UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells.

BACKGROUND: The high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive. METHOD: Expression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS. RESULT: We determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model. CONCLUSION: These findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.

MGMT inhibition regulates radioresponse in GBM, GSC, and melanoma.

Radiotherapy is the standard treatment for glioblastoma (GBM), but the overall survival rate for radiotherapy treated GBM patients is poor. The use of adjuvant and concomitant temozolomide (TMZ) improves the outcome; however, the effectiveness of this treatment varies according to MGMT levels. Herein, we evaluated whether MGMT expression affected the radioresponse of human GBM, GBM stem-like cells (GSCs), and melanoma. Our results indicated a correlation between MGMT promoter methylation status and MGMT expression. MGMT-producing cell lines ACPK1, GBMJ1, A375, and MM415 displayed enhanced radiosensitivity when MGMT was silenced using siRNA or when inhibited by lomeguatrib, whereas the OSU61, NSC11, WM852, and WM266-4 cell lines, which do not normally produce MGMT, displayed reduced radiosensitivity when MGMT was overexpressed. Mechanistically lomeguatrib prolonged radiation-induced γH2AX retention in MGMT-producing cells without specific cell cycle changes, suggesting that lomeguatrib-induced radiosensitization in these cells is due to radiation-induced DNA double-stranded break (DSB) repair inhibition. The DNA-DSB repair inhibition resulted in cell death via mitotic catastrophe in MGMT-producing cells. Overall, our results demonstrate that MGMT expression regulates radioresponse in GBM, GSC, and melanoma, implying a role for MGMT as a target for radiosensitization.

Fractionated irradiation induces radioresistant oral carcinoma cells with enhanced malignant phenotypes.

OBJECTIVE: The fact that certain oral carcinoma patients experience radiotherapy failure implies that a more radioresistant and aggressive phenotype of surviving cancer cells potentially occurs during treatment. Our study aimed to establish radioresistant oral cancer cells through a fractionated irradiation protocol that mimics clinically relevant radiotherapy dosing strategies and to investigate all-round alterations in the malignant phenotype. METHODS: Radioresistant oral carcinoma cells were generated by exposing Cal27 and Detroit 562 cells to 60 Gy radiation in 10 dose-escalating fractions and verified by cell immunofluorescence. Specific markers related to the epithelial-mesenchymal transition (EMT) process and the cancer stem cell (CSC) phenotype were assessed by Western blotting. Cell invasion and migration were evaluated using Matrigel-coated transwell and wound healing assays, respectively. Nontargeted metabolomics was used to mechanistically delineate the potential metabolic patterns linked to EMT and CSCs; the CSC phenotype was also examined by sphere formation assays and cell immunofluorescence. RESULTS: Radioresistant oral carcinoma cell lines were successfully established and validated. These cells exhibited enhanced EMT and increase in both cell invasion and migration. These radioresistant cells further demonstrated a high metabolic profile, notably marked by lipid metabolism reprogramming and functional enrichment of ATP-binding cassette (ABC) transporters. Consistently, enhanced CSC phenotype in radioresistant cells was confirmed by elevated expression of stemness markers and increased sphere-forming capacity. CONCLUSION: Radioresistant oral carcinoma cells subjected to fractionated radiation exhibit an augmented malignant phenotype. The metabolic characteristics linked to enhanced EMT and CSC phenotypes provide potential targets for improving radiotherapy in oral carcinoma.

Impact of Combination Therapy with Chemical Drugs and Megavoltage X-ray Exposure on Breast Cancer Stem Cells' Viability and Proliferation of MCF-7 and MDA-MB-231 Cell Lines.

BACKGROUND: Breast Cancer (BC) is a serious malignancy among women. However, chemotherapy is an important tool for cancer treatments, but the long-term use of chemotherapy drugs may lead to drug resistance and tumor recurrence. Since Breast Cancer Stem Cells (BCSCs) can be the main factor to induce BC treatment resistance and recurrence, investigation of BCSCs signaling pathways can be an effective modality to enhance cancer treatment efficiency. OBJECTIVE: In this study, the effect of metformin, SB203580, and takinib alone or in combination with radiotherapy on MCF-7 and MDA-MB-231 breast cancer cell lines was evaluated. METHODS: MCF-7 and MDA-MB-231 breast cancer cell lines were treated with metformin, SB203580, and takinib for 24 or 48 hours, followed by X-ray exposure. The MTT assay and flow cytometry analysis were performed to assess cell growth inhibition and cellular death, CXCr4 expression, and BCSCs, respectively. RESULTS: The results showed the combination of takinib/SB203580 with radiotherapy to remarkably reduce the CXCR4 expression and BCSCs levels in the MCF-7 cell line. Also, the concurrent administration of takinib/metformin/radiotherapy significantly reduced BCSCs and CXCR4 metastatic markers in the MDA-MB- 231 cells. Since the MAPK signaling pathway has an important role in inducing drug resistance and cell proliferation, the use of SB203580 as an inhibitor of p38 MAPK can improve breast cancer treatment. Furthermore, metformin and ionizing radiation by suppression of the mTOR signaling pathway can control AMPK activation and cellular proliferation. CONCLUSION: Anti-cancer and cytotoxic effects of metformin can be effective in this strategy. In conclusion, the combination of conventional chemotherapeutic drugs, including SB203580, metformin, and takinib with X-ray exposure can be a new approach to diminish the drug resistance of breast cancer.

Effects of Combined Exposure to Carbon Ions and Protons on the Pool of MCF-7 Breast Cancer Stem Cells In Vitro.

We studied the effects of single and combined action of protons and carbon ions 12C6+ on the pool of MCF-7 human breast cancer stem cells. Single irradiation with a beam of protons or carbon ions had no significant effects on the relative number of cancer stem cells (CSC). The effects of combined irradiation in a total equieffective dose of 4 Gy depended on the sequence of exposure to ionizing radiations: the relative number of CSC did not change after irradiation with carbon ions and then with protons, but increased in the case of the reverse sequence. The most favorable result, i.e. a decrease in the CSC pool, was observed in the case of sequential irradiation with carbon ions and protons and their equal contribution to total equieffective dose. In this case, the absolute number of CSC decreased by on average 2.1 times in comparison with the control (p<0.05). The revealed regularities are of interest for the further development of new methods of radiation therapy.

Targeting aldehyde dehydrogenase enzymes in combination with chemotherapy and immunotherapy: An approach to tackle resistance in cancer cells.

Modern cancer chemotherapy originated in the 1940s, and since then, many chemotherapeutic agents have been developed. However, most of these agents show limited response in patients due to innate and acquired resistance to therapy, which leads to the development of multi-drug resistance to different treatment modalities, leading to cancer recurrence and, eventually, patient death. One of the crucial players in inducing chemotherapy resistance is the aldehyde dehydrogenase (ALDH) enzyme. ALDH is overexpressed in chemotherapy-resistant cancer cells, which detoxifies the generated toxic aldehydes from chemotherapy, preventing the formation of reactive oxygen species and, thus, inhibiting the induction of oxidative stress and the stimulation of DNA damage and cell death. This review discusses the mechanisms of chemotherapy resistance in cancer cells promoted by ALDH. In addition, we provide detailed insight into the role of ALDH in cancer stemness, metastasis, metabolism, and cell death. Several studies investigated targeting ALDH in combination with other treatments as a potential therapeutic regimen to overcome resistance. We also highlight novel approaches in ALDH inhibition, including the potential synergistic employment of ALDH inhibitors in combination with chemotherapy or immunotherapy against different cancers, including head and neck, colorectal, breast, lung, and liver.

The Glycoprotein M6a Is Associated with Invasiveness and Radioresistance of Glioblastoma Stem Cells.

Systematic recurrence of glioblastoma (GB) despite surgery and chemo-radiotherapy is due to GB stem cells (GBSC), which are particularly invasive and radioresistant. Therefore, there is a need to identify new factors that might be targeted to decrease GBSC invasive capabilities as well as radioresistance. Patient-derived GBSC were used in this study to demonstrate a higher expression of the glycoprotein M6a (GPM6A) in invasive GBSC compared to non-invasive cells. In 3D invasion assays performed on primary neurospheres of GBSC, we showed that blocking GPM6A expression by siRNA significantly reduced cell invasion. We also demonstrated a high correlation of GPM6A with the oncogenic protein tyrosine phosphatase, PTPRZ1, which regulates GPM6A expression and cell invasion. The results of our study also show that GPM6A and PTPRZ1 are crucial for GBSC sphere formation. Finally, we demonstrated that targeting GPM6A or PTPRZ1 in GBSC increases the radiosensitivity of GBSC. Our results suggest that blocking GPM6A or PTPRZ1 could represent an interesting approach in the treatment of glioblastoma since it would simultaneously target proliferation, invasion, and radioresistance.

Association of ALDH1 with Response to Radiotherapy and Its Impact on Survival in Patients with Advanced Stage of Head and Neck Squamous Cell Carcinoma (HNSCC).

BACKGROUND: The presence of cancer stem-like cells within tumor microenvironment distinctly governs response to chemo-radiotherapy. The ALDH1 (Aldehyde dehydrogenase 1) has emerged as a cancer stem cell (CSC) marker in various tumors. The aim of the study was to examine the expression of ALDH1 in HNSCC patients undergoing radiotherapy to evaluate its correlation with clinicopathological parameter, treatment response and survival. METHODS: Expression of ALDH1 was evaluated by immunohistochemistry in 90 histopathologically confirmed HNSCC patients and 90 matched controls. The association between ALDH1 expression, clinicopathological parameters and treatment response was determined. RESULTS: The immunohistochemistry results showed that ALDH1 was consistently expressed in all the HNSCC specimens although at different intensities. On the other hand, control specimens did not show similar expression of ALDH1. ALDH1 expression demonstrated statistically significant association with tumor size (p<0.001), lymph node status (p<0.001), stage (p<0.001), grade (p<0.001) and treatment response (p<0.001). Multivariate ordinal logistic regression analysis indicated alcohol and ALDH1 as an independent predictor of responsiveness to radiotherapy in HNSCC patients. Multivariate Cox regression analysis indicated that lymph node status (p=0.020), grade (p=0.006) and recurrence (p=0.002) were potential independent predictors of overall survival. CONCLUSION: From previous studies, ALDH1 has been contemplated not only as a promising prognostic and diagnostic marker but also as a likely drug target. Our study gives new understanding regarding the association between ALDH1, cancer prognosis and radioresistance. Our findings suggest that ALDH1, lymph node status, grade and alcohol could be the viable targets for HNSCC and it also provides new prospects for radiotherapy sensitivity in HNSCC.

Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability.

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (-) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

The role of tumor acidification in aggressiveness, cell dissemination and treatment resistance of oral squamous cell carcinoma.

AIMS: To investigate the role of tumor acidification in cell behavior, migration, and treatment resistance of oral squamous cell carcinoma (OSCC). MAIN METHODS: The SCC4 and SCC25 cell lines were exposed to acidified (pH 6.8) cell culture medium for 7 days. Alternatively, a long-term acidosis was induced for 21 days. In addition, to mimic dynamic pH fluctuation of the tumor microenvironment, cells were reconditioned to neutral pH after experimental acidosis. This study assessed cell proliferation and viability by sulforhodamine B and flow cytometry. Individual and collective cell migration was analyzed by wound healing, time lapse, and transwell assays. Modifications of cell phenotype, EMT induction and stemness potential were investigated by qRT-PCR, western blot, and immunofluorescence. Finally, resistance to chemo- and radiotherapy of OSCC when exposed to acidified environmental conditions (pH 6.8) was determined. KEY FINDINGS: The exposure to an acidic microenvironment caused an initial reduction of OSCC cells viability, followed by an adaptation process. Acidic adapted cells acquired a mesenchymal-like phenotype along with increased migration and motility indexes. Moreover, tumoral extracellular acidity was capable to induce cellular stemness and to increase chemo- and radioresistance of oral cancer cells. SIGNIFICANCE: In summary, the results showed that the acidic microenvironment leads to a more aggressive and treatment resistant OSCC cell population.

Downregulation of SFRP2 facilitates cancer stemness and radioresistance of glioma cells via activating Wnt/ß-catenin signaling.

Secreted frizzled-related protein 2 (SFRP2) is a glycoprotein with frizzled-like cysteine-rich domain that binds with Wnt ligands or frizzled receptors to regulate Wnt signaling. SFRP2 is frequently hypermethylated in glioma patients, and analysis of TCGA data indicates that SFRP2 is one of the most downregulated genes in radiotherapy treated glioma patients. In the present study, we aimed to explore the potential function of SFRP2 in tumorigenesis and radioresistance of glioma. The RNA sequencing data of TCGA glioma samples were downloaded and analyzed. SFRP2 expression in 166 glioma patients was evaluated by qRT-PCR. The potential functions of SFRP2 in glioma were evaluated by loss-of-function assays and gain-of-function assays in glioma cell lines. We found that SFRP2 was downregulated in radiotherapy-treated glioma patients, and low SFRP2 expression was correlated with advanced tumor stage and poor prognosis. CRISP/Cas9-meidated SFRP2 knockdown promoted soft agar colony formation, cancer stemness and radioresistance of glioma cells, while enforced SFRP2 expression exhibited opposite effects. Moreover, Wnt/ß-catenin signaling was activated in radiotherapy treated glioma patients. SFRP2 knockdown activated Wnt/ß-catenin signaling in glioma cell lines, while overexpression of SFRP2 inhibited Wnt/ß-catenin activation. Besides, pharmacological inhibition of Wnt/ß-catenin signaling by XAV-939 abrogated the effects of SFRP2 knockdown on cancer stemness and radioresistance of glioma cells. Our data for the first time demonstrated a role of SFRP2 in radioresistance of glioma cells, and suggested that inhibition of Wnt/ß-catenin signaling might be a potential strategy for increasing radiosensitivity of glioma patients.

Comparative Study of the γH2AX Foci Forming in Human Lung Fibroblasts Incubated in Media Containing Tritium-Labeled Thymidine or Amino Acids.

We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation in a medium containing 3H-labeled thymidine or amino acids (glycine, alanine, and proline) with specific radioactivity from 100 to 400 MBq/liter. A linear dependence of changes in the number of γH2AX foci on the specific radioactivity of the medium was revealed. The quantitative yield of DNA double-strand breaks under the influence of 3H-thymidine was more than 2-fold higher than under the influence of 3H-labeled amino acids. Comparative analysis of the yields of DNA double-strand breaks during cell incubation with 3H-labeled amino acids showed that 3H-alanine produced more pronounced effect that 3H-proline, which is consistent with the data on the content of their non-radioactive analogs in chromatin proteins.

Effects of the Combined Treatment with a G-Quadruplex-Stabilizing Ligand and Photon Beams on Glioblastoma Stem-like Cells: A Magnetic Resonance Study.

Glioblastoma multiforme is a malignant primary brain tumor with a poor prognosis and high rates of chemo-radiotherapy failure, mainly due to a small cell fraction with stem-like properties (GSCs). The mechanisms underlying GSC response to radiation need to be elucidated to enhance sensitivity to treatments and to develop new therapeutic strategies. In a previous study, two GSC lines, named line #1 and line #83, responded differently to carbon ions and photon beams, with the differences likely attributable to their own different metabolic fingerprint rather than to radiation type. Data from the literature showed the capability of RHPS4, a G-quadruplex stabilizing ligand, to sensitize the glioblastoma radioresistant U251MG cells to X-rays. The combined metabolic effect of ligand #190, a new RHPS4-derivative showing reduced cardiotoxicity, and a photon beam has been monitored by magnetic resonance (MR) spectroscopy for the two GSC lines, #1 and #83, to reveal whether a synergistic response occurs. MR spectra from both lines were affected by single and combined treatments, but the variations of the analysed metabolites were statistically significant mainly in line #1, without synergistic effects due to combination. The multivariate analysis of ten metabolites shows a separation between control and treated samples in line #1 regardless of treatment type, while separation was not detected in line #83.

Downregulated CLIP3 induces radioresistance by enhancing stemness and glycolytic flux in glioblastoma.

BACKGROUND: Glioblastoma Multiforme (GBM) is a malignant primary brain tumor in which the standard treatment, ionizing radiation (IR), achieves a median survival of about 15 months. GBM harbors glioblastoma stem-like cells (GSCs), which play a crucial role in therapeutic resistance and recurrence. METHODS: Patient-derived GSCs, GBM cell lines, intracranial GBM xenografts, and GBM sections were used to measure mRNA and protein expression and determine the related molecular mechanisms by qRT-PCR, immunoblot, immunoprecipitation, immunofluorescence, OCR, ECAR, live-cell imaging, and immunohistochemistry. Orthotopic GBM xenograft models were applied to investigate tumor inhibitory effects of glimepiride combined with radiotherapy. RESULTS: We report that GSCs that survive standard treatment radiation upregulate Speedy/RINGO cell cycle regulator family member A (Spy1) and downregulate CAP-Gly domain containing linker protein 3 (CLIP3, also known as CLIPR-59). We discovered that Spy1 activation and CLIP3 inhibition coordinately shift GBM cell glucose metabolism to favor glycolysis via two cellular processes: transcriptional regulation of CLIP3 and facilitating Glucose transporter 3 (GLUT3) trafficking to cellular membranes in GBM cells. Importantly, in combination with IR, glimepiride, an FDA-approved medication used to treat type 2 diabetes mellitus, disrupts GSCs maintenance and suppresses glycolytic activity by restoring CLIP3 function. In addition, combining radiotherapy and glimepiride significantly reduced GBM growth and improved survival in a GBM orthotopic xenograft mouse model. CONCLUSIONS: Our data suggest that radioresistant GBM cells exhibit enhanced stemness and glycolytic activity mediated by the Spy1-CLIP3 axis. Thus, glimepiride could be an attractive strategy for overcoming radioresistance and recurrence by rescuing CLIP3 expression.

Gallotannin from Bouea macrophylla Seed Extract Suppresses Cancer Stem-like Cells and Radiosensitizes Head and Neck Cancer.

Cancer stem cells (CSCs) play a critical role in radiation resistance and recurrence. Thus, drugs targeting CSCs can be combined with radiotherapy to improve its antitumor efficacy. Here, we investigated whether a gallotannin extract from Bouea macrophylla seed (MPSE) and its main bioactive compound, pentagalloyl glucose (PGG), could suppress the stemness trait and further confer the radiosensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines. In this study, we evaluate the effect of MPSE or PGG to suppress CSC-like phenotypes and radiosensitization of HNSCC cell lines using a series of in vitro experiments, tumorsphere formation assay, colony formation assay, apoptosis assay, and Western blotting analysis. We demonstrate that MPSE or PGG is able to suppress tumorsphere formation and decrease protein expression of cancer stem cell markers. MPSE or PGG also enhanced the radiosensitivity in HNSCC cells. Pretreatment of cells with MPSE or PGG increased IR-induced DNA damage (γ-H2Ax) and enhanced radiation-induced cell death. Notably, we observed that pretreatment with MPSE or PGG attenuated the IR-induced stemness-like properties characterized by tumorsphere formation and the CD44 CSC marker. Our findings describe a novel strategy for increasing therapeutic efficacy for head and neck cancer patients using the natural products MPSE and PGG.

NK Cells Lose Their Cytotoxicity Function against Cancer Stem Cell-Rich Radiotherapy-Resistant Breast Cancer Cell Populations.

Cancer stem cells (CSCs) can be induced from differentiated cancer cells in the tumor microenvironment or in response to treatments and exhibit chemo- and radioresistance, leading to tumor recurrence and metastasis. We previously reported that triple negative breast cancer (TNBC) cells with acquired radioresistance exhibited more aggressive features due to an increased CSC population. Therefore, here, we isolated CSCs from radiotherapy-resistant (RT-R)-TNBC cells and investigated the effects of these CSCs on tumor progression and NK cell-mediated cytotoxicity. Compared to MDA-MB-231 and RT-R-MDA-MB-231 cells, CD24-/low/CD44+ cells isolated from RT-R-MDA-MB-231 cells showed increased proliferation, migration and invasion abilities, and induced expression of tumor progression-related molecules. Moreover, similar to MDA-MB-231 cells, CD24-/low/CD44+ cells recruited NK cells but suppressed NK cell cytotoxicity by regulating ligands for NK cell activation. In an in vivo model, CD24-/low/CD44+ cell-injected mice showed enhanced tumor progression and lung metastasis via upregulation of tumor progression-related molecules and altered host immune responses. Specifically, NK cells were recruited into the peritumoral area tumor but lost their cytotoxicity due to the altered expression of activating and inhibitory ligands on tumors. These results suggest that CSCs may cause tumor evasion of immune cells, resulting in tumor progression.

Transcriptional control of brain tumor stem cells by a carbohydrate binding protein.

Brain tumor stem cells (BTSCs) and intratumoral heterogeneity represent major challenges in glioblastoma therapy. Here, we report that the LGALS1 gene, encoding the carbohydrate binding protein, galectin1, is a key regulator of BTSCs and glioblastoma resistance to therapy. Genetic deletion of LGALS1 alters BTSC gene expression profiles and results in downregulation of gene sets associated with the mesenchymal subtype of glioblastoma. Using a combination of pharmacological and genetic approaches, we establish that inhibition of LGALS1 signaling in BTSCs impairs self-renewal, suppresses tumorigenesis, prolongs lifespan, and improves glioblastoma response to ionizing radiation in preclinical animal models. Mechanistically, we show that LGALS1 is a direct transcriptional target of STAT3 with its expression robustly regulated by the ligand OSM. Importantly, we establish that galectin1 forms a complex with the transcription factor HOXA5 to reprogram the BTSC transcriptional landscape. Our data unravel an oncogenic signaling pathway by which the galectin1/HOXA5 complex maintains BTSCs and promotes glioblastoma.

Silent FOSL1 Enhances the Radiosensitivity of Glioma Stem Cells by Down-Regulating miR-27a-5p.

Since few reports have mentioned the role of FOSL1 in the radiotherapy sensitivity of glioma, this study would dig deep into this aspect. Cancer stem cells (CSCs) isolated by magnetic bead assay were identified by microscopy, qRT-PCR and western blot. The number of apoptotic cells was counted 72 h after X-ray irradiation to evaluate the sensitivity of cancer cells to radiotherapy. The effects of radiotherapy, FOSL1 and miR-27a-5p on basic cell functions were detected by functional experiments. The expressions of FOSL1, apoptosis-related genes and miR-27a-5p were detected by qRT-PCR and western blot as needed. The differential expression of FOSL1 and the effect of miR-27a-5p on survival rate were analyzed using GEPIA and UALCAN, respectively. FOSL1 was found highly expressed in glioma cells and patients. The appearance of spherical cells and high expressions of CSC-related markers indicated the successful isolation of CSC-like cells. The increment of X-ray dose enhanced the sensitivity of cancer cells to radiotherapy. Radiotherapy down-regulated cell viability and the expressions of FOSL1 and Bcl-2, but up-regulated cell apoptosis and the expressions of cleaved caspase-3 and Bax, which could be partially reversed by overexpressed FOSL1 or further enhanced by shFOSL1. MiR-27a-5p was highly expressed in in patients with glioma, which was associated with poor prognosis, while shFOSL1-inhibited miR-27a-5p expression enhanced the sensitivity of glioma stem cells to radiotherapy. In vivo experiments further verified the results obtained from in vitro experiments. Silent FOSL1 strengthened the radiosensitivity of glioma by down-regulating miR-27a-5p.

Fractionated Irradiation Enhances Invasion and Migration by Inducing Epithelial-Mesenchymal Transition and Stemness-Like Properties in A549 Cells.

OBJECTIVE: Radioresistance-induced locoregional recurrence remains a major cause of low survival rates. However, the mechanism of treatment failure in these lung cancer patients has not been determined. In the current study, we tried to explore the potential molecular mechanism. METHODS: The fractionated irradiations were continued until the total concentration reached 80 Gy, and we established radioresistant subclones derived from A549 lines (designated as A549/R). The MTT assay, wound healing assay, transwell assay, and soft agar colony formation assay were employed to detect the proliferation, migration, invasion, and clonogenicity of the cells, respectively. Western blot and Fluorescence Activating Cell Sorter (FACS) indicated the expression of the markers. RESULTS: A549/R cells proliferated more slowly than the parental A549 cells. A significant acceleration in cell migration and invasion was revealed in A549/R cells compared with A549 cells. The expression levels of mesenchymal markers (N-cadherin, vimentin, claudin-1, and Snail) increased, while epithelial markers (E-cadherin and ß-catenin) decreased in A549/R cells. Meanwhile, the expression levels of stemness markers (Oct4, Notch1, and CD133) increased in A549/R cells, and A549/R cells showed more sphere-forming activity compared with A549 cells. CONCLUSION: Fractionated irradiation could promote epithelial-mesenchymal transition and enhance the migration, invasion, and stemness-like properties in A549 cells, elucidating the possible radioresistance mechanisms of the cancer cells.

Knockdown of the prognostic cancer stem cell marker Musashi-1 decreases radio-resistance while enhancing apoptosis in hormone receptor-positive breast cancer cells via p21WAF1/CIP1.

PURPOSE: While the stem cell marker Musashi-1 (MSI-1) has been identified as a key player in a wide array of malignancies, few findings exist on its prognostic relevance and relevance for cancer cell death and therapy resistance in breast cancer. METHODS: First, we determined prognostic relevance of MSI-1 in database analyses regarding multiple survival outcomes. To substantiate findings, MSI-1 was artificially downregulated in MCF-7 breast cancer cells and implications for cancer stem cell markers, cell apoptosis and apoptosis regulator p21, proliferation and radiation response were analyzed via flow cytometry and colony formation. Radiation-induced p21 expression changes were investigated using a dataset containing patient samples obtained before and after irradiation and own in vitro experiments. RESULTS: MSI-1 is a negative prognostic marker for disease-free and distant metastasis-free survival in breast cancer and tends to negatively influence overall survival. MSI-1 knockdown downregulated stem cell gene expression and proliferation, but increased p21 levels and apoptosis. Similar to the MSI-1 knockdown effect, p21 expression was strongly increased after irradiation and was expressed at even higher levels in MSI-1 knockdown cells after irradiation. Finally, combined use of MSI-1 silencing and irradiation reduced cancer cell survival. CONCLUSION: MSI-1 is a prognostic marker in breast cancer. MSI-1 silencing downregulates proliferation while increasing apoptosis. The anti-proliferation mediator p21 was upregulated independently after both MSI-1 knockdown and irradiation and even more after both treatments combined, suggesting synergistic potential. Radio-sensitization effects after combining radiation and MSI-1 knockdown underline the potential of MSI-1 as a therapeutic target.