Cancer Stem Cell-like Phenotype Research Articles

Breast Cancer Cell Detection and Characterization from Breast Milk-Derived Cells.

Radiologic techniques remain the main method for early detection for breast cancer and are critical to achieve a favorable outcome from cancer. However, more sensitive detection methods to complement radiologic techniques are needed to enhance early detection and treatment strategies. Using our recently established culturing method that allows propagation of normal and cancerous breast epithelial cells of luminal origin, flow cytometry characterization, and genomic sequencing, we show that cancer cells can be detected in breast milk. Cells derived from milk from the breast with cancer were enriched for CD49f+/EpCAM-, CD44+/CD24-, and CD271+ cancer stem-like cells (CSC). These CSCs carried mutations within the cytoplasmic retention domain of HDAC6, stop/gain insertion in MORF4L1, and deletion mutations within SWI/SNF complex component SMARCC2. CSCs were sensitive to HDAC6 inhibitors, BET bromodomain inhibitors, and EZH2 inhibitors, as mutations in SWI/SNF complex components are known to increase sensitivity to these drugs. Among cells derived from breast milk of additional ten women not known to have breast cancer, two of them contained cells that were enriched for the CSC phenotype and carried mutations in NF1 or KMT2D, which are frequently mutated in breast cancer. Breast milk-derived cells with NF1 mutations also carried copy-number variations in CDKN2C, PTEN, and REL genes. The approach described here may enable rapid cancer cell characterization including driver mutation detection and therapeutic screening for pregnancy/postpartum breast cancers. Furthermore, this method can be developed as a surveillance or early detection tool for women at high risk for developing breast cancer. SIGNIFICANCE: These findings describe how a simple method for characterization of cancer cells in pregnancy and postpartum breast cancer can be exploited as a surveillance tool for women at risk of developing breast cancer.

Overexpression of Cancer Upregulated Gene 2 (CUG2) Decreases Spry2 Through c-Cbl, Leading to Activation of EGFR and β-Catenin Signaling.

PurposeThe mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells.Materials and MethodsSpry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and β-catenin reporter assay were performed for downstream of Spry2 signaling.ResultsAlthough A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial–mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and β-catenin-NEK2-Yap1 signaling pathways.ConclusionCUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and β-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.

ALDH-1-positive cells exhibited a radioresistant phenotype that was enhanced with hypoxia in cervical cancer

BackgroundWe have previously found there was a small subpopulation of cells with cancer stem cell-like phenotype ALDH-1 in cervical cancer. Radiotherapy has been applied in most of the cervical cancer. However,the mechanisms underlying radioresistance still remained elusive. Our study is to explore whether ALDH+ cell promotes radioresistance by hypoxia.MethodsCells were respectively cultured in hypoxia and normoxia environment and analyzed for marker stability, and cell cycle distribution. Results: Cell growth, apoptosis, cell cycle, sphere formation were affected by hypoxia. ALDH-1 and CHK2 were upregulated after hypoxia.ConclusionsHere we show that ALDH-1 positive cells contribute to cervical carcinoma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of these cells is enriched after radiation in cervical carcinoma.

Hypoxia induces an endometrial cancer stem-like cell phenotype via HIF-dependent demethylation of SOX2 mRNA

Endometrial cancer stem cells (ECSCs) are stem-like cells endowed with self-renewal and differentiation abilities, and these cells are essential for cancer progression in endometrial cancer (EC). As hallmarks of the tumour microenvironment (TME), hypoxia and hypoxia-inducing factors (HIFs) give rise to the dysregulation of tumour stemness genes, such as SOX2. Against this backdrop, we investigated the regulatory mechanisms regulated by HIFs and SOX2 in ECSCs during EC development. Here, ECSCs isolated from EC cell lines and tissues were found to express stemness genes (CD133 and aldehyde dehydrogenase, ALDH1) following the induction of their ECSC expansion. Notably, m6A methylation of RNA and HIF-1α/2α-dependent AlkB homologue 5 (ALKBH5) participate in the regulation of HIFs and SOX2 in EC, as confirmed by the observations that mRNA levels of m6A demethylases and ALKBH5 significantly increase under hypoxic conditions in ECSCs. Moreover, hypoxia and high ALKBH5 levels restore the stem-like state of differentiated ECSCs and increase the ECSC-like phenotype, whereas the knockdown of HIFs or ALKBH5 significantly reduces their tumour initiation capacity. In addition, our findings validate the role of ALKBH5 in promoting SOX2 transcription via mRNA demethylation, thereby maintaining the stem-like state and tumorigenicity potential of ECSCs. In conclusion, these observations demonstrate a critical role for m6A methylation-mediated regulation of the HIF-ALKBH5-SOX2 axis during ECSC expansion in hypoxic TMEs.

PAK5 promotes the cell stemness ability by phosphorylating SOX2 in lung squamous cell carcinomas

Growing evidences suggest that the overexpression of p21-activated kinase 5 (PAK5) plays an important role in various tumor progression. However, the role of PAK5 and its downstream target gene(s) in lung squamous cell carcinomas (LUSC) are waiting to be elucidated. TCGA data were utilized to evaluate the expression levels of PAK5 in LUSC. We then explored the role of PAK5 in maintaining the stem-like phenotype of lung squamous cancer cells through RT-PCR, flow cytometry, oncosphere-forming assay. In addition, co-immunoprecipitation, western blotting and immunofluorescence assays were used to determine SOX2 as a novel effector of PAK5. Xenograft models in nude mice were established to explore the roles of PAK5 in lung cancer growth. In this study, we have shown that PAK5 is overexpressed in LUSC tissues. The absence of PAK5 abolishes self-renewal ability of LUSC cells by decreasing the expression and phosphorylation of SOX2 in vitro and in vivo. In xenograft models, knockdown or pharmacological inhibition of PAK5 suppressed the tumor growth and metastasis of lung squamous cancer cells in vivo. Taken together, our findings suggest that the PAK5-mediated SOX2 phosphorylation promoted the cancer stem cell-like phenotype of LUSC cells. PAK5 inhibition may be a promising target in the treatment of SOX2 positive lung squamous cell cancer.

Abstract PR05: Patient-derived tumoroids for exploration of the ovarian cancer stem cell regulation, chemoresistance, and tumor heterogeneity

Abstract Ovarian cancer is the leading cause of gynecologic malignancy, characterized by a high degree of heterogeneity and relapse leading to poor clinical outcomes. These outcomes are attributed in part to a population of cells termed cancer stem-like cells (CSCs), which are capable of repopulating tumors and are more chemoresistant and tumorigenic. These properties make CSCs a promising target for novel therapies; however, we lack a comprehensive understanding of how they are regulated by the tumor microenvironment. To address this, we have developed a tumoroid culture system wherein patient tumor cells are brought together with controlled ratios of mesenchymal stem cells (MSCs), endothelial cells (ECs), and peripheral blood mononuclear cells (PBMCs) within 384-well hanging drop arrays. This allows for comprehensive analysis of the surrounding cells in the tumor microenvironment and their influence on CSC populations and chemoresistance. The fine control of this culture system over the tumoroid cell composition coupled with the high-throughput nature of 384-well hanging drop also facilitates patient-specific high-throughput analysis of inter- and intrapatient heterogeneity and chemoresistance. Tumoroids were generated from 5 patient samples and characterized using flow cytometry detection of cell type proportions, immunofluorescence evaluation of cell localization, and progressive evaluation of cell localization with fluorescently labeled tumoroids. Patient-derived tumoroids were evaluated by single-cell RNA sequencing. Drug screening was performed on patient-derived tumoroids and control patient-derived spheroids using classic ovarian cancer therapies, carboplatin and paclitaxel, as well as three novel drugs. Additionally, tumor formation assay was performed in immunodeficient mice to compare the rate of tumor formation following injection of tumoroids versus spheroids. Finally, a mathematical model was developed to predict the evolution of cell populations within the tumoroids to facilitate further analysis. Using these methods, we successfully generated and characterized viable tumoroids with primary patient-derived tumor cells, MSCs, ECs, and PBMCs. Within these tumoroids, we observed heterogeneity both between and within patient samples reflective of clinical observations in ovarian cancers. Furthermore, we found increased CSC phenotypes in the tumoroids compared to patient-derived tumor cell-only spheroids. Patient-derived tumoroids also exhibited increased chemoresistance and tumorigenicity compared to spheroids generated with only tumor cells. Through thorough development and characterization of this patient-derived model, we present a novel 3D tumoroid model for comprehensive investigation of CSC regulation, chemoresistance, and heterogeneity in ovarian cancers, with the long-term goal of developing novel CSC targeting therapies and improving clinical outcomes. This abstract is also being presented as Poster B08. Citation Format: Micheal E. Bregenzer, Pooja Mehta, Arvind Rao, Karen McLean, Nouri Neamati, Ronald J. Buckanovich, Geeta Mehta. Patient-derived tumoroids for exploration of the ovarian cancer stem cell regulation, chemoresistance, and tumor heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR05.

PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC).

BackgroundsThe 2 isoforms of paired-related homeobox 1 (PRRX1), PRRX1A and PRRX1B, are critical in regulating several kinds of cancers, and figure prominently in the maintenance of stemness and progression of epithelial-mesenchymal transition (EMT). However their differential expression in non-small cell lung cancer (NSCLC) clinical samples and exact regulatory roles in cancer stem-like cells (CSCs) remain unknown.MethodsIn vitro and in vivo experiments were employed to investigate the molecular mechanism. Using CSCs, mouse models, and clinical tissues, we obtained a general picture of the relatively higher level of PRRX1A compared to PRRX1B, and PRRX1A thus promoting EMT and maintaining stemness of CSCs.ResultsPRRX1A but not PRRX1B was upregulated in lung cancer tissues and was positively correlated with TGF-β expression. In CSCs, overexpressed PRRX1A promoted malignant behaviors via transcriptional activation of TGF-β depending on TGF-β/TGF-βR signaling pathway. PRRX1A knockdown decreased self-renewal capacity accompanied by a decrease in stemness factor expression independent of the TGF-β/TGF-βR signaling pathway. Furthermore, PRRX1A was found to tightly bind to and stabilize SOX2. PRRX1A promoted sphere formation not only by enhancing stemness via stabilizing SOX2 but also by promoting cell proliferation.ConclusionsPRRX1A, but not PRRX1B, was demonstrated to have important roles in the regulation of the stemness and metastatic potential of lung cancer, which suggests the potential application of PRRX1A in cancer treatment.

Increased CD44 Expression and MEK Activity Predict Worse Prognosis in Gastric Adenocarcinoma Patients Undergoing Gastrectomy

PurposeWe have shown that activation of the receptor tyrosine kinase (RTK)-RAS pathway in gastric adenocarcinoma (GA) promotes acquisition of cancer stem-like cell (CSC) phenotypes including metastasis and chemotherapy resistance. Here, we evaluated the prognostic value of the CSC marker CD44 and the RTK-RAS activation marker phosphorylated MEK (p-MEK) in patients with resectable GA. MethodsCD44 and p-MEK were measured in tumors from GA patients who underwent curative-intent gastrectomy at Fujian Medical University Union Hospital (FMUUH, n = 134) and Memorial Sloan Kettering Cancer Center (MSKCC, n = 56). Overall survival (OS) was estimated by the Kaplan-Meier method, and multivariate analysis was performed by Cox proportional hazards regression modeling. ResultsDespite multiple significant differences in clinicopathologic characteristics between the FMUUH and MSKCC cohorts, high CD44 and high p-MEK expression were both independent negative prognostic factors for OS on univariate analysis in both cohorts (p < 0.05). Both factors were also significant on multivariate analysis when the cohorts were combined (p ≤ 0.003). On subgroup analysis, the 5-year OS of patients with both high CD44 and high p-MEK was 39.5–41.6% compared with 55.4–66.4% for patients with low CD44. High CD44 expression was associated with more advanced TNM stage in the FMUUH cohort and larger tumor size and undifferentiated histology in the MSKCC cohort. High p-MEK was associated with undifferentiated histology in the FMUUH cohort and larger tumor size in the MSKCC cohort. ConclusionsIncreased CD44 and p-MEK expression are predictive of worse OS in GA patients. Thus, targeting the RTK-RAS pathway may benefit patients with CD44-positive, RAS-activated GA by inhibiting metastasis and reversing chemotherapy resistance.

A cancer stem cell-like phenotype is associated with miR-10b expression in aggressive squamous cell carcinomas

BackgroundCutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa (RDEB), which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined.MethodsMiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression or knock-out of a dysregulated miRNA was assessed in migration and 3D-spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets.ResultsSeveral miRNAs were identified as dysregulated in cSCCs compared to control skin. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2.ConclusionThe discovery that miR-10b mediates an aspect of cancer stemness – that of enhanced tumor cell adhesion, known to facilitate metastatic colonization – provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.

RETRACTED ARTICLE: ERK1/2-Nanog signaling pathway enhances CD44(+) cancer stem-like cell phenotypes and epithelial-to-mesenchymal transition in head and neck squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCCs) harbor a subset of cells that are CD44(+) and present with malignancy and radiotherapy resistance. As a key regulator of self-renewal, Nanog expression not only determines cell fate in pluripotent cells but also mediates tumorigenesis in cancer cells; thus, we examined the role of Nanog in CD44(+) HNSCC. Three HNSCC cell lines, tumor xenografts, and patient tumors were examined. Nanog levels were significantly higher in CD44(+) HNSCC spheroids than in CD44(−) spheroids, and further increased when grown as spheroids to enrich for CSCs. CD44(+) spheroids showed a 3.4–7.5-fold increase in migration and invasion compared with CD44(−) spheroids and were resistant to radiation therapy, which was reversed by inhibiting Nanog. Nanog knockdown also decreased spheroid formation by 66.5–68.8%. Moreover, a phosphokinase array identified upregulated ERK1/2 signaling in CD44(+) HNSCC cells compared with that in CD44(−) cells. ERK1/2 signaling was found to regulate Nanog expression, aiding tumor progression, metastasis, and radiotherapy resistance. In xenograft models, the combination of radiation and Nanog or ERK1/2 inhibition inhibited tumor growth by 75.6% and 79.1%, respectively. In lung metastasis models, CD44(+) cells injected into the tail vein of mice led to significantly more lung metastases and higher Nanog expression level compared with that by ERK1/2-knockdown CD44(+) cells. Finally, in tumor tissues, CD44 and Nanog expression levels were correlated with tumorigenesis in HNSCC patients. Thus, targeting Nanog and the ERK1/2 signaling pathway may prevent or reverse CSC phenotypes and epithelial–mesenchymal transition that drive tumor progression, metastasis, and radiotherapy resistance in HNSCC.

EpCAM-independent isolation of circulating tumor cells with epithelial-to-mesenchymal transition and cancer stem cell phenotypes using ApoStream® in patients with breast cancer treated with primary systemic therapy.

BackgroundTumor cells with a mesenchymal phenotype and/or cancer stem-like cells (CSCs) are known to contribute to metastasis and drug resistance. Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) and CTCs reflecting a dedifferentiated CSC phenotype may not be detected using only an anti-EpCAM antibody to capture them. We used an antibody-independent CTC enrichment platform, ApoStream®, which does not rely on any antibody, including anti-EpCAM, to capture EMT- and CSC-CTCs in breast cancer patients who received neoadjuvant chemotherapy and correlated them to pathological complete response (pCR).MethodsBlood samples from newly diagnosed breast cancer patients were prospectively collected before neoadjuvant chemotherapy (T0), after chemotherapy but before surgery (T1), and after surgery (T2) and processed using ApoStream. CTCs detected were stained with additional markers to define 3 CTC subsets with the following phenotypes: epithelial CTCs (CK+, EpCAM+ or E-cadherin+), EMT-CTCs (β-catenin+ or vimentin+), and CSC-CTCs (CD44+ and CD24low).ResultsWe enrolled 55 patients, 47 of which had data for analysis. EMT-CTCs were detected in 57%, 62%, and 72% and CSC-CTCs in 9%, 22%, and 19% at the T0, T1, and T2 time points, respectively. Counts of epithelial (P = 0.225) and EMT (P = 0.522) phenotypes of CTCs at T0 did not significantly predict pCR. Moreover, no correlation between CTC count change and pCR was demonstrated.ConclusionsApoStream was successful in detecting EMT-CTCs among patients after neoadjuvant chemotherapy. However, EMT-/CSC-CTC counts did not correlate with pCR. Due to the small sample size and heterogeneity of this patient population, further study in a larger cohort of molecularly homogeneous patients is warranted.

KLF8 promotes cancer stem cell-like phenotypes in osteosarcoma through miR-429-SOX2 signaling.

Krüppel-like factor 8 (KLF8) regulates critical gene transcription associated with different types of cancer. A novel paradigm in tumor biology suggests that the initiation and progression of osteosarcoma (OS) are driven by osteosarcoma stem cell-like cells (OSCs), but the role and underlying mechanisms of KLF8 in OSCs are poorly elucidated. In this study, an obviously increased level of KLF8 is shown in 9 out of 10 primary OS tissues and is associated with the poor progression-free interval. Significantly, KLF8 expression in CD133+ OSCs is higher than that in CD133- counterparts. By knocking down KLF8 in CD133+ OSCs, we show that si-KLF8-OSCs can hardly form compact spheres. In the meantime, infection with si-KLF8 in CD133+ OSCs results in the downregulation of OCT4 and SOX2; increased adriamycin (ADM) sensitivity; and decreased tumorigenic potential in vivo. Mechanisms study demonstrates that KLF8 directly binds the miR-429 promoter region and regulates its expression transcriptionally. Furthermore, we indicate that miR-429 directly targets SOX2 to mediate cancer stem cell-like features in CD133+ OSCs. In the clinic, miR-429 levels are negatively associated with KLF8 levels in OS, suggesting that an elevated KLF8/miR-429 ratio may have clinical value as a predictive biomarker. In conclusion, targeting the KLF8-miR-429-SOX2 signaling pathway may provide an effective therapeutic approach to suppress the initiation and progression of OS.

GD2-targeted chimeric antigen receptor T cells prevent metastasis formation by elimination of breast cancer stem-like cells

ABSTRACT Expression of the disialoganglioside GD2 has been identified as a marker antigen associated with a breast cancer stem-like cell (BCSC) phenotype. Here, we report on the evaluation of GD2 as a BCSC-specific target antigen for immunotherapy. GD2 expression was confirmed at variable degree in a set of breast cancer cell lines, predominantly in triple-negative breast cancer (TNBC). To target GD2, we have generated novel anti-GD2 chimeric antigen receptors (GD2-CAR), based on single-chain variable fragments (scFv) derived from the monoclonal antibody (mAb) ch14.18, also known as dinutuximab beta. Expressed on T cells, GD2-CARs mediated specific GD2-dependent T-cell activation and target cell lysis. In contrast to previously described GD2-CARs, no signs of exhaustion by tonic signaling were found. Importantly, application of GD2-CAR expressing T cells (GD2-CAR-T) in an orthotopic xenograft model of TNBC (MDA-MB-231) halted local tumor progression and completely prevented lung metastasis formation. In line with the BCSC model, GD2 expression was only found in a subpopulation (4-6%) of MDA-MB-231 cells before injection. Significant expansion of GD2-CAR-T in tumor-bearing mice as well as T-cell infiltrates in the primary tumor and the lungs were found, indicating site-specific activation of GD2-CAR-T. Our data strongly support previous findings of GD2 as a BCSC-associated antigen. GD2-targeted immunotherapies have been extensively studied in human. In conclusion, GD2-CAR-T should be considered a promising novel approach for GD2-positive breast cancer, especially to eliminate disseminated tumor cells and prevent metastasis formation.

1943P - Monospecific antibody targeting of CDH11 inhibits epithelial-to-mesenchymal transition and represses cancer stem cell-like phenotype by up-regulating miR-335 in metastatic breast cancer, in vitro and in vivo

BackgroundMetastasis is a leading cause of breast cancer mortality. The induction of epithelial-to-mesenchymal transition (EMT) and complex oncogenic signaling is a vital step in the evolution of highly metastatic and therapeutically-intractable breast cancer; necessitating novel target discovery or development of therapeutics that target metastatic breast cells (MBCs). MethodsTo achieve this, this study employs a combination of in silico bioinformatics analyses, protein and transcript analyses, drug sensitivity assays, functional assays and animal studies. ResultsCDH11 as an inductor and/or facilitator of metastatic signaling, and biomarker of poor prognosis in MBCs. Furthermore, we showed that in the presence of CDH11-rich cancer-associated fibroblasts (CAFs), MCF7 and MDA-MB-231 MBC cell lines acquired enhanced metastatic phenotype with increased CDH11, β-catenin, vimentin, and fibronectin (FN) expression. exposure to anti-CDH11 antibody suppresses metastasis, reduces CDH11, FN and β-catenin expression, and abrogate the cancer stem cell (CSC)-like traits of MBC cells. Interestingly, ectopic expression of miR-335 suppressed CDH11, β-catenin and vimentin expression, in concert with attenuated metastatic and CSC potentials of the MBC cells; conversely, inhibition of miR-335 resulted in increased metastatic potential. Finally, corroborating the in silica and in vitro findings, in vivo assays showed that the administration of anti-CDH11 antibody or miR-335 mimic suppressed tumorigenesis and inhibited cancer metastasis. ConclusionsThese findings indicate that miR-335 mediates anti-CDH11 antibody therapy response and that an enhanced miR-335/CDH11 ratio elicits marked suppression of the MBC CSC-like and metastatic phenotypes, thus revealing a therapeutically-exploitable inverse correlation between CDH11-enhanced CSC-like and metastatic phenotype and miR-335 expression in MBCs. Thus, we highlight the therapeutic promise of humanized anti-CDH11 antibodies or miR-335-mimic, making a case for their clinical application as efficacious therapeutic option in patients with MBC. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Chronic Exposure to Chewing Tobacco Induces Metabolic Reprogramming and Cancer Stem Cell-Like Properties in Esophageal Epithelial Cells.

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.

Combined treatment with cisplatin and the tankyrase inhibitor XAV-939 increases cytotoxicity, abrogates cancer-stem-like cell phenotype and increases chemosensitivity of head-and-neck squamous-cell carcinoma cells

Cancer stem-like cells (CSCs) were reported to be linked with tumorigenesis, metastasis and resistant to chemo and radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study we investigated the role of CSCs in chemoresistance and abrogation of CSC mediated chemoresistance by combinatorial treatment with cisplatin and small molecule tankyrase inhibitor XAV-939. Two cisplatin-resistant HNSCC cells were generated by stepwise dose incremental strategy. We evaluated the chemoresistance, sphere forming capacity, extent of DNA damage and repair capacity in parental and cisplatin-resistant HNSCC cells. Furthermore, the abrogation of CSC mediated chemoresistance was evaluated in HNSCC cells with XAV-939 alone and in combination with cisplatin. It was observed that cisplatin-resistant HNSCC cell lines exhibited increase in chemoresistance, CSC phenotype and increased DNA repair capacity. We observed that combination of cisplatin and XAV-939 acts synergistically to abrogate chemoresistance by increasing DNA damage. Molecular docking study also revealed similar binding region that could contribute towards synergy predictions between cisplatin and XAV939. In conclusion, this study elucidated that combination of cisplatin and XAV-939 exerted cytotoxic and genotoxic effect to abrogate CSC mediated chemoresistance in HNSCC in synergistic manner.

Chemotherapy-Induced Extracellular Vesicle miRNAs Promote Breast Cancer Stemness by Targeting ONECUT2.

Cancer-secreted, extracellular vesicle (EV)-encapsulated miRNAs enable cancer cells to communicate with each other and with noncancerous cells in tumor pathogenesis and response to therapies. Here, we show that treatment with a sublethal dose of chemotherapeutic agents induces breast cancer cells to secrete EV with the capacity to stimulate a cancer stem-like cell (CSC) phenotype, rendering cancer cells resistance to therapy. Chemotherapy induced breast cancer cells to secrete multiple EV miRNAs, including miR-9-5p, miR-195-5p, and miR-203a-3p, which simultaneously targeted the transcription factor One Cut Homeobox 2 (ONECUT2), leading to induction of CSC traits and expression of stemness-associated genes, including NOTCH1, SOX9, NANOG, OCT4, and SOX2. Inhibition of these miRNAs or restoration of ONECUT2 expression abolished the CSC-stimulating effect of EV from chemotherapy-treated cancer cells. In mice bearing xenograft mammary tumors, docetaxel treatment caused elevations of miR-9-5p, miR-195-5p, and miR-203a-3p in circulating EV and decreased ONECUT2 expression and increased levels of stemness-associated genes. These effects following chemotherapy were diminished in tumors deficient in exosome secretion. In human breast tumors, neoadjuvant chemotherapy decreased ONECUT2 expression in tumor cells. Our results indicate a mechanism by which cancer cells communicate with each other and self-adapt to survive in response to cytotoxic treatment. Targeting these adaptation mechanisms along with chemotherapy, such as by blocking the EV miRNA-ONECUT2 axis, represents a potential strategy to maximize the anticancer effect of chemotherapy and to reduce chemoresistance in cancer management. SIGNIFICANCE: These findings reveal a critical mechanism of resistance to chemotherapy by which breast cancer cells secrete miRNA-containing extracellular vesicles to stimulate cancer stem cell-like features.

Abstract 4680: KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis

Abstract BACKGROUND: Our previous work showed that in a mouse model of gastric adenocarcinoma (GA) with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a triple conditional or Tcon mice) accelerates tumorigenesis and metastasis. The Receptor Tyrosine Kinase (RTK)-RAS signaling pathway is altered in 60% of GAs, and KRAS is amplified or mutated in 17% of GAs. There is some evidence that RTK-RAS signaling and oncogenic KRAS are important in the epithelial-to-mesenchymal transition (EMT) and maintenance of cancer stem-like cells (CSCs). We hypothesized that RTK-RAS activation in GA CSCs increases the metastatic potential of these cells. METHODS: The RTK-RAS pathway and KRAS were examined in nontransformed HFE-145 gastric epithelial cells, tumor-derived organoids derived from Tcon mice, human GA cell lines (AGS and KATOIII), AGS xenografts, and a tissue microarray of human GAs from 115 patients undergoing surgical resection. KRAS was inhibited using shRNA, and the RTK-RAS pathway was blocked using a MEK inhibitor PD0325901. RESULTS: Kras activity was much higher in GA cell lines grown as spheroids compared to GA cell lines grown monolayers. Metastasis following KRAS activation in GA cells results from stimulation of epithelial-to-mesenchymal transition (EMT) to cancer stem-like cells (CSCs). In organoids derived from our mouse model, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown in patient tumor-derived GA cell lines. KRAS inhibition in GA spheroid cells led to reduced flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. Supporting clinical relevance, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival in 115 GA patients. CONCLUSION: In this study, we show that the addition of oncogenic KRAS into gastric epithelial cells leads to EMT and acquisition of CSC phenotypes. Inhibition of KRAS in GA cell lines and in tumor derived organoids reverses EMT and inhibit CSC phenotypes. We conclude that metastasis following KRAS activation in GA cells likely results from stimulation of EMT and transition to CSCs. Citation Format: Changhwan Yoon, Jacob Till, Soo-Jeong Cho, Kevin Chang, Jian-Xian Lin, Sandra Ryeom, Sam Yoon. KRAS activation in gastric adenocarcinoma stimulates epithelial-to-mesenchymal transition to cancer stem-like cells and promotes metastasis [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 4680.

Abstract 4666: p21CIP1 promotes mammary cancer initiating cells via activation of Wnt/TCF1/CyclinD1 signaling

Abstract Cancer stem cells (CSCs) generate and sustain tumors due to tumor initiating potential, resulting in recurrence or metastasis. We showed previously that knockout of the cell cycle inhibitor, p21CIP1, in the PyMT mammary tumor model, inhibits metastasis; however the exact mechanism remained unknown. Here, we show a pivotal role for p21 in potentiating a cancer stem cell-like phenotype. p21 knockout in PyMT mammary tumor cells suppressed tumorsphere formation, ALDH1 expression, and tumor initiating potential, that were in turn rescued by p21 overexpression. Consistent with these effects, p21 loss caused mesenchymal to epithelial transition that was accompanied by downregulation of Slug, Sox9, basal keratins and upregulation of luminal keratins and Claudin-3. Furthermore, p21 knockout suppressed Wnt/β-catenin signaling which was due to TCF1 downregulation. In turn, TCF1 knockdown in PyMT cells, attenuated Wnt signaling and tumorsphere formation. Interestingly, p21 rescue in p21 knockout cells, caused dramatic upregulation of Cyclin D1, consistent with known inhibition of Cyclin D1 nuclear export by p21. These data suggest that p21 promotes a cancer stem like phenotype via Wnt-TCF1 signaling along with direct upregulation of Cyclin D1 by p21. Citation Format: Rachel B. Hazan. p21CIP1 promotes mammary cancer initiating cells via activation of Wnt/TCF1/CyclinD1 signaling [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 4666.

Highlights of This Issue

Mechanistic studies of patient-derived tumor tissues are often limited by sample size and tumor cell content. Methods for culturing primary cells ex vivo over a feeder layer are suitable for certain applications, but lack utility in highly sensitive assays like single cell sequencing due to potential contamination by cells from the feeder layer. Here, Prasad and colleagues detail a novel method of propagating dissociated primary cells in tissue culture using defined growth medium with inhibitors of ROCK, TGFβ, and BMP signaling. This method supports the expansion of a variety of cell types, including stromal, luminal, basal, and stem cell subpopulations that are compatible with cell sorting applications and downstream -omics analysis. This method maximizes the utility of primary tissue samples, and broadens the horizon for ex vivo analyses.Mouse models of hepatocellular carcinoma (HCC) often fail to capture the intricacies of human disease, which may influence the clinical applicability of data derived from these models. Here, Friemel, Frick, and colleagues demonstrate the feasibility of using genomic, morphologic, and immunohistochemical analyses to stratify genetically engineered mouse models along eitiologic criteria, matching them with phenotypes observed in human HCC. The authors posit that this data will allow for better alignment of mouse models with HCC subtypes, thereby increasing their clinical relevance.Hypoxia-inducible factor (HIF)-1α is a key regulator of the cellular response to hypoxia. HIF-1α plays a major role in hypoxic tumor metabolism and owns the potential to be targeted for therapy. However, the mechanism by which cancer cells can adapt to HIF-1α deletion is poorly understood. Integrated metabolomic, proteomic, and transcriptomic analysis by Valli and colleagues has now revealed that HIF-1α-knockout colon cancer cells upregulate creatine metabolism and glucose transporter GLUT14 in response to hypoxic stress. Their data elucidate that colon cancer cell hypoxic metabolism can bypass HIF-1α deletion, provide new insight into the mechanisms underlying hypoxic stress responses, and highlight potential avenues for targeted therapy in colorectal cancer.The cell cycle inhibitor p21CIP1 has well-known roles in regulating cellular proliferation, but previous work has shown that it also exerts pro-metastatic effects in mammary tumor models via an unknown mechanism. Here, Benard and colleagues have defined the role of p21CIP1 in promoting cancer stem cell-like phenotypes, demonstrating that its expression is associated with increased Wnt/β-catenin pathway activity through TCF1 and Cyclin D1. Knockout of p21 was concordantly associated with reduction of cancer stem cell properties, inhibition of tumorsphere formation, and suppression of tumor-initiating potential in vivo. The data suggest that, despite its classical role in suppressing cell cycle progression, p21 is also a strong promoter of cancer stem cell properties in mammary cells.