Proliferation Of Breast Cancer Stem Cells Research Articles

Bestatin attenuates breast cancer stemness by targeting puromycin-sensitive aminopeptidase

Breast cancer is a prevalent malignant tumor among women with an increasing incidence rate annually. Breast cancer stem cells (BCSCs) are integral in impeding tumor advancement and addressing drug resistance. Bestatin serves as an adjuvant chemotherapy, triggering apoptosis in cancer cells. In this study, the effects of bestatin on sorted BCSCs from breast cancer cell lines have been studied. Our results indicated that bestatin inhibits the migration and proliferation of breast cancer cells by reducing the stemness of BCSCs both in vitro and in vivo. Puromycin-sensitive aminopeptidase is implicated in the process through the regulation of cell cycle, resulting in heightened cell apoptosis and diminished cell proliferation of BCSCs. Our study suggest that targeting cancer stem cell may offer a promising approach in breast cancer treatment, presenting noval therapeutic strategies for patients with breast cancer.

Correction for: Gomisin M2 from Baizuan suppresses breast cancer stem cell proliferation in a zebrafish xenograft model.

Correction for: Gomisin M2 from Baizuan suppresses breast cancer stem cell proliferation in a zebrafish xenograft model.

Downregulation of the long noncoding RNA DSCR9 (Down syndrome critical region 9) delays breast cancer progression by modulating microRNA-504-5p-dependent G protein-coupled receptor 65.

Possible roles of long noncoding RNAs (lncRNAs) in cancer stem cells (CSCs) have often been reported. Here, we focused on the regulatory function of the lncRNA Down syndrome critical region 9 (DSCR9) in breast cancer stem cells (BCSCs). Through bioinformatics analysis, DSCR9, microRNA-504-5p (miR-504-5p), and G protein-coupled receptor 65 (GPR65) were identified as targets implicated in breast cancer development. Then, clinical tissue samples, breast cancer cells, and isolated BCSCs were used to determine the expression of DSCR9, miR-504-5p, and GPR65. The results confirmed the overexpression of DSCR9 and GPR65 but low expression of miR-504-5p in breast cancer tissues and cells as well as in BCSCs. Following mechanistic investigation, it was found that DSCR9 targeted miR-504-5p, and that silencing DSCR9 inhibited the proliferation of BCSCs by elevating the expression of miR-504-5p. Additionally, miR-504-5p targeted GPR65 and inhibited its expression. Moreover, GPR65 activated the MEK/ERK signaling pathway to regulate BCSC proliferation. Finally, animal study verified that depletion of DSCR9 inhibited the proliferation of BCSCs in vivo and that BCSC proliferation was restored by overexpression of GPR65. Altogether, our findings revealed that DSCR9 elevated GPR65 expression by targeting miR-504-5p to exacerbate breast cancer, highlighting a new treatment modality for breast cancer.

Brain infiltration of breast cancer stem cells is facilitated by paracrine signaling by inhibitor of differentiation 3 to nuclear respiratory factor 1.

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood-brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.

Lysophosphatidic Acid Promotes the Expansion of Cancer Stem Cells via TRPC3 Channels in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC.

Analysis of translation factors using public database of breast cancer patients.

Breast cancer is originated from cancer stem cells (CSCs). Since CSCs are responsible for the chemoresistance and cancer relapse, it is essential to develop novel therapeutic drugs for the eradication of CSC. We have previously reported that translation factor X is involved in the proliferation of breast CSCs. In the present study, we examined the role of translation factor X in the xenograft experiments and clinical samples. First, we examined the effect of translation factor X on tumorigenicity in nude mice. Overexpression of factor X increased tumorigenicity of breast CSCs. Next, we analyzed the expression levels of translation factor X using clinical samples from patient with breast cancer. By analyzing RNA-seq data of breast cancer patients from the National Center for Biotechnology Information, translation factor X was highly expressed in breast cancer tissue samples, compared with normal tissue and early neoplasia. Finally, we analyzed cumulative survival analysis from The Cancer Genome Atlas. Kaplan-Meier analysis showed that mRNA level of translation factor X was reversely correlated with the overall survival time in patients with breast cancer. These results suggest the translation factor X can be used to predict breast cancer outcomes.

Effect of Cell Culture Medium on the Proliferation and Stemness of CD24-/CD44+ Human Breast Cancer Stem Cells

BACKGROUND: Cancer stem cells (CSCs) is defined as tumor initiating cells within tumor that maintain stemness properties and tumorigenicity. Extracellular pH of CSCs in in vitro condition is important for supporting cell proliferation which may also regulate the expression of stemness markers such as OCT4. This work aimed to examine the effect of cell culture media on the proliferation and stemness of human breast cancer stem cells (BCSCs).METHODS: Human CD24-/CD44+ BCSCs were grown in Dulbecco's Modified Eagle Medium/F-12 (DMEM/F-12) with 15mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), without HEPES and adjusted to pH 7.4, or without HEPES but pH was not adjusted. BCSCs were grown under standard conditions for various days. Viable cell number was measured using trypan blue exclusion, whereas proliferation rate using MTS assay. OCT4 mRNA and protein were analyzed using quantitative real time PCR (qRT-PCR) and Western Blot assay, respectively. In vitro tumorigenic activity was determined using mammosphere formation unit (MFU) assay.RESULTS: Our results showed a higher viable cell number and proliferation of BCSCs in DMEM/F-12 HEPES (-) compared to HEPES (+) medium until 4 day incubation. OCT4 mRNA and protein level, as well as MFU of BCSCs were significantly higher in HEPES (-) compared to HEPES (+) medium on day 2.CONCLUSION: DMEM/F-12 medium without HEPES facilitates CD24-/CD44+ BCSCs to have higher proliferation and stemness on day 2 incubation compared to those with HEPES.KEYWORDS: breast cancer, cancer stem cell, OCT4, stemness, proliferation

Increased lipogenesis is critical for self-renewal and growth of breast cancer stem cells: Impact of omega-3 fatty acids.

Aberrant lipid metabolism has recently been recognized as a new hallmark of malignancy, but the characteristics of fatty acid metabolism in breast cancer stem cells (BCSC) and potential interventions targeting this pathway remain to be addressed. Here, by using the in vitro BCSC models, mammosphere‐derived MCF‐7 cells and HMLE‐Twist‐ER cells, we found that the cells with stem cell‐like properties exhibited a very distinct profile of fatty acid metabolism compared with that of their parental cancer cells, characterized by increased lipogenesis, especially the activity of stearoyl‐CoA desaturase 1 (SCD1) responsible for the production of monounsaturated fatty acids, and augmented synthesis and utilization of the omega‐6 arachidonic acid (AA). Suppression of SCD1 activity by either enzyme inhibitors or small interfering RNA (siRNA) knockdown strikingly limited self‐renewal and growth of the BCSC, suggesting a key role for SCD1 in BCSC proliferation. Furthermore, elevated levels of SCD1 and other lipogenic enzymes were observed in human breast cancer tissues relative to the noncancer tissues from the same patients and correlated with the pathological grades. Interestingly, treatment of BCSC with omega‐3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, effectively downregulated the expression of the lipogenic enzymes and markedly suppressed BCSC self‐renewal and growth. Dietary supplementation of nude mice bearing BCSC‐derived tumors with omega‐3 fatty acids also significantly reduced their tumor load. These findings have demonstrated that increased lipogenesis is critical for self‐renewal and growth of BCSC, and that omega‐3 fatty acids are effective in targeting this pathway to exert their anticancer effect.

The C-X-C Motif Chemokine Ligand 1 Sustains Breast Cancer Stem Cell Self-Renewal and Promotes Tumor Progression and Immune Escape Programs.

Breast cancer (BC) mortality is mainly due to metastatic disease, which is primarily driven by cancer stem cells (CSC). The chemokine C-X-C motif ligand-1 (CXCL1) is involved in BC metastasis, but the question of whether it regulates breast cancer stem cell (BCSC) behavior is yet to be explored. Here, we demonstrate that BCSCs express CXCR2 and produce CXCL1, which stimulates their proliferation and self-renewal, and that CXCL1 blockade inhibits both BCSC proliferation and mammosphere formation efficiency. CXCL1 amplifies its own production and remarkably induces both tumor-promoting and immunosuppressive factors, including SPP1/OPN, ACKR3/CXCR7, TLR4, TNFSF10/TRAIL and CCL18 and, to a lesser extent, immunostimulatory cytokines, including IL15, while it downregulates CCL2, CCL28, and CXCR4. CXCL1 downregulates TWIST2 and SNAI2, while it boosts TWIST1 expression in association with the loss of E-Cadherin, ultimately promoting BCSC epithelial-mesenchymal transition. Bioinformatic analyses of transcriptional data obtained from BC samples of 1,084 patients, reveals that CXCL1 expressing BCs mostly belong to the Triple-Negative (TN) subtype, and that BC expression of CXCL1 strongly correlates with that of pro-angiogenic and cancer promoting genes, such as CXCL2-3-5-6, FGFBP1, BCL11A, PI3, B3GNT5, BBOX1, and PTX3, suggesting that the CXCL1 signaling cascade is part of a broader tumor-promoting signaling network. Our findings reveal that CXCL1 functions as an autocrine growth factor for BCSCs and elicits primarily tumor progression and immune escape programs. Targeting the CXCL1/CXCR2 axis could restrain the BCSC compartment and improve the treatment of aggressive BC.

Divergence of Intracellular Trafficking of Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 3 in MCF-7 Breast Cancer Cells and MCF-7-Derived Stem Cell-Enriched Mammospheres.

Breast cancer MCF-7 cell-line-derived mammospheres were shown to be enriched in cells with a CD44+/CD24– surface profile, consistent with breast cancer stem cells (BCSC). These BCSC were previously reported to express key sphingolipid signaling effectors, including pro-oncogenic sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1P3). In this study, we explored intracellular trafficking and localization of SphK1 and S1P3 in parental MCF-7 cells, and MCF-7 derived BCSC-enriched mammospheres treated with growth- or apoptosis-stimulating agents. Intracellular trafficking and localization were assessed using confocal microscopy and cell fractionation, while CD44+/CD24- marker status was confirmed by flow cytometry. Mammospheres expressed significantly higher levels of S1P3 compared to parental MCF-7 cells (p < 0.01). Growth-promoting agents (S1P and estrogen) induced SphK1 and S1P3 translocation from cytoplasm to nuclei, which may facilitate the involvement of SphK1 and S1P3 in gene regulation. In contrast, pro-apoptotic cytokine tumor necrosis factor α (TNFα)-treated MCF-7 cells demonstrated increased apoptosis and no nuclear localization of SphK1 and S1P3, suggesting that TNFα can inhibit nuclear translocation of SphK1 and S1P3. TNFα inhibited mammosphere formation and induced S1P3 internalization and degradation. No nuclear translocation of S1P3 was detected in TNFα-stimulated mammospheres. Notably, SphK1 and S1P3 expression and localization were highly heterogenous in mammospheres, suggesting the potential for a large variety of responses. The findings provide further insights into the understanding of sphingolipid signaling and intracellular trafficking in BCs. Our data indicates that the inhibition of SphK1 and S1P3 nuclear translocation represents a novel method to prevent BCSCs proliferation.

DR-5 and DLL-4 mAb Functionalized SLNs of Gamma-Secretase Inhibitors- An Approach for TNBC Treatment.

Triple-negative breast cancer (TNBC) is the most aggressive and heterogeneous cancer subtypes. High rates of metastasis, poor prognosis, and drug resistance are the major problems associated with TNBC. The current chemotherapeutics eliminate only the bulk tumor cells (non-BCSCs) and do not affect breast cancer stem cells (BCSCs). The BCSCs which are left behind after chemotherapy is reported to promote recurrence and metastasis of TNBC. Death receptor-5 (DR-5) is exclusively expressed in TNBCs and mediates the extrinsic pathway of apoptosis. DR-5, therefore, can be exploited for targeted drug delivery and to induce apoptosis. Gamma-secretase mediated Notch signaling in BCSCs regulates its proliferation, differentiation, and metastasis. The endogenous ligand, Delta-like ligand 4 (DLL4), is reported to activate this Notch signaling in TNBC. Blocking this signaling pathway using both gamma-secretase inhibitors (GSIs) and DLL4 monoclonal antibody (mAb) may produce synergistic benefits. Further, the GSIs (DAPT, LY-411575, RO4929097, MK0752, etc.) suffer from poor bioavailability and off-target side effects such as diarrhea, suppression of lymphopoiesis, headache, hypertension, fatigue, and ventricular dysfunctions. In this hypothesis, we discuss Solid lipid nanoparticles (SLNs) based drug delivery systems containing GSIs and surface modified with DR-5 and DLL4 monoclonal antibodies (mAb) to effectivity target and treat TNBC. The delivery system is designed to deliver the drug cargo precisely to TNBCs through its DR-5 receptors and hence expected to reduce the off-target side effects of GSIs. Further, DLL4 mAb and GSIs are expected to act synergistically to block the Notch signal mediated BCSCs proliferation, differentiation, and metastasis.

IL-8の産生を介したリゾホスファチジン酸によるトリプルネガティブ型乳癌幹細胞の増殖

Triple-negative breast cancer (TNBC) is a highly aggressive cancer with fewer effective targeted therapy. Since growing evidence suggests that TNBC is originated from breast cancer stem cells (BCSCs), it is required to elucidate the molecular mechanism of BCSC proliferation for new drug development. We have previously reported that a lipid mediator lysophosphatidic acid (LPA) increased BCSCs via Ca2+ signaling pathway. In this study, we examined whether calcineurin/NFAT pathway is involved in the LPA-induced increase in BCSCs. We found that LPA stimulation increased the transcriptional activation of NFAT. The calcineurin inhibitor cyclosporine A inhibited both LPA-increased NFAT activation and increase in BCSCs. We next examined the downstream signaling pathway. To identify NFAT target gene which is involved in the LPA-induced increase in BCSCs, we performed RNA-sequencing using MDA-MB-231 cells. We identified that 428 transcripts were upregulated by LPA by two fold or more. Among them, we focused on proinflammatory cytokine IL-8 which promoter contains NFAT consensus sequence. We found that LPA increased IL-8 production in MDA-MB-231 cells. In addition, a selective IL-8 receptor antagonist inhibited the LPA-induced increase in BCSCs. These results suggested that LPA increases BCSC through the NFAT-mediated IL-8 production.

Anticancer Effect of Metformin in Herceptin-Conjugated Liposome for Breast Cancer.

Metformin (MET) is an anti-diabetic drug effective against breast cancer, targeting breast cancer stem cells (BCSCs). MET-encapsulating liposome (LP-MET) and Herceptin-conjugated LP-MET (Her-LP-MET) were evaluated for their anti-cancer effect in vitro and in vivo. Size and zeta potentials of LP-MET and Her-LP-MET were suitable for enhanced permeability and retention effects. Her-LP-MET yielded greater inhibition of BCSC proliferation in vitro than free MET or LP-MET, as well as a dose-dependent long-term anti-proliferation effect. Further, the anti-migration effect of Her-LP-MET on BCSCs was superior to that of MET or LP-MET, and was enhanced when used in concert with doxorubicin (DOX). In a mouse model, Her-LP-MET combined with free DOX was more effective than free MET, free DOX, or Her-LP-MET. Moreover, Her-LP-MET combined with free DOX yielded tumor remission, whereas free DOX alone resulted in metastasis or death. As such, Her-LP-MET formulation is expected to provide a new therapeutic modality targeting BCSCs.

A standardized suppresses breast cancer cell proliferation by regulating the expression of EphA2 antisense RNA-mRNA axis independently of micro RNA

AbstractBackground: A standardized extract of cultured Lentinula edodes mycelia (ECLM), an extract from cultured Lentinula edodes, has been reported to suppress breast cancer stem cell proliferation by regulating microRNA (miR) expression. Natural antisense RNAs (ASs), a type of protein non-coding RNA, can regulate the expression of protein-coding genes by acting as a competing endogenous RNA (ceRNA) that adsorbs miRNAs, resulting in the prevention of mRNA degradation, and can also form a transient RNA duplex with mRNA. EphA2, a receptor tyrosine kinase, is typically expressed at low levels in normal epithelial cells, whereas its overexpression has been widely observed in numerous solid tumors and is associated with cell transformation, primary tumor initiation, and tumor progression. Objective: This study aimed to investigate the effect of ECLM on the expression of both EphA2 mRNA and endogenous AS to this mRNA, which could negatively affect human breast carcinoma cell proliferation. Methods: We used MCF7 and MDA-MB-231 human breast carcinoma cells, which were sub-cultured three times in the presence of optimized concentrations of ECLM. The effect of ECLM on the expression of EphA2 AS and mRNA was analyzed by RT-qPCR. miRNAs targeting both EphA2 AS and EphA2 mRNA and their RNA miR response elements (MREs) were predicted and analyzed by RT-qPCR and luciferase reporter assays. Results: ECLM suppressed the proliferation of MCF7 and MDA-MB-231 cells in a dose-dependent manner. In cells for which proliferation was negatively affected by ECLM, EphA2 AS and mRNA expression was also significantly inhibited by ECLM. Although neutralization of miR-335 led to the de-repression of both EphA2 AS and mRNA, results did not fully support the possibility that EphA2 AS might function as a ceRNA to regulate EphA2 mRNA levels. Conclusion: ECLM suppressed the proliferation of breast carcinoma cells in a specific dose-dependent manner. This suppressive effect was associated with a concordant reduction in both EphA2 AS and mRNA expression. These effects were not thought to occur via the reported ceRNA effect. These results thus suggest that ECLM could regulate EphA2 AS and mRNA expression by forming a transient RNA duplex formation, thereby stabilizing EphA2 mRNA. Keywords: ECLM, regulatory RNA, antisense RNA, microRNA, EphA2

The Hedgehog Signaling Pathway: A Viable Target in Breast Cancer?

The hedgehog (Hh) pathway plays a key role in embryonic development and stem cell programs. Deregulation of the Hh pathway is a key driver of basal cell carcinoma, and therapeutic targeting led to approval of Hh inhibitor, vismodegib, in the management of this cancer. The Hh pathway is implicated in other malignancies including hormone receptor (HR+) positive and triple negative breast cancer (TNBC). Hh signaling, which is activated in human mammary stem cells, results in activation of glioma-associated oncogene (GLI) transcription factors. High GLI1 expression correlates with worse outcomes in breast cancer. Non-canonical GLI1 activation is one mechanism by which estrogen exposure promotes breast cancer stem cell proliferation and epithelial–mesenchymal transition. Tamoxifen resistant cell lines show aberrant activation of Hh signaling, and knockdown of Hh pathway inhibited growth of tamoxifen resistant cells. As in other cancers Hh signaling is activated by the PI3K/AKT pathway in these endocrine resistant cell lines. Hh pathway activation has also been reported to mediate chemotherapy resistance in TNBC via various mechanisms including paracrine signaling to tumor micro-environment and selective proliferation of cancer stem cells. Co-activation of Hh and Wnt signaling pathways is a poor prognostic marker in TNBC. Early phase clinical trials are evaluating the combination of smoothened (SMO) inhibitors and chemotherapy in TNBC. In addition to SMO inhibitors like vismodegib and sonidegib, which are in clinical use for basal cell carcinoma, GLI1 inhibitors like GANT58 and GANT61 are in preclinical drug development and might be an effective mechanism to overcome drug resistance in breast cancer. Gene signatures predictive of Hh pathway activation could enrich for patients likely to respond to these agents.

MiR-1 inhibits the proliferation of breast cancer stem cells by targeting EVI-1.

PurposeBreast cancer stem cells (BCSCs) have been regarded as the key factor for treatment failure in breast cancer. The abnormal expression of miRNAs plays a significant role in different tumor types. However, the role of miR-1 in breast cancer remains poorly understood. The purpose of this study was to evaluate the effects of miR-1 on the proliferation and apoptosis of BCSCs.Materials and methodsCD44+/CD24−/low/epithelial-specific antigen+ BCSCs were isolated by flow cytometry. Real-time PCR and Western blotting were used to determine the expression of miRNAs, mRNAs, and epithelial–mesenchymal transition (EMT)-related genes. Cell proliferation and apoptosis were measured using the Cell Counting Kit-8 assay and Annexin V-fluorescein isothiocyanate flow cytometry, respectively. Luciferase reporter assay was used to verify whether miR-1 targeted ecotropic virus integration-1 (EVI-1). The role of miR-1 in breast cancer in vivo was evaluated using BCSCs xenograft mouse models.ResultsIn this study, we demonstrated that miR-1 was significantly downregulated in breast cancer tissues compared to the adjacent non-tumor tissues. The luciferase reporter assay verified that EVI-1 was a direct target of miR-1, and upregulation of miR-1 negatively correlated with the expression of EVI-1 in BCSCs at both the transcriptional and posttranslational levels. Furthermore, overexpression of miR-1 inhibited BCSCs proliferation and promoted apoptosis, which was reversed by the overexpression of EVI-1. In addition, we demonstrated that aberrant expression of miR-1 could regulate EMT-related genes in BCSCs. Finally, immunohistochemical staining demonstrated that EVI-1 expression was decreased in BCSCs tumors following intra-tumoral miR-1 agomir treatment compared to the control group.ConclusionmiR-1 can negatively regulate the expression of EVI-1 and, thus, affect BCSCs proliferation, apoptosis, and EMT-related markers. Taken together, these findings demonstrate that miR-1 could be employed as a therapeutic strategy in the treatment of breast cancer.

MiR-140-5p inhibits the proliferation and enhances the efficacy of doxorubicin to breast cancer stem cells by targeting Wnt1.

MicroRNAs (miRNAs) are a group of small non-coding single-stranded RNAs molecules, the dysregulation of which plays a critical role in the initiation and biological progression of malignancies. The current study demonstrated that miR-140-5p was frequently downregulated in breast cancer stem cells (BCSCs), and miR-140-5p mimics could inhibit the proliferation of BCSCs. Moreover, Wnt1 was a direct target of miR-140-5p, as was proved by luciferase reporter assays. miR-140-5p mimics could downregulate the wnt1 mRNA and protein levels in MCF-7 and MDA-MB-231 cells. Furthermore, miR-140 mimics could enhance the sensitivity of BCSCs to doxorubicin (Dox) through the Wnt1/ABCB1 pathway both in vitro and vivo. Our findings have presented a novel miRNA-mediated regulatory network for BCSCs, which may provide a potential therapeutic target for breast cancer.

Abstract 1128: Exosomal ID3 is pro-metastatic through guiding NRF1-induced breast cancer stem cells across the blood-brain-barrier

Abstract Nuclear respiratory factor 1 (NRF1) and inhibitor of differentiation protein 3 (ID3) are transcription regulating proteins. Recently we have uncovered a novel oncogenic function of NRF1 in breast cancer development and progression. In this study, we tested the postulate that exposure to PCB153 contributes in ID3 over-expressing endothelial stem cells (EndSCs) chaperoning and guiding of mesenchymal NRF1 breast cancer stem cells (BCSCs) across the blood-brain barrier. BCSCs/EndSCs were subjected to functional gain/loss of ID3 and/or NRF1 to test if PCB153 [1ng/ml] exposure produces NRF/ID3 signals regulating lineage specific BCSCs organ entry. First, we tested whether NRF1 promotes transmigration of breast cancer using a 3D blood-brain barrier (BBB) model consisting of breast tumor initiating cells (BTICs) or MDAMB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BTICs and mesenchymal enriched MDAMB231 BCSCs to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1+ BCSCs with ID3+ EndSCs was detected. ID3 EndSCs promoted the transmigration of BCSCs through the BBB. We found differential effects by endocrine disruptors, namely PCB153 and PCB77. The treatment with PCB153 showed increased growth of NRF1+ BCSCs tumor spheroids. PCB153 also induced the tumor cell adhesion to microvascular endothelium and transendothelial migration of BCSCs. The exosomal ID3 released from endothelial cells helped the mesenchymal NRF1+ BCSCs to cross through blood brain barriers. Xenograft experiments showed that ID3+ brain EndSCs not only supported the growth of estrogen treated NRF1+ BCSC tumorospheroids, but guided them to the brain in zebrafish. These findings show for the first time a key role for ID3 and NRF1 by which specific circulatory EndSCs accompany a particular sub-type of BCSCs to distant metastatic sites where they most likely facilitate the seeding, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents that prevent the spread of breast cancer to the brain. This work was, in part, supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jayanta K. Das, Mayur Doke, Alok Deoraj, Quentin Felty, Deodutta Roy. Exosomal ID3 is pro-metastatic through guiding NRF1-induced breast cancer stem cells across the blood-brain-barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1128.

Anticancer effect of curcumin on breast cancer and stem cells

Numerous studies have shown that curcumin, a natural compound, exerts anticancer effects by inhibiting cancer cell proliferation and metastasis and by inducing cell cycle arrest and apoptosis. In particular, curcumin exhibits potent inhibitory effects on breast cancer, the most prevalent type of cancer among women worldwide. It has low maximal inhibitory concentration for breast cancer cell lines that express the hormone receptor ER and sensitizes cell lines to anticancer drugs. Moreover, it can induce apoptosis in cell lines independently of hormone receptor expression. In addition, curcumin inhibits the proliferation of breast cancer stem cells (BCSC), an important factor that influences cancer recurrence. The inhibition of BCSC proliferation suppresses metastasis and reattachment, ultimately limiting tumor formation. A xenograft study similarly showed that curcumin exerts tumor-suppression effects on cancer cells and cancer stem cells. Therefore, curcumin is a potential anticancer compound, and its concurrent application with other anticancer drugs appears promising.

PDGF-AB rich-trombocyte lysate supplementation from breast cancer patients increased the proliferation of breast cancer stem cells

Background: Thrombocytosis in breast cancer (BC) patient was thought to play a role in the invasiveness of breast cancer stem cells (BCSCs). Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. This study was aimed to analyze the effect of platelet lysate (PL) as well as its PDGF-AB content as a tumor microenvironment on (CD24-/CD44+) BCSC proliferation.Methods: This was an experimental study that treated culture of BCSCs with PL from breast cancer (BC) patients or healthy donors. Venous blood from all subjects were subjected to prior hematology test and then processed to obtain platelet rich plasma (PRP). Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL at concentrations of 0.01% (v/v) was supplemented into DMEM-F12 medium and used for culturing BCSCs (CD24-/CD44+ cells). After 48 hours, total cell count, population doubling time (PDT), and cell viability were calculated and their correlation with platelet count and PDGF-AB levels were analyzed.Results: BC patients (n=5) had higher platelet counts and PDGF-AB levels in PL compared to healthy donors (n=15), (p=0.02). PL from BC patients could stimulate the proliferation of BCSCs higher than healthy donors (p&lt;0.001) and showed lower PDT value (p=0.001). Cell proliferation and PDT showed strong correlation with PDGF-AB level. This observation suggests that PDGF-AB has a role on BCSCs proliferation. PL showed no effect on BCSCs viability.Conclusion: Breast cancer patient platelet lysate stimulated BCSC proliferation.