Breast Cancer Stem Cell Population Research Articles

Abstract PO4-27-12: Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen, aromatase inhibitors, AI, or fulvestrant) in ER+ breast cancer (BC) could be due to survival of breast cancer stem cells (BCSCs). BCSCs contribute to relapse of ER+ BC. We discovered a novel and potent anti-BCSC gene, Death Associated Protein 6 (DAXX) through a pre-surgical biomarker window study combining ET plus a Notch inhibitor [MK-0752, a g-secretase inhibitor (GSI)]. We also found that ET enhances the BCSC population by decreasing DAXX protein levels. In this current study, we measured DAXX protein levels in ER+ PDX tumors and human tumor tissues treated with ET. We determined the mechanism by which ET regulates DAXX expression at the level of protein stability. The goal of this study was to identify novel therapeutic strategies to prevent ET-mediated degradation of the DAXX protein, eliminate BCSCs, and prevent ER+ recurrence. Methods: ER+ BC cell lines (MCF-7 and T47D), an ER+ PDX BCM 5097 tumor line, and human breast cancer tumor samples were used. The ER+ PDX tumor (BCM 5097) was implanted into NSG mice and passaged into female athymic, nude mice-supplemented with an estradiol capsule. After one week, the estradiol capsule was removed from 10 mice, to mimic the use of AI. The remaining 10 mice retained the estradiol capsule. Tumor area was measured weekly up to 50 weeks. Western blotting detected ERa, the stem cell factor Notch4, and DAXX levels. Immunohistochemistry (IHC) detected DAXX protein levels. ER+ human tumor samples were collected prior to and after neoadjuvant treatment with ET (N=11) or chemotherapy (N=14) and stained for the DAXX protein. Correlations were made between DAXX and Ki67. Full length and deletion mutants of DAXX were expressed in ER+ BC cells. BCSCs were measured using the mammosphere forming assay. Liquid chromatography followed by mass spectrometry were used to detect phosphorylated residues of DAXX in response to ET. Kinases that phosphorylate these residues on DAXX were identified using PhosphoSite.org. ER+ cells were treated with kinase inhibitors for AURKA (alisertib), AURKB (barasertib), CK1 (CK-IN-1), or CK2 (CX-4945) and DAXX protein was detected by western blotting. BCSC survival was measured using mammosphere forming assay. Based on results from the kinase inhibitor screen, barasertib was selected for pre-clinical testing in mice. ER+ MCF-7 xenograft tumors were treated with vehicle or barasertib in vivo up to 120 days. Results: DAXX protein levels decreased in ER+ PDX tumors in response to ET. Similar trends were detected in human tumor tissue after ET or chemotherapy alone. DAXX protein levels inversely correlated with Ki67 in ER+ human tumor samples. Analysis of DAXX deletion mutants demonstrated that amino acids 400-740 of the DAXX protein were required to inhibit BCSC survival. This region of the DAXX protein had high levels of phosphorylated residues in response to ET. Only the AURKB (barasertib) inhibitor increased DAXX protein expression and inhibited BCSCs in a DAXX-dependent manner. Barasertib inhibited growth of ER+ MCF-7 tumor xenografts compared to the vehicle control. After treatment ceased, tumor recurrence was measured up to 120 days. Regrowth of tumors was partially delayed in response to barasertib. Conclusions: ET decreased DAXX protein levels in ER+ PDX and human tumors. Downregulation of the DAXX protein by ET was through activation of AURKB and hyper-phosphorylation of DAXX which resulted in protein degradation and enhanced survival of BCSCs. Therefore, Inhibition of AURKB using barasertib partially restored DAXX expression, inhibited BCSCs, and delayed tumor recurrence. A combination approach of ET plus an AURKB inhibitor might be a novel therapeutic strategy to prevent ER+ breast cancer relapse by increasing DAXX in order to eradicate BCSCs. Support: Breast Cancer Research Foundation Citation Format: Clodia Osipo, Debra Wyatt, Kathy Albain. Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-12.

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.

MiR-29a-KLF4 signaling inhibits breast tumor initiation by regulating cancer stem cells

Cancer stem cells (CSCs) are known for their potent ability to drive tumor initiation and recurrence, yet the molecular mechanisms regulating CSCs are still unclear. Our study found a positive correlation between increased levels of miR-29a and better survival rates in early-stage breast cancer patients, but a negative correlation in late-stage patients, suggesting a dual function of miR-29a in regulating breast cancer. Furthermore, miR-29a showed significant downregulation in the ALDH+ breast cancer stem cell population compared to non-stem cancer cells. Overexpression of miR-29a in human breast cancer cells reduced the proportion of CSCs, suppressed their ability to form mammospheres, and inhibited the expression of stemness genes SOX2, KLF4, and hTERT in vitro. Conversely, knockdown of miR-29a in breast cancer cells showed opposite effects. Tumor xenograft experiments revealed that miR-29a overexpression significantly inhibited tumorigenesis initiated by MDA-MB-231 cell transplantation in nude mice. We further demonstrated that Krüppel-like factor 4 (KLF4), a key gene that regulates cell stemness, was a direct target of miR-29a in breast cancer cells. miR-29a suppressed the expression of KLF4 at both mRNA and protein levels. Reintroduction of KLF4 into breast cancer cells rescued the miR-29a-induced CSC suppression phenotype. In summary, our study is the first to demonstrate that miR-29a-KLF4 signaling inhibits breast tumor initiation by regulating CSCs, which provides novel therapeutic targets for preventing breast tumor initiation.

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.

Chronic treatment with TNF-α, alone and in combination with Takinib, SB203580 and metformin induce cell death in breast cancer

Breast cancer (BC) is the most common malignancy, and the largest cause of cancer death among women. The interactions between tumor cells and tumor micro environmental factors have a major impact on tumor progression. One of the critical pro-inflammatory cytokines present in breast cancer tumor microenvironment is TNF-α. The aim of this study was to evaluate the long-term effect of TNF-α (1 week) along with p38 or TAK1 inhibitors as well as metformin on induction of cellular death, cancer stem cell and expression of metastatic marker CXCR4. MCF-7 and MDA-MB-231 cells were treated with TNF-α for one week and then were treated with combination of Takinib, SB203580 or Metformin; after all treatments were done, cell proliferation, cellular death, surface expression of CXCR4, CD44 and CD24 were determined. The results showed that treatment with TNF-α alone or in combination with Takinib, SB203580 and metformin elevated induction of cellular death in both cell lines compared to the control group. TNF-α also increased CXCR4 expression in MCF-7 cells, but it reduced its expression in the MDA-MB-231 cells. Also, breast cancer stem cells (BCSCs) population decreased in MDA-MB-231 cells treated with TNF-α alone or in combination with SB203580 and metformin. Although, in MCF-7 cells only combination of TNF-α and Takinib reduced BCSCs population in a time dependent manner. Altogether, we showed that TNF-α alone or in combination with other treatments can affect the progression of breast cancer.

Soloxolone methyl induces apoptosis and oxidative/ER stress in breast cancer cells and target cancer stem cell population.

One of the most prevalent malignancies in women and one of the leading causes of cancer-related death is breast cancer. There is a need for new treatment approaches and drugs for breast cancer. Many studies show the high potential of triterpene compounds and their semisynthetic derivatives as anticancer agents due to their ability to induce apoptosis and suppress tumorigenesis. The effects of soloxolone methyl (SM), a semisynthetic derivative of 18-H-glycyrrhetinic acid, on the cytotoxicity and apoptosis of human breast cancer cell line (T-47D) and cancer stem cell (CSCs) population (mammospheres; CD44+/CD24-antigen) derived from breast cancer cells, were examined in this work. The ATP assay was used to determine SM growth-inhibitory effects. Fluorescent staining, caspase-cleaved cytokeratin 18, and flow cytometry analysis were used to determine the mode of the cell death. In addition, cell death was investigated at protein and gene levels by Western Blotting and PCR, respectively. SM resulted in cytotoxicity in a time and dose dependent manner via ROS production and ER stress in T-47D cells in 2 models. The mode of cell death was apoptosis, evidenced by phosphatidylserine exposure, caspase activation, and bax overexpression. In mammospheres as 3D model, SM decreased stem cell properties and induced cell death. Taken together, SM may be a promising agent in the treatment of breast cancer, especially due to its antigrowth activity on CSCs.

Estrogen Receptor β4 Regulates Chemotherapy Resistance and Induces Cancer Stem Cells in Triple Negative Breast Cancer.

Triple Negative Breast Cancer (TNBC) has the worst prognosis among all breast cancers, and survival in patients with recurrence is rarely beyond 12 months due to acquired resistance to chemotherapy, which is the standard of care for these patients. Our hypothesis is that Estrogen Receptor β1 (ERβ1) increases response to chemotherapy but is opposed by ERβ4, which it preferentially dimerizes with. The role of ERβ1 and ERβ4 in influencing chemotherapy sensitivity has never been studied before. CRISPR/CAS9 was used to truncate ERβ1 Ligand Binding Domain (LBD) and knock down the exon unique to ERβ4. We show that the truncated ERβ1 LBD in a variety of mutant p53 TNBC cell lines, where ERβ1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERβ4 knockdown cell line was sensitized to Paclitaxel. We further show that ERβ1 LBD truncation, as well as treatment with ERβ1 antagonist 2-phenyl-3-(4-hydroxyphenyl)-5,7-bis(trifluoromethyl)-pyrazolo[1,5-a] pyrimidine (PHTPP), leads to increase in the drug efflux transporters. Hypoxia Inducible Factors (HIFs) activate factors involved in pluripotency and regulate the stem cell phenotype, both in normal and cancer cells. Here we show that the ERβ1 and ERβ4 regulate these stem cell markers like SOX2, OCT4, and Nanog in an opposing manner; and we further show that this regulation is mediated by HIFs. We show the increase of cancer cell stemness due to ERβ1 LBD truncation is attenuated when HIF1/2α is knocked down by siRNA. Finally, we show an increase in the breast cancer stem cell population due to ERβ1 antagonist using both ALDEFLUORTM and SOX2/OCT4 response element (SORE6) reporters in SUM159 and MDA-MB-231 cell lines. Since most TNBC cancers are ERβ4 positive, while only a small proportion of TNBC patients are ERβ1 positive, we believe that simultaneous activation of ERβ1 with agonists and inactivation of ERβ4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemotherapy resistant TNBC patients.

Growth and invasion inhibition of T47D ductal carcinoma cells by the association of docetaxel with a bioactive agent in neutral nanosuspension.

Introduction: The approach for drug delivery has impressively developed with the emergence of nanosuspension, particularly the targeted nanoemulsions (NEs). It can potentially improve the bioavailability of drugs, enhancing their therapeutic efficiency. This study aims to examine the potential role of NE as a delivery system for the combination of docetaxel (DTX), a microtubule-targeting agent, and thymoquinone (TQ) in the treatment of human ductal carcinoma cells T47D. Methods: NEs were synthesized by ultra-sonication and characterized physically by dynamic light scattering (DLS). A sulforhodamine B assay was performed to evaluate cytotoxicity, and a flow cytometry analysis for cell cycle, apoptosis, autophagy, and cancer stem cell evaluations. A quantitative polymerase chain reaction further assessed the epithelial-mesenchymal transition gene expirations of SNAIL-1, ZEB-1, and TWIST-1. Results: The optimal sizes of blank-NEs and NE-DTX+TQ were found at 117.3 ± 8 nm and 373 ± 6.8 nm, respectively. The synergistic effect of the NE-DTX+TQ formulation significantly inhibited the in vitro proliferation of T47D cells. It caused a significant increase in apoptosis, accompanied by the stimulation of autophagy. Moreover, this formulation arrested T47D cells at the G2/M phase, promoted the reduction of the breast cancer stem cell (BCSC) population, and repressed the expression of TWIST-1 and ZEB-1. Conclusion: Co-delivery of NE-DTX+TQ may probably inhibit the proliferation of T47D via the induction of apoptosis and autophagy pathways and impede the migration by reducing the BCSC population and downregulating TWIST-1 expression to decrease the epithelial-to-mesenchymal transition (EMT) of breast cancer cells. Therefore, the study suggests the NE-DTX+TQ formula as a potential approach to inhibit breast cancer growth and metastasis.

Modulatory Effect of Vitamin C on Hypoxia Induced Breast Cancer Stem Cells.

Eliminating cancer stem cells (CSCs) is a challenge because of their enhanced resistance to anti-cancer drugs. Vitamin C, which is insufficient in patients with higher stages of cancer, has been gaining attention as a potential treatment for human malignancies. Hence this study aimed to analyze the effect of high-dose vitamin C treatment on the gene expression level of HIF-1α, NF-κB1, BAX, and DNMT1 in the MCF7 cells undergoing hypoxia, as an inducer of CSCs characteristics. As a result, vitamin C could be possibly used as a promising therapeutic adjuvant. Here we first analyzed the breast CSC population alteration in MCF7 cells following hypoxia induction. Then, we evaluated the impact of vitamin C treatment on the gene expression level of four stemness-related genes in hypoxic MCF7 cells. Our results indicate that vitamin C could reduce proliferation and stemness states in CSCs possibly by induction of apoptotic markers such as BAX, along with attenuating stemness markers, including NF-κB1, and DNMT1 gene expressions. According to our findings, vitamin C administration would become a new approach to avoiding the stimulation of CSCs during cancer therapies.

Dual Function of CCAT2 in Regulating Luminal Subtype of Breast Cancer Depending on the Subcellular Distribution.

Breast cancer is the most common cancer in women around the world. Emerging evidence has indicated the important roles that non-coding RNAs play in regulating tumor development and progression in breast cancer. Herein, we found a dual function of long non-coding RNA (LncRNA) CCAT2 in the luminal subtype of breast cancer, depending on its subcellular distribution. CCAT2 showed an overall downregulation in the tumor tissues from luminal breast cancer patients. Transient overexpression of CCAT2 in the luminal subtype of breast cancer cell MCF-7 or T47D significantly suppressed cell proliferation in vitro and inhibited tumor growth in vivo. Gene expression analysis of cancer stem cell markers including OCT4, NANOG, h-TERT, SOX2 and KLF4; flow cytometry analysis of breast cancer stem cell population, and mammosphere formation assay demonstrated inhibition of cancer cell stemness with transient transfection of CCAT2 in which exogenous CCAT2 mainly distributed in the cytoplasm and regulated miR-221-p27 signaling via RNA sequence interaction. However, overexpression of CCAT2 in MCF-7 cells through pMX retroviral nuclear expression vector accumulated CCAT2 in the nucleus, leading to upregulation of OCT4-PG1, a pseudogene of stem gene OCT4, thereby promoting the cancer cell stemness. In conclusion, the current study, for the first time, revealed a dual function of lncRNA CCAT2 as a tumor suppressor or oncogene depending upon its subcellular distribution. It also demonstrated the regulatory mechanism of cytoplasmic CCAT2 in suppressing tumorigenesis in the luminal subtype of breast cancer.

Adolescent- and adult-initiated alcohol exposure in mice differentially promotes tumorigenesis and metastasis of breast cancer.

Alcohol exposure increases the risk of breast cancer. Alcohol consumption by adolescents is a serious social and public health issue. This study investigated the impact of adolescent alcohol consumption on mammary tumorigenesis and progression and compared it to that of adult alcohol exposure in animal models. Female adolescent (5 weeks) and adult (8 weeks) MMTV-Wnt1 mice were exposed to alcohol either chronically or acutely. For chronic alcohol exposure, animals were fed a liquid diet containing 6.7% ethanol for 23 weeks. For acute exposure, animals were treated with ethanol (2.5g/kg, 25% w/v) via intraperitoneal (IP) injection for 15 days. In control animals, the tumor latency was 18.5 to 22 weeks. Both chronic and acute alcohol exposure in adolescent mice significantly shortened the tumor latency to 9.5 and 8.4 weeks, respectively. However, adult-initiated alcohol exposure had little effect on the tumor latency. Both adolescent- and adult-initiated alcohol exposure significantly increased lung metastasis. Adolescent-initiated alcohol exposure but not adult-initiated alcohol exposure increased the breast cancer stem cell population. Adolescent-initiated alcohol exposure significantly altered the proliferation of mammary epithelial cells, ductal growth, and the formation of terminal end buds in the mammary glands. Adolescent-initiated alcohol exposure but not adult-initiated alcohol exposure increased estradiol levels in the blood. Acute adolescent alcohol exposure also significantly increased blood progesterone levels. Furthermore, adolescent-initiated alcohol exposure activated PAK1 and p38γ MAPK, critical regulators of mammary tumorigenesis and aggressiveness, respectively, while adult-initiated alcohol exposure activated only p38γ MAPK. In addition, both adolescent and adult alcohol exposure significantly decreased the levels of a prognostic marker miR200b. Adolescent-initiated alcohol exposure enhanced both tumorigenesis and aggressiveness of mammary tumors, while adult-initiated alcohol exposure mainly promoted tumor metastasis. Thus, adolescent mice were more sensitive than adult mice in response to alcohol-induced tumor promotion.

NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification.

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.

Targeting Triple Negative Breast Cancer Stem Cells by Heat Shock Protein 70 Inhibitors.

Simple SummaryTriple negative breast cancer (TNBC) has been a leading cause of cancer deaths among women and limited treatment options are currently available. Consequently, developing the promising therapy for TNBC remains an unmet medical need. The aim of this study was to assess the effect of Hsp70 inhibitors on breast cancer stem cells (BCSCs) in TNBC cells. The present study demonstrated the potent in vitro and in vivo anti-BCSCs activity of two Hsp70 inhibitors, compound 1 and 6, in TNBC cells. Our findings suggest that targeting Hsp70 could be an attractive strategy for eliminating BCSCs populations in TNBC.Triple negative breast cancer (TNBC) is considered the most aggressive breast cancer with high relapse rates and poor prognosis. Although great advances in the development of cancer therapy have been witnessed over the past decade, the treatment options for TNBC remain limited. In this study, we investigated the effect and potential underlying mechanism of the Hsp70 inhibitors, compound 1 and compound 6, on breast cancer stem cells (BCSCs) in TNBC cells. Our results showed that compound 1 and 6 exhibited potent tumor suppressive effects on cell viability and proliferation, and effectively inhibited BCSC expansion in TNBC cells. Reminiscent with the effect of Hsp70 inhibitors, Hsp70 knockdown effectively suppressed mammosphere formation and the expressions of BCSCs surface markers. Mechanistically, evidence showed that the Hsp70 inhibitors inhibited BCSCs by down-regulating β-catenin in TNBC cells. Moreover, we used the Hsp70 inhibitors treated TNBC cells and a stable Hsp70 knockdown clone of MDA-MB-231 cells to demonstrate the in vivo efficacy of Hsp70 inhibition in suppressing tumorigenesis and xenograft tumor growth. Together, these findings suggest the potential role of Hsp70 as a target for TNBC therapy and foster new therapeutic strategies to eliminate BCSCs by targeting Hsp70.

Role of stiffness and physico-chemical properties of tumour microenvironment on breast cancer cell stemness

Several physico-chemical properties of the tumour microenvironment (TME) are dysregulated during tumour progression, such as tissue stiffness, extracellular pH and interstitial fluid flow. Traditional preclinical models, although useful to study biological processes, do not provide sufficient control over these physico-chemical properties, hence limiting the understanding of cause-effect relationships between the TME and cancer cells. Breast cancer stem cells (B-CSCs), a dynamic population within the tumour, are known to affect tumour progression, metastasis and therapeutic resistance. With their emerging importance in disease physiology, it is essential to study the interplay between above-mentioned TME physico-chemical variables and B-CSC marker expression. In this work, 3D in vitro models with controlled physico-chemical properties (hydrogel stiffness and composition, perfusion, pH) were used to mimic normal and tumour breast tissue to study changes in proliferation, morphology and B-CSC population in two separate breast cancer cell lines (MCF-7 and MDA-MB 231). Cells encapsulated in alginate-gelatin hydrogels varying in stiffness (2-10 kPa), density and adhesion ligand (gelatin) were perfused (500 µL/min) for up to 14 days. Physiological (pH 7.4) and tumorigenic (pH 6.5) media were used to mimic changes in extracellular pH within the TME. We found that both cell lines have distinct responses to changes in physico-chemical factors in terms of proliferation, cell aggregates size and morphology. Most importantly, stiff and dense hydrogels (10 kPa) and acidic pH (6.5) play a key role in B-CSCs dynamics, increasing both epithelial (E-CSCs) and mesenchymal cancer stem cell (M-CSCs) marker expression, supporting direct impact of the physico-chemical microenvironment on disease onset and progression. Statement of significanceCurrently no studies evaluate the impact of physico-chemical properties of the tumour microenvironment on breast cancer stem cell (B-CSC) marker expression in a single in vitro model and at the same time. In this study, 3D in vitro models with varying stiffness, extracellular pH and fluid flow are used to recapitulate the breast tumour microenvironment to evaluate for the first time their direct effect on multiple breast cancer phenotypes: cell proliferation, cell aggregate size and shape, and B-CSC markers. Results suggest these models could open new ways of monitoring disease phenotypes, from the early-onset to progression, as well as being used as testing platforms for effective identification of specific phenotypes in the presence of relevant tumour physico-chemical microenvironment.

Transforming Growth Factor Beta Promotes the Expansion of Cancer Stem Cells &lt;i&gt;via&lt;/i&gt; S1PR3 by Ligand-Independent Notch Activation

Growing evidence suggests that cancer originates from cancer stem cells (CSCs), which can be identified by aldehyde dehydrogenase (ALDH) activity-based flow cytometry. However, the regulation of CSC growth is not fully understood. In the present study, we investigated the effects of Transforming Growth Factor-β (TGFβ) in breast CSC expansion. Stimulation with TGFβ increased the ALDH-positive breast CSC population via the phosphorylation of sphingosine kinase 1 (SphK1), a sphingosine-1-phosphate (S1P)-producing enzyme, and subsequent S1P-mediated S1P receptor 3 (S1PR3) activation. These data suggest that TGFβ promotes breast CSC expansion via the ALK5/SphK1/S1P/S1PR3 signaling pathway. Our findings provide new insights into the role of TGFβ in the regulation of CSCs.

Downregulation of miRNA-21 and cancer stem cells after chemotherapy results in better outcome in breast cancer patients.

Epigenetic modifications have been observed as a decline in miRNA-21 expression and breast cancer stem cell (CSC) population after 3 cycles of standard chemotherapy. The epigenetic response (miRNAs expression) and CSCs are also correlated in patients with Breast Cancer. In patients who tolerated chemotherapy well, miRNA-21 (non-coding RNA) expression decreased significantly after three cycles of chemotherapy. The miRNA-21 expression in breast cancer tissue was quantified by quantitative PCR (real-time PCR) using the standard protocol. In addition, breast CSCs (CD44+/CD24-) were also decreased in these patients. The miRNA-21 regulates cell division, proliferation, and autophagy of cancerous cells (as it targets phosphatase and tensin homolog/AKT/transcription factor EB/programmed cell death 4/autophagy-related protein 5 and chemotherapy also produces similar effects), thereby contributing to these benefits. Therefore, when all of the targets on genes have been explored by mimic miRNA, chemotherapy combined with anti-miRNA21 therapy may prove useful in the care of cancer patients.

Molecular Mechanisms Underlying the Anti-Breast Cancer Stem Cell Activity of Pterocladia capillacea and Corallina officinalis Polysaccharides.

This study aimed to appraise the activity of Pterocladia capillacea and Corallina officinalis polysaccharides against Breast Cancer Stem Cells (BCSCs). P. capillacea and C. officinalis polysaccharides were characterized to be sulfated polysaccharide-protein complexes. Cytotoxicity of the polysaccharides against MDA-MB-231 and MCF-7 cell lines along with their impact on CD44+/CD24- and aldehyde dehydrogenase 1(ALDH1) positive BCSC population were determined. Their effect on gene expression of CSC markers, Wnt/β-catenin and Notch signaling pathways was evaluated. P. capillacea and C. officinalis polysaccharides inhibited the growth of breast cancer cells and reduced BCSC subpopulation. P. capillacea polysaccharides significantly down-regulated OCT4, SOX2, ALDH1A3 and vimentin in MDA-MB-231 as well as in MCF-7 cells except for vimentin that was up-regulated in MCF-7 cells. C. officinalis polysaccharides exhibited similar effects except for OCT4 that was up-regulated in MDA-MB-231 cells. Significant suppression of Cyclin D1 gene expression was noted in MDA-MB-231 and MCF-7 cells treated with P. capillacea or C. officinalis polysaccharides. β-catenin and c-Myc genes were significantly down-regulated in MDA-MB-231 cells treated with C. officinalis and P. capillacea polysaccharides, respectively, while being up-regulated in MCF-7 cells treated with either of them. Additionally, P. capillacea and C. officinalis polysaccharides significantly down-regulated Hes1 gene in MCF-7 cells despite increasing Notch1 gene expression level. However, significant down-regulation of Notch1 gene was observed in MDA-MB-231 cells treated with P. capillacea polysaccharides. Collectively, this study provides evidence for the effectiveness of P. capillacea and C. officinalis polysaccharides in targeting BCSCs through interfering with substantial signaling pathways contributing to their functionality.

Abstract P5-12-11: NSDHL knockdown decreases tightly cohesive tumorsphere formation and breast cancer stem cell population

Abstract Background: Cholesterol biosynthesis pathway is commonly elevated or dysregulated in cancer cells and high cholesterol levels are associated with cancer progression. NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an enzyme involved in cholesterol biosynthesis, has been implicated in maintaining cancer stem cell, but its role in regulating breast cancer stem cells (BCSC) remains unclear. This study aimed to uncover the molecular mechanisms by which NSDHL regulates BCSC properties.Methods: The knockdown of NSDHL was induced by an introduction of siRNA/shRNA into MCF-7 cells. A non-adherent 3D culture method based on polymer-X coating was applied for growing tumorspheres under serum-free condition. 3’ quant mRNA-sequencing, qRT-PCR, western blot, immunofluorescent staining, ELISA, flow cytometry and ALDEFLUOR assay were carried out. Immunodeficient NSG mice were used for orthotropic xenograft tumor models by injecting tumorspheres.Result: NSDHL knockdown reduced a tightly cohesive sphere formation. RNA-seq data revealed a significant upregulation of 253 DEGs and downregulation of 364 DEGs with &amp;gt;2-fold change in NSDHL knockdown tumorsphere. The KEGG pathway analysis showed that DEGs were mainly enriched in TGF-β signaling pathway and cell cycle. NSDHL knockdown reduced the population of BCSCs with CD44+/CD24- phenotype and high ALDH activity as well as luminal progenitors with CD49f+/EpCAM+ phenotype along with decrease in TGF-β 1 and 3 and GLI1 in tumorsphere. In orthotropic xenograft tumor models, NSDHL knockdown strongly suppressed the tumor initiation and growth.Conclusions: Our findings reveal that NSDHL has an important role in maintaining BCSC population and tumor initiating capacity, suggesting NSDHL as an attractive therapeutic target to eliminate BCSCs, and thus preventing breast cancer initiation and progression. Citation Format: So-Hyun Yoon, Hoe Suk Kim, Junhyuk Song, Sunkyu Kwon, Seungyeon Ryu, Moonjou Baek, Sangeun Lee, Han-Byoel Lee, Hyeong-Gon Moon, Dong-Young Noh, Sangyong Jon, Wonshik Han. NSDHL knockdown decreases tightly cohesive tumorsphere formation and breast cancer stem cell population [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-11.

The Prostaglandin E2 Pathway and Breast Cancer Stem Cells: Evidence of Increased Signaling and Potential Targeting.

Culprits of cancer development, metastasis, and drug resistance, cancer stem cells (CSCs) are characterized by specific markers, active developmental signaling pathways, metabolic plasticity, increased motility, invasiveness, and epithelial-mesenchymal transition. In breast cancer, these cells are often more prominent in aggressive disease, are amplified in drug-resistant tumors, and contribute to recurrence. For breast cancer, two distinct CSC populations exist and are typically defined by CD44+/CD24- cell surface marker expression or increased aldehyde dehydrogenase (ALDH) activity. These CSC populations share many of the same properties but also exhibit signaling pathways that are more active in CD44+/CD24- or ALDH+ populations. Understanding these CSC populations and their shared or specific signaling pathways may lead to the development of novel therapeutic strategies that will improve breast cancer patient outcomes. Herein, we review the current evidence and assess published patient tumor datasets of sorted breast CSC populations for evidence of heightened prostaglandin E2 (PGE2) signaling and activity in these breast CSC populations. PGE2 is a biologically active lipid mediator and in cancer PGE2 promotes tumor progression and poor patient prognosis. Overall, the data suggests that PGE2 signaling is important in propagating breast CSCs by enhancing inherent tumor-initiating capacities. Development of anti-PGE2 signaling therapeutics may be beneficial in inhibiting tumor growth and limiting breast CSC populations.

Breast cancer stem cell population in different molecular subtypes of breast cancer.

Breast cancer heterogeneity is well documented and to some extent is attributed to the presence of cancer stem cells (CSCs). Breast cancer stem cells are identified by the presence of cell surface molecule CD44 and absence of CD24. In the present study a flowcytometric analysis was done to study the expression distribution of CSC phenotype of CD44+/CD24-/low, among different molecular subtypes of breast cancer and to find a correlation with clinicopathological features. CSCs were observed in all the molecular subtypes of breast cancer. The highest population of CSCs was noted in luminal B (3.4%), followed by TNBC (1.7%), and Her-2 subtype (1.6%). The least number of CD44+/CD24- cells were seen in Luminal A subgroup (1.3%). Existence of cancer stem cells in all the subtypes may suggest the possibility of failure of current therapies in treatment of patients.