Cancer stem cells (CSCs) have unique properties, including self-renewal, differentiation, and chemoresistance. In this study, we found that p21-activated kinase (PAK1) inhibitor (Group I, PAK inhibitor, IPA-3) and inactivator (ivermectin) treatments inhibit cell proliferation and that tumor growth of PAK1-knockout cells in a mouse model is significantly reduced. IPA-3 and ivermectin inhibit CSC formation. PAK1 physically interacts with Janus Kinase 2 (JAK2), and JAK2 inhibitor (TG101209) treatment inhibits mammosphere formation and reduces the nuclear PAK1 protein level. PAK1 interacts with signal transducer and activator of transcription 3 (Stat3), and PAK1 and Stat3 colocalize in the nucleus. We show through electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and reporter assays that the PAK1/Stat3 complex binds to the IL-6 promoter and regulates the transcription of the IL-6 gene. Inhibition of PAK1 and JAK2 in mammospheres reduces the nuclear pStat3 and extracellular IL-6 levels. PAK1 inactivation inhibits CSC formation by decreasing pStat3 and extracellular IL-6 levels. Our results reveal that JAK2/PAK1 dysregulation inhibits the Stat3 signaling pathway and CSC formation, the PAK1/Stat3 complex regulates IL-6 gene expression, PAK1/Stat3 signaling regulates CSC formation, and PAK1 may be an important target for treating breast cancer.

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.

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 CD44^high/CD24^low 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.

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 (CD44^high/CD24^low) 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.

Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)’s Integrated Colony Enumerator (NICE) with a screening of protein kinase library. Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated. Automated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IκB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-β1-induced migration, colony formation and EMT of MCF-7 cells. We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.

Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.