Formation Of Breast Cancer Stem Cells Research Articles

Hypoxia-induced circSTT3A enhances serine synthesis and promotes H3K4me3 modification to facilitate breast cancer stem cell formation

Hypoxia is a key feature of tumor microenvironment that contributes to the development of breast cancer stem cells (BCSCs) with strong self-renewal properties. However, the specific mechanism underlying hypoxia in BCSC induction is not completely understood. Herein, we provide evidence that a novel hypoxia-specific circSTT3A is significantly upregulated in clinical breast cancer (BC) tissues, and is closely related to the clinical stage and poor prognosis of patients with BC. The study revealed that hypoxia-inducible factor 1 alpha (HIF1α)-regulated circSTT3A has a remarkable effect on mammosphere formation in breast cancer cells. Mechanistically, circSTT3A directly interacts with nucleotide-binding domain of heat shock protein 70 (HSP70), thereby facilitating the recruitment of phosphoglycerate kinase 1 (PGK1) via its substrate-binding domain, which reduces the ubiquitination and increases the stability of PGK1. The enhanced levels of PGK1 catalyze 1,3-diphosphoglycerate (1,3-BPG) into 3-phosphoglycerate (3-PG) leading to 3-PG accumulation and increased serine synthesis, S-adenosylmethionine (SAM) accumulation, and trimethylation of histone H3 lysine 4 (H3K4me3). The activation of the H3K4me3 contributes to BCSCs by increasing the transcriptional level of stemness-related factors. Especially, our work reveals that either loss of circSTT3A or PGK1 substantially suppresses tumor initiation and tumor growth, which dramatically increases the sensitivity of tumors to doxorubicin (DOX) in mice. Injection of PGK1-silenced spheroids with 3-PG can significantly reverse tumor initiation and growth in mice, thereby increasing tumor resistance to DOX. In conclusion, our study sheds light on the functional role of hypoxia in the maintenance of BCSCs via circSTT3A/HSP70/PGK1-mediated serine synthesis, which provides new insights into metabolic reprogramming, tumor initiation and growth. Our findings suggest that targeting circSTT3A alone or in combination with chemotherapy has potential clinical value for BC management.

Amino acid profile changes during enrichment of spheroid cells with cancer stem cell properties in MCF-7 and MDA-MB-231 cell lines.

Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis. This study aimed to evaluate the changes in the amino acid profile in MCF-7 and MDA-MB-231 cells during spheroid formation to discover the specific metabolic properties in CSCs. MCF-7 and MDA-MB-231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high-performance liquid chromatography (HPLC). The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF-7 and MDA-MB-231 spheroids (histidine and serine, respectively). Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.

Abstract 1039: TrkA and JAK2-STAT3 pathway crosstalk promotes breast cancer stem cells in HER2-enriched and triple-negative breast cancers

Abstract Breast cancer is the most commonly diagnosed cancer in American women and accounts for ~15% of cancer-related deaths. Despite the current standard of care, metastatic HER2-enriched breast cancer and triple-negative breast cancer remain difficult to treat due to poor response to treatment, lack of actionable targets, or eventual acquired resistance. The high mortality rate of metastatic HER2-enriched breast cancers and triple-negative breast cancers highlights the need for novel actionable targets for improved response to therapeutic intervention. Through datamining of publicly available breast cancer patient datasets, we recently found that Tropomyosin receptor kinase A (TrkA) and Janus kinase 2 (JAK2)-STAT3 pathways are co-activated and co-enriched in HER2-enriched breast cancers and triple-negative breast cancers (Cancers 10:2340-2360, 2021). We also found that TrkA, a receptor tyrosine kinase, directly interacts with and phosphorylates STAT3 on Y705 residue, resulting in STAT3 activation, nuclear translocation, and increased transcription of STAT3 target genes SOX2 and c-MYC, which are associated with breast cancer stem cell formation as well as tumor recurrence and metastasis. In this study, we aimed to further characterize the effects of the TrkA-JAK2/STAT3 pathway crosstalk on breast cancer stem cells. Our data showed that overexpression of TrkA enhances mammosphere formation and ALDH activity of breast cancer cells, which are further enhanced upon co-overexpression of STAT3. Our study further revealed that TrkA and JAK2 inhibitors synergize to reduce breast cancer stemness since co-inhibition of TrkA and JAK2 significantly reduces the CD44high/CD24low cell subpopulation, mammosphere formation, and ALDH activity to a greater extent compared to vehicle or monotherapies. Western blot analysis of breast cancer cells treated with TrkA and/or JAK2 inhibitors showed that co-inhibition of these two kinases significantly reduces levels of p-STAT3 (Y705), as well as stemness markers SOX2, MYC, and CD44. Taken together, these findings suggest that TrkA cooperates with the JAK2-STAT3 signaling pathway to promote breast cancer stem cells through increasing expression of SOX2, c-MYC, and CD44, and that co-inhibition of TrkA and JAK2 significantly suppressed breast cancer stemness, suggesting its future utility as a promising new treatment modality for patients with HER2-enriched breast cancers and triple-negative breast cancers. Citation Format: Angelina T. Regua, Dongqin Zhu, Daniel L. Doheny, Grace L. Wong, Sara G. Manore, Calvin J. Wagner, Austin Arrigo, Mariana Najjar, Hui-Wen Lo. TrkA and JAK2-STAT3 pathway crosstalk promotes breast cancer stem cells in HER2-enriched and triple-negative breast cancers [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 1039.

Key Epigenetic Events Involved in the Maintenance of Breast Cancer Stem Cells.

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors and play significant roles in tumorigenesis, metastasis, resistance to treatment, and relapse. They are defined by self-renewal and multi-lineage differentiation, and aggressiveness. Epigenetic modifications, including DNA methylation and acetylation, histone modifications, and non-coding. RNAs (ncRNAs), are partly responsible for CSC potentials and are involved in the modification of key components of crucial pathways such as Notch and Wnt signaling in breast cancer. In this review, we present an overview of the pathways and epigenetic events that lead to the transformation of mammary gland stem cells to breast CSCs (BCSCs). Based on the data presented here, important pathways such as TGF-β/SMAD2 and Wnt/β-catenin and epigenetic modifications, including histone modifications, DNA methylations, and microRNAs, play important roles in BCSC formation and maintenance. Epigenetic events can alter the expression of genes and functional RNAs, resulting in tumor initiation and progression. Thus, a better understanding of epigenetic modifications involved in BCSC maintenance signaling pathways may help to eliminate or suppress BCSCs and overcome cancer by generating more effective and efficient therapeutic agents.

Inhibitory Effects of Tangeretin, A Citrus Peel-Derived Flavonoid, on Breast Cancer Stem Cell Formation through Suppression of Stat3 Signaling.

Breast cancer stem cells (BCSCs) are responsible for tumor chemoresistance and recurrence. Targeting CSCs using natural compounds is a novel approach for cancer therapy. A CSC-inhibiting compound was purified from citrus extracts using silica gel, gel filtration and high-pressure liquid chromatography. The purified compound was identified as tangeretin by using nuclear magnetic resonance (NMR). Tangeretin inhibited cell proliferation, CSC formation and tumor growth, and modestly induced apoptosis in CSCs. The frequency of a subpopulation with a CSC phenotype (CD44+/CD24−) was reduced by tangeretin. Tangeretin reduced the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3). Our results in this study show that tangeretin inhibits the Stat3 signaling pathway and induces CSC death, indicating that tangeretin may be a potential natural compound that targets breast cancer cells and CSCs.

Potential Role of MSC/Cancer Cell Fusion and EMT for Breast Cancer Stem Cell Formation.

Solid tumors comprise of maturated cancer cells and self-renewing cancer stem-like cells (CSCs), which are associated with various other nontumorigenic cell populations in the tumor microenvironment. In addition to immune cells, endothelial cells, fibroblasts, and further cell types, mesenchymal stroma/stem-like cells (MSC) represent an important cell population recruited to tumor sites and predominantly interacting with the different cancer cells. Breast cancer models were among the first to reveal distinct properties of CSCs, however, the cellular process(es) through which these cells are generated, maintained, and expanded within neoplastic tissues remains incompletely understood. Here, we discuss several possible scenarios that are not mutually exclusive but may even act synergistically: fusion of cancer cells with MSC to yield hybrid cells and/or the induction of epithelial-mesenchymal transition (EMT) in breast cancer cells by MSC, which can relay signals for retrodifferentiation and eventually, the generation of breast CSCs (BCSCs). In either case, the consequences may be promotion of self-renewal capacity, tumor cell plasticity and heterogeneity, an increase in the cancer cells’ invasive and metastatic potential, and the acquisition of resistance mechanisms towards chemo- or radiotherapy. While specific signaling mechanisms involved in each of these properties remain to be elucidated, the present review article focusses on a potential involvement of cancer cell fusion and EMT in the development of breast cancer stem cells.

Disruption of the NF-κB/IL-8 Signaling Axis by Sulconazole Inhibits Human Breast Cancer Stem Cell Formation

Breast cancer stem cells (BCSCs) are tumor-initiating cells that possess the capacity for self-renewal. Cancer stem cells (CSCs) are responsible for poor outcomes caused by therapeutic resistance. In our study, we found that sulconazole—an antifungal medicine in the imidazole class—inhibited cell proliferation, tumor growth, and CSC formation. This compound also reduced the frequency of cells expressing CSC markers (CD44high/CD24low) as well as the expression of another CSC marker, aldehyde dehydrogenase (ALDH), and other self-renewal-related genes. Sulconazole inhibited mammosphere formation, reduced the protein level of nuclear NF-κB, and reduced extracellular IL-8 levels in mammospheres. Knocking down NF-κB expression using a p65-specific siRNA reduced CSC formation and secreted IL-8 levels in mammospheres. Sulconazole reduced nuclear NF-κB protein levels and secreted IL-8 levels in mammospheres. These new findings show that sulconazole blocks the NF-κB/IL-8 signaling pathway and CSC formation. NF-κB/IL-8 signaling is important for CSC formation and may be an important therapeutic target for BCSC treatment.

Estrogen receptor coactivator Mediator Subunit 1 (MED1) as a tissue-specific therapeutic target in breast cancer.

Breast cancer, one of the most frequent cancer types, is a leading cause of death in women worldwide. Estrogen receptor (ER) α is a nuclear hormone receptor that plays key roles in mammary gland development and breast cancer. About 75% of breast cancer cases are diagnosed as ER-positive; however, nearly half of these cancers are either intrinsically or inherently resistant to the current anti-estrogen therapies. Recent studies have identified an ER coactivator, Mediator Subunit 1 (MED1), as a unique, tissue-specific cofactor that mediates breast cancer metastasis and treatment resistance. MED1 is overexpressed in over 50% of human breast cancer cases and co-amplifies with another important breast cancer gene, receptor tyrosine kinase HER2. Clinically, MED1 expression highly correlates with poor disease-free survival of breast cancer patients, and recent studies have reported an increased frequency of MED1 mutations in the circulating tumor cells of patients after treatment. In this review, we discuss the biochemical characterization of MED1 and its associated MED1/Mediator complex, its crosstalk with HER2 in anti-estrogen resistance, breast cancer stem cell formation, and metastasis both in vitro and in vivo. Furthermore, we elaborate on the current advancements in targeting MED1 using state-of-the-art RNA nanotechnology and discuss the future perspectives as well.

TGFβ promotes breast cancer stem cell self-renewal through an ILEI/LIFR signaling axis.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFβ signaling pathway by 3’-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFβ stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates to enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo. We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo. These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

Triterpene Acid (3-O-p-Coumaroyltormentic Acid) Isolated From Aronia Extracts Inhibits Breast Cancer Stem Cell Formation through Downregulation of c-Myc Protein.

Cancer stem cells (CSCs) are drug-resistant and radiation-resistant cancer cells that are responsible for tumor progression and maintenance, cancer recurrence, and metastasis. Targeting breast CSCs with phytochemicals is a new paradigm for cancer prevention and treatment. In this study, activity-guided fractionation from mammosphere formation inhibition assays, repeated chromatographic preparations over silica gel, preparatory thin layer chromatography, and HPLC using aronia extracts led to the isolation of one compound. Using 1H and 13C 2-dimensional nuclear magnetic resonance (NMR) as well as electrospray ionization (ESI) mass spectrometry, the isolated compound was identified as 3-O-p-coumaroyltormentic acid. This compound inhibits breast cancer cell proliferation and mammosphere formation in a dose-dependent manner and reduces the CD44high/CD24low subpopulation and aldehyde dehydrogenase (ALDH)-expressing cell population as well as the expression of the self-renewal-related genes CD44, SOX2, and OCT4. 3-O-p-Coumaroyltormentic acid preferentially reduced the protein levels of c-Myc, which is a CSC survival factor, by inducing c-Myc degradation. These findings indicate the novel utilization of 3-O-p-coumaroyltormentic acid for breast cancer therapy via disruption of c-Myc protein, which is a CSC survival factor.

Catechol derived from aronia juice through lactic acid bacteria fermentation inhibits breast cancer stem cell formation via modulation Stat3/IL-6 signaling pathway.

Cancer stem cells (CSCs) as a subpopulation of cancer cells are drug-resistant and radiation-resistant cancer cells to be responsible for tumor progress, maintenance and recurrence of cancer, and metastasis. This study isolated and investigated a new cancer stem cell (CSC) inhibitor derived from lactic acid fermentation products using culture broth with 2% aronia juice. The anti-CSC activity of aronia-cultured broth was significantly higher than that of the control. Activity-guided fractionation and repeated chromatographic preparation led to the isolation of one compound. Using nuclear magnetic resonance and ESI mass spectrometry, we identified the isolated compound as catechol. In this study, we report that aronia-fermented catechol has a novel inhibitory effect on human breast CSCs. Catechol inhibited breast cancer cell proliferation and mammosphere formation in a dose-dependent manner. This compound reduced the CD44high /CD24low subpopulation, ALDH-expressing cell population and the self-renewal-related genes nanog, sox2, and oct4. Catechol preferentially reduced mRNA transcripts and protein levels of Stat3 and did not induce c-Myc degradation. These findings support the novel utilization of catechol for breast cancer therapy via the Stat3/IL-6 signaling pathway. Our results suggest that catechol can be used for breast cancer therapy and that Stat3 expression is a marker of CSCs. Catechol inhibited Stat3 signaling by reducing Stat3 expression and secreted IL-6, a CSC survival factor. These findings support the novel utilization of catechol for breast cancer therapy via Stat3/IL-6 signaling.

The Notch Pathway in Breast Cancer Progression.

Objective Notch signaling pathway is a vital parameter of the mammalian vascular system. In this review, the authors summarize the current knowledge about the impact of the Notch signaling pathway in breast cancer progression and the therapeutic role of Notch's inhibition. Methods The available literature in MEDLINE, PubMed, and Scopus, regarding the role of the Notch pathway in breast cancer progression was searched for related articles from about 1973 to 2017 including terms such as “Notch,” “Breast Cancer,” and “Angiogenesis.” Results. Notch signaling controls the differentiation of breast epithelial cells during normal development. Studies confirm that the Notch pathway has a major participation in breast cancer progression through overexpression and/or abnormal genetic type expression of the notch receptors and ligands that determine angiogenesis. The cross-talk of Notch and estrogens, the effect of Notch in breast cancer stem cells formation, and the dependable Notch overexpression during breast tumorigenesis have been studied enough and undoubtedly linked to breast cancer development. The already applied therapeutic inhibition of Notch for breast cancer can drastically change the course of the disease. Conclusion Current data prove that Notch pathway has a major participation and multiple roles during breast tumor progression. Inhibition of Notch receptors and ligands provides innovative therapeutic results and could become the therapy of choice in the next few years, even though further research is needed to reach safe conclusions.

Quercetin‑3‑methyl ether suppresses human breast cancer stem cell formation by inhibiting the Notch1 and PI3K/Akt signaling pathways.

Breast cancer is a leading cause of mortality among women with cancer worldwide. Quercetin‑3‑methyl ether, a natural compound occurring in various plants, has been indicated to have potent anticancer activity. Breast cancer cell growth and survival were examined by CCK‑8 and colony formation assay, whilst cell cycle and apoptosis were determined by flow cytometry. Cell invasion and migration were assessed by wound‑healing assay and Transwell assay. Cancer stem cell formation was analyzed by mammosphere formation assay and related signaling pathways were detected by western blotting. In the present study, it was observed that treatment with quercetin‑3‑methyl ether significantly inhibited cell growth, induced apoptosis and cell cycle arrest at the G2‑Mphase, and suppressed invasion and migration in human breast cancer cells, including the triple negative MDAMB‑231 cell line, and the estrogen receptor‑positive/progesterone receptor‑positive/human epidermal growth factor receptor2‑negative MCF‑7 and T47D cell lines. This compound also markedly suppressed the epithelial‑mesenchymal transition process as evidenced by the upregulated expression of E‑cadherin, and the concomitant downregulated expression of vimentin and MMP‑2. Furthermore, it was demonstrated that quercetin‑3‑methyl ether treatment inhibited mammosphere formation and the expression of the stemness‑related genes, SRY‑box2 and Nanog. Mechanistically, this compound decreased the expression of Notch1, and induced the phosphorylation of phosphoinositide3‑kinase (PI3K) and Akt. It also attenuated the human insulin growth factor1‑induced phosphorylation of PI3K, Akt and glycogen synthase kinaseβ. Additionally, the combination of quercetin‑3‑methyl ether and a secretase inhibitor (DAPT) exhibited additive suppression of the expression of Notch1, PI3K, Akt and mammalian target of rapamycin and a more marked inhibitory effect on cell proliferation and colony formation compared with either drug alone. Treatment with quercetin‑3‑methyl ether alone markedly suppressed the levels of tri‑methyl histoneH3 (Lys27), but had no effect on the expression of enhancer of zeste homolog2. Overall, these findings indicated that quercein‑3‑methyl ether may be a potential therapeutic compound for the treatment of triple negative and hormone‑sensitive breast cancer.

Interleukin‐like EMT Inducer ILEI mediates breast cancer stem cell formation and tumorigenesis through LIF receptor signaling

During the epithelial‐mesenchymal transition (EMT), cells undergo a switch from a polarized, epithelial state to a more fibroblastic and mesenchymal phenotype. This process is associated with the formation of highly tumorigenic cancer stem cells (CSC). Our lab has identified a regulatory mechanism by which Transforming Growth Factor‐β (TGFβ) induces EMT at the translational step of gene expression. Among gene products controlled by this post‐translational mechanism is the cytokine Interleukin‐like EMT Inducer (ILEI), a secreted factor belonging to the FAM3 family that is involved in EMT induction and tumorigenesis. The biochemical mechanism of the ILEI signaling and identification of an ILEI receptor has remained elusive. Preliminary studies from our lab indicate that modulation of ILEI in several cell lines results in altered stemness potential, giving insight into the possible role of ILEI in the formation of CSCs. The basis of this study aims to investigate the mechanism of the ILEI signaling pathway and its contribution to CSC formation. In order to identify potential ILEI binding partners, we utilized yeast two‐hybrid screening and observed that ILEI interacts with the 190kD leukemia inhibitory factor receptor (LIFR). LIFR serves as a signaling platform for several cytokine ligands and forms intricate complexes with an array of other ligand‐specific co‐receptors during its activation. Our data indicates that ILEI functions through LIFR, inducing downstream CSC phenotypes by activating PI3k/Akt and Jak/STAT signaling pathways. Insight into the TGFβ/ILEI/LIFR pathway may serve as a platform for development of a novel targeted therapy against metastatic breast cancer.Support or Funding InformationNIH F31CA213627NIH R01CA555536This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNAs, a subpopulation of regulators, are involved in breast cancer progression through regulating breast cancer stem cells.

Cancer stem cells (CSCs; also known as tumor-initiating cells) are essential effectors of tumor progression due to their self-renewal capacity, differentiation potential, tumorigenic ability and resistance to chemotherapy, all of which contribute to cancer relapse, metastasis and a poor prognosis. Breast cancer stem cells (BCSCs) have been identified to be involved in the processes of BC initiation, growth and recurrence. MicroRNAs (miRNAs) are a class of non-coding small RNAs of 19-23 nucleotides in length that regulate gene expression at the post-transcriptional level through various mechanisms, and serve critical roles in cancer progression. miRNAs have been demonstrated to elicit effects on BCSCs characteristics via the targeting of oncogenes or tumor suppressor genes. The present study focused on the effect of miRNAs on BCSC, including BCSC formation, self-renewal and differentiation, by which miRNAs may inhibit BCSC invasion and metastasis, modulate clonogenicity and tumorigenicity of BCSCs as well as regulate chemotherapy resistance to BC. Through an improved understanding of the association between BCSCs and miRNAs, a novel and safer therapeutic target for BC may be identified.

VEGF-A/Neuropilin 1 Pathway Confers Cancer Stemness via Activating Wnt/β-Catenin Axis in Breast Cancer Cells

Background/Aims: Targeting cancer stem cells (CSCs) is emerging as a promising method for cancer treatment. We previously indicated that knockdown of Neuropilin 1(NRP-1) could inhibit breast cancer cell proliferation. Here, we continue exploring the roles and mechanisms of VEGF-A/NRP-1 axis in breast CSCs formation. Methods: qRT-PCR was used to detect the levels of VEGF-A and NRP-1 in breast cancer sphere cells and wild-type cells. Mammospheres formation, flow cytometry, soft agar colony and tumor formation assays were performed to evaluate the effects of VEGF-A/NRP-1 on breast cancer stemness. Further HUVECs tube formation, cell invasion assays were carried out to detect the effects of VEGF-A/NRP-1 on breast cancer spheres-induced angiogenesis. Finally, Annexin V/PI apoptosis and CCK8 assays were used to detect the effects of VEGF-A/NRP-1 on chemoresistance. Results: Overexpression of VEGF-A or NRP-1 conferred CSCs-related traits in MCF-7 cells, while knockdown of VEGF-A or NRP-1 reduced CSCs-related traits in MDA-MB-231 cells in vitro and in vivo. Notably, VEGF-A acted in a NRP-1 dependent way. Mechanistically, the VEGF-A/NRP-1 axis conferred CSCs phenotype via activating Wnt/β-catenin pathway. Conclusion: our results suggest that VEGF-A/NRP-1 axis could confer CSCs-related traits and chemoresistance.

Abstract 5197: Quantitative phosphorproteomics identified GPER-initiated PKA signal transduction event in breast cancer stem cell

Abstract Human breast cancer stem cells (BCSC) identified recently have the ability to initiate neoplastic growth with differentiation and display higher resistance to chemotherapy and radiotherapy. The allure of targeting this specific group of cells to reduce mortality of disease has attracted research efforts toward BCSC. Although some unique properties of BCSC have been deciphered, the molecular mechanisms are still poorly understood. Here, we applied in-house developed techniques to quantitatively compare the phosphoproteomic profiles between BCSC and non-cancer stem cells (nonCSC) derived from a xenograft of human breast cancer. For quantitative analysis, we utilized a SEMI-strategy for multiplexed label-free quantitation combining pH/acid-controlled IMAC chromatography and LC-MS/MS for relative quantitation of site-specific phosphorylation. This strategy effectively increases the phosphopeptide quantitation coverage. The differentially expressed phosphoproteins were mapped to multiple signaling pathways including NOTCH, CDK/ERK and JAK-STAT pathways, which potentially orchestrate the self renewal and stemness of BCSC. We also demonstrated ∼2 fold greater expression of GPER in BCSC than non-BCSC population, and GPER signaling could induce PKA-mediated phosphorylation of BAD in BCSC. Silencing of GPER via RNAi or mutation of phosphorylation sites of BAD led to reduced proliferation to 40.8 and 53.2 % of control, respectively, and the RNAi silencing of GPER decreased the mammosphere formation of BCSC to 43.5 % of control. These findings implies the importance of GPER and its downstream PKA pathway to the maintenance of stemness characteristics of BCSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5197. doi:1538-7445.AM2012-5197

Abstract 4365: Glucosylceramide synthase promotes cancer stem cells accumulating in chemotherapy

Abstract Cancer stem cells initiate tumorigenesis and are the causes of metastasis and relapse. Cancer stem cells, rather than other differentiated cells in tumors display resistance to cytotoxicity of anticancer drugs, but we do not know whether by-product of chemotherapy can induce cancer stem cells leading to chemotherapy failure. Glucosylceramide synthase (GCS) is a limiting enzyme regulating the synthesis of glycosphingolipids that play an essential role in the maintenance of embryonic stem cells. The purpose of this study was to identify and characterize the effect of ceramide glycosylation in the formation and maintenance of breast cancer stem cells (BCSCs). We have found that GCS overexpression was interrelated to the increment BCSCs and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cells. The BCSCs with CD24-/CD44+/ESA+ phenotype was increased by 5-fold in MCF-7/Dox cells as compared to MCF-7 cells. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs more than 2-fold in MCF-7/Dox cells. In the soft agar colony formation assay, MCF-7/Dox cells showed significantly higher colonies formed as compare to the MCF-7 cells. BCSCs sorted from MCF-7/Dox cells displayed significantly higher GCS enzyme activity more than 3-fold and formed 2-fold more colonies, as compare with other non-stem cell subsets. The BCSCs cells showed significantly higher tumorigenicity and metastasibility as compared to non-stem cells (CD24-/CD44-) in athymic nude mice. More interestingly, doxorubicin treatment (1 mg/kg, once a week, and 40 days) considerably increased BCSCs 2-fold in tumors, and MBO-asGCS treatment eliminated the tumor growth and reduced metastasis due to reduction of BCSCs more than 2-fold in vivo. Comparison of glycosphingolipids and gene profile of stem cells indicated GCS or globo-series glycosphingolipids upregulates FGF1, FGFR1, ALDH2, ISL1, MSX1, SIGMAR1, PPARD and downregulates CXCL12, FRAT1, CCND1 to accumulate BCSCs during the course of chemotherapy. These results, for the first time, demonstrate that ceramide glycosylation by GCS accumulates BCSCs formation and causes aggressive tumor progression. Silencing of GCS that eliminates BCSCs can prevent and treat drug-resistant tumors and relapse. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4365. doi:10.1158/1538-7445.AM2011-4365