Cancer Stem Cell-like Properties Research Articles

Biological and clinical significance of the AGE-RAGE axis in the aggressiveness and prognosis of prostate cancer.

Dietary factors and chronic hyperglycemia are linked to the formation of advanced glycation end products (AGEs) and prostate cancer (PCa) risk. The activation of the receptor for AGEs (RAGE) acts as a bridge between various RAGE ligands and certain malignancies. This study showed that the interaction of AGEs and RAGE promoted PCa cell proliferation, invasion, and autophagy-mediated survival in response to chemotherapeutic agents. RAGE-overexpressed PCa cells underwent epithelial-mesenchymal transition and showed increased cancer stem cell-like properties. In mouse xenograft models, RAGE-overexpressed cells showed more substantial tumorigenic capacity than parental cells, whereas RAGE knockdown decreased tumorigenicity. The clinical data validated a positive correlation between high AGE and RAGE expressions with poor clinical outcomes. Our findings suggest that the AGE-RAGE axis facilitates PCa progression and aggressiveness. Prostatic AGEs and RAGE expression levels are associated with PCa prognosis. Adherence to a reduced-AGE diet and targeting RAGE are potential approaches to complement and synergize with the current PCa therapies.

SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma

Tumor lineage plasticity, considered a hallmark of cancer, denotes the phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. However, the underlying molecular events remain largely elusive. Our recent study identified CD133/Prom1 in hepatocellular carcinoma (HCC) tumors to mark proliferative tumor-propagating cells with cancer stem cell-like properties, that follow a dedifferentiation trajectory towards a more embryonic state. Here we show SPINK1 to strongly associate with CD133 + HCC, and tumor dedifferentiation. Enhanced transcriptional activity of SPINK1 is mediated by promoter binding of ELF3, which like CD133, is found to increase following 5-FU and cisplatin treatment; while targeted depletion of CD133 will reduce both ELF3 and SPINK1. Functionally, SPINK1 overexpression promotes tumor initiation, self-renewal, and chemoresistance by driving a deregulated EGFR-ERK-CDK4/6-E2F2 signaling axis to induce dedifferentiation of HCC cells into their ancestral lineages. Depleting SPINK1 function by neutralizing antibody treatment or in vivo lentivirus-mediated Spink1 knockdown dampens HCC cancer growth and their ability to resist chemotherapy. Targeting oncofetal SPINK1 may represent a promising therapeutic option for HCC treatment.

Up-regulation of RNA m6A methyltransferase like-3 expression contributes to arsenic and benzo[a]pyrene co-exposure-induced cancer stem cell-like property and tumorigenesis

While arsenic or BaP alone exposure can cause lung cancer, studies showed that arsenic plus BaP co-exposure displays a significantly stronger lung tumorigenic effect. However, the underlying mechanism has not been well understood. Studies showed that RNA molecules are chemically modified. The most frequently occurring RNA modification in eukaryotic messenger RNAs is the N6-methyladenosine (m6A) methylation. This study aimed to determine whether arsenic plus BaP exposure alters RNA m6A methylation and its role in lung tumorigenic effect of arsenic plus BaP exposure. Human bronchial epithelial cells transformed by exposure to arsenic or BaP alone, and arsenic plus BaP and mouse xenograft tumorigenesis models were used in this study. It was found that arsenic plus BaP exposure-transformed cells have significantly higher levels of RNA m6A methylation than arsenic or BaP alone exposure-transformed human bronchial epithelial cells. Western blot analysis showed that arsenic plus BaP exposure greatly up-regulates the m6A writer methyltransferase like-3 (METTL3) expression levels in cultured cells and mouse lung tissues. METTL3 knockdown in cells transformed by arsenic plus BaP exposure drastically reduced their RNA m6A methylation levels. Functional studies revealed that METTL3 knockdown in cells transformed by arsenic plus BaP exposure greatly reduces their anchorage-dependent and -independent growth, cancer stem cell characters and tumorigenesis. The findings from this study suggest that arsenic plus BaP co-exposure causes epitranscriptomic dysregulation, which may contribute significantly to arsenic plus BaP co-exposure-caused synergistic lung tumorigenic effect.

Downregulation of TAB182 promotes cancer stem-like cell properties and therapeutic resistance in triple-negative breast cancer cells

TAB182 participates in DNA damage repair and radio-/chemosensitivity regulation in various tumors, but its role in tumorigenesis and therapeutic resistance in breast cancer remains unclear. In the current paper, we observed that triple-negative Breast Cancer (TNBC), a highly aggressive type of breast cancer, exhibits a lower expression of TAB182. TAB182 knockdown stimulates the proliferation, migration, and invasion of TNBC cells. Our study first obtained RNA-seq data to explore the cellular functions mediated by TAB182 at the genome level in TNBC cells. A transcriptome analysis and in vitro experiments enabled us to identify that TAB182 downregulation drives the enhanced properties of cancer stem-like cells (CSCs) in TNBC cells. Furthermore, TAB182 deletion contributes to the resistance of cells to olaparib or cisplatin, which can be rescued by silencing GLI2, a gene downstream of cancer stemness-related signaling pathways. Our results reveal a novel function of TAB182 as a potential negative regulator of cancer stem-like properties and drug sensitivity in TNBC cells, suggesting that TAB182 may be a tumor suppressor gene and is associated with increased therapeutic benefits for TNBC patients.

Transcriptome and Lipidomic Analysis Suggests Lipid Metabolism Reprogramming and Upregulating SPHK1 Promotes Stemness in Pancreatic Ductal Adenocarcinoma Stem-like Cells.

Cancer stem cells (CSCs) are considered to play a key role in the development and progression of pancreatic ductal adenocarcinoma (PDAC). However, little is known about lipid metabolism reprogramming in PDAC CSCs. Here, we assigned stemness indices, which were used to describe and quantify CSCs, to every patient from the Cancer Genome Atlas (TCGA-PAAD) database and observed differences in lipid metabolism between patients with high and low stemness indices. Then, tumor-repopulating cells (TRCs) cultured in soft 3D (three-dimensional) fibrin gels were demonstrated to be an available PDAC cancer stem-like cell (CSLCs) model. Comprehensive transcriptome and lipidomic analysis results suggested that fatty acid metabolism, glycerophospholipid metabolism, and, especially, the sphingolipid metabolism pathway were mostly associated with CSLCs properties. SPHK1 (sphingosine kinases 1), one of the genes involved in sphingolipid metabolism and encoding the key enzyme to catalyze sphingosine to generate S1P (sphingosine-1-phosphate), was identified to be the key gene in promoting the stemness of PDAC. In summary, we explored the characteristics of lipid metabolism both in patients with high stemness indices and in novel CSLCs models, and unraveled a molecular mechanism via which sphingolipid metabolism maintained tumor stemness. These findings may contribute to the development of a strategy for targeting lipid metabolism to inhibit CSCs in PDAC treatment.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/β-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/β-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.

Isolation of tumor stem-like cells from primary laryngeal squamous cell carcinoma cells (FD-LS-6).

The development of efficient treatments for laryngeal squamous cell carcinoma (LSCC) is hindered by the lack of applicable tumor cell lines and animal models of the disease, especially those related to cancer stem-like cells (CSCs). CSCs play critical roles in tumor propagation and pathogenesis whereas no CSCs lines have been developed to date. In this study, we establish an LSCC cell line (FD-LS-6) from primary LSCC tumor tissue (not experienced single-cell cloning) and adapted a culturing condition for the expansion of potential stem cells (EPSCs) to isolate CSCs from FD-LS-6. We successfully derived novel CSCs and named them as LSCC sphere-forming cells (LSCSCs) which were subsequently characterized for their CSC properties. We showed that LSCSCs shared many properties of CSCs, including CSC marker, robust self-renewal capacity, tumorigenesis ability, potential to generate other cell types such as adipocytes and osteoblasts, and resistance to chemotherapy. Compared to parental cells, LSCSCs were significantly more potent in forming tumors in vivo in mice and more resistant to chemotherapy. LSCSCs have higher expressions of epithelial-mesenchymal transition proteins and chemotherapy resistance factors, and exhibit an activated COX2/PEG2 signaling pathway. Altogether, our work establishes the first CSCs of LSCC (FD-LS-6) and provides a tool to study tumorigenesis and metastasis of LSCC and help the development of anticancer therapies.

Notum enhances gastric cancer stem-like cell properties through upregulation of Sox2 by PI3K/AKT signaling pathway

PurposeConsiderable evidence suggests that tumor cells with stemness features contribute to initiation, progression, recurrence of gastric cancer (GC) and resistance to therapy, but involvement of underlying regulators and mechanisms remain largely unclear. However, the clinical significance and biological function of Notum in GC tumor sphere formation and tumorigenesis remain unclear.MethodsBioinformatics analysis, RT-qPCR, western blot and imunohistochemistry staining were applied to characterize Notum expression in GC specimens. The early diagnostic value of Notum was analyzed by logistic regression analysis method. Cancer stemness assays were used in Notum knockdown and overexpressing cells in vitro and in vivo. RNA-seq was employed to reveal the downstream effectors of Notum.ResultsNotum is highly expressed in early stage of GC patients and stem-like GC cells. For discriminating the early-stage and advanced GC patients, the joint analysis had a better diagnostic value. Overexpression of Notum markedly increased stemness features of GC cells to promote tumor sphere formation and tumorigenesis. Conversely, Notum knockdown attenuated the stem-like cell properties in vitro and in vivo. Mechanically, Notum upregulates Sox2 through activating the PI3K/AKT signaling pathway. Notum inhibitor Caffeine exhibited a potent inhibitory effect on stemness features by impairing the PI3K/AKT signaling pathway activity and targeting Sox2.ConclusionOur findings confer a comprehensive and mechanistic function of Notum in GC tumor sphere formation and tumorigenesis that may provide a novel and promising target for early diagnosis and clinical therapy of GC.

SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity

CSCs (Cancer stem cells) with distinct metabolic features are considered to cause HCC (hepatocellular carcinoma) initiation, metastasis and therapeutic resistance. Here, we perform a metabolic gene CRISPR/Cas9 knockout library screen in tumorspheres derived from HCC cells and find that deletion of SCARB2 suppresses the cancer stem cell-like properties of HCC cells. Knockout of Scarb2 in hepatocytes attenuates HCC initiation and progression in both MYC-driven and DEN (diethylnitrosamine)-induced HCC mouse models. Mechanistically, binding of SCARB2 with MYC promotes MYC acetylation by interfering with HDCA3-mediated MYC deacetylation on lysine 148 and subsequently enhances MYC transcriptional activity. Screening of a database of FDA (Food and Drug Administration)-approved drugs shows Polymyxin B displays high binding affinity for SCARB2 protein, disrupts the SCARB2-MYC interaction, decreases MYC activity, and reduces the tumor burden. Our study identifies SCARB2 as a functional driver of HCC and suggests Polymyxin B-based treatment as a targeted therapeutic option for HCC.

Ubiquitin-specific peptidase 5 facilitates cancer stem cell-like properties in lung cancer by deubiquitinating β-catenin

BackgroundLung cancer has the highest mortality rate in the world, and mounting evidence suggests that cancer stem cells (CSCs) are associated with poor prognosis, recurrence, and metastasis of lung cancer. It is urgent to identify new biomarkers and therapeutic targets for targeting lung CSCs.MethodsWe computed the single-sample gene set enrichment analysis (ssGSEA) of 1554 Reactome gene sets to identify the mRNA expression-based stemness index (mRNAsi)-associated pathways using the genome-wide RNA sequencing data of 509 patients from The Cancer Genome Atlas (TCGA) cohort of lung adenocarcinoma (LUAD). Phenotypic effects of ubiquitin-specific peptidase 5 (USP5) on the CSC-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Cycloheximide chase assay, co-immunoprecipitation assay, and deubiquitination assay were performed to confirm the effect of USP5 on the deubiquitination of β-catenin.ResultsWe demonstrated that USP5 expression were positively correlated with the stemness-associated signatures and poor outcomes in lung cancer specimens. Silencing of endogenous USP5 reduced CSC-like characteristics, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo. Furthermore, USP5 interacted with β-catenin, which resulted in deubiquitination, stabilization of β-catenin, and activation of Wnt/β-catenin pathway. Accordingly, expression of USP5 was positively correlated with the enrichment score of the Wnt/TCF pathway signature in human lung cancer. Silencing of β-catenin expression suppressed USP5-enhancing sphere formation. Targeting USP5 with the small molecule WP1130 promoted the degradation of β-catenin, and showed great inhibitory effects on sphere formation, migration, and invasion. Finally, we identified a poor-prognosis subset of tumors characterized by high levels of USP5, Wnt signaling score, and Stemness score in both TCGA-LUAD and Rousseaux_2013 datasets.ConclusionsThese findings reveal a clinical evidence for USP5-enhanced Wnt/β-catenin signaling in promoting lung cancer stemness and metastasis, implying that targeting USP5 could provide beneficial effects to improve lung cancer therapeutics.

Orai3 Calcium Channel Contributes to Oral/Oropharyngeal Cancer Stemness through the Elevation of ID1 Expression.

Emerging evidence indicates that intracellular calcium (Ca2+) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca2+ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation.

SPDEF enhances cancer stem cell-like properties and tumorigenesis through directly promoting GALNT7 transcription in luminal breast cancer

BackgroundLuminal breast cancer (BC) is the predominant subtype of breast cancer with a sustained risk of late recurrence and death. Understanding the molecular mechanisms for the oncogenesis of luminal BC would improve the prognosis for this large subset of patients. SPDEF was reported to be dysregulated in breast cancers. However, the biological functions and underlying molecular mechanism of SPDEF in luminal BC remains largely unknown. The aim of the present study was to elucidate the potential roles of SPDEF underlying subtype-specific functions in BC, especially in luminal subtypes.MethodsThe expressions and clinicopathological characteristics of SPDEF in luminal BC patients were evaluated bioinformatically. In vitro and in vivo assays were performed to investigate the oncogenic function and stemness maintenance of SPDEF in luminal BC. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were conducted to determine the transcription regulation of GALNT7 by SPDEF. GALNT7 levels in serum from luminal BC patients were further detected by enzyme-linked immunosorbent assay (ELISA).ResultsSPDEF is markedly upregulated in luminal BC and positively associated with tumor progression and poor prognosis. Furthermore, we confirmed that SPDEF enhanced the proliferation, migration, invasion and stemness of luminal BC cells in vitro as well the tumorigenicity in vivo. Mechanistically, we demonstrated the stimulative effect of SPDEF on the progression and stemness of luminal BC, which is mediated by its directly transcriptional target GALNT7. Clinically, we verified that the GALNT7 can be used as a noninvasive diagnostic marker. Noteworthy, the combined detection of serum GALNT7 and traditional tumor markers can enhance diagnostic accuracy thus is of vital importance in the early diagnosis of luminal BC.ConclusionsOur study reveals a novel mechanism by which SPDEF transcriptionally activates GALNT7 via directly binding to its promoter to promote cell proliferation, motility and stemness, and led to luminal BC tumorigenesis and poor prognosis.

CDK7-YAP-LDHD axis promotes D-lactate elimination and ferroptosis defense to support cancer stem cell-like properties

Reprogrammed cellular metabolism is essential for maintaining cancer stem cells (CSCs) state. Here, we report that mitochondrial D-lactate catabolism is a necessary initiating oncogenic event during tumorigenesis of esophageal squamous cell carcinoma (ESCC). We discover that cyclin-dependent kinase 7 (CDK7) phosphorylates nuclear Yes-associated protein 1 (YAP) at S127 and S397 sites and enhances its transcription function, which promotes D-lactate dehydrogenase (LDHD) protein expression. Moreover, LDHD is enriched significantly in ESCC-CSCs rather than differentiated tumor cells and high LDHD status is connected with poor prognosis in ESCC patients. Mechanistically, the CDK7-YAP-LDHD axis helps ESCC-CSCs escape from ferroptosis induced by D-lactate and generates pyruvate to satisfy energetic demands for their elevated self-renewal potential. Hence, we conclude that esophageal CSCs adopt a D-lactate elimination and pyruvate accumulation mode dependent on CDK7-YAP-LDHD axis, which drives stemness-associated hallmarks of ESCC-CSCs. Reasonably, targeting metabolic checkpoints may serve as an effective strategy for ESCC therapy.

Tspan15-ADAM10 signalling enhances cancer stem cell-like properties and induces chemoresistance via Notch1 activation in ICC.

Notch1 activation promotes ICC progression and is associated with chemoresistance; however, therapies directly targeting Notch1 showed severe adverse effects. Notch1 activation is mediated by ADAM10, a molecular scissor that separates the target protein from its substrates in the cell membrane. Tspan15 regulates ADAM10 function, but the role of Tspan15 in ICC progression is unclear. Tspan15, ADAM10, and Notch1 expression and activation in fresh surgical specimens from 80 ICC patients and ICC cells were evaluated by immunohistochemistry, RT-PCR, western blotting, and flow cytometry. Tspan15 expression was increased in ICC compared with adjacent liver tissue, and high Tspan15 expression was an independent factor for poor prognosis. In ICC with high Tspan15 expression, vascular invasion, lymph node metastasis, and haematogenous recurrence were increased. Tspan15 was co-expressed with ADAM10 in ICC, and associated with the expression of stemness and EMT markers. In ICC cells, Tspan15 induced ADAM10 activation by mediating the translocation of activated m-ADAM10 from the cytoplasm to the surface of the cell membrane, which further activated Notch1 by separating the intracellular domain of Notch1 from its extracellular domain, leading to enhancement of CSC-like properties and EMT. This signalling was associated with enhanced chemoresistance against gemcitabine and cisplatin. Inhibition of Tspan15 or ADAM10 is a promising therapeutic strategy in ICC, as Tspan15 or ADAM10 knockdown or treatment with ADAM10 inhibitor reduced chemoresistance and invasiveness by suppressing Notch1-mediated CSC-like properties and EMT. Tspan15-ADAM10-Notch1 signalling is associated with aggressive tumour progression and poor prognosis in ICC.

Stem Cell Properties of Gastric Cancer Stem-Like Cells under Stress Conditions Are Regulated via the c-Fos/UCH-L3/β-Catenin Axis.

Gastric cancer stem-like cells (GCSCs) possess stem cell properties, such as self-renewal and tumorigenicity, which are known to induce high chemoresistance and metastasis. These characteristics of GCSCs are further enhanced by autophagy, worsening the prognosis of patients. Currently, the mechanisms involved in the induction of stemness in GCSCs during autophagy remain unclear. In this study, we compared the cellular responses of GCSCs with those of gastric cancer intestinal cells (GCICs) whose stemness is not induced by autophagy. In response to glucose starvation, the levels of β-catenin and stemness-related genes were upregulated in GCSCs, while the levels of β-catenin declined in GCICs. The pattern of deubiquitinase ubiquitin C-terminal hydrolase-L3 (UCH-L3) expression in GCSCs and GCICs was similar to that of β-catenin expression depending on glucose deprivation. We also observed that inhibition of UCH-L3 activity reduced β-catenin protein levels. The interaction between UCH-L3 and β-catenin proteins was confirmed, and it reduced the ubiquitination of β-catenin. Our results suggest that UCH-L3 induces the stabilization of β-catenin, which is required to promote stemness during autophagy activation. Also, UCH-L3 expression was regulated by c-Fos, and the levels of c-Fos increased in response to autophagy activation. In summary, our findings suggest that the inhibition of UCH-L3 during nutrient deprivation could suppress stress resistance of GCSCs and increase the survival rates of gastric cancer patients.

LIMP-2 enhances cancer stem-like cell properties by promoting autophagy-induced GSK3β degradation in head and neck squamous cell carcinoma

Cancer stem cell-like cells (CSCs) play an integral role in the heterogeneity, metastasis, and treatment resistance of head and neck squamous cell carcinoma (HNSCC) due to their high tumor initiation capacity and plasticity. Here, we identified a candidate gene named LIMP-2 as a novel therapeutic target regulating HNSCC progression and CSC properties. The high expression of LIMP-2 in HNSCC patients suggested a poor prognosis and potential immunotherapy resistance. Functionally, LIMP-2 can facilitate autolysosome formation to promote autophagic flux. LIMP-2 knockdown inhibits autophagic flux and reduces the tumorigenic ability of HNSCC. Further mechanistic studies suggest that enhanced autophagy helps HNSCC maintain stemness and promotes degradation of GSK3β, which in turn facilitates nuclear translocation of β-catenin and transcription of downstream target genes. In conclusion, this study reveals LIMP-2 as a novel prospective therapeutic target for HNSCC and provides evidence for a link between autophagy, CSC, and immunotherapy resistance.

EIF3a sustains non-small cell lung cancer stem cell-like properties by promoting YY1-mediated transcriptional activation of β-catenin

Cancer stem cells (CSCs) are the leading cause of recurrence and poor prognosis in non-small cell lung cancer (NSCLC). Eukaryotic translation initiation factor 3a (eIF3a) participates in many tumor development processes, such as metastasis, therapy resistance, and glycolysis, all of which are closely associated with the presence of CSCs. However, whether eIF3a maintains NSCLC-CSC-like properties remains to be elucidated. In this study, eIF3a was highly expressed in lung cancer tissues and was linked to poor prognosis. eIF3a was also highly expressed in CSC-enriched spheres compared with adherent monolayer cells. Moreover, eIF3a is required for NSCLC stem cell-like traits maintenance in vitro and in vivo. Mechanistically, eIF3a activates the Wnt/β-catenin signaling pathway, promoting the transcription of cancer stem cell markers. Specifically, eIF3a promotes the transcriptional activation of β-catenin and mediates its nuclear accumulation to form a complex with T cell factor 4 (TCF4). However, eIF3a has no significant effect on protein stability and translation. Proteomics analysis revealed that the candidate transcription factor, Yin Yang 1 (YY1), mediates the activated effect of eIF3a on β-catenin. Overall, the findings of this study implied that eIF3a contributes to the maintenance of NSCLC stem cell-like characteristics through the Wnt/β-catenin pathway. eIF3a is a potential target for the treatment and prognosis of NSCLC.

TMT-based quantitative proteomic analysis of spheroid cells of endometrial cancer possessing cancer stem cell properties

BackgroundCancer stem cells (CSCs) play an important role in endometrial cancer progression and it is potential to isolate CSCs from spheroid cells. Further understanding of spheroid cells at protein level would help find novel CSC markers.MethodsSpheroid cells from endometrial cancer cell lines, Ishikawa and HEC1A, exhibited increased colony forming, subsphere forming, chemo-drug resistance, migration, invasion ability and tumorigenicity, verifying their cancer stem-like cell properties. The up-regulated CD90, CD117, CD133 and W5C5 expression also indicated stemness of spheroid cells. TMT-based quantitative proteomic analysis was performed to explore the potential alterations between parent cells and cancer stem-like spheroid cells. HK2-siRNA was transfected to Ishikawa and HEC1A cells to explore the roles and molecular mechanism of HK2 in endometrial cancer.ResultsWe identified and quantified a total of 5735 proteins and 167 overlapped differentially expressed proteins of two cell types, 43 proteins were up-regulated and 124 were down-regulated in spheroid cells comparing with parent cells. KEGG pathway revealed a significant role of HIF-1 pathway in spheroid cells. qRT-PCR and western blot results of GPRC5A, PFKFB3 and HK2 of HIF-1 pathway confirmed their elevated expressions in spheroid cells which were consistent with proteomic results. HK2 promoted cancer stemness in endometrial cancer.ConclusionThese findings indicate that spheroid cells from endometrial cancer cell lines possess cancer stem-like cell properties and enrich CSCs. HIF-1 pathway is activated in endometrial cancer stem-like spheroid cells.

Kruppel-like factor 8 regulates triple negative breast cancer stem cell-like activity.

Breast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties. Triple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice. Here, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells. These results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically.

Abstract 5805: Ebastine targets cancer stem cell-like properties and metastasis in triple-negative breast cancer by binding focal adhesion kinase

Abstract Triple-negative breast cancer (TNBC) exhibits an aggressive behavior associated with poor prognosis due to the absence of established molecular targets. Focal adhesion kinase (FAK) is a major determinant and participates in the acquisition of migration and invasion, as well as the maintenance of breast cancer stem cell (BCSC)-like traits in TNBC. We sought to investigate the effect of ebastine, a second-generation antihistamine on apoptosis, FAK activation, BCSC subpopulations and metastasis in TNBC in vitro and in vivo. TCGA dataset analysis revealed that mRNA levels of FAK were highly expressed in TNBC compared to other breast cancer subtypes. We found that ebastine binds to the tyrosine kinase domain of FAK, which blocks phosphorylation at the Y397 and Y576/577 residues and subsequent inactivation of SRC. Ebastine-induced apoptosis was associated with attenuation of JAK2/STAT3 and MEK/ERK signaling in TNBC cells. Kinetic analysis revealed a concentration-dependent impairment of cell migration in the presence of ebastine in MDA-MB-231 and 4T1 cells in vitro. Ebastine targets BCSC-like cell populations as evidenced by a sharp decline in the expression of the BCSC markers ALDH1, CD44 and CD49f and suppression of mammosphere-forming capacity. Ebastine administration led to a significant reduction in the growth ebastine of BCSC-enriched 4T1 mammospheres orthotopically injected into the mammary glands of Balb/c mice. Ebastine administration significantly impeded angiogenesis and distant metastasis while reducing MMP-2/-9 levels in circulating blood in vivo. Importantly, biochemical analysis in mice serum showed that ebastine had no effect on liver and kidney function. Our findings suggest that EBA may be an effective therapeutic repositioning candidate for the simultaneous targeting of multiple survival signaling pathways for the treatment of molecularly heterogeneous TNBC. Further investigation of ebastine as an anti-metastatic agent for the treatment of TNBC is warranted. Citation Format: Juyeon Seo, Minsu Park, Dongmi Ko, Seongjae Kim, Soeun Park, Kee Dal Nam, Yong Koo Kang, So Ra Seuk, Jaeyoun Park, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. Ebastine targets cancer stem cell-like properties and metastasis in triple-negative breast cancer by binding focal adhesion kinase. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5805.