistically, the knockdown of YTHDF1 inhibited the expression of SIAH2, thereby downregulating the Hippo pathway, which inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells.The current findings revealed the regulatory mechanism of YTHDF1 in TNBC and clarified the role of the YTHDF1/SIAH2 axis in TNBC drug resistance and stemness. This could provide new insights into the vital role of targeting YTHDF1/SIAH2 to suppress drug resistance and stemness in TNBC cells.

LncRNA LUCAT1/ELAVL1/LIN28B/SOX2 Positive Feedback Loop Promotes Cell Stemness in Triple-Negative Breast Cancer.

Background Triple-negative breast cancer (TNBC), as a subtype of breast cancer (BC), features an aggressive nature. Long noncoding RNAs (lncRNAs) are proved to get involved in the processes of cancers. lncRNA lung cancer associated transcript 1 (LUCAT1) has been reported in multiple cancers. The role of LUCAT1 in TNBC and its latent regulatory mechanism were investigated. Methods RT-qPCR was performed to examine LUCAT1 expression. Functional experiments were implemented to disclose the role of LUCAT1 in TNBC. The underlying regulatory mechanism of LUCAT1 in TNBC was explored by chromatin immunoprecipitation (ChIP), RNA-binding protein immunoprecipitation (RIP), luciferase reporter, and RNA pull-down assays. Results LUCAT1 is significantly overexpressed in TNBC cells. LUCAT1 interference impedes cell stemness in TNBC cells. SRY-box transcription factor 2 (SOX2) is an active transcription factor of LUCAT1. LUCAT1 recruits ELAV-like RNA binding protein 1 (ELAVL1) protein to stabilize lin-28 homolog B (LIN28B) mRNA, thereby further modulating SOX2 expression, which forms a positive feedback loop. Conclusion The lncRNA LUCAT1/ELAVL1/LIN28B/SOX2 positive feedback loop promotes cell stemness in TNBC. The exploration of the mechanisms underlying TNBC stemness might be beneficial to TNBC treatment.

Tamoxifen suppresses brain metastasis of estrogen receptor-deficient breast cancer by skewing microglia polarization and enhancing their immune functions

BackgroundBrain metastasis of breast cancer exhibits exceedingly poor prognosis, and both triple negative (TN) and Her2+ subtypes have the highest incidence of brain metastasis. Although estrogen blockers are considered to be ineffective for their treatment, recent evidence indicates that estrogen blockade using tamoxifen showed certain efficacy. However, how estrogen affects brain metastasis of triple negative breast cancer (TNBC) remains elusive.MethodsTo examine the effect of estrogen on brain metastasis progression, nude mice were implanted with brain metastatic cells and treated with either estrogen supplement, tamoxifen, or ovariectomy for estrogen depletion. For clinical validation study, brain metastasis specimens from pre- and post-menopause breast cancer patients were examined for microglia polarization by immunohistochemistry. To examine the estrogen-induced M2 microglia polarization, microglia cells were treated with estrogen, and the M1/M2 microglia polarization was detected by qRT-PCR and FACS. The estrogen receptor-deficient brain metastatic cells, SkBrM and 231BrM, were treated with conditioned medium (CM) derived from microglia that were treated with estrogen in the presence or absence of tamoxifen. The effect of microglia-derived CM on tumor cells was examined by colony formation assay and sphere forming ability.ResultsWe found that M2 microglia were abundantly infiltrated in brain metastasis of pre-menopausal breast cancer patients. A similar observation was made in vivo, when we treated mice systemically with estrogen. Blocking of estrogen signaling either by tamoxifen treatment or surgical resection of mice ovaries suppressed M2 microglial polarization and decreased the secretion of C-C motif chemokine ligand 5, resulting in suppression of brain metastasis. The estrogen modulation also suppressed stemness in TNBC cells in vitro. Importantly, estrogen enhanced the expression of signal regulatory protein α on microglia and restricted their phagocytic ability.ConclusionsOur results indicate that estrogen promotes brain metastasis by skewing polarity of M2 microglia and inhibiting their phagocytic ability, while tamoxifen suppresses brain metastasis by blocking the M2 polarization of microglia and increasing their anti-tumor phagocytic ability. Our results also highlight a potential therapeutic utility of tamoxifen for treating brain metastasis of hormone receptor-deficient breast cancer.

The molecular underpinning of geminin-overexpressing triple-negative breast cancer cells homing specifically to lungs.

Triple-negative breast cancer (TNBCs) display lung metastasis tropism. However, the mechanisms underlying this organ-specific pattern remains to be elucidated. We sought to evaluate the utility of blocking extravasation to prevent lung metastasis. To identify potential geminin overexpression-controlled genetic drivers that promote TNBC tumor homing to lungs, we used the differential/suppression subtractive chain (D/SSC) technique. A geminin overexpression-induced lung metastasis gene signature consists of 24 genes was discovered. We validated overexpression of five of these genes (LGR5, HAS2, CDH11, NCAM2, and DSC2) in worsening lung metastasis-free survival in TNBC patients. Our data demonstrate that LGR5-induced β-catenin signaling and stemness in TNBC cells are geminin-overexpression dependent. They also demonstrate for the first-time expression of RSPO2 in mouse lung tissue only and exacerbation of its secretion in the circulation of mice that develop geminin overexpressing/LGR5+-TNBC lung metastasis. We identified a novel extravasation receptor complex, consists of CDH11, CD44v6, c-Met, and AXL on geminin overexpressing/LGR5+-TNBC lung metastatic precursors, inhibition of any of its receptors prevented geminin overexpressing/LGR5+-TNBC lung metastasis. Overall, we propose that geminin overexpression in normal mammary epithelial (HME) cells promotes the generation of TNBC metastatic precursors that home specifically to lungs by upregulating LGR5 expression and promoting stemness, intravasation, and extravasation in these precursors. Circulating levels of RSPO2 and OPN can be diagnostic biomarkers to improve risk stratification of metastatic TNBC to lungs, as well as identifying patients who may benefit from therapy targeting geminin alone or in combination with any member of the newly discovered extravasation receptor complex to minimize TNBC lung metastasis.

Abstract PS16-19: Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer

Abstract p53 mutations occur in 80% of triple-negative breast cancer (TNBC) cases. Several types of p53 mutations have been reported, among which gain of function mutations have been associated with increased cancer stem cells, chemotherapy resistance, and disease relapse in several malignancies. Particularly, hotspot p53 mutations (mutations at amino acid location 157, 175, 248, 249, 273 predict worse overall survival in some cancer types. We have previously reported that Ganglioside GD2, a cell surface glycosphingolipid, identifies breast cancer stem cells and promotes tumorigenesis. We have also reported that GD3 synthase, a key enzyme which catalyzes the rate-limiting step of the GD2 biosynthesis pathway, is significantly upregulated in breast tumors with p53 mutations. Here, we hypothesize that mutant p53 promotes stemness in TNBC cells by regulating GD3 synthase and GD2 expression. To identify specific p53 mutations that contribute to TNBC stemness, we stratified TNBC cell lines (N=18) based on p53 mutation status including mutation type, location, and domain affected. We measured GD2 and GD3 synthase expression in the available TNBC cell lines by flow cytometry and RT-PCR, respectively. We found that GD3 synthase expression is significantly upregulated in TNBC cell lines with p53 hotspot mutations compared to cell lines with other p53 mutations (Median relative expression 0.0114 compared to 0.0003, p=0.005). Similarly, we found that GD2 expression is significantly higher in TNBC cell lines with hotspot mutations compared to non-hotspot counterparts (Median GD2+ cells 15.2% compared to 0.97%, p= 0.013) Additionally, GD2 and GD3 synthase are upregulated in basal-type TNBC cell lines and TCGA patient samples compared to other TNBC molecular subtypes. Interestingly, we also found that GD2 expression is not always directly correlated with GD3S expression, suggesting that GD2 synthase expression is also a key factor in the regulation of GD2 expression. To examine the effect of mutant p53 protein levels on TNBC stemness, we measured p53 expression in all the available TNBC cell lines by western blot, and found that p53 protein levels do not correlate with GD2 or GD3S expression. To validate these findings, we analyzed RPPA data from the MD Anderson Cancer Cell Lines Project and found similar results. These data suggest that it is the type of p53 mutation, but not the amount of p53 protein in the cells that determines GD2 and GD3 synthase expression in TNBC cells. For instance, p53 hotspot mutations involving amino acid position 248 have been shown to be strongly associated with increased cancer stemness. We found that cell lines with p53 mutations at this specific location have significantly higher GD3 synthase expression compared to other mutant p53 forms. We are currently examining the direct role of mutant p53 in the regulation of GD3S expression by stabilizing mutant p53 using MDM2 inhibitors as well as stable knockdown of p53 gene in multiple TNBC cell lines to establish a clear link between mutant p53 and stem cell marker expression in TNBC cell lines. In conclusion, stem cell markers are highly expressed in p53 mutant TNBC cell lines compared to wild type p53 counterparts. Specifically, cell lines with hotspot p53 mutations significantly correlate with increased TNBC stemness. The type of p53 mutation, rather than level of its expression correlates with stem cell marker expression in TNBC cells. Citation Format: Fouad El-Dana, Bin Yuan, Stanley Ly, Vivek Anand, Venkata Lokesh Battula. Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-19.

LIPH promotes metastasis by enriching stem-like cells in triple-negative breast cancer.

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24− stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44+/CD24− stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.

Abstract P2-06-02: Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative breast cancer

Abstract Background: Leukemia inhibitory factor (LIF) and its receptor LIFR are over-expressed in multiple solid tumors and play a key role in tumor growth, progression, and resistance to standard anti-cancer treatments. Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of breast cancer (BCa) mortality. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BCa patients. LIF signaling also promotes maintenance of stem cells. Therefore, targeting the LIF/LIFR axis may have therapeutic utility in TNBC. Methods: We rationally designed a small organic molecule (EC359) that emulates the LIF/LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Direct binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and microscale thermophoresis technique (MST) assays. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: Molecular docking studies showed that EC359 interacts at the LIF/LIFR binding interface. SPR and MST studies confirmed direct interaction of EC359 to LIFR. EC359 reduced the growth of TNBC cells with high potency (IC50 50-100nM) and promoted apoptosis. Further, EC359 treatment reduced invasion and stemness of TNBC cells. EC359 activity is dependent on the expression levels of LIFR and showed little or no activity on TNBC cells that have low levels of LIFR or ER+ve BCa cells. Further, EC359 significantly reduced the viability of cisplatin and taxane-resistant TNBC cells and enhanced the efficacy of HDAC inhibitors. Mechanistic and biochemical studies showed that EC359 interacts with LIFR and effectively blocking LIF/LIFR interactions. EC359 also blocked LIFR interactions with other LIFR ligands such as oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. EC359 treatment attenuated the activation of LIF/LIFR driven pathways including STAT3, mTOR, AKT, and MAPK. RNA-seq analysis identified regulation of apoptosis as one of the important pathway modulated by EC359. In TNBC xenograft and PDX assays, EC359 significantly reduced tumor progression. Further, using human primary BCa PDEX cultures, we demonstrated that EC359 has the potential to substantially reduce the proliferation of human BCa. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel targeted therapeutic agent that inhibits LIF/LIFR oncogenic signaling in TNBC via a unique mechanism of action. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. (DOD BCRP grant #BC170312) Citation Format: Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, Li M, Pratap UP, Altwegg KA, Li X, Srinivasan U, Ma S, Chang A, Riveros AC, Zhang KY, Dileep KV, Pan X, Murali R, Bajda M, Raj G, Brenner A, Manthati V, Rao M, Tekmal RR, Nair HB, Nickisch KJ, Vadlamudi RK. Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-02.

Embryonic stem cell secreted factors decrease invasiveness of triple-negative breast cancer cells through regulome modulation

ABSTRACTStem cell microenvironments decrease the invasiveness of cancer cells, and elucidating the mechanisms associated with disease regression could further the development of targeted therapies for aggressive cancer subtypes. To this end, we applied an emerging technology, TRanscriptional Activity CEll aRray (TRACER), to investigate the reprogramming of triple-negative breast cancer (TNBC) cells in conditions that promoted a less aggressive phenotype. The repressive environment was established through exposure to mouse embryonic stem cell conditioned media (mESC CM). Assessment of carcinogenic phenotypes indicated that mESC CM exposure decreased proliferation, invasion, migration, and stemness in TNBC cells. Protein expression analysis revealed that mESC CM exposure increased expression of the epithelial protein E-cadherin and decreased the mesenchymal protein MMP9. Gene expression analysis showed that mESC CM decreased epithelial to mesenchymal transition (EMT) markers fibronectin, vimentin, and Snail. Over a period of 6 d, TRACER quantified changes in activity of 11 transcription factors (TFs) associated with oncogenic progression. The EMT profile was decreased in association with the activity of 7 TFs (Smad3, NF-κΒ, MEF2, GATA, Hif1, Sp1, and RXR). Further examination of Smad3 and GATA expression and phosphorylation revealed that mESC CM exposure decreased noncanonical Smad3 phosphorylation and Smad3-mediated gene expression, increased GATA3 expression and phosphorylation, and resulted in a synergistic decrease in migration of GATA3 overexpressing MDA-MB-231 cells. Collectively, the application of TRACER to examine TF activity associated with the transition of cancer cells to a less aggressive phenotype, as directed by mESC CM, identified novel mechanistic events linking the embryonic microenvironment to both favorable changes and cellular plasticity in TNBC cell phenotypes.

Abstract 4767: Identifying CDCA7 as a novel regulator of tumor-initiating cells in triple-negative breast cancer

Abstract Tumor-initiating cells (TICs) are a small fraction of cancer cells that stands at the apex of the tumor hierarchy. Given their tumor-initiating ability and resistance to conventional chemotherapies, TICs are considered potential therapeutic targets for preventing the relapse and metastasis of malignancies. Our group has identified a novel gene, Cell division cycle associated 7 (CDCA7), as an important regulator of TICs in triple-negative breast cancer (TNBC). CDCA7 is a target gene of MYC and NOTCH. Ectopic expression of CDCA7 has been demonstrated to induce neoplastic transformation in vitro and tumorigenesis in vivo. Despite its potential tumor-initiating ability, CDCA7 has not yet been well-studied in breast cancer. We found that CDCA7 was highly expressed in TNBC compared to other subtypes of breast cancer according to the RNA-sequencing data from The Cancer Genome Atlas. Furthermore, our Kaplan-Meier analysis of multiple datasets demonstrated that higher expression of CDCA7 was associated with poor prognosis in breast cancer. When we knocked down CDCA7 by siRNA in TNBC cell lines including SUM149, HCC1937, MDA-MB-231, and MDA-MB-436, CD44+/CD24- stem-like cell population and primary and secondary mammosphere formation were significantly decreased. In addition to suppressing the stem-like properties, knockdown of CDCA7 also inhibited the invasion and migration of breast cancer cells. These phenotypes indicated the inhibition of epithelial-to-mesenchymal transition (EMT), which was in accordance with the up-regulation of E-Cadherin and down-regulation of Vimentin after CDCA7 knockdown in TNBC cell lines. In conclusion, our results have demonstrated that CDCA7 is an important regulator of TICs in TNBC. Loss-of-function of CDCA7 leads to the inhibition of EMT and stemness in TNBC cells. Citation Format: Chang-Ching Lin, Miao-Chia Lo, Rebecca Moody, Nicholas Stevers, Samantha Tinsley, Max Wicha, Duxin Sun. Identifying CDCA7 as a novel regulator of tumor-initiating cells 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 4767. doi:10.1158/1538-7445.AM2017-4767

Abstract P4-16-02: EF24 targets radiation-induced NFkB-dependent stemness in triple negative breast cancer cells

Abstract Prognosis of patients with late stage BCa still remains poor, mostly due to development of chemoradioresistance followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability, and other stem cell-like properties were proposed to be responsible for resistance, relapse and progression of BCa. We have shown that EF24 alleviates radiation (IR)-orchestrated NFkB mediated clonal expansion. Herein, we investigated the potential of EF24 in the regulation of IR-induced NFkB dependent stemness in triple negative breast cancer cells (TNBC). MDA-MB-231 cells exposed to mock-IR or IR (2Gy) with/without EF24 were examined for transcriptional alterations of 93 EMT, CSCs self-renewal, pluoripotentcy maintenance and other stem cell markers. NFkB (p50/p65) overexpression (with or without EF24) and RelA siRNA knockout (with IR) approach were used to delineate the role of IR-induced NFkB and the selective NFkB targeting of EF24 in this setting. Nanog, Sox-2, ABCG-2, N-Cadherin, POU5F1 and Myc expression was examined with immunoblotting. IR profoundly increased the transactivation of 86 stem-cell related molecules in TNBCs that are involved in cell survival. Interestingly, muting IR-induced NFkB attenuated 85 of those genes. Notably, EF24 suppressed identical 85 genes reproducing the inhibitory signature of NFkB muting. Coherently, activating NFkB induced 87 stem-cell related molecules in TNBC and of which 86 genes were completely suppressed with EF24. Alterations in the cellular expression levels of Nanog, Sox-2, ABCG-2, N-Cadherin, POU5F1 and Myc validates the potential of EF24 in mitigating IR-induced stemness in TNBC. Together these data demonstrates, at least in TNBC cells, IR-induced NFkB mediates increased stem-like characteristics and further imply that EF24 may alleviate stemness by selectively targeting IR-induced NFkB. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-02.