Ganoderma lucidum extract (GLE) impairs breast cancer stem cells by targeting the STAT3 pathway.
ABSTRACT: The aggressive nature of triple negative breast cancer (TNBC) may be explained in part by the presence of breast cancer stem cells (BCSCs), a subpopulation of cells, which are involved in tumor initiation, progression, metastasis, recurrence, and therapy resistance. The signal transducer and activator of transcription 3 (STAT3) pathway participates in the development and progression of BCSCs, but its role in TNBC remains unclear. Here, we report that Ganoderma lucidum extract (GLE), a medicinal mushroom with anticancer activity, acts on BCSCs in vitro and in TNBC pre-clinical animal tumor models by downregulating the STAT3 pathway. We show that GLE significantly reduces TNBC cell viability, and down-regulates total and phosphorylated STAT3 expression. This is consistent with the reduction of OCT4, NANOG and SOX2 expression, reduction in the BCSC population by loss of the ALDH1 and CD44+/CD24- population, the deformation of mammospheres, and the strong reduction in animal tumor volume and tumor weight. Analysis of the BCSC compartment in tumors revealed that GLE decreases the STAT3 pathway and the expression of OCT4, NANOG, and SOX2 in BCSCs. These findings demonstrate that the anti-cancer activity of GLE targets BCSCs of TNBC through the downregulation of the STAT3 pathway.
Project description:Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells (BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal, recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected to silica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) for isolation of one compound, based on activity-guided purification using tumorsphere formation assays. The purified compound was identified as betavulgarin, using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) mass spectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, and mammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24- subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4. This compound decreased the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sex determining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest that betavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicating betavulgarin might be an anticancer agent against breast cancer cells and BCSCs.
Project description:Breast cancer stem-like cells (BCSC) are implicated in cancer recurrence and metastasis of triple-negative breast cancer (TNBC). We have recently discovered that ganglioside GD2 expression defines BCSCs and that ST8SIA1 regulates GD2 expression and BCSC function. In this report, we show that ST8SIA1 is highly expressed in primary TNBC; its expression is positively correlated with the expression of several BCSC-associated genes such as BCL11A, FOXC1, CXCR4, PDGFR?, SOX2, and mutations in p53. CRISPR knockout of ST8SIA1 completely inhibited BCSC functions, including in vitro tumorigenesis and mammosphere formation. Mechanistic studies discovered activation of the FAK-AKT-mTOR signaling pathway in GD2+ BCSCs, and its tight regulation by ST8SIA1. Finally, knockout of ST8SIA1 completely blocked in vivo tumor growth and metastasis by TNBC cells. In summary, these data demonstrate the mechanism by which ST8SIA1 regulates tumor growth and metastasis in TNBC and identifies it as a novel therapeutic target.
Project description:Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.
Project description:Nodal signaling plays several vital roles in the embryogenesis process. However, its reexpression in breast cancer is correlated with cancer progression, metastasis and poor prognosis. Recently, Nodal has also been reported to regulate self-renewal capacity in pancreatic cancer. This study aimed to explore the role of Nodal in breast cancer stem cells (BCSCs) and the underlying mechanisms. Therefore, the immunohistochemistry staining of Nodal in 135 human breast cancer cases was performed to analyzed the relationship of Nodal signaling, clinical outcomes and BCSC marker. And the results showed that high Nodal expression was positively correlated with poor prognosis and BCSC marker expression in breast cancer samples. We further assessed the effects of Nodal in regulating the BCSC properties in breast cancer cell lines and xenografts. Then, SB431542 was administered in vitro and in vivo to explore the function of the Smad2/3 pathway. And we demonstrated that Nodal signaling up-regulated the expression of ALDH1, CD44, CD133, Sox2, Oct4 and Nanog by activating the Smad2/3 pathway, thereby enhancing the tumorigenicity and sphere-forming ability of breast cancer cells. Furthermore, treatment with SB431542 could inhibit the properties of BCSCs in vitro and in vivo. In conclusion, these findings indicate that Nodal signaling may play a vital role in maintaining the BCSC phenotype in breast cancer and serve as a potential target to explore BCSC-specific therapies.
Project description:Breast cancer stem cells (BCSCs) are intrinsically chemoresistant and capable of self-renewal. Following chemotherapy, patients can develop minimal residual disease due to BCSCs which can repopulate into a relapsed tumor. Therefore, it is imperative to co-target BCSCs along with the bulk tumor cells to achieve therapeutic success and prevent recurrence. So, it is vital to identify actionable molecular targets against both BCSCs and bulk tumor cells. Previous findings from our lab and others have demonstrated that inhibition of the emerging drug target eIF4A with Rocaglamide A (RocA) was efficacious against triple-negative breast cancer cells (TNBC). RocA specifically targets the pool of eIF4A bound to the oncogenic mRNAs that requires its helicase activity for their translation. This property enables specific targeting of tumor cells. The efficacy of RocA against BCSCs is unknown. In this study, we postulated that eIF4A could be a vulnerable node in BCSCs. In order to test this, we generated a paclitaxel-resistant TNBC cell line which demonstrated an elevated level of eIF4A along with increased levels of cancer stemness markers (ALDH activity and CD44), pluripotency transcription factors (SOX2, OCT4, and NANOG) and drug transporters (ABCB1, ABCG2, and ABCC1). Furthermore, genetic ablation of eIF4A resulted in reduced expression of ALDH1A1, pluripotency transcription factors and drug transporters. This pointed out that eIF4A is likely associated with selected set of proteins that are critical to BCSCs, and hence targeting eIF4A may eliminate BCSCs. Therefore, we isolated BCSCs from two TNBC cell lines: MDA-Bone-Un and SUM-159PT. Following RocA treatment, the self-renewal ability of the BCSCs was significantly reduced as determined by the efficiency of the formation of primary and secondary mammospheres. This was accompanied by a reduction in the levels of NANOG, OCT4, and drug transporters. Exposure to RocA also induced cell death of the BCSCs as evaluated by DRAQ7 and cell viability assays. RocA treatment induced apoptosis with increased levels of cleaved caspase-3. Overall, we identified that RocA is effective in targeting BCSCs, and eIF4A is an actionable molecular target in both BCSCs and bulk tumor cells. Therefore, anti-eIF4A inhibitors could potentially be combined synergistically with existing chemo-, radio- and/or immunotherapies.
Project description:AIM:To identify and characterize cancer stem cells (CSC) in moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC). METHODS:Four micrometer-thick, formalin-fixed, paraffin-embedded MDBMSCC samples from six patients underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers, NANOG, OCT4, SALL4, SOX2, and pSTAT3; cancer stem cell marker, CD44; squamous cell carcinoma (SCC) marker, EMA; and endothelial marker, CD34. The transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, STAT3, and CD44 were studied using NanoString gene expression analysis and colorimetric in situ hybridization (CISH) for NANOG, OCT4, SOX2, SALL4, and STAT3. RESULTS:Diaminobenzidine and immunofluorescent (IF) IHC staining demonstrated the presence of (1) an EMA(+)/CD44(+)/SOX2(+)/SALL4(+)/OCT4(+)/pSTAT3(+)/NANOG(+) CSC subpopulation within the tumor nests; (2) an EMA(-)/CD44(-)/CD34(-)/SOX2(+)/OCT4(+)/pSTAT3(+)/NANOG(+) subpopulation within the stroma between the tumor nests; and (3) an EMA(-)/CD44(-)/CD34(+)/SOX2(+)/SALL4(+)/OCT4(+)/pSTAT3(+)/NANOG(+) subpopulation on the endothelium of the microvessels within the stroma. The expression of CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG was confirmed by the presence of mRNA transcripts, using NanoString analysis and NANOG, OCT4, SOX2, SALL4, and STAT3 by CISH staining. CONCLUSION:This study demonstrated a novel finding of three separate CSC subpopulations within MDBMSCC: (1) within the tumor nests expressing EMA, CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG; (2) within the stroma expressing SOX2, SALL4, OCT4, pSTAT3, and NANOG; and (3) on the endothelium of the microvessels within the stroma expressing CD34, SOX2, SALL4, OCT4, pSTAT3, and NANOG.
Project description:Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs) able to regrow a tumor while their progeny lack this ability. Recently, several groups reported enrichment for BCSCs when breast cancers were subjected to classic anticancer treatment. However, the underlying mechanisms leading to this enrichment are incompletely understood. Using non-BCSCs sorted from patient samples, we found that ionizing radiation reprogrammed differentiated breast cancer cells into induced BCSCs (iBCSCs). iBCSCs showed increased mammosphere formation, increased tumorigenicity, and expressed the same stemness-related genes as BCSCs from nonirradiated samples. Reprogramming occurred in a polyploid subpopulation of cells, coincided with re-expression of the transcription factors Oct4, sex determining region Y-box 2, Nanog, and Klf4, and could be partially prevented by Notch inhibition. We conclude that radiation may induce a BCSC phenotype in differentiated breast cancer cells and that this mechanism contributes to increased BCSC numbers seen after classic anticancer treatment.
Project description:The purpose of the study was to investigate the role of the signal transducer and activator of transcription 3 (STAT3), signal transduction protein in regulating the biological characteristics of stem cells in cervical carcinoma. Overexpressed plasmid of STAT3 was constructed and used to transfect SiHa into cervical carcinoma cells. STAT3-targeted specific siRNA was designed and produced. The effects of STAT3 upregulation (or inhibition) on the expression of NANOG, OCT4 and SOX2 markers of stem cells were measured, using western blot analysis and RT-qPCR. In addition, the tumor sphere experiment was also conducted to detect the formation of tumor spheres after the intervention of expression of STAT3 and the expression of STAT3, NANOG, OCT4 and SOX2 was detected in 35 cases of cervical carcinoma tissues and 31 cases of normal cervical tissues using immunohistochemistry. We determined whether the STAT3 overexpression plasmid was successfully constructed using enzyme digestion, PCR for bacterium solution, western blot analysis and RT-qPCR and found that the plasmid met the requirements of subsequent procedures. Compared with the empty plasmid group and STAT3 low expression group, the mRNA and protein expression of markers of stem cells, OCT4, SOX2 and NANOG were significantly elevated in the STAT3 overexpression group with statistically significant differences (P<0.05), the formation ratio of tumor spheres in the STAT3 overexpression group was also significantly higher than those in the other two groups (P<0.05). The positive expression of STAT3, OCT4, NANOG and SOX2 in the cervical squamous carcinoma group was also markedly higher than that in the chronic cervicitis group (P<0.05). This study led us to a conclusion that STAT3 can regulate the characteristics of stem cells in cervical carcinoma, and STAT3, NANOG, OCT4 and SOX2 are highly expressed in cervical squamous carcinoma, thus able to promote the progression of cervical carcinoma.
Project description:AIM:To identify and characterize cancer stem cells (CSCs) in moderately differentiated lip squamous cell carcinoma (MDLSCC). METHOD:MDLSCC samples underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for squamous cell carcinoma marker EMA, CSC marker CD44 and embryonic stem cell markers NANOG, octamer-binding transcription factor 4 (OCT4), spalt-like transcription factor 4 (SALL4), sex-determining region Y-box 2 (SOX2), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Immunofluorescent IHC staining was performed on two MDLSCC samples. Western blotting (WB) was used to confirm the expression of the aforementioned proteins and their transcription activation was investigated using NanoString and RT-qPCR. RESULTS:IHC staining demonstrated the presence of (1) an EMA+/CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4- CSC subpopulation within the tumor nests (TNs); (2) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4- CSC subpopulation; and (3) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4+ CSC subpopulation within the stroma, between the TNs. NanoString and RT-qPCR confirmed the presence of mRNA for CD44, SALL4, STAT3, SOX2, and OCT4, and WB confirmed the presence of NANOG, pSTAT3, SOX2, and OCT4. CONCLUSION:This study demonstrates three putative CSC subpopulations within MDLSCC.
Project description:AIM:To identify and characterize cancer stem cells (CSC) in glioblastoma multiforme (GBM). METHODS:Four-micrometer thick formalin-fixed paraffin-embedded GBM samples from six patients underwent 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers NANOG, OCT4, SALL4, SOX2, and pSTAT3. IF IHC staining was performed to demonstrate co-expression of these markers with GFAP. The protein expression and the transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, and STAT3 were investigated using Western blotting (WB) and NanoString gene expression analysis, respectively. RESULTS:DAB and IF IHC staining demonstrated the presence of a CSC population expressing NANOG, OCT4, SOX2, SALL4, and pSTAT3 with the almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4, within GBM. The expression of NANOG, SOX2 and, pSTAT3 but, not OCT and SALL4, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of NANOG, OCT4, SALL4, STAT3, and SOX2 in GBM. CONCLUSION:This study demonstrated a population of CSCs within GBM characterized by the expression of the CSC markers NANOG, SALL4, SOX2, pSTAT3 and OCT4 at the protein and mRNA levels. The almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4 would support the putative existence of a stem cell hierarchy within GBM.