Acquisition Of Cancer Stem Cell Properties Research Articles

Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

Diallyl trisulfide inhibited tobacco smoke-mediated bladder EMT and cancer stem cell marker expression via the NF-κB pathway in vivo.

ObjectiveThis study examined the effect of the NF-κB pathway on tobacco smoke-elicited bladder epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC) marker expression in vivo. The effect of diallyl trisulfide (DATS) treatment was also examined.MethodsBALB/c mice were exposed to tobacco smoke and treated with an NF-κB inhibitor and DATS. Western blotting, quantitative real-time PCR, and immunohistochemical staining were used to detect the changes of relevant indices.ResultsPhosphorylated inhibitor of kappa-B kinase alpha/beta expression and p65 and p50 nuclear transcription were increased by tobacco smoke exposure, whereas inhibitor of kappa-B expression was decreased. In addition, tobacco smoke reduced the expression of epithelial markers but increased that of mesenchymal and CSC markers. Our study further demonstrated that tobacco smoke-mediated EMT and CSC marker expression were attenuated by inhibition of the NF-κB pathway. Moreover, DATS reversed tobacco smoke-induced NF-κB pathway activation, EMT, and the acquisition of CSC properties in bladder tissues.ConclusionsThese data suggested that the NF-κB pathway regulated tobacco smoke-induced bladder EMT, CSC marker expression, and the protective effects of DATS.

Polychlorinated biphenyl quinone induced the acquisition of cancer stem cells properties and epithelial-mesenchymal transition through Wnt/β-catenin

Polychlorinated biphenyls (PCBs) are persistent industrial pollutants that have been linked to breast cancer progression. However, their molecular mechanism(s) are currently unclear. Our previous assessment suggested that the highly reactive PCB metabolite 2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone (PCB29-pQ) induces the metastasis of breast cancer. Here, our data illustrate that PCB29-pQ increases cancer stem cell (CSC) marker expression, resulting in an increase in the epithelial-mesenchymal transition (EMT) in MDA-MB-231 breast cancer cells; further, the Wnt/β-catenin pathway also becomes activated by PCB29-pQ. When the Wnt/β-catenin pathway is inhibited, the promotion of CSC properties and EMT by PCB29-pQ were accordingly reversed. In addition, the overproduction of reactive oxygen species (ROS) mediated by PCB29-pQ plays a key role in Wnt/β-catenin activation. Collectively, our current data designated the regulatory role of Wnt/β-catenin in PCB29-pQ-triggered acquisition of CSC properties and EMT.

MicroRNA-93 targets WASF3 and functions as a metastasis suppressor in breast cancer.

Cancer cells with cancer stem cell (CSC) properties initiate both primary tumor formation and metastases at distant sites. Acquisition of CSC properties is highly associated with epigenetic alterations, including those mediated by microRNAs (miRNAs). We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, we found that the expression levels of 3 miRNAs (miR‐25, miR‐93, and miR‐106b) in the miR‐106b‐25 cluster were much lower in the CD44+ human cancer cells metastasized to the liver than those at the primary site. Constitutive overexpression of miR‐93 suppressed invasive ability and 3D‐organoid formation capacity of breast cancer cells in vitro and significantly suppressed their metastatic ability to the liver in vivo. Wiskott‐Aldrich syndrome protein family member 3 (WASF3), a regulator of both cytoskeleton remodeling and CSC properties, was identified as a functional target of miR‐93: overexpression of miR‐93 reduced the protein level of WASF3 in breast cancer cells and WASF3 rescued the miR‐93‐mediated suppression of breast cancer cell invasion. These findings suggest that miR‐93 functions as a metastasis suppressor by suppressing both invasion ability and CSC properties in breast cancers.

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition in Cancer Metastasis.

The cancer stem cell (CSC) concept stands for undifferentiated tumor cells with the ability to initiate heterogeneous tumors. It is also relevant in metastasis and can explain how metastatic tumors mirror the heterogeneity of primary tumors. Cellular plasticity, including the epithelial-to-mesenchymal transition (EMT), enables the generation of CSCs at different steps of the metastatic process including metastatic colonization. In this review, we update the concept of CSCs and provide evidence of the existence of metastatic stem cells (MetSCs). In addition, we highlight the nuanced understanding of EMT that has been gained recently and the association of mesenchymal-to-epithelial transition (MET) with the acquisition of CSCs properties during metastasis. We also comment on the computational approaches that have profoundly influenced our understanding of CSCs and EMT; and how these studies and new experimental technologies can yield a deeper understanding of the biological aspects of metastasis.

Abstract LB-260: Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths in U.S. men, disproportionately affecting African American men and certain Hispanic/Latino populations. Patients with advanced PCa are treated with androgen deprivation therapy (ADT) and docetaxel (DTX)-based chemotherapy with effective but transient results due to therapy resistance. While the role of glucocorticoid receptor (GR) in promoting ADT resistance has been well characterized, comparatively little is known about its contribution to PCa stemness and chemoresistance. Our group demonstrated recently that DTX-resistance is associated with upregulation of a stemness transcriptomic program and enhanced cancer stem cell (CSC) properties in PCa cells. We also demonstrated that GR signaling upregulates the DTX-resistance associated proteins Clusterin and LEDGF/p75 in a panel of PCa cells. The proto-oncogene β-catenin promotes CSC survival in many cancers, including PCa, and several studies have demonstrated a crosstalk between AR and β-catenin signaling pathways contributing to ADT resistance. Given the structural similarities between AR and GR and the importance of GR in therapy resistance, this study was designed to investigate the interaction between GR and β-catenin and its contribution to CSC survival and DTX resistance. Using a panel of PCa cell lines including PC3, DU145, 22rv1, and their corresponding DTX-resistant cell lines, we showed by quantitative immunoblotting that GR is upregulated and β-catenin is activated in PC3 and 22rv1 DTX-resistant cells compared to sensitive cells. Co-immunoprecipitation studies revealed interaction between endogenous GR and β-catenin in these cells. We next assessed the effect of the potent GR ligand dexamethasone (Dex) on CSC maintenance, and observed that Dex treatment of PC3 cells increased tumorsphere number and expression of established CSC markers measured by multicolor flow cytometry. To identify potential GR target genes that could mediate the acquisition of CSC properties and DTX resistance, we performed RNA-sequencing analysis in four different Dex-treated and -untreated PCa cell lines. This analysis revealed several candidate GR target genes differentially regulated in a cell-type dependent manner. To determine the effects of the GR/β-catenin interaction on downstream GR target genes and β-catenin target genes, we performed immunoblotting of specific candidate target genes on lysates from cells treated with Dex alone or in combination with COR-10829 (selective GR inhibitor, and MSAB (β-catenin inhibitor). Interestingly, we also observed that the dual blockade of GR and β-catenin markedly reduced tumorsphere formation in DTX-resistant cells, a phenomenon not observed with either GR or β-catenin inhibition alone. In summary, our findings revealed a novel interaction between GR and β-catenin in PCa cells, and suggest that co-targeting the GR/β-catenin signaling axis in advanced PCa patients could be a potential strategy to overcome therapy resistance. Citation Format: Shannalee R. Martinez, Carlos J. Diaz Osterman, Xin S. Chen, Charles Wang, Lubo Zhang, Carlos A. Casiano. Role of the glucocorticoid receptor/beta-catenin interaction in prostate cancer stem cell survival and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-260.

The CD147-HYALURONAN Axis in Cancer.

CD147 (basigin; EMMPRIN), hyaluronan, and hyaluronan receptors (e.g., CD44) are intimately involved in several phenomena that underlie malignancy. A major avenue whereby they influence tumor progression is most likely their role in the characteristics of cancer stem cells (CSCs), subpopulations of tumor cells that exhibit chemoresistance, invasiveness, and potent tumorigenicity. Both CD147 and hyaluronan have been strongly implicated in chemoresistance and invasiveness, and may be drivers of CSC characteristics, since current evidence indicates that both are involved in epithelial-mesenchymal transition, a crucial process in the acquisition of CSC properties. Hyaluronan is a prominent constituent of the tumor microenvironment whose interactions with cell surface receptors influence several signaling pathways that lead to chemoresistance and invasiveness. CD147 is an integral plasma membrane glycoprotein of the Ig superfamily and cofactor in assembly and activity of monocarboxylate transporters (MCTs). CD147 stimulates hyaluronan synthesis and interaction of hyaluronan with its receptors, in particular CD44 and LYVE-1, which in turn result in activation of multiprotein complexes containing members of the membrane-type matrix metalloproteinase, receptor tyrosine kinase, ABC drug transporter, or MCT families within lipid raft domains. Multivalent hyaluronan-receptor interactions are essential for formation or stabilization of these lipid raft complexes and for downstream signaling pathways or transporter activities. We conclude that stimulation of hyaluronan-receptor interactions by CD147 and the consequent activities of these complexes may be critical to the properties of CSCs and their role in malignancy. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.

Glycoprotein nmb Is Exposed on the Surface of Dormant Breast Cancer Cells and Induces Stem Cell-like Properties.

Glycoprotein nmb (GPNMB) is a type I transmembrane protein that contributes to the initiation and malignant progression of breast cancer through induction of epithelial-mesenchymal transition (EMT). Although it is known that EMT is associated with not only cancer invasion but also acquisition of cancer stem cell (CSC) properties, the function of GPNMB in this acquisition of CSC properties has yet to be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine the correlation between GPNMB and CSC properties in breast cancer cells. Three-dimensional sphere cultures induced higher expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than the 2D monolayer cultures. Three-dimensional culture also induced cell surface expression of GPNMB on limited numbers of cells in the spheres, whereas the 2D cultures did not. Therefore, we isolated cell surface-GPNMBhigh and -GPNMBlow cells from the spheres. Cell surface-GPNMBhigh cells expressed high levels of CSC genes and EMT-TF genes, had significantly higher sphere-forming frequencies than the cell surface-GPNMBlow cells, and showed no detectable levels of proliferation marker genes. Similar results were obtained from transplanted breast tumors. Furthermore, wild-type GPNMB, but not mutant GPNMB (YF), which lacks tumorigenic activity, induced CSC-like properties in breast epithelial cells. These findings suggest that GPNMB is exposed on the surface of dormant breast cancer cells and its activity contributes to the acquisition of stem cell-like properties.Significance: These findings suggest that cell surface expression of GPNMB could serve as a marker and promising therapeutic target of breast cancer cells with stem cell-like properties. Cancer Res; 78(22); 6424-35. ©2018 AACR.

VEGF-neuropilin-2 signaling promotes stem-like traits in breast cancer cells by TAZ-mediated repression of the Rac GAP β2-chimaerin.

The role of vascular endothelial growth factor (VEGF) signaling in cancer is not only well known in the context of angiogenesis but also important in the functional regulation of tumor cells. Autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears to be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression, and drug resistance. Therefore, understanding the mechanisms involved in VEGF-mediated support of CSCs is critical to successfully treating cancer patients. The expression of the Hippo effector TAZ is associated with breast CSCs and confers stem cell-like properties. We found that VEGF-NRP2 signaling contributed to the activation of TAZ in various breast cancer cells, which mediated a positive feedback loop that promoted mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, thus leading to TAZ activity. In a complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) β2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, the TAZ-mediated repression of β2-chimaerin resulted in sustained Rac1 activity in CSCs. Depletion of β2-chimaerin in non-CSCs increased Rac1 activity, TAZ abundance, and mammosphere formation. Analysis of a breast cancer patient database revealed an inverse correlation between β2-chimaerin and TAZ expression in tumors. Our findings highlight an unexpected role for β2-chimaerin in a feed-forward loop of TAZ activation and the acquisition of CSC properties.

ZEB1 confers stem cell-like properties in breast cancer by targeting neurogenin-3.

Cancer stem cells (CSCs) are a subpopulation of cancer cells believed to be implicated in cancer initiation, progression, and recurrence. Here, we report that ectopic expression of zinc finger E-box binding homeobox 1 protein (ZEB1) results in the acquisition of CSC properties by breast cancer cells, leading to tumor initiation and progression in vitro and in vivo. The neurogenin 3 gene (Ngn3) is a bona fide target of ZEB1, and its repression is a key factor contributing to ZEB1-induced cancer cell stemness. ZEB1 suppressed Ngn3 transcription by forming a ZEB1/DNA methyltransferase (DNMT)3B/histone deacetylase 1 (HDAC1) complex on the Ngn3 promoter, leading to promoter hypermethylation and gene silencing. The rescue of Ngn3 expression attenuated ZEB1-induced cancer stemness and symmetric CSC division. Immunohistological analysis of human breast cancer specimens revealed a strong inverse relationship between ZEB1 and NGN3 protein expression. Thus, our findings suggest ZEB1-mediated silencing of Ngn3 is required for breast tumor initiation and maintenance. Targeted therapies against the ZEB1/Ngn3 axis may be highly valuable for the prevention and treatment of breast cancer.

Abstract 1059: EGF induces epithelial-mesenchymal transition and cancer stem cell properties in human oral squamous cell carcinoma

Abstract Head and neck cancer is one of the 10 most common cancers worldwide. Despite advances in therapeutic research in the last few decades, morbidity and mortality remain unchanged. Recurrent and metastatic oral squamous cell carcinoma (OSCC) of the head and neck may have correlated with epithelial-mesenchymal transition (EMT), a property acquired by cancer stem cells (CSCs). In our study, SCC-1 cells (a human tongue cancer cell line) treated with epidermal growth factor (EGF) underwent EMT as confirmed by morphological changes and expression of EMT-related markers using Western blot, immunocytostaining, and flow cytometric analyses. Our data showed that, in comparison with non-treated cells, EGF treatment induced SCC-1 cells to undergo EMT, characterized by spindle-shaped mesenchymal-like morphology, an altered suppression of E-cadherin and promotion of vimentin expression, and an enhanced invasive capacity. Our results also indicated that EGF significantly enriched the fraction of CD44+/CD24low/- CSCs in SCC-1 cells, from 7∼8% to 25∼29%, and simultaneously increased the expression of aldehyde dehydrogenase 1 (ALDH1), a common marker for stem cells. Moreover, the purified CD44+/CD24low/- CSC population displayed stem cell properties associated with EMT phenotypes and increased migration and invasion abilities in vitro. Using a murine orthotopic tongue cancer model transplanted with stably GFP-expressing SCC-1 cells (GFP-SCC-1), we confirmed that EGF can promote tumor metastasis to cervical lymph nodes, and simultaneously augmented CD44 and ALDH1 expression in these invasive tumor cells. Taken together, we have demonstrated that EGF-induced EMT process is closely associated with the acquisition of CSC properties in human OSCC, which may contribute to the increased distant metastasis and recurrence of head and neck cancer. Further elucidation of the underlying mechanisms will facilitate development of new generation of effective therapies that aim to specifically target CSCs and eliminate the “root” of cancer recurrence. This work was supported by National Institute of Health Research Grant, R01DE 019932, Oral and Maxillofacial Surgery Foundation Research Support, 002894, and the Schoenleber funding support. Citation Format: Qilin Xu, Qunzhou Zhang, Anh Le. EGF induces epithelial-mesenchymal transition and cancer stem cell properties in human oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1059. doi:10.1158/1538-7445.AM2014-1059

Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties.

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1, verteporfin, significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population.