Expression Of Cancer Stem Cell Markers Research Articles

Abstract B035: Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer

Abstract Cancer stem cells (CSCs) are a subpopulation of cancer cells that are responsible for disease recurrence and chemoresistance. The mechanisms by which CSCs arise in endometrial cancer are not fully understood and elucidating key molecular events may provide insight into therapeutic targets. CD73, a cell surface 5’nucleotidase that generates adenosine, is frequently downregulated in endometrial tumorigenesis, and CD73 loss is associated with aggressive disease and poor patient outcomes. In developing and characterizing CRISPR/Cas-9 models for CD73, we discovered CD73 deletion (CD73−/−) in HEC-1-A cells causes growth arrest and prolonged survival which are features suggestive of a stemness phenotype. Thus, we hypothesized that loss of CD73 is critical for promoting CSCs in endometrial cancer. To evaluate CSC characteristics, we performed MTT and spheroid assays and interrogated CSC marker protein expression (e.g., CD44 and NOTCH3). CD73−/− HEC-1-A cells formed larger spheroids and expressed high levels of CD44 and NOTCH3 compared to wild-type (CD73+/+) cells. To assess whether loss of CD73 enzymatic activity is responsible for the enrichment of CSCs, CD73+/+ HEC-1-B and HEC-50 cells were treated with AoPCP, a catalytic inhibitor of CD73. Similar to genetic deletion of CD73 in HEC-1-A cells, AoPCP treatment resulted in HEC-1-B and HEC-50 cells forming larger spheroids and expressing high CD44 and NOTCH3. These data indicate that loss of CD73 enzyme activity is important to the enrichment of CSCs. In contrast, HEYA8 ovarian cancer cells showed the opposite effects, wherein treatment with AoPCP decreased spheroid size. These differences support the emerging paradigm that CD73 has tissue- and cancer-specific roles. As CD73 is a major node in purine metabolic signaling, we further hypothesized that loss of CD73 activity may be promoting CSC enrichment via changes to metabolic pathways. Mass spectrometry metabolomics revealed significantly different profiles of CD73+/+ and CD73−/− HEC-1-A cells. Conditioned media experiments showed CD73−/−-conditioned media induces growth arrest in HEC-1-A wild-type cells, whereas the growth arrest and CSC marker expression enrichment of CD73−/− cells was abrogated by CD73+/+-conditioned media or daily media replenishment. Metabolomic profiling of CD73+/+ and CD73−/− HEC-1-A cells with/without daily media replenishment revealed metabolic pathways, such as NAD+ biosynthesis, lipid metabolism, and sugar alcohol synthesis, are up-regulated by CD73 deficiency and parallels the enrichment of CSCs. Current studies are dissecting these metabolic dependencies in supporting endometrial cancer cell stemness. Altogether, these studies provide novel insight into how CSCs arise in endometrial cancer and why CD73 loss is detrimental in this disease. Citation Format: Emily M. Rabjohns, Blake R. Rushing, Sayali Joseph, Cyrus Vaziri, Jessica L. Bowser. Evidence for CD73 loss promoting cancer cell stemness via metabolic reprogramming in endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B035.

Effect of Pioglitazone and Cetuximab on Colon Cancer Stem-like Cell (CCSLCs) Properties.

One of the main reasons for cancer resistance to chemotherapy is the presence of cancer stem cells (CSCs) in cancer tissues. It is also believed that CSCs are the unique originators of all tumor cells. On the other hand, the Epithelial-Mesenchymal Transition pathway (EMT) can act as the main agent of metastasis. Therefore, it is possible that targeting CSCs as well as the EMT pathway could help in cancer therapy. Considering that CSCs constitute only a small percentage of the total tumor mass, enrichment before study is necessary. In our previous study, CSCs were enriched in the human colon cancer cell line HT29 by induction of EMT. These CSC-enriched HT29 cells with mesenchymal morphology were named "HT29-shE". In the present study, these cells were used to investigate the effect of pioglitazone (Pio) and Cetuximab (Cet) in order to find CSC and EMT targeting agents. The viability and IC50 rate of cells treated with different concentrations of Pio and Cet were evaluated using the MTT test. EMT and CSC markers and cell morphology were assessed in Pio and Cet treated and untreated HT29-shE cells using flow cytometry, realtime PCR, immunocytochemistry, and microscopic monitoring. The findings showed that Pio and Cet at concentrations of 250 μM and 40 μg/ml, respectively, decrease cell viability by 50%. Also, they were able to reduce the expression of CSC markers (CD133 and CD44) in the CSC enriched HT29 cell line. Furthermore, Pio and Cet could efficiently reduce the expression of vimentin as a mesenchymal marker and significantly upregulate the expression of E-cadherin as an epidermal marker of EMT and its reverse mesenchymal-- to-epithelial transition (MET). In addition, the mesenchymal morphology of HT29-shE changed into epithelial morphology after Cet treatment. Pio and Cet could inhibit EMT and reduce CSC markers in the EMT induced/CSC enriched cell line. We expect that focus on finding EMT/CSC-targeting agents like these drugs can be helpful for cancer treatment.

Fbxw7 suppresses carcinogenesis and stemness in triple-negative breast cancer through CHD4 degradation and Wnt/β-catenin pathway inhibition

BackgroundCancer stem cells (CSCs) are a small population of cells in tumor tissues that can drive tumor initiation and promote tumor progression. A small number of previous studies indirectly mentioned the role of F-box and WD repeat domain-containing 7 (FBXW7) as a tumor suppressor in Triple-negative breast cancer (TNBC). However, few studies have focused on the function of FBXW7 in cancer stemness in TNBC and the related mechanism.MethodsWe detected FBXW7 by immunohistochemistry (IHC) in 80 TNBC patients. FBXW7 knockdown and overexpression in MD-MBA-231 and HCC1937 cell models were constructed. The effect of FBXW7 on malignant phenotype and stemness was assessed by colony assays, flow cytometry, transwell assays, western blot, and sphere formation assays. Immunoprecipitation-Mass Spectrometry (IP-MS) and ubiquitination experiments were used to find and verify potential downstream substrate proteins of FBXW7. Animal experiments were constructed to examine the effect of FBXW7 on tumorigenic potential and cancer stemness of TNBC cells in vivo.ResultsThe results showed that FBXW7 was expressed at low levels in TNBC tissues and positively correlated with prognosis of TNBC patients. In vitro, FBXW7 significantly inhibited colony formation, cell cycle progression, cell migration, EMT process, cancer stemness and promotes apoptosis. Further experiments confirmed that chromodomain-helicase-DNA-binding protein 4 (CHD4) is a novel downstream target of FBXW7 and is downregulated by FBXW7 via proteasomal degradation. Moreover, CHD4 could promote the nuclear translocation of β-catenin and reverse the inhibitory effect of FBXW7 on β-catenin, and ultimately activate the Wnt/β-catenin pathway. Rescue experiments confirmed that the FBXW7-CHD4-Wnt/β-catenin axis was involved in regulating the maintenance of CSC in TNBC cells. In animal experiments, FBXW7 reduced CSC marker expression and suppressed TNBC cell tumorigenesis in vivo.ConclusionsTaken together, these results highlight that FBXW7 degrades CHD4 protein through ubiquitination, thereby blocking the activation of the Wnt/β-catenin pathway to inhibit the stemness of TNBC cells. Thus, targeting FBXW7 may be a promising strategy for therapeutic intervention against TNBC.

Inhibition of MEK Signaling Attenuates Cancer Stem Cell Activity in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Evaluation of BMP-2 as a differentiating and radiosensitizing agent for colorectal cancer stem cells.

Despite effective clinical responses, a large proportion of patients undergo resistance to radiotherapy. The low response rate to current treatments in different stages of colorectal cancer depends on the prominent role of stem cells in cancer. In the present study, the role of BMP-2 as an ionizing radiation-sensitive factor in colorectal cancer cells was investigated. A sphere formation assay was used for the enrichment of HCT-116 cancer stem cells (CSCs). The effects of combination therapy (BMP-2+ radiation) on DNA damage response (DDR), proliferation, and apoptosis were evaluated in HCT-116 and CSCs. Gene expressions of CSCs and epithelial-mesenchymal transition (EMT) markers were also evaluated. We found that the sphere formation assay showed a significant increase in the percentage of CSCs. Moreover, expression of CSCs markers, EMT-related genes, and DNA repair proteins significantly decreased in HCT-116 cells compared to the CSCs group after radiation. In addition, BMP-2 promoted the radiosensitivity of HCT-116 cells by decreasing the survival rate of the treated cells at 2, 4, and 6 Gy compared to the control group in HCT-116 cells. Our findings indicated that BMP-2 could affect numerous signaling pathways involved in radioresistance. Therefore, BMP-2 ‎ can be considered an appealing therapeutic target for the treatment of radioresistant human colorectal cancer.

Significant Association of PD‐L1 With CD44 Expression and Patient Survival: Avenues for Immunotherapy and Cancer Stem Cells Downregulation in Pancreatic Cancers

Background: Pancreatic cancers are known for their aggressive nature. This aggressiveness may be attributed to the presence of cancer stem cells (CSCs), which promote relapse, metastasis, and resistance to chemotherapy. Targeting CSCs is essential to reverse this aggressiveness in pancreatic malignancies. Literature highlights the association of PD‐L1 expression with CSCs in various cancers, suggesting immunotherapy as a promising therapeutic approach. This study is aimed at investigating the potential of immunotherapy in pancreatic cancers by examining its association with selected CSC marker expression.Method: A retrospective cohort study was conducted involving 56 patients with confirmed diagnoses of pancreatic cancers at Aga Khan University Hospital from January 2015 to October 2022. After exclusions, based on refusal to provide consent or incomplete follow‐up data, 38 patients were enrolled in the study. Immunohistochemistry was performed on formalin‐fixed paraffin‐embedded (FFPE) tumor tissue samples to assess the expression of CSC markers (CD133, CD44, and L1CAM) and immune checkpoint inhibitor marker (PD‐L1). Statistical analysis was employed to determine associations between marker expression, clinical factors, and overall survival.Results: The study revealed that 86.8% of pancreatic cancer cases exhibited positive PD‐L1 expression. Moreover, a significant association of PD‐L1 expression was observed with the presence of CD44 protein (p = 0.030), as well as with the overall survival of patients (p = 0.023).Conclusion: Our findings show a significant association of PD‐L1 with CD44 marker expression as well as patient survival. This research shows the potential to serve as the foundation for investigating the efficacy of immunotherapy in reducing CD44‐expressing CSCs in pancreatic cancer, potentially enhancing patient outcomes.

ZNF217-activated Notch signaling mediates sulforaphane-suppressed stem cell properties in colorectal cancer

Cancer stem cells (CSCs) are known to contribute to the progression of colorectal cancer (CRC). However, understanding of the molecular mechanisms and key factors involved in CRC is still insufficient to identify therapeutic targets against colorectal CSCs. In an effort to identify such mechanisms, we conducted bioinformatics analyses to evaluate the expression patterns in tumor and normal colorectal tissues, leading us to focus on the role of the ZNF217/Notch1 axis in mediating stem cell properties in CRC. Our findings revealed that ZNF217 overexpression activated self-renewal ability, expression of colorectal CSC markers, and Notch signaling in CRC. Dual-luciferase reporter assay suggested a role for ZNF217 in targeting Notch1 to activate Notch signaling. We observed that the promotional effects of Notch signaling, as well as CSC markers, under ZNF217 overexpression were attenuated after Notch1 knockdown. In addition to in vitro data, our in vivo results confirmed the inhibitory effect of sulforaphane on the tumorigenicity of CSCs, depicted the suppressive role of sulforaphane on colorectal CSCs mediated by the ZNF217/Notch1 axis, thereby providing new targetable vulnerabilities and therapeutic strategies for CRC.

Self-Renewal Inhibition in Breast Cancer Stem Cells: Moonlight Role of PEDF in Breast Cancer.

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells' self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells' in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers.

Effect of Matrix Stiffness and Hepatocyte Growth Factor on Small Cell Lung Cancer Cells in Decellularized Extracellular Matrix-Based Hydrogels.

Small cell lung cancer (SCLC) is one of lethal cancers resulting in very low 5-year-survival rate. Although its clinical treatment largely relies on chemotherapy, SCLC cell physiology in three-dimenstional (3D) matrix has been less explored. In this work, the tumor microenvironment is reconstructed with decellularized porcine pulmonary extracellular matrix (dECM) with hyaluronic acid. To modulate matrix stiffness, the methacrylate groups are introduced into both dECM and hyaluronic acid, followed by photocrosslinking with photoinitiator. The stiffness of the resulting dECM-based hydrogel covers the stiffness of normal or cancerous tissue with varying dECM content. The proliferation and cancer stem cell marker expression of encapsulated SCLC cells are promoted in a compliant hydrogel matrix, which has a low shear modulus similar to that of the normal tissue. The hepatocyte growth factor (HGF) that induces SCLC cell invasion and chemoresistance markedly increases invasiveness and gene expression levels of CD44 and Sox2 in the hydrogel matrix. In addition, HGF treatment causes higher resistance against anticancer drugs (cisplatin and paclitaxel) in the 3D microenvironment. These findings indicate that malignant SCLC can be recapitulated in a pulmonary dECM-based matrix.

Kindlin-2 regulates colonic cancer stem-like cells survival and self-renewal via Wnt/β-catenin mediated pathway

BackgroundCancer Stem Cells (CSCs) have emerged as a critical mediator in recurrence and resistance in cancers. Kindlin-isoform (1 and 2) binds with cytoplasmic β-tail of integrin and are essential co-activators of integrin function. Given their important function in regulating cancer hallmarks such as cell proliferation, invasion, migration, and metastasis, we hypothesize that it might play a critical role in CSC growth, survival, and self-renewal of colon cancer. Materials and methodsUsing knockdown approaches, we inhibited Kindlin-2 expression in HCT116 and HT29 colon cancer cells. Extreme limiting dilution and self-renewal assay were performed to measure the role of Kindlin in colonic CSC. Standard methods such as qRT-PCR and western blotting were carried out to understand the signaling cascade by which Kindlin regulates CSC marker expression and downstream targets. ResultsOur data show isoform-specific upregulation of Kindlin-2 in colonic CSCs. The silencing of Kindlin-2 reduces colonosphere formation, decreases CSC size, and self-renewal marker genes such as CD-133, CXCR-4, LGR-5, and C-MYC. Kindlin-2 silencing reduces colonosphere proliferation, invasion, and migration of colonic CSCs. Mechanistically, Kindlin-2 silencing reduces the expression, and nuclear localization of β-catenin, and decreases β-catenin target genes such as C-MYC, cyclin D1, DKK-1, and Snail-1. ConclusionOur study delineates the isoform-specific activity of Kindlin-2 in regulating Colonic CSC. Isoform-specific targeting of Kindlin-2 may be a novel strategy to tackle this devastating disease.

Fabrication of a Three-Dimensional Spheroid Culture System for Oral Squamous Cell Carcinomas Using a Microfabricated Device.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24-48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

Combining microfluidic chip and low-attachment culture devices to isolate oral cancer stem cells

Background/purposeCancer stem cells (CSCs) are widely recognized as key drivers of cancer initiation, progression, and therapeutic resistance. Microfluidic chip technology offers a promising approach for CSC isolation and study. This study investigated the efficacy of a microfluidic chip-based method for isolating single cells from oral cancer cell lines characterized by high stem-like phenotypes. Specifically, the study focused on examining the sphere-forming capability and the expression of CSC markers, including aldehyde dehydrogenase 1A1 (ALDH1A1), CD44, and CD133, in isolated cell clones from OECM-1 and SAS cell lines. Materials and methodsOral cancer cell lines were subjected to isolation using a microfluidic chip. The captured single cells were cultured to assess their sphere-forming capacity in ultra-low binding culture. Furthermore, the protein expression levels of ALDH1A1, CD44, and CD133 in the isolated cell clones were analyzed using western blotting. ResultsThe microfluidic chip-assisted isolation method significantly enhanced the sphere-forming capability of both OECM-1 and SAS cell clones compared to their parent cell lines. Moreover, the expression levels of CSC markers ALDH1A1, CD44, and CD133 were upregulated in the microfluidic chip-assisted isolated cell clones, indicating a higher stem-like phenotype. ConclusionThis study demonstrates the effectiveness of the microfluidic chip-based approach in isolating oral cancer cell clones with elevated stem-like characteristics. This method offers a valuable tool for further investigation of CSCs and their role in cancer progression, as well as future therapy development for oral cancers.

Androgen receptor promotes cell stemness via interacting with co-factor YAP1 in gastric cancer

Cancer stem cells (CSCs) have been proposed to explain tumor relapse and chemoresistance in various types of cancers, and androgen receptor (AR) has been emerged as a potential regulator of stemness in cancers. However, the underlying mechanism of AR-regulated CSCs properties and chemoresistance in gastric cancer (GC) remains unknown. Here, we shown that AR is upregulated in GC tissues and correlates with poor survival rate and CSCs phenotypes of GC patients. According to our experimental data, overexpression of AR upregulated the expression of CSCs markers and this was consistent with the result concluded from data analysis that the expression of AR was positively correlated with CD44 in GC patients. In addition, AR overexpression obviously enhanced the tumor sphere formation ability and chemoresistance of GC cells in vitro. Whereas these effects were attenuated by inhibition of AR. These results were further validated in vivo that MGC-803 cells overexpressing AR had stronger properties to initiate gastric tumorigenesis than the control cells, and inhibition of AR increased the chemosensitivity of GC cells. Mechanically, AR upregulated CD44 expression by directly binding to its promoter region and Yes-associated protein 1 (YAP1) served as the co-factor of AR, which was demonstrated by the fact that the promoting effects of AR on GC cells stemness were partially counteracted by YAP1 knockdown. Thus, this study revealed that AR facilitates CSCs properties and chemoresistance of GC cells via forming complex with YAP1and indicates a potential therapeutic approach to GC patients.

Hepatoma Derived Growth Factor (HDGF) Targeting as a Therapeutic Approach for Radio-Naive and Radioresistant Hepatocellular Carcinoma

The role of radiotherapy in treating hepatocellular carcinoma (HCC) has gained importance. Hepatoma-derived growth factor (HDGF) is a nuclear growth factor involved in the development of HCC and its overexpression has been linked to tumor progression and radiation treatment failure. The aim of this study is to investigate the potential therapeutic benefits of HDGF-targeting treatment in combination with or without radiation for both radio-naive and radioresistant HCC cells. A series of radiation doses (2Gy, 4Gy, 8Gy, 12Gy, and 20Gy in one fraction) were administered to Huh7 human HCC cells. The gene expression profiles of radiation-treated Huh7 cells were analyzed. The radioresistant Huh7 cell line (Huh7-IR) was established by repeatedly irradiating with 2-week intervals with 8 Gy of X-ray to a total dose of 80 Gy. After acquiring radioresistance, the expression levels of HDGF, cancer stem cell (CSC) markers, and epithelial-mesenchymal transition (EMT) factors were validated. The pShuttle-shHDGF plasmid with adenovirus vector was generated for HDGF targeting treatment (HDGF knockdown). The effect of the HDGF targeting treatment on tumorigenic processes and cell cycle distribution in the radio-naive and radioresistant cells with or without irradiation were explored. After radiation treatment, qPCR analysis showed that the mRNA expression of HDGF, CSC markers (CD133, CD44, and ABCG2), and EMT markers (TWIST and SNAIL) increased over time, with the most significant upregulation observed between 48-72 hours. In the radioresistant Huh7-IR cells, HDGF, CSC markers (CD133 and CD44), and the EMT transcriptional factor (TWIST) were significantly higher than in the parental Huh7 cells. The Huh7-IR cells also showed a significant arrest in the G0/G1 phase. X-ray radiation alone significantly reduced cell proliferation and invasion in the Huh7 cells compared to the control group. The HDGF targeting treatment significantly enhanced the X-ray-mediated inhibition of colony formation and cell invasion in both the Huh7 and Huh7-IR cells. Targeting HDGF leads to cell cycle arrest (G2/M phase) in Huh7 and Huh7-IR cells. The HDGF targeting treatment in combination with X-ray irradiation results in extensive G2/M phase arrest. These results indicate the crucial role of HDGF in radiation therapy for radio-naive and radioresistant HCC cells. Targeting HDGF effectively suppressed colony formation and invasion, and enhanced cell cycle arrest in the G2/M phase in combination with radiation. HDGF may serve as a promising therapeutic target for improving HCC management strategies. Further investigation is required to fully comprehend the role of HDGF in improving radiation therapy efficacy for HCC.

Modulation of Viability, Proliferation, and Stemness by Rosmarinic Acid in Medulloblastoma Cells: Involvement of HDACs and EGFR.

Medulloblastoma (MB) is a heterogeneous group of malignant pediatric brain tumors, divided into molecular groups with distinct biological features and prognoses. Currently available therapy often results in poor long-term quality of life for patients, which will be afflicted by neurological, neuropsychiatric, and emotional sequelae. Identifying novel therapeutic agents capable of targeting the tumors without jeopardizing patients' quality of life is imperative. Rosmarinic acid (RA) is a plant-derived compound whose action against a series of diseases including cancer has been investigated, with no side effects reported so far. Previous studies have not examined whether RA has effects in MB. Here, we show RA is cytotoxic against human Daoy (IC50 = 168μM) and D283 (IC50 = 334μM) MB cells. Exposure to RA for 48h reduced histone deacetylase 1 (HDAC1) expression while increasing H3K9 hyperacetylation, reduced epidermal growth factor (EGFR) expression, and inhibited EGFR downstream targets extracellular-regulated kinase (ERK)1/2 and AKT in Daoy cells. These modifications were accompanied by increased expression of CDKN1A/p21, reduced expression of SOX2, and a decrease in proliferative rate. Treatment with RA also reduced cancer stem cell markers expression and neurosphere size. Taken together, our findings indicate that RA can reduce cell proliferation and stemness and induce cell cycle arrest in MB cells. Mechanisms mediating these effects may include targeting HDAC1, EGFR, and ERK signaling, and promoting p21 expression, possibly through an increase in H3K9ac and AKT deactivation. RA should be further investigated as a potential anticancer agent in experimental MB.

Expression of the Embryonic Cancer Stem Cells' Biomarkers SOX2 and OCT3/4 in Oral Leukoplakias and Squamous Cell Carcinomas: A Preliminary Study.

Cancer stem cells (CSCs) are incriminated for initiating the process of carcinogenesis either de novo or through the transformation of oral potentially malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of this study was to detect the expression of embryonic-type CSC markers OCT3/4 and SOX2 in OSCCs and oral leukoplakias (OLs), the most common of OPMDs. The study type is experimental, and the study design is characterized as semiquantitative research, which belongs to the branch of experimental research. The experiment was conducted in the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece. This study focuses on the semiquantitative immunohistochemical (IHC) pattern of expression of CSCs protein-biomarkers SOX2 and OCT3/4, in paraffin embedded samples of 21 OSCCs of different grades of differentiation and 30 cases of OLs with different grades of dysplasia, compared to five cases of normal oral mucosa in both terms of cells' stain positivity and intensity. Statistical analysis wasperformed through SPSS 2017 Pearson Chi-square and the significance level was set at 0.05 (p=0.05). The expression of the respective genes of SOX2 and OCT3/4 was studied through quantitative polymerase chain reaction (qPCR), in paraffin-embedded samples of 12 cases of OLs with mild/non dysplasia and 19 cases moderately/poorly differentiated OSCCs(n=19) and five normal mucosa using the Independent Paired T-test. The genes SOX2 and Oct3/4 were expressed in all examined cases although no statistically significant correlations among normal, OL and OSCC, were established. A nuclear/membrane staining of OCT3/4 was noticed only in three out of 21 OSCCs but in none of OLs or normal cases (without statistical significance). A characteristic nuclear staining of SOX2 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. SOX2 was significantly detected in the OSCCs group (strong positivity in 17/21) than in the OL group (30 cases, mostly mildly stained) (p-value=0.007), and the normal oral epithelium (mild stained, p=0.065). Furthermore, SOX2 was overexpressed in well differentiated OSCCs group (5/OSCCs, strongly stained) rather than in mildly dysplastic and non-dysplastic OLs samples (14/OLs, mildly stained) (p-value =0.035). The characteristic expression of SOX2 but not of OCT3/4 in OLs' and OSCCs' lesions suggests the presence of neoplastic cells with certain CSC characteristics whose implication in the early stages of oral tumorigenesiscould be further evaluated. The clinical use of SOX2, as prognostic factor, requires further experimental evaluation in larger number of samples.

Putative Cancer Stem Cell Markers are Frequently Expressed by Melanoma Cells in Vitro and in Situ but are also Present in Benign Differentiated Cells.

Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Cancer Stem Cell Markers-Clinical Relevance and Prognostic Value in High-Grade Serous Ovarian Cancer (HGSOC) Based on The Cancer Genome Atlas Analysis.

Cancer stem cells (CSCs) may contribute to an increased risk of recurrence in ovarian cancer (OC). Further research is needed to identify associations between CSC markers and OC patients' clinical outcomes with greater certainty. If they prove to be correct, in the future, the CSC markers can be used to help predict survival and indicate new therapeutic targets. This study aimed to determine the CSC markers at mRNA and protein levels and their association with clinical presentation, outcome, and risk of recurrence in HGSOC (High-Grade Serous Ovarian Cancer). TCGA (The Cancer Genome Atlas) database with 558 ovarian cancer tumor samples was used for the evaluation of 13 CSC markers (ALDH1A1, CD44, EPCAM, KIT, LGR5, NES, NOTCH3, POU5F1, PROM1, PTTG1, ROR1, SOX9, and THY1). Data on mRNA and protein levels assessed by microarray and mass spectrometry were retrieved from TCGA. Models to predict chemotherapy response and survival were built using multiple variables, including epidemiological data, expression levels, and machine learning methodology. ALDH1A1 and LGR5 mRNA expressions indicated a higher platinum sensitivity (p = 3.50 × 10-3; p = 0.01, respectively). POU5F1 mRNA expression marked platinum-resistant tumors (p = 9.43 × 10-3). CD44 and EPCAM mRNA expression correlated with longer overall survival (OS) (p = 0.043; p = 0.039, respectively). THY1 mRNA and protein levels were associated with worse OS (p = 0.019; p = 0.015, respectively). Disease-free survival (DFS) was positively affected by EPCAM (p = 0.004), LGR5 (p = 0.018), and CD44 (p = 0.012). In the multivariate model based on CSC marker expression, the high-risk group had 9.1 months longer median overall survival than the low-risk group (p < 0.001). ALDH1A1, CD44, EPCAM, LGR5, POU5F1, and THY1 levels in OC may be used as prognostic factors for the primary outcome and help predict the treatment response.

Inducing Cytotoxicity in Colon Cancer Cells and Suppressing Cancer Stem Cells by Dolasetron and Ketoprofen through Inhibition of RNA Binding Protein PUM1.

Clinical trials of new drugs often face a high failure rate of approximately 45 percent due to safety and toxicity concerns. Repurposing drugs with well-established safety profiles becomes crucial in addressing this challenge. Colon cancer ranks as the third most prevalent cancer and the second leading cause of cancer related mortality worldwide. This study focuses on the RNA-binding protein pumilio1 (PUM1), a member of the PUF family involved in post-transcriptional gene expression regulation. By utilizing molecular docking techniques and FDA-approved drugs, potential inhibitors against PUM1 were identified. Notably, dolasetron and ketoprofen demonstrated promising results, exhibiting strong binding affinity, hydrophobic interactions, and favorable chemical reactivity according to Conceptual-DFT calculations. Both compounds effectively reduced cell viability, with IC50 values of 150 µM and 175 µM, respectively and shows long term inhibitory effects as seen by reduced in number of colonies. Moreover, they exhibited inhibitory effects on colon cancer stem cells, as indicated by reduced colonospheroid size and numbers. Apoptosis is induced by these compounds and has triggered activation of executioner caspase 3/7 in HCT116 cells which is evident through a caspase 3/7 assay and AO/EB staining, while the non-toxic effect of these compounds was evident from viability against non-cancerous cell line and hemolysis assay. Additionally, the treatment group showed a significant decrease in PUM1 and cancer stem cell markers expression compared to the control group. In conclusion, this study highlights the potential of targeting PUM1 as a novel approach to colon cancer treatment. Dolasetron and ketoprofen demonstrate promise as effective anti-cancer and anti-cancer stem cell drugs, inducing apoptosis in colon cancer cells through inhibition of PUM1.

Activin and Hepatocyte Growth Factor Promotes Colorectal Cancer Stemness and Metastasis through FOXM1/SOX2/CXCR4 Signaling.

Cancer stem cells (CSCs) are believed to drive tumor development and metastasis. Activin and hepatocyte growth factor (HGF) are important cytokines with the ability to induce cancer stemness. However, the effect of activin and HGF combination treatment on CSCs is still unclear. In this study, we sequentially treated colorectal cancer cells with activin and HGF and examined CSC marker expression, self-renewal, tumorigenesis, and metastasis. The roles of forkhead box M1 (FOXM1) and sex-determining region Y-box 2 (SOX2), two stemness-related transcription factors, in activin/HGF-induced aggressive phenotype were explored. Activin and HGF treatment increased the expression of CSC markers and enhanced sphere formation in colorectal cancer cells. The tumorigenic and metastatic capacities of colorectal cancer cells were enhanced upon activin and HGF treatment. Activin and HGF treatment preferentially promoted stemness and metastasis of CD133+ subpopulations sorted from colorectal cancer cells. FOXM1 was upregulated by activin and HGF treatment, and the knockdown of FOXM1 blocked activin/HGF-induced stemness, tumorigenesis, and metastasis of colorectal cancer cells. Similarly, SOX2 was silencing impaired sphere formation of activin/HGF-treated colorectal cancers. Overexpression of SOX2 rescued the stem cell-like phenotype in FOXM1-depleted colorectal cancer cells with activin and HGF treatment. Additionally, the inhibition of FOXM1 via thiostrepton suppressed activin/HGF-induced stemness, tumorigenesis and metastasis. Sequential treatment with activin and HGF promotes colorectal cancer stemness and metastasis through activation of the FOXM1/SOX2 signaling. FOXM1 could be a potential target for the treatment of colorectal cancer metastasis.