Formation Of Cancer Stem Cells Research Articles

Mechanism of DAPK1 for Regulating Cancer Stem Cells in Thyroid Cancer.

Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine (Ser/Thr) protein kinase and is characteristically downregulated in metastatic cancer. Several studies showed that DAPK1 is involved in both the early and late stages of cancer. DAPK1 downregulation is elaborately controlled by epigenetic, transcriptional, posttranscriptional, and posttranslational processes. DAPK1 is known to regulate not only cancer cells but also stromal cells. Recent studies showed that DAPK1 was involved not only in tumor suppression but also in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) formation in colon and thyroid cancers. CSCs are major factors in determining cancer aggressiveness in cancer metastasis and treatment prognosis by influencing EMT. However, the molecular mechanism involved in the regulation of cancer cells by DAPK1 remains unclear. In particular, little is known about the existence of CSCs and how they are regulated in papillary thyroid carcinoma (PTC) among thyroid cancers. In this review, we describe the molecular mechanism of CSC regulation by DAPK1 in PTC progression.

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

STAT3 mediates cancer stem‐like tumorsphere formation and PD‐L1 expression to contribute radioresistance in HBV‐positive hepatocellular carcinoma

AbstractWe proposed that cancer stem cells (CSCs) survived and presented resistance to radiotherapy (RT) in hepatocellular carcinoma (HCC) cells. Interleukin 6 (IL‐6) has been reported to be particularly involved in HCC tumorigenesis. Therefore, we intended to validate that IL‐6 downstream STAT3‐mediated CSCs formation and immune checkpoint PD‐L1 expression in HCC, thus contributing to radioresistance. HBV‐positive HCC tumorspheres were formed and exposed with X‐ray irradiation, cell viability of which was measured consequently. Specific inhibitors targeting EGFR (by gefitinib), STAT3 (by BBI608), and HCC‐targeted therapy sorafenib were investigated to suppress tumorsphere formation. Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) was used for detecting STAT3‐downstream PD‐L1 and anti‐apoptosis MCL1 and BCL2 gene expression in the PLC5‐derived tumorspheres and STAT3‐knockdown PLC5. We found that RT significantly inhibited HBV‐positive Hep3B and PLC5 cell viability but not for HCC‐derived stem‐like tumorspheres cultured by EGF, IL‐6, bFGF, and HGF. It revealed that tumorspheres presented radioresistance compared with the parental cells. Specifically, RT induces IFNs, EGF, and IL‐6 expression, resulting in STAT3 phosphorylation. Kaplan–Meier plotter indicated that highly EGF (p = .0024), IL‐6 (p = .12), and FGF2 (p = .0041) were associated with poor survival probability in patients with HBV‐positive HCC. We further demonstrated that BBI608 and sorafenib significantly suppressed PLC5 cell viability and PLC5‐derived tumorsphere formation. To investigate the mechanism of CSC‐presented radioresistance, STAT3 and STAT3‐downstream genes, including PD‐L1 and anti‐apoptosis MCL1 and BCL2, were detected using qPCR. We demonstrated higher STAT3, PD‐L1, MCL1, and BCL2 in Hep3B‐ and PLC5‐derived CSCs compared to PLC5. In addition, knockdown of STAT3 reduced cell proliferation in PLC5 cells, resulting in down‐regulation of IL‐6‐mediated PD‐L1 and BCL‐2. Meanwhile, we found that knockdown of STAT3 significantly improved RT‐mediated suppression of tumorsphere formation. In conclusion, we found that CSCs presented radioresistance and figured out which may be mediated by STAT3 in HBV‐positive HCC.

Abstract LB049: Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway

Abstract Lung cancer is the second most common cancer in both men and women in the United States. Even though treatments including chemotherapy, targeted therapy, and immunotherapy are available, lung cancer has been the leading cause of cancer deaths. The discovery and evaluation of new alternative medications targeting lung cancer are tremendously important for reducing lung cancer mortality. Phytochemicals are very desirable chemotherapeutic agents because of their greater safety profile. Isorhapontigenin (ISO), one of the oral bioavailable dietary stilbenes, can be found in various natural resources. Our result showed that ISO treatment suppressed the invasion and migration of human lung cancer cell lines A549, H23, and H1299 cells. Long non-coding RNA Maternally Expressed Gene 3 (MEG3), a tumor suppressor, was downregulated or lost in various primary human tumor tissues and cancer cell lines. The low expression of MEG3 is correlated with poor prognosis in non-small cell lung cancer (NSCLC). Our results showed that ISO treatment elevated MEG3 levels in these lung cancer cell lines. One of the major genetic causes of lung cancer is mutations in the epidermal growth factor receptor (EGFR) receptor tyrosine kinase, resulting in activation of EGFR which activates several signaling pathways involved in cell proliferation, drug sensitivity, and angiogenesis. Our results showed that ISO decreased the phosphorylation and protein levels of EGFR in A549 cells. Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, regulates the activation of several genes involved in cell proliferation, angiogenesis, invasion, and metastasis. Our results showed that ISO decreased HIF-1α protein levels. Overexpression of MEG3 reduced p-EGFR and HIF-1α, indicating MEG3 negatively regulated EGFR and HIF-1α. Our data also showed that inhibition of EGFR by its tyrosine inhibitor decreased HIF-1α, suggesting that EGFR positively regulates HIF-1α. In cancer cells, Epithelial-Mesenchymal Transition(EMT) endows cells with increased invasiveness and tumor-initiating capacity, contributing to metastasis and resistance to therapeutics. Slug, one of the EMT transcription factors maintains cell plasticity during carcinogenesis. Our results showed that ISO treatment decreased Slugprotein levels in A549 and H23 cells. Transcription factor SOX2 regulates cancer cell proliferation, EMT, migration, invasion, metastasis, tumor initiation, cancer stem cell formation as well as resistance to apoptosis and therapy. Our results showed that ISO decreased SOX2 expression. Knockdown of MEG3 increased the expressions of Slug and SOX2, indicating that MEG3 negatively regulates Slug and SOX2. In conclusion, ISO elevated MEG3, inhibiting EGFR, HIF-1α, Slug, and SOX2, suppressing the migration and invasion of lung cancer cells. Citation Format: Sophia Shi, Zhuo Zhang, Jingxia Li, Huailu Tu, Max Costa. Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB049.

Cancer stem cell, chromosomal instability, and cancer immunity

Cancer stem cells (CSCs) are a unique population of tumor cells with stem cell-like properties. They are believed to be involved in drug resistance, potential therapy failure, tumor relapse after treatment, and ultimately reduced overall survival of cancer patients. One of the causal factors that may lead to CSC formation is chromosomal instability (CIN), a dynamic event leading to numerical and structural changes in the chromosomes. The CIN is also proposed to aid the maintenance of CSCs, contribute towards their heterogeneity, and facilitate their immune escape. However, the role of CIN in the modulation of the immune system in tumors remains contradictory. Studies have revealed that it can lead to both activation and suppression of the immune system. Previous literature suggests that the CIN, CSCs, and cancer immunity (3Cs), interact with and complement each other to create a pro-tumor environment. However, the mechanisms underlying such an interaction are poorly understood. So, in this review article, an attempt has been made to understand the nature of the interaction between the triad of CIN, CSC, and the immune response in tumors and some of the pathways governing the same. Understanding the above may be a positive step towards the complete cure for malignant diseases.

Abstract 4755: Extracellular GRP78 inhibition reverses drug and immune resistance in cancer cells

Abstract CBT300 was created from Creative BioTherapeutics’ discovery that drug resistant recurrent cancer cells up-regulate a survival pathway that results in the expression of extracellular Glucose-Regulated Protein 78 (ecGRP78) in the tumor microenvironment (TME). This TME ecGRP78 binds to cancer and immune cell surfaces, which induces a cascade of events to increase drug resistance, immune suppression, and cancer stem cell (CSC) formation. CBT300 targets surface bound ecGRP78 that has been found on breast, lung, ovarian, prostate, melanoma, multiple myeloma, colon, pediatric and adult brain tumors. We can now show that inhibition of ecGRP78 can a) induce apoptosis of drug resistant tumor cells, b) eliminate drug and immune resistance showing synergistic effects with chemotherapy and immunotherapy, c) decrease the amount of chemotherapy about 90% in combination with CBT300. Recent publications show that ecGRP78 is found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, cell surface bound ecGRP78 is important for many aspects of cancer development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. Recently, it has been shown that increased ecGRP78 expression in metastatic breast cancer, glioblastoma and multiple myeloma patients was significantly associated with later stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival. In studies to help understand how ecGRP78 causes tumor progression and drug and immune resistance, we discovered a novel ecGRP78 binding transmembrane protein on TNBC, and brain cancer cells called Receptor Tyrosine Kinase Orphan Receptor-1 (ROR1). Using the GRP78 binding domain from ROR1 and a human Fc domain, we created a biologic fusion protein that is a potent and specific ecGRP78 inhibitor called CBT300. We now show that CBT300’s elimination of csGRP78 destabilizes and removes oncofetal proteins ROR1, and Cripto, and checkpoint protein PD-L1 from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. Our results with CBT300 demonstrate proof of concept data in vitro and in vivo for our novel ecGRP78 inhibitor on several drug resistant cancers demonstrating the potential to provide a major advance in the treatment of drug resistant cancers either alone or in combination with lower doses of chemotherapy. Exploiting a novel mechanism of action with a non-toxic, efficacious and cost effective biologic therapy that has shown increased survival in recurrent cancers will bring new hope for these patients. Citation Format: Donald J. Davidson, Jorryn Zelek, Elizabeth Stolarik, Mariah Thigpen, Anita Davidson, DPT. Extracellular GRP78 inhibition reverses drug and immune resistance in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4755.

Abstract 2978: Extracellular ATP induced senescence in human lung cancer cells

Abstract In tumors, extracellular ATP (eATP) concentrations are 1,000 to 10,000 times higher than is found in normal tissues and eATP is up taken through macropinocytosis leading to a large increase in intracellular ATP levels. eATP is also linked to the TGF-β signaling pathway as exocytosis of ATP is induced that is then used in an autocrine fashion activating the P2 × 7 purinergic receptor1. Through these mechanisms, eATP has also been recently shown to induce EMT, CSC formation, and drug resistance1. One of the mechanisms through which these processes may be induced is a senescent cell precursor2. Senescence is a generally irreversible epigenetically controlled process characterized by a complete exit from the cell cycle in response to either DNA damage, stressful conditions, or aging. Recent mounting evidence has shown that senescence can be a cancer promoting mechanism by entering this state in response to stress such as during chemotherapy, but they can utilize it to enhance their malignant capabilities, including the ability to escape their senescent state with heightened stem cell characteristics, which can lead to a higher chance of relapse after treatment2. Senescent cells have recently been named a hallmark of cancer3. This study shows the time dependent induction of a senescent like state in human lung cancer A549 cells after only 2 hours of treatment with 0.5mM eATP, the concentration found in the tumor microenvironment, through analysis of transcriptional markers using total polyA RNAseq as well as chromogenic and flow cytometric assays for senescence associated beta-galactosidase activity. Additionally, inhibitors of macropinocytosis and purinergic receptors have been used to gain insight into the mechanisms of action that this pathway takes. This demonstrates a novel use of ATP within the tumor microenvironment for aiding in cancer progression and drug resistance. After EMT, CSC formation, and drug resistance, senescence is added to the long list of eATP mediated cancer promoting processes. 1. Song J, Qian Y, Evers M, Nielsen CM, Chen X. Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors. IJMS. 2022;23(23):14770. doi:10.3390/ijms232314770 2. Zhang D, Monteiro MJ, Liu J, Gu W. Mechanisms of cancer stem cell senescence: Current understanding and future perspectives. Clin Exp Pharma Physio. 2021;48(9):1185-1202. doi:10.1111/1440-1681.13528 3. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discovery. 2022;12(1):31-46. doi:10.1158/2159-8290.CD-21-1059 Citation Format: Ryan A. Ward, Arabella Hunt, Nora Anderson, Xiaozhuo Chen. Extracellular ATP induced senescence in human lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2978.

Anti-Tumor Efficacy of Oleuropein-Loaded ZnO/Au Mesoporous Silica Nanoparticle in 5-FU-Resistant Colorectal Cancer Cells.

5-fluorouracil (5-FU) is a first-line chemotherapeutic agent used to treat colorectal cancer (CRC). However, 5-FU induces drug resistance and activation of cancer stem cells (CSCs). In the present study, we designed a novel biocompatible nanomedicine system with high efficacy and biocompatibility by synthesizing mesoporous silica nanoparticle (MSN)-structured ZnO and gold ions. Oleuropein (OLP) is a phenolic compound derived from olive leaves that exerts anti-cancer effects. Therefore, we synthesized OLP-loaded ZnO/Au MSNs (ZnO/Au/OLP MSNs) and examined their anti-cancer effects on 5-FU-resistant CRC cells. ZnO/Au MSNs were synthesized and functionalized, and their physical and chemical compositions were characterized using UV-visible spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). Their effects were assessed in terms of cellular proliferation capacity, migration and invasion ability, colony-forming ability, spheroid-forming ability, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. ZnO/Au MSNs were mostly composed of various ions containing ZnO and gold ions, had a spheroid phenotype, and exhibited no cytotoxicity. ZnO/Au/OLP MSNs reduced cell viability and CSC formation and induced apoptosis of 5-FU-resistant CRC cells via necrosis via ROS accumulation and DNA fragmentation. ZnO/Au/OLP MSNs exhibited anti-cancer activity by upregulating necrosis. These results revealed that ZnO/Au/OLP MSNs are a novel drug delivery system for 5-FU CRC therapy.

Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

Hypoxia in solid tumors is an important source of chemoresistance that can determine poor patient prognosis. Such chemoresistance relies on the presence of cancer stem cells (CSCs), and hypoxia promotes their generation through transcriptional activation by HIF transcription factors. We used ovarian cancer (OC) cell lines, xenograft models, OC patient samples, transcriptional databases, induced pluripotent stem cells (iPSCs) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Here, we show that hypoxia induces CSC formation and chemoresistance in ovarian cancer through transcriptional activation of the PLD2 gene. Mechanistically, HIF-1α activates PLD2 transcription through hypoxia response elements, and both hypoxia and PLD2 overexpression lead to increased accessibility around stemness genes, detected by ATAC-seq, at sites bound by AP-1 transcription factors. This in turn provokes a rewiring of stemness genes, including the overexpression of SOX2, SOX9 or NOTCH1. PLD2 overexpression also leads to decreased patient survival, enhanced tumor growth and CSC formation, and increased iPSCs reprograming, confirming its role in dedifferentiation to a stem-like phenotype. Importantly, hypoxia-induced stemness is dependent on PLD2 expression, demonstrating that PLD2 is a major determinant of de-differentiation of ovarian cancer cells to stem-like cells in hypoxic conditions. Finally, we demonstrate that high PLD2 expression increases chemoresistance to cisplatin and carboplatin treatments, both in vitro and in vivo, while its pharmacological inhibition restores sensitivity. Altogether, our work highlights the importance of the HIF-1α-PLD2 axis for CSC generation and chemoresistance in OC and proposes an alternative treatment for patients with high PLD2 expression.

Diverse temporal and spatial mechanisms work, partially through Stanniocalcin-1, V-ATPase and senescence, to activate the extracellular ATP-mediated drug resistance in human cancer cells.

Resistance to drug therapies is associated with a large majority of cancer-related deaths. ATP-binding cassette (ABC) transporter-mediated drug efflux, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs), glutathione (GSH), senescence, and vacuole-type ATPase (V-ATPase) all contribute to the resistance. We recently showed that extracellular ATP (eATP) induces and regulates EMT, CSC formation, and ABC transporters in human cancer cells and tumors. eATP also consistently upregulates Stanniocalcin-1 (STC1), a gene that significantly contributes to EMT, CSC formation, and tumor growth. We also found that eATP enhances drug resistance in cancer cells through eATP internalization mediated by macropinocytosis, leading to an elevation of intracellular ATP (iATP) levels, induction of EMT, and CSC formation. However, these factors have never been systematically investigated in the context of eATP-induced drug resistance. In this study, we hypothesized that eATP increases drug resistance via inducing ABC efflux, EMT, CSCs, STC1, and their accompanied processes such as GSH reducing activity, senescence, and V-ATPase. RNA sequencing, metabolomics, gene knockdown and knockout, and functional assays were performed to investigate these pathways and processes. Our study results showed that, in multiple human cancer lines, eATP induced genes involved in drug resistance, elevated ABC transporters' efflux activity of anticancer drugs; generated transcriptomic and metabolic profiles representing a drug resistant state; upregulated activities of GSH, senescence, and V-ATPase to promote drug resistance. Collectively, these newly found players shed light on the mechanisms of eATP-induced as well as STC1- and V-ATPase-mediated drug resistance and offer potential novel targets for combating drug resistance in cancers.

Hypoxia-induced circSTT3A enhances serine synthesis and promotes H3K4me3 modification to facilitate breast cancer stem cell formation

Hypoxia is a key feature of tumor microenvironment that contributes to the development of breast cancer stem cells (BCSCs) with strong self-renewal properties. However, the specific mechanism underlying hypoxia in BCSC induction is not completely understood. Herein, we provide evidence that a novel hypoxia-specific circSTT3A is significantly upregulated in clinical breast cancer (BC) tissues, and is closely related to the clinical stage and poor prognosis of patients with BC. The study revealed that hypoxia-inducible factor 1 alpha (HIF1α)-regulated circSTT3A has a remarkable effect on mammosphere formation in breast cancer cells. Mechanistically, circSTT3A directly interacts with nucleotide-binding domain of heat shock protein 70 (HSP70), thereby facilitating the recruitment of phosphoglycerate kinase 1 (PGK1) via its substrate-binding domain, which reduces the ubiquitination and increases the stability of PGK1. The enhanced levels of PGK1 catalyze 1,3-diphosphoglycerate (1,3-BPG) into 3-phosphoglycerate (3-PG) leading to 3-PG accumulation and increased serine synthesis, S-adenosylmethionine (SAM) accumulation, and trimethylation of histone H3 lysine 4 (H3K4me3). The activation of the H3K4me3 contributes to BCSCs by increasing the transcriptional level of stemness-related factors. Especially, our work reveals that either loss of circSTT3A or PGK1 substantially suppresses tumor initiation and tumor growth, which dramatically increases the sensitivity of tumors to doxorubicin (DOX) in mice. Injection of PGK1-silenced spheroids with 3-PG can significantly reverse tumor initiation and growth in mice, thereby increasing tumor resistance to DOX. In conclusion, our study sheds light on the functional role of hypoxia in the maintenance of BCSCs via circSTT3A/HSP70/PGK1-mediated serine synthesis, which provides new insights into metabolic reprogramming, tumor initiation and growth. Our findings suggest that targeting circSTT3A alone or in combination with chemotherapy has potential clinical value for BC management.

Hyperbaric Oxygen Therapy Adjuvant Chemotherapy and Radiotherapy through Inhibiting Stemness in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor in adults. Despite the advances in GBM treatment, outcomes remain poor, with a 2-year survival rate of less than 5%. Hyperbaric oxygen (HBO) therapy is an intermittent, high-concentration, short-term oxygen therapy used to increase cellular oxygen content. In this study, we evaluated the effects of HBO therapy, alone or combined with other treatment modalities, on GBM in vitro and in vivo. In the in vitro analysis, we used a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess the effects of HBO therapy alone, a colony formation assay to analyze the effects of HBO therapy combined with radiotherapy and with temozolomide (TMZ), and a neurosphere assay to assess GBM stemness. In the in vivo analysis, we used immunohistochemical staining and in vivo bioluminescence imaging to assess GBM stemness and the therapeutic effect of HBO therapy alone or combined with TMZ or radiotherapy, respectively. HBO therapy did not affect GBM cell viability, but it did reduce the analyzed tumors' ability to form cancer stem cells. In addition, HBO therapy increased GBM sensitivity to TMZ and radiotherapy both in vitro and in vivo. HBO therapy did not enhance tumor growth and exhibited adjuvant effects to chemotherapy and radiotherapy through inhibiting GBM stemness. In conclusion, HBO therapy shows promise as an adjuvant treatment for GBM by reducing cancer stem cell formation and enhancing sensitivity to chemotherapy and radiotherapy.

Tumor microenvironment-induced tumor cell plasticity: relationship with hypoxic stress and impact on tumor resistance.

The role of tumor interaction with stromal components during carcinogenesis is crucial for the design of efficient cancer treatment approaches. It is widely admitted that tumor hypoxic stress is associated with tumor aggressiveness and thus impacts susceptibility and resistance to different types of treatments. Notable biological processes that hypoxia functions in include its regulation of tumor heterogeneity and plasticity. While hypoxia has been reported as a major player in tumor survival and dissemination regulation, the significance of hypoxia inducible factors in cancer stem cell development remains poorly understood. Several reports indicate that the emergence of cancer stem cells in addition to their phenotype and function within a hypoxic tumor microenvironment impacts cancer progression. In this respect, evidence showed that cancer stem cells are key elements of intratumoral heterogeneity and more importantly are responsible for tumor relapse and escape to treatments. This paper briefly reviews our current knowledge of the interaction between tumor hypoxic stress and its role in stemness acquisition and maintenance. Our review extensively covers the influence of hypoxia on the formation and maintenance of cancer stem cells and discusses the potential of targeting hypoxia-induced alterations in the expression and function of the so far known stem cell markers in cancer therapy approaches. We believe that a better and integrated understanding of the effect of hypoxia on stemness during carcinogenesis might lead to new strategies for exploiting hypoxia-associated pathways and their targeting in the clinical setting in order to overcome resistance mechanisms. More importantly, at the present time, efforts are oriented towards the design of innovative therapeutical approaches that specifically target cancer stem cells.

Differential gene expression of immunity and inflammation genes in colorectal cancer using targeted RNA sequencing.

Colorectal cancer (CRC) is a heterogeneous disease caused by molecular changes, as driver mutations, gene methylations, etc., and influenced by tumor microenvironment (TME) pervaded with immune cells with both pro- and anti-tumor effects. The studying of interactions between the immune system (IS) and the TME is important for developing effective immunotherapeutic strategies for CRC. In our study, we focused on the analysis of expression profiles of inflammatory and immune-relevant genes to identify aberrant signaling pathways included in carcinogenesis, metastatic potential of tumors, and association of Kirsten rat sarcoma virus (KRAS) gene mutation. A total of 91 patients were enrolled in the study. Using NGS, differential gene expression analysis of 11 tumor samples and 11 matching non-tumor controls was carried out by applying a targeted RNA panel for inflammation and immunity genes containing 475 target genes. The obtained data were evaluated by the CLC Genomics Workbench and R library. The significantly differentially expressed genes (DEGs) were analyzed in Reactome GSA software, and some selected DEGs were used for real-time PCR validation. After prioritization, the most significant differences in gene expression were shown by the genes TNFRSF4, IRF7, IL6R, NR3CI, EIF2AK2, MIF, CCL5, TNFSF10, CCL20, CXCL11, RIPK2, and BLNK. Validation analyses on 91 samples showed a correlation between RNA-seq data and qPCR for TNFSF10, RIPK2, and BLNK gene expression. The top differently regulated signaling pathways between the studied groups (cancer vs. control, metastatic vs. primary CRC and KRAS positive and negative CRC) belong to immune system, signal transduction, disease, gene expression, DNA repair, and programmed cell death. Analyzed data suggest the changes at more levels of CRC carcinogenesis, including surface receptors of epithelial or immune cells, its signal transduction pathways, programmed cell death modifications, alterations in DNA repair machinery, and cell cycle control leading to uncontrolled proliferation. This study indicates only basic molecular pathways that enabled the formation of metastatic cancer stem cells and may contribute to clarifying the function of the IS in the TME of CRC. A precise identification of signaling pathways responsible for CRC may help in the selection of personalized pharmacological treatment.

Ionizing radiation normalizes the features of active breast cancer stromal fibroblasts and suppresses their paracrine pro-carcinogenic effects

BackgroundRadiotherapy is an important therapeutic strategy for breast cancer patients through reducing the chances of recurrence and metastasis, which are fueled by cancer-associated fibroblasts (CAFs). Thereby, we addressed here the effect of various doses of X-rays on breast CAFs and their adjacent counterparts. MethodsWe have used WST1 and annexin V-associated with flow cytometry to test the cytotoxic effects of X-rays. Immunoblotting and ELISA was used to assess the expression/secretion of various proteins. Immunohistochemistry was utilized to determine the level of β-galactosidase and Ki-67. Sphere formation assay was used to test the ability of breast cancer cells to form tumorspheres. Orthotopic tumor xenografts were also used to evaluate the effect of X-ray-treated breast stromal fibroblasts on breast cancer tumor growth in vivo. ResultsBreast stromal fibroblasts showed high resistance to X-rays. While the low dose (5 Gy) inhibited cell proliferation and the active features of CAFs, the higher doses (16 and 50 Gy) promoted senescence. However, this was not accompanied by the senescence-associated secretory phenotype (SASP), but rather a reduction in the synthesis/secretion of various cancer-associated cytokines. Additionally, X-rays suppressed the features of active breast stromal fibroblasts, and their paracrine pro-carcinogenic effects. The ablative dose (16 Gy) inhibited the capacity of active stromal fibroblasts to promote the pro-metastatic processes epithelial-to-mesenchymal transition, the formation of cancer stem cells, as well as the growth of humanized orthotopic breast tumor xenografts. ConclusionTogether, these findings indicate that X-rays can normalize the features of active breast stromal fibroblasts through promoting senescence without SASP.

Transcriptional factor BRD4 promotes the stemness of esophageal cancer by activating the nuclear PD-L1/RelB axis.

Esophageal cancer (EC) is a prevalent malignancy associated with therapeutic resistance and poor prognosis. This study investigates the role of programmed death-ligand 1 (PD-L1) in esophageal cancer stem cell (ECSC) formation. ECSCs were enriched and characterized using various assays. We found that both PD-L1 and bromodomain-containing protein 4 (BRD4) were upregulated in ECSCs, promoting their stemness. Inhibiting BRD4 suppressed ECSC markers expression and sphere formation. Furthermore, BRD4 inhibitors downregulated membrane and nuclear PD-L1 levels, with knockdown of PD-L1 inhibiting ECSC formation. PD-L1 degraders also affected PD-L1 and its downstream effector RelB expression. Moreover, inhibiting RelB influenced sphere formation through interleukin-6 expression. This study reveals the critical role of the BRD4/nuclear PD-L1/RelB axis in ECSC formation, highlighting nuclear PD-L1 as a potential immunotherapeutic target for refractory EC.

Monofunctional dimetallic Ru(η6-arene) complexes inhibit NOTCH1 signaling pathway and synergistically enhance anticancer effect in combination with cisplatin or vitamin C

ONS donor ligands L1-L4 were utilized in the preparation of monofunctional dimetallic Ru(η6-arene) complexes (C1–C4). These ONS donor ligand based novel tricoordinated Ru(II) complexes bearing η6-arene co-ligand were prepared for the first time. The current methodology resulted in excellent isolated yields and these complexes were characterized in detail by different spectroscopic and spectrometric techniques. The structures of C1–C2 and C4 were characterized in solid state by single crystal X-ray analysis. The in vitro anticancer analyses showed these novel complexes suppressed the growth of breast (MCF-7), liver (HepG2) and lung (A549) cancer cells. C2 suppressed the growth of these cells in dose-dependent manner revealed form the MTT and crystal violet cell viability assays. Moreover, C2 was observed the most potent complex that was used further in detailed mechanistic analyses in cancer cells. C2 showed good cytotoxic activity at 10 μM dose level as compared to cisplatin or oxaliplatin in these cancer cells. We observed morphological changes in cancer cells upon treatment with C2. Moreover, C2 suppressed the invasion and migration ability of cancer cells. C2 induced cellular senescence to retard cell growth and suppressed the formation of cancer stem cells. Importantly, C2 showed synergistic anticancer effect in combination with cisplatin and Vitamin C to further inhibit cell growth which suggested the potential role of C2 in cancer therapy. Mechanistically, C2 inhibited NOTCH1 dependent signaling pathway to suppress cancer cell invasion, migration and cancer stem cells formation. Thus, these data suggested potential role of C2 in cancer therapy by targeting NOTCH1-dependent signaling to suppress tumorigenesis. The results obtained in this study for these novel monofunctional dimetallic Ru(η6-arene) complexes showed their high anticancer potency and this study will pave to further cytotoxicity exploration on this class of complexes.

Latest Findings on the Effect of Gastrointestinal Microecology Remodeling of Tumor Microenvironment on Tumor Stemness

Gastrointestinal microecology (GM) system is composed of normal gut microbiota and its living environment. The impact of GM on human health and many diseases has been widely studied. The impact of GM system on tumors is mainly reflected in the remodeling of the tumor microenvironment (TME). TME, a special microenvironment that tumors live in, can regulate the characteristics of tumor cells and affect the occurrence and development of tumors through intercellular contact and the secretion of cytokines. At present, cancer stem cell (CSC) model is considered an important theory that explains the origin and malignant progression of tumors. The formation and proliferation of CSC usually represent increased tumor invasion, metastasis, and chemotherapy resistance, resulting in poor clinical prognosis in patients. Therefore, it is important to study the role and mechanism through which GM system affects the acquisition of CSC characteristics through remodeling TME, thereby affecting tumor invasion, metastasis, and chemotherapy resistance. Studies on this topic are of great significance for clinical understanding of tumor malignant progression and improving tumor treatment outcomes. However, due to the low content of single bacteria in the gastrointestinal model, high heterogeneity, and difficulty in tracing distant metastasis, there are still great limitations in the previous research. Herein, we reviewed the research progress in the effect of GM remodeling of TME on the acquisition of tumor stemness, tumor invasion and metastasis, and the resistance to chemotherapy.

Amino acid profile changes during enrichment of spheroid cells with cancer stem cell properties in MCF-7 and MDA-MB-231 cell lines.

Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis. This study aimed to evaluate the changes in the amino acid profile in MCF-7 and MDA-MB-231 cells during spheroid formation to discover the specific metabolic properties in CSCs. MCF-7 and MDA-MB-231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high-performance liquid chromatography (HPLC). The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF-7 and MDA-MB-231 spheroids (histidine and serine, respectively). Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.

An Isoxazoloquinone Derivative Inhibits Tumor Growth by Targeting STAT3 and Triggering Its Ubiquitin-Dependent Degradation.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with shorter five-year survival than other breast cancer subtypes, and lacks targeted and hormonal treatment strategies. The signal transducer and activator of transcription 3 (STAT3) signaling is up-regulated in various tumors, including TNBC, and plays a vital role in regulating the expression of multiple proliferation- and apoptosis-related genes. By combining the unique structures of the natural compounds STA-21 and Aulosirazole with antitumor activities, we synthesized a class of novel isoxazoloquinone derivatives and showed that one of these compounds, ZSW, binds to the SH2 domain of STAT3, leading to decreased STAT3 expression and activation in TNBC cells. Furthermore, ZSW promotes STAT3 ubiquitination, inhibits the proliferation of TNBC cells in vitro, and attenuates tumor growth with manageable toxicities in vivo. ZSW also decreases the mammosphere formation of breast cancer stem cells (BCSCs) by inhibiting STAT3. We conclude that the novel isoxazoloquinone ZSW may be developed as a cancer therapeutic because it targets STAT3, thereby inhibiting the stemness of cancer cells.