Stemness Properties Research Articles

Abstract 2447: XPC suppresses cancer stem cells via inhibiting STAT1-mediated expression of SOX2 in NSCLC

Abstract Xeroderma pigmentosum group C (XPC) is a DNA repair factor mainly involved in nucleotide excision repair but also has functions beyond its role in DNA repair. More than 60% lung adenocarcinoma patients exhibit XPC copy number deletions, and among these patients, low XPC expression is correlated with a poor outcome, indicating that XPC may play a critical role in preventing lung cancer progression. Here, we revealed an inverse relationship between XPC expression and the abundance of cancer stem cell (CSC) subpopulation in non-small cell lung cancer (NSCLC). We demonstrated that knockdown of XPC leads to larger CSC subpopulations in lung cancer cells, while overexpression of XPC can inhibit the stemness properties of these cells. The RNA-seq analysis suggested that XPC can suppress the expression of SOX2, a critical stem cell-specific transcription factor. This finding was further validated in multiple NSCLC cell lines at both mRNA and protein levels. To further investigate the mechanism underlying XPC-mediated suppression of SOX2 expression, we performed quantitative proteomics combined with immuno-affinity purification and found that XPC can interact with STAT1, which is a transcription factor of SOX2. Moreover, XPC can inversely regulate the activation of STAT1 (pSTAT1-Y701), indicating that XPC may suppress SOX2 expression via inhibiting the STAT1 signaling. Taken together, we identified a novel mechanism behind poor outcomes of NSCLC patients with haploinsufficiency XPC. Low level of XPC in lung cancer cells de-represses the activity of STAT1, which further promotes the expression of stem cell-specific gene SOX2 and facilitates the maintenance of CSC subpopulations. Given that CSCs are believed to contribute to tumor progression and chemoresistance, enhanced stemness and expanded CSC populations in XPC haploinsufficiency NSCLC could be responsible for the poor outcome of these patients. Citation Format: Na Li, Xuetao Bai, Shurui Cai, Ananya Banerjee, Yajing Yang, Qianyun Ge, Linzhou Wang, Qi-En Wang. XPC suppresses cancer stem cells via inhibiting STAT1-mediated expression of SOX2 in NSCLC [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 2447.

Abstract 6040: The CHST11-CP-iron axis contributes to NSCLC stemness

Abstract The emerging advances in cancer therapies have changed the landscape for the treatment, but there are limited treatments options for advanced non-small-cell lung cancer (NSCLC). Cancer stem cell-induced chemoresistance and tumor metastasis accounts for treatment failure and recurrence in NSCLC. Additionally, the up-regulated glycosaminoglycan-chondroitin sulfate (CS) biosynthesis pathway contributes to the mesenchymal state of lung cancer and related to unfavorable prognosis. Previously, we found carbohydrate sulfotransferase 11 (CHST11), a key CS biosynthetic enzyme, was associated with tumor progression and metastasis in NSCLC as well as lung fibrosis. Furthermore, ceruloplasmin (CP) acted as a downstream effector of CHST11 through interferon-γ signaling pathway stimulation and increased NSCLC metastatic features. However, the linkage between metabolic dysregulation and NSCLC stemness remains unclear. Here, we found not only overexpression of CHST11 but also CS-A treatment, the metabolite of CHST11, promoted NSCLC tumor sphere formation, the expression of cancer stemness markers, and transcriptional activities of stemness-related pathways. Iron was a necessary ion in upregulating cancer stemness properties such as tumor-initiation, metastasis and chemoresistance. We found both CHST11 and iron enhanced NSCLC tumorigenesis. Moreover, iron chelation disrupted CHST11-CP axis induced-stemness. The combination of iron chelator with chemotherapies showed the synergistic effect on antitumor as well as anti-cancer stemness and further promoted apoptosis. Our findings implicate that the CHST11-CP-iron axis facilitates tumor progression and cancer stemness in NSCLC. A regimen that combines an iron chelator and chemotherapies targeting cancer stem cells will benefit for not only preventing NSCLC tumorigenesis but cancer recurrence. Citation Format: Li-Jie Li, Wei-Min Chang, Michael Hsiao. The CHST11-CP-iron axis contributes to NSCLC stemness. [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 6040.

Abstract 3516: Metastatic colonization requires a proliferative pause linked to vascular co-option

Abstract The physical interaction between metastasis-initiating cells and the pre-existing capillary network (a process known as vascular co-option) is critical during the initial stages of multi-organ metastasis in cancer. As such, this process might provide an opportunity to prevent metastasis. As part of the process of vascular co-option, we observed that brain metastatic cells in the perivascular niche temporarily enter into a novel cell state characterized by a decreased proliferation before resuming their aggressive growth to colonize the organ. Transcriptomic analysis of co-opting metastatic cells confirmed downregulation of MYC signatures, mitotic cell cycle and increased stemness properties. By focusing on one of the top upregulated transcription factors in co-opting cells: MXD4, a MYC antagonist; we have been able to dissect the relevance of this cellular state, that we termed proliferative pause, both respect to the maintenance of the interaction with the vasculature and to the ability to generate macrometastases. As such, targeting MXD4 in lung adenocarcinoma and triple-negative breast cancer metastatic models reduced multi-organ metastases to a level that translates into increased overall survival. Mechanistically, this obliged proliferative pause is linked to a cellular response to the increasing environmental pressure involved in organ colonization. For instance, we found that crossing the blood-brain barrier induced an increased DNA damage due to mechanical constrains leading to nuclear deformation. However, the MXD4-dependent proliferative pause during vascular co-option allows metastatic cells to repair this damage to continue the colonization process. Given the enormous potential to prevent metastasis and our findings dissecting the proliferative pause status, we developed a therapeutic strategy to target vascular co-opting cells. As part of the molecular profile of co-opting cells, we validated their high dependency on Bcl proteins. As such, we have used a Bcl-2 inhibitor (obatoclax) permeable to the blood-brain barrier to target these cells in preventive scenarios. Beyond the preventive therapeutic assays in vivo, we have applied additional clinically-relevant models where preventive strategies could easily translate into the clinical practice. As such, obatoclax post-surgery provided a survival benefit by preventing relapse, as the cells left behind after the local therapy are vascular co-opting cells. Furthermore, our national network of brain metastasis (RENACER) provided us with fresh neurosurgeries and, in a limited cohort of 10 surgeries with extended resections, we were able to identify invasive fronts with metastatic cells co-opting the vasculature. The use of obatoclax to target these cancer cells, which are the seeds of relapse post-surgery, confirmed that targeting vascular co-option could be a novel strategy to prevent metastasis in a clinically relevant situation. Citation Format: Pedro García-Gómez, Diana Retana, Pablo Sanz-Martínez, Irene Salgado-Crespo, Carolina Hernández-Oliver, Maria Isabel García, Oliva Sánchez, Kevin Troulé-Lozano, Verona Villar-Cerviño, Miguel Lafarga-Coscojuela, Fátima Al-Shahrour, RENACER Red Nacional de Metástasis Cerebral, Manuel Valiente. Metastatic colonization requires a proliferative pause linked to vascular co-option [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 3516.

376 From Core to Margin: Intratumoral Differences in Cancer Hallmarks Depend on Spatial Localization of Cells in Glioblastoma

INTRODUCTION: Glioblastoma (GBM) carries a dismal prognosis due to its invasive nature. Studies show that GBMs have transcriptionally heterogeneous cell populations within the same tumor. However, information regarding the biological behavior of different intratumoral populations has never been demonstrated in vitro. METHODS: GBM patients who underwent resective surgery were consented for research. MRI-guided images were registered during resection to localize the area where samples were obtained. Two GBM cell lines were established from the same patient for each case: margin and core cell line. Proliferation, migration, and stemness experiments were performed in vitro and intracranial implantation of tumors was then carried out to evaluate tumor behavior in vivo. RESULTS: 10 cell lines from 5 patients were established from the margin and the core. There were no differences in cell proliferation. Sphere area and limiting dilution assay (p < 0.01) show that cells derived from the margin have greater stemness capabilities. Cells from the margin have higher migratory abilities in transwell migration assay (p < 0.01 in 4/5 patients) and in real-time cell invasion monitoring (p < 0.01). Trend which was later noted in sphere invasion assay. In vivo studies demonstrate that both cell populations were capable of tumor engraftment with no differences in tumor growth by bioluminescence (p > 0.05). Nonetheless, tumors from the marginal area demonstrated a more diffuse phenotype and greater extension when compared core-derived tumors on histopathology . CONCLUSIONS: There is a large degree of behavioral heterogeneity between cells from the tumor core versus the margin. GBM cells from the margin have higher migration and stemness properties when compared to their core counterparts in vitro; suggesting that peripheral cells carry more aggressive phenotypes that could be responsible for treatment resistance and tumor recurrence

Use of adipose derived stem cells in Treacher Collins syndrome.

Treacher Collins syndrome (TCS) is a rare congenital disorder of craniofacial development. TCS occurs with an incidence of 1:50,000, and more than 60% of TCS cases have no previous family history and arise as the result of de novo mutations. The high rate of de novo mutations, together with the extreme variability in the degree to which individuals can be affected, makes the provision of genetic counseling extremely complicated. Consequently, every case of TCS is unique and needs to be assessed individually. Patients with TCS frequently undergo multiple reconstructive surgeries from birth through adulthood, which rarely are fully corrective in the long-term. The nascent field of regenerative medicine offers the promise to improve some of these treatments. In particular, structural fat grafting (SFG) seems to be a good strategy not only to restore the normal volume and contour of the face, but also to provide a source of adipose-derived stem cells (ADSCs) with a multilineage differentiation potential. In this work, we present genetical analyses of ADSC affected by TCS. ADSCs from were analyzed for their stemness properties and shared many characteristics with those of a healthy subject. Screening of the genome of the TCS patient using array-Comparative Genomic Hybridization allowed us to identify some chromosomal imbalances that are probably associated with TCS. We found that some alterations, involving the TIMELESS gene, were usually associated with embryonic stem cells. With the aim to improve the final results, we need to consider combining knowledge of genetic alterations and expression profiles as a fundamental step before starting with surgical procedures.

Taxonomic Characteristics and Phytochemicals of Centaurea bruguieriana (DC.) Hand-Mazz (Asreraceae) From Western Desert of Iraq

In the present study, morphological, anatomical, and palynological characteristics of Centaurea bruguieriana (DC.) Hand.-Mazz. (Asteraceae) from the western desert of Iraq were investigated. The Phytochemicals of this species were also studied. Morphological features of different plant parts ( stem, leaf, inflorescence, florets, bracts fruit, and pollen ) were reported. Anatomical properties of stem and leaf (including frequency of stomata, glandular and non-glandular hairs) were studied, based on cross-sections and stripped off the epidermi. Active contents (or phytochemicals) of the studied species, such as flavonoids, alkaloids, glycosides, and tannins were determined.

Characterization of mesenchymal stem cells in human fetal bone marrow by single-cell transcriptomic and functional analysis

Bone marrow mesenchymal stromal/stem cells (MSCs) are a heterogeneous population that can self-renew and generate stroma, cartilage, fat, and bone. Although a significant progress has been made toward recognizing about the phenotypic characteristics of MSCs, the true identity and properties of MSCs in bone marrow remain unclear. Here, we report the expression landscape of human fetal BM nucleated cells (BMNCs) based on the single-cell transcriptomic analysis. Unexpectedly, while the common cell surface markers such as CD146, CD271, and PDGFRa used for isolating MSCs were not detected, LIFR+PDGFRB+ were identified to be specific markers of MSCs as the early progenitors. In vivo transplantation demonstrated that LIFR+PDGFRB+CD45-CD31-CD235a- MSCs could form bone tissues and reconstitute the hematopoietic microenvironment (HME) effectively in vivo. Interestingly, we also identified a subpopulation of bone unipotent progenitor expressing TM4SF1+CD44+CD73+CD45-CD31-CD235a-, which had osteogenic potentials, but could not reconstitute HME. MSCs expressed a set of different transcription factors at the different stages of human fetal bone marrow, indicating that the stemness properties of MSCs might change during development. Moreover, transcriptional characteristics of cultured MSCs were significantly changed compared with freshly isolated primary MSCs. Our cellular profiling provides a general landscape of heterogeneity, development, hierarchy, microenvironment of the human fetal BM-derived stem cells at single-cell resolution.

A Self-Propagating c-Met-SOX2 Axis Drives Cancer-Derived IgG Signaling That Promotes Lung Cancer Cell Stemness.

Sialylated cancer IgG activates c-Met-SOX2 signaling to promote stemness properties in cancer cells and can be targeted to suppress tumor growth.

Analysis of skeletal stem cells by renal capsule transplantation and ex vivo culture systems.

Skeletal stem cells residing in the suture mesenchyme are responsible for proper development, homeostasis, and injury repair of the craniofacial skeleton. These naïve cells are programmed to differentiate into osteoblast cell types and mediate bone formation via an intramembranous ossification mechanism. The simplicity of this system also offers great advantages to studying osteoblastogenesis compared to the appendicular and axial skeletons. Recent studies utilizing genetically based cell tracing have led to the identification of skeletal stem cell populations in craniofacial and body skeletons. Although the genetic analysis indicates these cells behave like stem cells in vivo, not all of them have been thoroughly examined by stem cell isolation and stem cell-mediated tissue generation. As regeneration is an integral part of stem cell characteristics, it is necessary to further analyze their ability to generate tissue at the ectopic site. The establishment of an ex vivo culture system to maintain the stemness properties for extended periods without losing the regenerative ability is also pertinent to advance our knowledge base of skeletal stem cells and their clinical applications in regenerative medicine. The purpose of this review is to discuss our recent advancements in analyses of skeletal stem cells using renal capsule transplantation and sphere culture systems.

Adipocytes reprogram prostate cancer stem cell machinery.

It is now well-established that an obese condition correlates with a higher risk of prostate cancer (PCa). A crosstalk between adipose tissue and PCa has been observed but is still poorly characterized. Herein, we demonstrated that 3T3-L1 adipocyte conditioned media (CM) could endow PC3 and DU145 PCa cells with stemness properties, by stimulating their sphere formation ability and promoting CD133 and CD44 expression. Moreover, after exposure to adipocyte CM both PCa cell lines underwent partial epithelial-to-mesenchymal transition (EMT), with E-/N-cadherin switch and Snail upregulation. Specifically, these changes in PC3 and DU145 cell phenotype were accompanied by increased tumor clonogenic activity and survival, as well as by enhanced invasion, anoikis resistance and matrix metalloproteinase (MMP) production. Finally, adipocyte CM-treated PCa cells exhibited reduced responsiveness to both docetaxel and cabazitaxel, demonstrating greater chemoresistance. Overall, these data indicate that adipose tissue can effectively contribute to PCa aggressiveness by reprogramming the cancer stem cell (CSC) machinery. Adipocytes endow prostate cancer cells with stem-like properties and mesenchymal traits, increasing their tumorigenicity, invasion and chemoresistance.

Silicon nanowire array overcomes chemotherapeutic resistance by inducing the differentiation of breast cancer stem cells.

Currently, traditional cancer treatment strategies are greatly challenged by the existence of cancer stem cells (CSCs), which are root cause of chemotherapy resistance. Differentiation therapy presents a novel therapeutic strategy for CSC-targeted therapy. However, there are very few studies on the induction of CSCs differentiation so far. Silicon nanowire array (SiNWA) with many unique properties is considered to be an excellent material for various applications ranging from biotechnology to biomedical applications. In this study, we report the SiNWA differentiates MCF-7-derived breast CSCs (BCSCs) into non-CSCs by modulating the morphology of cells. In vitro, the differentiated BCSCs lose the stemness properties and thus become sensitive to chemotherapeutic drugs, eventually leading to the death of BCSCs. Therefore, this work suggests a potential approach for overcoming chemotherapeutic resistance.

NAD pool as an antitumor target against cancer stem cells in head and neck cancer

SummaryHead and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that affect different anatomical locations. Despite this heterogeneity, HNSCC treatment depends on the anatomical location, TNM stage and resectability of the tumor. Classical chemotherapy is based on platinum-derived drugs (cisplatin, carboplatin and oxaliplatin), taxanes (docetaxel, paclitaxel) and 5-fluorouracil1. Despite advances in HNSCC treatment, the rate of tumor recurrence and patient mortality remain high. Therefore, the search for new prognostic identifiers and treatments targeting therapy-resistant tumor cells is vital. Our work demonstrates that there are different subgroups with high phenotypic plasticity within the CSC population in HNSCC. CD10, CD184, and CD166 may identify some of these CSC subpopulations with NAMPT as a common metabolic gene for the resilient cells of these subpopulations. We observed that NAMPT reduction causes a decrease in tumorigenic and stemness properties, migration capacity and CSC phenotype through NAD pool depletion. However, NAMPT-inhibited cells can acquire resistance by activating the NAPRT enzyme of the Preiss-Handler pathway. We observed that coadministration of the NAMPT inhibitor with the NAPRT inhibitor cooperated inhibiting tumor growth. The use of an NAPRT inhibitor as an adjuvant improved NAMPT inhibitor efficacy and reduced the dose and toxicity of these inhibitors. Therefore, it seems that the reduction in the NAD pool could have efficacy in tumor therapy. This was confirmed by in vitro assays supplying the cells with products of inhibited enzymes (NA, NMN or NAD) and restoring their tumorigenic and stemness properties. In conclusion, the coinhibition of NAMPT and NAPRT improved the efficacy of antitumor treatment, indicating that the reduction in the NAD pool is important to prevent tumor growth.

Abstract P6-11-04: Novel roles of Inosine Monophosphate Dehydrogenase 2 (IMPDH2) in TNBC doxorubicin resistance

Abstract Triple negative breast cancer (TNBC) is one of the major subtypes of breast cancer, being associated with the lowest survival rate after metastasis occurs. TNBC patients face limited therapeutic options, relying mostly on a combination of chemotherapies (anthracycline, taxanes and cyclophosphamide). However, around 40% of patients treated with chemotherapy relapse due to onset of chemoresistance, contributing to the dismal survival of this aggressive subtype. Therefore, understanding the molecular mechanisms underlying chemoresistance is critical to identify better therapeutic target options. Guanosine triphosphate (GTP) is important for several biological processes, including cell proliferation. We and others have shown that inosine 5’-monophosphate dehydrogenase 2 (IMPDH2), the rate limiting enzyme in the de novo synthesis of GTP, is important in migration and invasion of cancer, including TNBC, however the role it plays in chemoresistance remains to be elucidated. Our preliminary data revealed that IMPDH2 expression levels modulate doxorubicin sensitivity in different TNBC cell lines. Moreover, we have generated MDA-MB-231 cells that are resistant to doxorubicin (referred to as DoxoR), and revealed IMPDH2 to be upregulated in DoxoR cells, at both protein and mRNA levels as well as having higher activity. Consistently, DoxoR cells have ~50% more GTP than their naïve counterpart and, interestingly, are twice more sensitive to treatment with Ribavirin, a well-tolerated FDA approved IMPDH inhibitor, suggesting that Ribavirin could be repurposed for the treatment of chemoresistant TNBC. As expected, RNA sequencing of DoxoR cells revealed increased stemness properties when compared to naïve cells, which we confirmed experimentally in tumor sphere formation assays. Importantly, knockdown of IMPDH2 reverted the ability of DoxoR cells to form bigger tumor spheres, as well as reduced the IC50 of doxorubicin to that of naïve cells. Therefore, our data suggests that increased IMPDH2 and GTP levels in resistant setting could be a potential new vulnerability to be leveraged therapeutically to suppress and/or prevent the growth of chemoresistant lesions. Citation Format: Tatiane da Silva Fernandes, Anna Bianchi-Smiraglia. Novel roles of Inosine Monophosphate Dehydrogenase 2 (IMPDH2) in TNBC doxorubicin resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-04.

Effect of Mir-4270 Inhibitor and Mimic on Viability and Stemness in Gastric Cancer Stem-Like Cells Derived from MKN-45 Cell Line.

MicroRNAs (miRNAs) are significant regulatory factors in stem cell proliferation, and change in miRNA expression influences the cancer stem cell viability and gene expression. Herein, we evaluated the effect of the hsa-miR-4270 inhibitor and its mimic on the expression of stem cell markers in gastric cancer (GC) stem-like cells. GC stem-like cells were isolated from the MKN-45 cell line by a non-adherent surface system. The cells were confirmed by differentiation assays using dexamethasone and insulin as adipogenesis-inducing agents and also Staurosporine as a neural-inducing agent. Isolated GC stem-like cells were treated with different concentrations (0, 15, 20, 25, 30, 40, 50, and 60 nM) of hsa-miR-4270 inhibitor and its mimic. The quantity of cell viability was determined by trypan blue method. Transcription of the stem cell marker genes, including CD44, OCT3/4, SOX2, Nanog, and KLF4, was evaluated by real-time RT-PCR. The results showed that GC stem-like cells were differentiated into both adipose cells using dexamethasone and insulin and neural cells by Staurosporine. Treatment of GC stem-like cells with hsa-miR-4270 inhibitor decreased cell viability and downregulated OCT3/4, CD44, and Nanog to 86%, 79%, and 91% respectively. Also, SOX2 and KLF4 were overexpressed to 8.1- and 1.94-folds, respectively. However, hsa-miR-4270 mimic had opposite effects on the cell viability and gene expression of the stem cell markers. The effect of hsa-miR-4270 inhibitor and its mimic on the expression of the stem cell markers in GCSCs indicated that hsa-miR-4270 stimulates the stemness property of GCSCs, likely through stimulating the development of gastric stem cells.

Osteogenic and Adipogenic Differentiation Potential of Oral Cancer Stem Cells May Offer New Treatment Modalities

(1) Treatment failure of oral squamous cell carcinoma (OSCC) is generally due to the development of therapeutic resistance caused by the existence of cancer stem cells (CSCs), a small cell subpopulation with marked self-renewal and differentiation capacity. Micro RNAs, notably miRNA-21, appear to play an important role in OSCC carcinogenesis. Our objectives were to explore the multipotency of oral CSCs by estimating their differentiation capacity and assessing the effects of differentiation on stemness, apoptosis, and several miRNAs' expression. (2) A commercially available OSCC cell line (SCC25) and five primary OSCC cultures generated from tumor tissues obtained from five OSCC patients were used in the experiments. Cells harboring CD44, a CSC marker, were magnetically separated from the heterogeneous tumor cell populations. The CD44+ cells were then subjected to osteogenic and adipogenic induction, and the specific staining was used for differentiation confirmation. The kinetics of the differentiation process was evaluated by qPCR analysis of osteogenic (Bone Morphogenetic Protein-BMP4, Runt-related Transcription Factor 2-RUNX2, Alkaline Phosphatase-ALP) and adipogenic (Fibroblast Activation Protein Alpha-FAP, LIPIN, Peroxisome Proliferator-activated Receptor Gamma-PPARG) markers on days 0, 7, 14, and 21. Embryonic markers (Octamer-binding Transcription Factor 4-OCT4, Sex Determining Region Y Box 2-SOX2, and NANOG) and micro RNAs (miRNA-21, miRNA-133, and miRNA-491) were also correspondingly evaluated by qPCR. An Annexin V assay was used to assess the potential cytotoxic effects of the differentiation process. (3) Following differentiation, the levels of markers for the osteo/adipo lineages showed a gradual increase from day 0 to day 21 in the CD44+ cultures, while stemness markers and cell viability decreased. The oncogenic miRNA-21 also followed the same pattern of gradual decrease along the differentiation process, while tumor suppressor miRNA-133 and miRNA-491 levels increased. (4) Following induction, the CSCs acquired the characteristics of the differentiated cells. This was accompanied by loss of stemness properties, a decrease of the oncogenic and concomitant, and an increase of tumor suppressor micro RNAs.

IOX-1 suppresses metastasis of osteosarcoma by upregulating histone H3 lysine trimethylation

New therapeutic approaches are needed for metastatic osteosarcoma (OS), as survival rates remain low despite surgery and chemotherapy. Epigenetic changes, such as histone H3 methylation, play key roles in many cancers including OS, although the underlying mechanisms are not clear. In this study, human OS tissue and OS cell lines displayed lower levels of histone H3 lysine trimethylation compared with normal bone tissue and osteoblast cells. Treating OS cells with the histone lysine demethylase inhibitor 5-carboxy-8-hydroxyquinoline (IOX-1) dose-dependently increased histone H3 methylation and inhibited cellular migratory and invasive capabilities, suppressed matrix metalloproteinase expression, reversed epithelial-to-mesenchymal transition by increasing levels of epithelial markers E-cadherin and ZO-1 and decreasing the expression of mesenchymal markers N-cadherin, vimentin, and TWIST, and also reduced stemness properties. An analysis of cultivated MG63 cisplatin-resistant (MG63-CR) cells revealed lower histone H3 lysine trimethylation levels compared with levels in MG63 cells. Exposing MG63-CR cells to IOX-1 increased histone H3 trimethylation and ATP-binding cassette transporter expression, potentially sensitizing MG63-CR cells to cisplatin. In conclusion, our study suggests that histone H3 lysine trimethylation is associated with metastatic OS and that IOX-1 or other epigenetic modulators present promising strategies to inhibit metastatic OS progression.

Zinc finger and SCAN domain containing 1, ZSCAN1, is a novel stemness-related tumor suppressor and transcriptional repressor in breast cancer targeting TAZ.

Cancer stem cells (CSCs) targeted therapy holds the potential for improving cancer management; identification of stemness-related genes in CSCs is necessary for its development. The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets were used for survival analysis. ZSCAN1 correlated genes was identified by Spearman correlation analysis. Breast cancer stem-like cells (BCSLCs) were isolated by sorting CD44+CD24- cells from suspension cultured breast cancer (BC) spheroids. The sphere-forming capacity and sphere- and tumor-initiating capacities were determined by sphere formation and limiting dilution assays. The relative gene expression was determined by qRT-PCR, western blot. Lentivirus system was used for gene manipulation. Nuclear run-on assay was employed to examine the levels of nascent mRNAs. DNA pull-down and Chromatin immunoprecipitation (ChIP) assays were used for determining the interaction between protein and target DNA fragments. Luciferase reporter assay was used for evaluating the activity of the promoter. ZSCAN1 is aberrantly suppressed in BC, and this suppression indicates a bad prognosis. Ectopic expression of ZSCAN1 inhibited the proliferation, clonogenicity, and tumorigenicity of BC cells. ZSCAN1-overexpressing BCSLCs exhibited weakened stemness properties. Normal human mammary epithelial (HMLE) cells with ZSCAN1 depletion exhibited enhanced stemness properties. Mechanistic studies showed that ZSCAN1 directly binds to -951 ~ -925bp region of WWTR1 (encodes TAZ) promoter, inhibits WWTR1 transcription, thereby inhibiting the stemness of BCSCs. Our work thus revealed ZSCAN1 as a novel stemness-related tumor suppressor and transcriptional repressor in BC.

BRCA1 interactors, RAD50 and BRIP1, as prognostic markers for triple-negative breast cancer severity.

Introduction: BRIP1 (BRCA1-interacting protein 1) is one of the major interacting partners of BRCA1, which plays an important role in repair by homologous recombination (HR). This gene is mutated in around 4% of cases of breast cancer; however, its mechanism of action is unclear. In this study, we presented the fundamental role of BRCA1 interactors BRIP1 and RAD50 in the development of differential severity in triple-negative breast cancer (TNBC) among various affected individuals. Methods: We have analyzed the expression of DNA repair-related genes in different BC cells using Real-time PCR and western blotting analysis and assessed changes in stemness property and proliferation through Immunophenotyping. We have performed cell cycle analysis to see the defect in checkpoints and also immunofluorescence assay to confirm the accumulation of gamma-H2AX and BRCA1 foci and subsequent incidence. We have performed a severity analysis using TCGA data sets for comparing the expression in MDA-MB-468 MDA-MB-231 and MCF7 cell line. Results: We showed that in some TNBC cell lines such as MDA-MB-231, the functioning of both BRCA1/TP53 is compromised. Furthermore, the sensing of DNA damage is affected. Due to less damage-sensing capability and low availability of BRCA1 at the damage sites, the repair by HR becomes inefficient, leading to more damage. Accumulation of damage sends a signal for over activation of NHEJ repair pathways. Over expressed NHEJ molecules with compromised HR and checkpoint conditions lead to higher proliferation and error-prone repair, which increases the mutation rate and corresponding tumour severity. The in-silico analysis of the TCGA datasets with gene expression in the deceased population showed a significant correlation of BRCA1 expression with overall survival (OS) in TNBCs (0.0272). The association of BRCA1 with OS became stronger with the addition of BRIP1 expression (0.000876**). Conclusion: The severity phenotypes were more in cells having compromised BRCA1-BRIP1 functioning. Since the OS is directly proportional to the extent of severity, the data analysis hints at the role of BRIP1 in controlling the severity of TNBC.

Luteolin directly binds to KDM4C and attenuates ovarian cancer stemness via epigenetic suppression of PPP2CA/YAP axis

Long-term use of low-toxic natural products holds the promise for eradicating cancer stem cells. In this study, we report that luteolin, a natural flavonoid, attenuates the stemness of ovarian cancer stem cells (OCSCs) by directly binding to KDM4C and epigenetic suppression of PPP2CA/YAP axis. Ovarian cancer stem like cells (OCSLCs) isolated by suspension culture and CD133 + ALDH+ cell sorting was employed as OCSCs model. The maximal non-toxic dose of luteolin suppressed stemness properties, including sphere-forming capacity, the expression of OCSCs markers, sphere-initiating and tumor-initiating capacities, as well as the percentage of CD133 + ALDH+ cells of OCSLCs. Mechanistic study showed that luteolin directly binds to KDM4C, blocks KDM4C-induced histone demethylation of PPP2CA promoter, inhibits PPP2CA transcription and PPP2CA-mediated YAP dephosphorylation, thereby attenuating YAP activity and the stemness of OCSLCs. Furthermore, luteolin sensitized OCSLCs to traditional chemotherapeutic drugs in vitro and in vivo. In summary, our work revealed the direct target of luteolin and the underlying mechanism of the inhibitory effect of luteolin on the stemness of OCSCs. This finding thus suggests a novel therapeutic strategy for eradicating human OCSCs driven by KDM4C.

Intracellular Delivery of Anti-Kirsten Rat Sarcoma Antibodies Mediated by Polymeric Micelles Exerts Strong In Vitro and In Vivo Anti-Tumorigenic Activity in Kirsten Rat Sarcoma-Mutated Cancers.

The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.