RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24^−/low versus CD24^+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24^−/low and CD24^+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.     

单细胞测序技术及其在乳腺癌研究中的应用

乳腺癌是起源于乳腺各级导管和乳腺上皮细胞,由增生到不典型增生而逐步发展成原位癌、早期浸润癌至浸润性癌的一种恶性肿瘤。传统高通量测序技术对乳腺癌的研究主要是鉴定与乳腺癌发生发展相关的"驱动基因",但是对于乳腺癌基因组结构变化以及亚克隆的鉴定等存在一定的局限性,并且忽略了乳腺癌肿瘤细胞之间的异质性。近年来兴起的单细胞测序技术是以单个细胞为研究对象,对基因拷贝和基因表达等数据进行分析,探究乳腺癌的细胞组成、细胞状态和细胞命运,绘制乳腺癌生态系统图谱,对临床患者进行精准分层,为实现个体化治疗提供支持。同时,还可以揭示乳腺癌与T细胞、巨噬细胞等免疫细胞间的相关性,为发现乳腺癌新的治疗靶点、免疫检查点等提供参考。本文对单细胞测序技术及其在乳腺癌研究中的应用和研究进展进行了综述,以期为单细胞测序技术的进一步发展提供参考,同时为理解乳腺癌的发病机制和免疫治疗提供基础支持。     

Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

Breast cancer stem cells (BCSCs) have been considered responsible for cancer progression, recurrence, metastasis and drug resistance. However, the mechanisms by which cells acquire self‐renewal and chemoresistance properties are remaining largely unclear. Herein, we evaluated the role of miR‐708 and metformin in BCSCs, and found that the expression of miR‐708 is significantly down‐regulated in BCSCs and tumour tissues, and correlates with chemotherapy response and prognosis. Moreover, miR‐708 markedly inhibits sphere formation, CD44⁺/CD24^? ratio, and tumour initiation and increases chemosensitivity of BCSCs. Mechanistically, miR‐708 directly binds to cluster of differentiation 47 (CD47), and regulates tumour‐associated macrophage‐mediated phagocytosis. On the other hand, CD47 is essential for self‐renewal, tumour initiation and chemoresistance of BCSCs, and correlates with the prognosis of breast cancer patients. In addition, the anti‐type II diabetes drug metformin are found to be involved in the miR‐708/CD47 signalling pathway. Therefore, our study demonstrated that miR‐708 plays an important tumour suppressor role in BCSCs self‐renewal and chemoresistance, and the miR‐708/CD47 regulatory axis may represent a novel therapeutic mechanism of metformin in BCSCs.     

Advance in metabolism and target therapy in breast cancer stem cells

Breast cancer, like many other cancers, is believed to be driven by a population of cells that display stem cell properties. Recent studies suggest that cancer stem cells (CSCs) are essential for tumor progression, and tumor relapse is thought to be caused by the presence of these cells. CSC-targeted therapies have also been proposed to overcome therapeutic resistance in breast cancer after the traditional therapies. Additionally, the metabolic properties of cancer cells differ markedly from those of normal cells. The efficacy of metabolic targeted therapy has been shown to enhance anti-cancer treatment or overcome therapeutic resistance of breast cancer cells. Metabolic targeting of breast CSCs (BCSCs) may be a very effective strategy for anti-cancer treatment of breast cancer cells. Thus, in this review, we focus on discussing the studies involving metabolism and targeted therapy in BCSCs.     

microRNA‐128‐3p overexpression inhibits breast cancer stem cell characteristics through suppression of Wnt signalling pathway by down‐regulating NEK2

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray‐based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA‐128‐3p (miR‐128‐3p). Herein, this study aimed to characterize the tumour‐suppressive role of miR‐128‐3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44^＋CD24^?/low cells were selected for subsequent experiments. After verification of the target relationship between miR‐128‐3p and NEK2, the relationship among miR‐128‐3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR‐128‐3p on proliferation, migration, invasion and self‐renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain‐ and loss‐of‐function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down‐regulation of miR‐128‐3p. Overexpression of miR‐128‐3p was found to inhibit proliferation, migration, invasion, self‐renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down‐regulating NEK2. In summary, this study indicates that miR‐128‐3p inhibits the stem‐like cell features of BCSCs via inhibition of the Wnt signalling pathway by down‐regulating NEK2, which provides a new target for breast cancer treatment.     

CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44^high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44^high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44^high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44^high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.     

Identification of Tumor Endothelial Cells with High Aldehyde Dehydrogenase Activity and a Highly Angiogenic Phenotype

Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDH^high TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDH^high/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDH^low TECs, ALDH^high TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDH^high TECs than that in ALDH^low TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDH^high TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.     

Breast cancer stem cells

Breast cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell-associated markers and have a high capacity for tumor generation in vivo. Identification of BCSCs from tumor samples or breast cancer cell lines has been based mainly on CD44(+)/CD24(-/low) or ALDH(+) phenotypes. BCSCs isolation has allowed the analysis of the molecular mechanisms involved in their origin, self-renewal, differentiation into tumor cells, resistance to radiation therapy and chemotherapy, and invasiveness and metastatic ability. Molecular genetic analysis using knockout animals and inducible transgenics has identified NF-κB, c-Jun, p21(CIP1), and Forkhead-like-protein Dach1 involvement in BCSC expansion and fate. Clinical analyses of BCSCs in breast tumors have found a correlation between the proportion of BCSCs and poor prognosis. Therefore, new therapies that specifically target BCSCs are an urgent need. We summarize recent evidence that partially explain the biological characteristics of BCSCs.     

Hierarchical heterogeneity in mammary tumors and its regulation by autophagy

Intra-tumor heterogeneity can be attributed in part to the ability of tumor cells to acquire traits associated with less differentiated cells. In MMTV-PyMT mammary tumors, this hierarchical heterogeneity can be illustrated with the use of ITGB1/CD29^hi ITGB3/CD61⁺ markers to enrich for mammary stem-like cells and ALDH⁺ to identify luminal progenitor-like cells. Macroautophagy/autophagy appears to be important for maintaining the cancer stem-like traits of both these populations. Interestingly, the regulation of these distinct cancer stem-like cells by autophagy occurs through EGFR-STAT3 and TGFB/TGF-β-SMAD pathways, respectively. These findings indicate that autophagy plays a significant role in cancer stem-like cells, and distinct cancer stem-like cells within a tumor may require different treatment modalities.     

An atlas of inter- and intra-tumor heterogeneity of apoptosis competency in colorectal cancer tissue at single-cell resolution

Cancer cells’ ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells, and quantification of 18 cell lineage and apoptosis proteins. We identified an enrichment for BCL2 in immune, and BAK, SMAC, and XIAP in cancer cells. Ordinary differential equation-based modeling of apoptosis sensitivity at single-cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. Systems modeling at single-cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, but showed significant inter- and intra-tumor heterogeneity.Subject terms: Cancer microenvironment, Tumour heterogeneity     

Curcumin Improves the Tumoricidal Effect of Mitomycin C by Suppressing ABCG2 Expression in Stem Cell-Like Breast Cancer Cells

Cancer cells with stem cell–like properties contribute to the development of resistance to chemotherapy and eventually to tumor relapses. The current study investigated the potential of curcumin to reduce breast cancer stem cell (BCSC) population for sensitizing breast cancer cells to mitomycin C (MMC) both in vitro and in vivo. Curcumin improved the sensitivity of paclitaxel, cisplatin, and doxorubicin in breast cancer cell lines MCF-7 and MDA-MB-231, as shown by the more than 2-fold decrease in the half-maximal inhibitory concentration of these chemotherapeutic agents. In addition, curcumin sensitized the BCSCs of MCF-7 and MDA-MB-231 to MMC by 5- and 15-fold, respectively. The BCSCs could not grow to the fifth generation in the presence of curcumin and MMC. MMC or curcumin alone only marginally reduced the BCSC population in the mammospheres; however, together, they reduced the BCSC population in CD44⁺CD24^−/low cells by more than 75% (29.34% to 6.86%). Curcumin sensitized BCSCs through a reduction in the expression of ATP-binding cassette (ABC) transporters ABCG2 and ABCC1. We demonstrated that fumitremorgin C, a selective ABCG2 inhibitor, reduced BCSC survival to a similar degree as curcumin did. Curcumin sensitized breast cancer cells to chemotherapeutic drugs by reducing the BCSC population mainly through a reduction in the expression of ABCG2.     

Determining Duration of HER2-Targeted Therapy Using Stem Cell Extinction Models

Introduction

Trastuzumab dramatically improves survival in breast cancer patients whose tumor overexpresses HER2. A subpopulation of cells in human breast tumors has been identified with characteristics of cancer stem cells. These breast cancer stem-like cells (BCSCs) rely on HER2 signaling for self-renewal, suggesting that HER2-targeted therapy targets BCSCs even when the bulk of the tumor does not overexpress HER2. In order to guide clinical trials examining HER2-targeted therapy in the adjuvant setting, we propose a mathematical model to examine BCSC population dynamics and predict optimal duration of therapy.

Methods

Varying the susceptibility of BCSCs to HER2-targeted therapy, we quantify the average time to extinction of BCSCs. We expand our model using stochastic simulation to include the partially differentiated tumor cells (TCs) that represent bulk tumor population and examine effects of plasticity on required duration of therapy.

Results

Lower susceptibility of BCSCs and increased rates of dedifferentiation entail longer extinction times, indicating a need for prolonged administration of HER2-targeted therapy. We predict that even when therapy does not appreciably reduce tumor size in the advanced cancer setting, it will eventually eradicate the tumor in the adjuvant setting as long as there is at least a modest effect on BCSCs.

Conclusions

We anticipate that our results will inform clinical trials of targeted therapies in planning the duration of therapy needed to eradicate BCSCs. Our predictions also address safety, as longer duration of therapy entails a greater potential impact on normal stem cells that may also be susceptible to stem cell-targeted therapies.     

Resveratrol Inhibits Breast Cancer Stem-Like Cells and Induces Autophagy via Suppressing Wnt/β-Catenin Signaling Pathway

Resveratrol, a natural polyphenolic compound, is abundantly found in plant foods and has been extensively studied for its anti-cancer properties. Given the important role of CSCs (Cancer Stem Cells) in breast tumorigenesis and progression, it is worth investigating the effects of resveratrol on CSCs. The article is an attempt to investigate the effects of resveratrol on breast CSCs. Resveratrol significantly inhibits the proliferation of BCSCs (breast cancer stem-like cells) isolated from MCF-7 and SUM159, and decreased the percentage of BCSCs population, consequently reduced the size and number of mammospheres in non-adherent spherical clusters. Accordingly, the injection of resveratrol (100 mg/kg/d) in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice effectively inhibited the growth of xenograft tumors and reduced BCSC population in tumor cells. After the reimplantation of primary tumor cells into the secondary mice for 30 d, all 6 control inoculations produced tumors, while tumor cells derived from resveratrol-treated mice only caused 1 tumor of 6 inoculations. Further studies by TEM (Transmission electron microscopy) analysis, GFP-LC3-II puncta formation assay and western blot for LC3-II, Beclin1 and Atg 7, showed that resveratrol induces autophagy in BCSCs. Moreover, resveratrol suppresses Wnt/β-catenin signaling pathway in BCSCs; over-expression of β-catenin by transfecting the plasmid markedly reduced resveratrol-induced cytotoxicity and autophagy in BCSCs. Our findings indicated that resveratrol inhibits BCSCs and induces autophagy via suppressing Wnt/β-catenin signaling pathway.     

Breast cancer stem cells: The role of sex steroid receptors

Breast cancer(BC) is the most common cancer among women, and current available therapies often have high success rates. Nevertheless, BC might acquire drug resistance and sometimes relapse. Current knowledge about the most aggressive forms of BC points to the role of specific cells with stem properties located within BC, the so-called "BC stem cells"(BCSCs). The role of BCSCs in cancer formation, growth, invasiveness, therapy resistance and tumor recurrence is becoming increasingly clear. The growth and metastatic properties of BCSCs are regulated by different pathways, which are only partially known. Sex steroid receptors(SSRs), which are involved in BC etiology and progression, promote BCSC proliferation, dedifferentiation and migration. However, in the literature,there is incomplete information about their roles. Particularly, there are contrasting conclusions about the expression and role of the classical BC hormonal biomarkers, such as estrogen receptor alpha(ERα), together with scant,albeit promising information concerning ER beta(ERβ) and androgen receptor(AR) properties that control different transduction pathways in BCSCs. In this review, we will discuss the role that SRs expressed in BCSCs play to BC progression and recurrence and how these findings have opened new therapeutic possibilities.     

Single-cell analysis of tumors: Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.