Cancer Stem Cell Population Research Articles

Nitric oxide facilitates the S-nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality, with tumour heterogeneity, fueled by cancer stem cells (CSCs), intricately linked to treatment resistance. Therefore, it is imperative to advance therapeutic strategies targeting CSCs in NSCLC. In this study, we utilized RNA sequencing to investigate metabolic pathway alterations in NSCLC CSCs and identified a crucial role of nitric oxide (NO) metabolism in governing CSC stemness, primarily through modulation of the Notch1 protein. Mechanistically, NO-induced S-nitrosylation of Notch1 facilitated its interaction with the deubiquitylase UCHL1, leading to increased Notch1 protein stability and enhanced CSC stemness. By inhibiting NO synthesis and downregulating UCHL1 expression, we validated the impact of NO on the Notch signalling pathway and CSC stemness. Importantly, targeting NO effectively reduced CSC populations within patient-derived organoids (PDOs) during radiotherapy. This mechanism presents a promising therapeutic target to surmount radiotherapy resistance in NSCLC treatment.

Leveraging Neural Crest-Derived Tumors to Identify NF1 Cancer Stem Cell Signatures.

Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes individuals to develop benign and malignant tumors of the nerve sheath. Understanding the signatures of cancer stem cells (CSCs) for NF1-associated tumors may facilitate the early detection of tumor progression. Background: Neural crest cells, the cell of origin of NF1-associated tumors, can initiate multiple tumor types, including melanoma, neuroblastoma, and schwannoma. CSCs within these tumors have been reported; however, identifying and targeting CSC populations remains a challenge. Results: This study aims to leverage existing studies on neural crest-derived CSCs to explore markers pertinent to NF1 tumorigenesis. By focusing on the molecular and cellular dynamics within these tumors, we summarize CSC signatures in tumor maintenance, progression, and treatment resistance. Conclusion: A review of these signatures in the context of NF1 will provide insights into NF1 tumor biology and pave the way for developing targeted therapies and improving treatment outcomes for NF1 patients.

Veratridine, a plant-derived alkaloid, suppresses the hyperactive Rictor-mTORC2 pathway: a new targeted therapy for primary and metastatic colorectal cancer.

Despite considerable advances to improve colorectal cancer (CRC) survival over the last decade, therapeutic challenges remain due to the rapid metastatic dissemination of primary tumors and screening limitations. Meanwhile, the rise of CRC in younger adults (Early-onset CRC), commonly diagnosed with a metastatic form of the disease, shows the pressing need to develop more effective targeted therapies to decrease the high mortality rates associated with metastatic disease. Hyperactivation of the Rictor-mTORC2-AKT signaling pathway drives key metastatic players in diverse malignant tumors, including early- and late-onset colorectal cancer. Selective mTORC2 inhibitors are becoming a potential treatment strategy for CRC due to the therapeutic limitations of mTORC1 inhibitors. Veratridine (VTD), a lipid-soluble alkaloid extracted from Liliaceae plants, can transcriptionally increase UBXN2A, which induces 26S proteasomal degradation of the Rictor protein, a key member in the mTORC2 complex. Destabilization of Rictor protein by VTD decreases Akt phosphorylation on Ser 473 , which is responsible for metastatic signaling downstream of the mTORC2 pathway in diverse malignant tumors. VTD decreases the population of metastatic colon cancer stem cells and functions as an angiogenesis inhibitor. VTD effectively reduces the spheroid growth rate and restricts cell migration. Live cell migration and invasion assays alongside biomechanical-force-based experiments revealed that VTD suppresses colon cancer cell invasiveness and the ensuing risk of tumor metastasis. A CRC mouse model that mimics the natural stages of human sporadic CRC revealed that VTD treatment significantly decreases tumor growth in a UBXN2A-dependent manner. This study showed a novel mechanistic connection between a ubiquitin-like protein and mTORC2-dependent migration and invasion in CRC tumors. This study revealed the therapeutic benefit of selective inhibition of Rictor in CRC, particularly in tumors with a hyperactive Rictor-mTORC2 signaling pathway. Finally, this study opened a new platform for repurposing VTD, a supplemental anti-hypertension molecule, into an effective targeted therapy in CRC tumors.

Development of a novel N14-substituted antitumor evodiamine derivative with inhibiting heat shock protein 70 in non-small cell lung cancer

Notwithstanding the latest advancements in anticancer therapy, non-small cell lung cancer (NSCLC) remains a prominent contributor to cancer-associated mortality worldwide. Therefore, effective anti-cancer agents are required for the treatment of NSCLC. We previously demonstrated that the natural alkaloid evodiamine efficiently suppressed lung cancer cells and lung cancer stem-like cell populations by suppressing heat shock protein 70 (Hsp70). This finding inspired us to formulate evodiamine-based anti-cancer compounds against NSCLC. In this study, we synthesized a series of evodiamine derivatives with substitutions at the N14 position. EV206 was chosen for further study because it was the most effective among the 22 evodiamine derivatives at stopping H1299 cell growth. EV206 treatment efficiently suppressed cell viability and colony formation in both attached cells and in soft agar, even in those carrying drug resistance, by inducing apoptosis. The effectiveness of EV206 is approximately ten times greater than that of evodiamine. Normal cell viability was marginally affected by EV206 treatment. Additionally, EV206 efficiently decreased the cancer stem cell (CSC) population in the NSCLC cells. EV206 reduced the growth of H460 xenograft tumors without exhibiting toxic effects. These data implied that EV206 has the potential to be an effective Hsp70-targeting anticancer drug with low toxicity.

Unveiling the Potential of Hyptis Capitata and Hyptis Brevipes Compounds in Overcoming Apoptosis Resistance and Inducing Targeted Cell Cycle Arrest in Breast Cancer Stem Cells

Breast cancer stem cells (CSC), as a kind of tumor cells, are able to regenerate themselves, leading to apoptosis resistance and cancer relapse. In this study, we investigated the potential of Hyptis Capitata and Hyptis Brevipes compounds in preventing apoptosis resistance and inducing targeted cell cycle arrest in CSC. The cytotoxic effects of the compounds were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and the cell cycle distribution and apoptosis induction were assessed through flow cytometry analysis. The phytochemical characterization of Hyptis bravipes extract (HBE) and Hyptis capitata extract (HCE) conducted under thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis. The results revealed that Hyptis Brevipes extract (HBE) exhibited strong cytotoxicity towards CSCs, with an IC50 value of 89 µg/mL. In contrast, Hyptis Capitata extract (HCE) showed weak growth inhibition, with an IC50 value greater than 500 µg/mL. Subsequent analysis demonstrated that HBE induced cell cycle arrest in the G2/M and S phases and significantly increased apoptosis in the CSC population. Furthermore, phytochemical characterization using TLC and HPLC revealed that HBE and HCE share similar constituent compounds, with HBE exhibiting a more diverse compound content and stronger cytotoxic effects. These findings provide valuable insights into the potential of Hyptis Capitata and Hyptis Brevipes compounds as targeted agents against breast cancer stem cells and contribute to understanding their chemical profiles and pharmacological properties. HIGHLIGHTS Hyptis brevipes extract induced cell cycle arrest in G2/M and S phases. Cytotoxicity of Hyptis brevipes leading to apoptosis induction on CSC Hyptis brevipes significantly targeted Breast Cancer Stem Cells GRAPHICAL ABSTRACT

Dysgerminomas: germ cell tumors exhibit high expression of PD-L1 and associated with high TILs and good prognosis

Ovarian germ cell tumors (OVGCTs) account for 28% of all diagnosed ovarian cancers, and malignant germ cell tumors specifically account for approximately 13% of diagnosed ovarian cancers in Saudi Arabia. Although most germ cell tumor patients have a high survival rate, patients who experience tumor recurrence have a poor prognosis and present with more aggressive and chemoresistant tumors. The use of immunotherapeutic agents such as PD-L1/PD-1 inhibitors for OVGCTs remains very limited because few studies have described the immunological characteristics of these tumors. This study is the first to investigate PD-L1 expression in ovarian germ cell tumors and explore the role of PD-L1 expression in tumor microenvironment cells and genetic alterations. A total of 34 ovarian germ cell tumors were collected from pathology archives. The collected tumor tissues included ten dysgerminomas, five yolk sac tumors, five immature teratomas, and one mature teratoma, and the remaining samples were mixed germ cell tumors. The tumors were analyzed using immunohistochemical analysis to determine PD-L1 expression, immune cell infiltration and cancer stem cell populations and their correlation with clinical outcome. Furthermore, the genetic alterations in different subtypes of germ cell tumors were correlated with PD-L1 expression and clinical outcome. Datasets for testicular germ cells (TGCTs) were retrieved from The Cancer Genome Atlas (TCGA) and analyzed using cBioPortal (cbioportal.org) and Gene Expression Profiling Interactive Analysis (GEPIA). Compared with yolk sac tumors, dysgerminomas highly express PD-L1 and are associated with high levels of tumor infiltrating lymphocytes (TILs) and stem cell markers. In addition, compared with PD-L1-negative yolk sac tissue, dysgerminomas/seminomas with high PD-L1 expression are associated with more genetic alterations and a better prognosis. Our findings will contribute to the knowledge about the potential benefits of ovarian cancer immunotherapy in specific subsets of germ cell tumor patients and the risk factors for resistance mediated by tumor microenvironment cells.

KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines

Glioblastoma is a deadly tumor which possesses glioblastoma stem cell populations involved in temozolomide (TMZ) resistance. To gain insight into the mechanisms of self-renewing and therapy-resistant cancer stem cells, subcellular proteomics was utilized to identify proteins whose expression is enriched in U251-derived glioblastoma stem-like cells. The KH RNA Binding Domain Containing, Signal Transduction Associated 3, KHDRBS3, was successfully identified as a gene up-regulated in the cancer stem cell population compared with its differentiated derivatives. Depletion of KHDRBS3 by RNA silencing led to a decrease in cell proliferation, neurosphere formation, migration, and expression of genes involved in glioblastoma stemness. Importantly, TMZ sensitivity can be induced by the gene knockdown. Collectively, our results highlight KHDRBS3 as a novel factor associated with self-renewal of glioblastoma stem-like cells and TMZ resistance. As a consequence, targeting KHDRBS3 may help eradicate glioblastoma stem-like cells.

Engineered Biomimetic Nanovesicles Synergistically Remodel Folate-Nucleotide and γ-Aminobutyric Acid Metabolism to Overcome Sunitinib-Resistant Renal Cell Carcinoma.

Reprogramming of cellular metabolism in tumors promoted the epithelial-mesenchymal transition (EMT) process and established immune-suppressive tumor microenvironments (iTME), leading to drug resistance and tumor progression. Therefore, remodeling the cellular metabolism of tumor cells was a promising strategy to overcome drug-resistant tumors. Herein, CD276 and MTHFD2 were identified as a specific marker and a therapeutic target, respectively, for targeting sunitinib-resistant clear cell renal cell carcinoma (ccRCC) and its cancer stem cell (CSC) population. The blockade of MTHFD2 was confirmed to overcome drug resistance via remodeling of folate-nucleotide metabolism. Moreover, the manganese dioxide nanoparticle was proven here by a high-throughput metabolome to be capable of remodeling γ-aminobutyric acid (GABA) metabolism in tumor cells to reconstruct the iTME. Based on these findings, engineered CD276-CD133 dual-targeting biomimetic nanovesicle EMφ-siMTHFD2-MnO2@Suni was designed to overcome drug resistance and terminate tumor progression of ccRCC. Using ccRCC-bearing immune-humanized NPG model mice, EMφ-siMTHFD2-MnO2@Suni was observed to remodel folate-nucleotide and GABA metabolism to deactivate the EMT process and reconstruct the iTME thereby overcoming the drug resistance. In the incomplete-tumor-resection recurrence model and metastasis model, EMφ-siMTHFD2-MnO2@Suni reduced recurrence and metastasis in vivo. This work thus provided an innovative approach that held great potential in the treatment of drug-resistant ccRCC by remodeling cellular metabolism.

Bisphenol A-induced oxidative stress increases the production of ovarian cancer stem cells in mice

Bisphenol A (BPA) belongs to the endocrine disruptor chemicals (EDCs) causing various reproductive disorders in females. We analysed the toxic effects of BPA in the uterus and ovaries. The BPA was administered orally with the repeated low dose (LD, 1 mg/kg) and high dose (HD, 5 mg/kg) of body weight on alternate days for 4 months via oral gavage to Swiss mice. BPA administration decreases body weight, ovarian weight and size at LD, but increases ovarian weight and size at HD. The uterus weight, length, and diameter were increased in both the treated groups. The histopathological data show decreased ovarian follicle size, epithelial hyperplasia, and lymphocytic infiltration in the ovary. The BPA-treated uterus shows increased vascularization, atrophied endometrium and myometrium, and endometrial hyperplasia (EH) with aberrant glandular growth. The cancer stem cells (CSCs) in the ovaries were identified based on staining with anti-mouse CD44 and anti-mouse CD133 antibodies and analysed by flow cytometry. Three different populations of ovarian CSCs: CD44+CD133-, CD44+CD133+, and CD44−CD133+, can be recognised based on the intensity of these receptors. CD44+CD133- and CD44+CD133+ cell percentages were increased in BPA-treated groups. CD44−CD133+ were increased in LD but decreased in HD. The BPA administration also induces ROS production, which decreases the expression of antioxidant genes Superoxide dismutase 1 (SOD1), Superoxide dismutase 2 (SOD2), Catalase (CAT), Glutathione peroxidase 1 (GPX1), and Forkhead box O3 (FOXO3) in ovarian cells. In conclusion, BPA exposure induced an inflammatory response, increased CSC proportions, induced ROS, and decreased antioxidant responses in the ovaries.

Abstract B086: Patient-derived pancreatic tumor organoids as a tool to evaluate cancer stem cell populations and their role in therapeutic resistance

Abstract Introduction: Pancreatic ductal adenocarcinoma remains one of the most lethal forms of cancer with a five-year survival rate of less than 15%. Regardless of surgery eligibility, most patients receive systemic chemo-radiation therapy, which includes FOLFIRINOX or gemcitabine and nab-paclitaxel, often followed by radiation therapy (RT) with or without 5-FU. Unfortunately, patients’ tumors frequently develop resistance to these therapies, and it’s often unclear as to why. One potential source of resistance and recurrence is cancer stem cell (CSC) perseverance following treatment. Purpose: The purpose of this study was to characterize and treat three patient-derived pancreatic tumor organoids (IDs: 8510, 7800, and 11777) with RT and LD50 doses of relevant chemotherapy drugs to evaluate the treatment response. Furthermore, we sought to examine the impact of individual and combination treatments (i.e., chemo-RT) on the population of CSCs in these organoids. Methods: Organoids were cultured in 3D BME gel domes and grown in an NCI-recommended media formulation. Genome sequencing of 505 relevant genes was performed using a PGDx kit. The dose responses of three organoids to radiation (2-12 Gy) and several chemotherapy approaches were determined via MTT assay. CSCs were identified as those cells staining positive for markers SOX2 and OCT4. The population of CSCs before and after chemotherapy, RT, and combined chemo-RT were evaluated with immunofluorescence, western blotting, and flow cytometry. Results: The data revealed a variation in the responses of tumor organoids to treatments, as demonstrated by different LD50 doses. For instance, tumor organoid 8510 exhibited higher tolerance to 5-FU and FOLFIRINOX treatments compared to organoid 7800. Interestingly, organoid 11777 showed resistance to RT, unlike organoids 8510 and 7800. Clinical genomic sequencing revealed that organoid 11777 showed unique variants of several genes known to be involved with DNA repair, including PIM1, RAD54L, and SLX4, as well as mutations in the ARID1b and TGFBR2 genes. The latter two genes may have implications for radiation resistance and could explain the unique resistance of organoid 11777 to RT. Further investigation is warranted. Regarding the population of CSCs, the immunofluorescence data has confirmed the presence of SOX2 and OCT4 in these organoids, and the immunoblot data showed an upregulation of SOX2 and OCT4 in tumor organoids treated with radiation alone. Flow cytometry analysis confirmed that the percentage of CSCs increased following radiation treatment; however, the CSC population was slightly decreased following treatment with FOLFIRINOX and significantly decreased after combination treatment with FOLFIRINOX and RT. Conclusions: Patient-derived pancreatic tumor organoids can be used as a surrogate to provide molecular insights and therapeutic sensitivity profiles in individual patients. Additionally, while the data suggests a link between CSCs and RT resistance, the combination approach results in a more pronounced inhibition of the CSC population. Citation Format: Zachery L Keepers, Sanjit Roy, William Ryan, Binny Bhandary, Narottam Lamichhane, France Carrier, Ramaswamy K Iyer, Jason K Molitoris, William F Regine, Hem D Shukla. Patient-derived pancreatic tumor organoids as a tool to evaluate cancer stem cell populations and their role in therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B086.

LncRNA BASP1-AS1 is a positive regulator of stemness and pluripotency in human SH-SY5Y neuroblastoma cells

Neuroblastoma is the most common extra-cranial solid tumor diagnosed mostly in children below the age of five years and comprises of about 15 % of all paediatric cancer deaths. Tumor initiating cancer stem cells (CSCs) can be targeted for better treatment approaches. BASP1-AS1 is a long non coding (Lnc) RNA that is a divergent LncRNA for its coding gene brain abundant membrane attached signal protein 1 (BASP1). We had earlier demonstrated it to be expressed in foetus derived human neural progenitor cells (hNPCs), where it was a positive regulator of BASP1 and was critical for neural differentiation. In this study, we have investigated the role of BASP1-AS1 in CSCs derived from the human neuroblastoma cell line SH-SY5Y. We cultured SH-SY5Y cells on Poly-d-Lysine coated flasks in serum free media supplemented with growth factors, which led to the enrichment of CSCs as determined by marker expression. When grown on ultra-low attachment flasks, these cells formed CSCs enriched neurospheres. We examined the effects of BASP1-AS1 siRNA mediated knockdown on CSCs enriched SH-SY5Y cells and SH-SY5Y derived neurospheres. BASP1-AS1 knockdown decreased the levels of the corresponding gene BASP1 and the rate of cell proliferation of CSCs enriched cells along with low expression of Ki67. It also reduced the mRNA levels of stem cell and pluripotency gene markers (CD133, CD44, c-KIT, SOX2, OCT4 and NANOG), as also Wnt 2 and the Wnt pathway effector β catenin. It also abrogated the formation of neurospheres in ultra-low attachment flasks. A similar effect on proliferation and stemness related properties was seen on BASP1 knockdown. BASP1-AS1 and its related pathways may provide a point of intervention for the CSCs population in neuroblastoma.

ALDH1A3 is the switch that determines the balance of ALDH+ and CD24−CD44+ cancer stem cells, EMT-MET, and glucose metabolism in breast cancer

Plasticity is an inherent feature of cancer stem cells (CSCs) and regulates the balance of key processes required at different stages of breast cancer progression, including epithelial-to-mesenchymal transition (EMT) versus mesenchymal-to-epithelial transition (MET), and glycolysis versus oxidative phosphorylation. Understanding the key factors that regulate the switch between these processes could lead to novel therapeutic strategies that limit tumor progression. We found that aldehyde dehydrogenase 1A3 (ALDH1A3) regulates these cancer-promoting processes and the abundance of the two distinct breast CSC populations defined by high ALDH activity and CD24−CD44+ cell surface expression. While ALDH1A3 increases ALDH+ breast cancer cells, it inversely suppresses the CD24−CD44+ population by retinoic acid signaling-mediated gene expression changes. This switch in CSC populations induced by ALDH1A3 was paired with decreased migration but increased invasion and an intermediate EMT phenotype. We also demonstrate that ALDH1A3 increases oxidative phosphorylation and decreases glycolysis and reactive oxygen species (ROS). The effects of ALDH1A3 reduction were countered with the glycolysis inhibitor 2-deoxy-D-glucose (2DG). In cell culture and tumor xenograft models, 2DG suppresses the increase in the CD24−CD44+ population and ROS induced by ALDH1A3 knockdown. Combined inhibition of ALDH1A3 and glycolysis best reduces breast tumor growth and tumor-initiating cells, suggesting that the combination of targeting ALDH1A3 and glycolysis has therapeutic potential for limiting CSCs and tumor progression. Together, these findings identify ALDH1A3 as a key regulator of processes required for breast cancer progression and depletion of ALDH1A3 makes breast cancer cells more susceptible to glycolysis inhibition.

NNMT overexpression is an adverse prognostic factor in uterine leiomyosarcoma.

Uterine leiomyosarcomas (uLMS) are extremely rare high-grade tumors with a poor prognosis. Their etiopathogenesis remains largely unknown. The uterus is the most frequent site for LMS. uLMS and uterine leiomyoma (uLM) must frequently be differentiated in patients with a uterine mass. Nicotinamide N-methyltransferase (NNMT), a cytoplasmic protein, is involved in the progression and spread of a variety of cancer types. The expression of NNMT in a mesenchymal malignancy was not examined previously. This study represents the first investigation into NNMT expression in uLMS, uLM and benign uterine myometrium and correlates NNMT overexpression with worse prognosis in uLMS. The expression of NNMT was investigated by immunohistochemistry on formalin-fixed paraffin-embedded tissue of uLMS in 31 patients, uLM in seven patients and benign myometrial in 31 patients. The expression of NNMT in uLMS was markedly higher than in uLM and normal myometrial tissue (p < 0.001). The expression of NNMT in early stage uLMS was lower than in advanced stage disease (p = 0.034). NNMT expression was an independent prognostic factor in predicting recurrence-free survival in uLMS (p = 0.037). NNMT can aid in the preoperative differentiation of uLMS and uLM. The consequences of NNMT overexpression, such as the activation and inactivation of oncoproteins and tumor suppressor proteins, respectively, as well as the enrichment of the cancer stem cell population, overlap with the major mechanisms responsible for poor prognosis in mesenchymal tumors. NNMT may be investigated further in the context of antitumor treatment in patients with mesenchymal malignancies.

Unlocking the Therapeutic Potential of Oral Cancer Stem Cell-Derived Exosomes.

Oral cancer (OC) presents a significant global health burden with rising incidence rates. Despite advancements in diagnosis and treatments, the survival rate for OC patients, particularly those with advanced or recurrent disease, remains low at approximately 20%. This poor prognosis is often due to a small population of cancer stem cells (CSCs) that are capable of self-renewal and immune evasion, playing pivotal roles in proliferation, tumor initiation, progression, metastasis, and therapy resistance. Exosomes, which are nano-sized extracellular vesicles (EVs), have emerged as crucial mediators of cell-to-cell communication within the tumor microenvironment (TME). These vesicles carry diverse molecules such as DNA, RNA, proteins, lipids, and metabolites, influencing various cellular processes. Emerging evidence suggests that CSC-derived EVs significantly promote tumor progression and metastasis and maintain the balance between CSCs and non-CSCs, which is vital for intracellular communication within the TME of oral cancer. Recent reports indicate that oral cancer stem cell-derived EVs (OCSC-EVs) influence stemness, immune evasion, metastasis, angiogenesis, tumor reoccurrence, and drug resistance. Understanding OCSC-EVs could significantly improve oral cancer diagnosis, prognosis, and therapy. In this mini-review, we explore OCSC-derived exosomes in oral cancer, examining their potential as diagnostic and prognostic biomarkers that reflect CSC characteristics, and delve into their therapeutic implications, emphasizing their roles in tumor progression and therapy resistance. However, despite their promising potential, several challenges remain, including the need to standardize isolation and characterization methods and to elucidate exosome-mediated mechanisms. Thus, a comprehensive understanding of OCSC-EVs could pave the way for innovative therapeutic strategies that have the potential to improve clinical outcomes for OC patients.

The Peptidoglycan Recognition Protein 1 confers immune evasive properties on pancreatic cancer stem cells

ObjectivePancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant ‘stem-like’ cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending...

Radio-miRs: a comprehensive view of radioresistance-related microRNAs.

Radiotherapy is a key treatment option for a wide variety of human tumors, employed either alone or alongside with other therapeutic interventions. Radiotherapy uses high-energy particles to destroy tumor cells, blocking their ability to divide and proliferate. The effectiveness of radiotherapy is due to genetic and epigenetic factors that determine how tumor cells respond to ionizing radiation. These factors contribute to the establishment of resistance to radiotherapy, which increases the risk of poor clinical prognosis of patients. Although the mechanisms by which tumor cells induce radioresistance are unclear, evidence points out several contributing factors including the overexpression of DNA repair systems, increased levels of reactive oxygen species, alterations in the tumor microenvironment, and enrichment of cancer stem cell populations. In this context, dysregulation of microRNAs or miRNAs, critical regulators of gene expression, may influence how tumors respond to radiation. There is increasing evidence that miRNAs may act as sensitizers or enhancers of radioresistance, regulating key processes such as the DNA damage response and the cell death signaling pathway. Furthermore, expression and activity of miRNAs have shown informative value in overcoming radiotherapy and long-term radiotoxicity, revealing their potential as biomarkers. In this review, we will discuss the molecular mechanisms associated with the response to radiotherapy and highlight the central role of miRNAs in regulating the molecular mechanisms responsible for cellular radioresistance. We will also review radio-miRs, radiotherapy-related miRNAs, either as sensitizers or enhancers of radioresistance that hold promise as biomarkers or pharmacological targets to sensitize radioresistant cells.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of the Long Noncoding RNA HOTAIR and Lysine Methyltransferase EZH2.

Persistence of cancer stem cells (CSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse in ovarian cancer, the fifth leading cause of cancer-related death among US women. HOXC transcript antisense RNA (HOTAIR) is a long noncoding RNA (lncRNA) overexpressed in high-grade serous ovarian cancer and linked to chemoresistance. However, HOTAIR impacts chromatin dynamics in ovarian CSCs and how this oncogenic lncRNA contributes to drug resistant disease are incompletely understood. Here we generated HOTAIR knock-out (KO) high-grade serous ovarian cancer cell lines using paired CRISPR guide RNA design to investigate the function of HOTAIR. We show loss of HOTAIR function re-sensitized ovarian cancer cells to platinum treatment and decreased the population of ovarian CSCs. Furthermore, HOTAIR KO inhibited development of stemness-related phenotypes, including spheroid formation ability, as well as expression of key stemness-associated genes ALDH1A1, NOTCH3, SOX9, and PROM1. HOTAIR KO altered both the cellular transcriptome and chromatin accessibility landscape of multiple oncogenic-associated genes and pathways, including the NF-kB pathway. HOTAIR functions as an oncogene by recruiting enhancer of zeste 2 (EZH2) to catalyze H3K27 tri-methylation to suppress downstream tumor suppressor genes, and it was of interest to inhibit both HOTAIR and EZH2. In vivo, combining a HOTAIR inhibitor with an EZH2 inhibitor and platinum chemotherapy decreased tumor formation and increased survival. These results suggest a key role for HOTAIR in ovarian CSCs and malignant potential. Targeting HOTAIR in combination with epigenetic therapies may represents therapeutic strategy to ameliorate ovarian cancer progression and resistance to platinum-based chemotherapy.

PP2A B55α inhibits epithelial-mesenchymal transition via regulation of Slug expression in non-small cell lung cancer

PP2A B55α, encoded by PPP2R2A, acts as a regulatory subunit of the serine/threonine phosphatase PP2A. Despite a frequent loss of heterozygosity of PPP2R2A in cases of non-small cell lung cancer (NSCLC), research on PP2A B55α′s functions remains limited and controversial. To investigate the biological roles of PP2A B55α, we conducted bulk RNA-sequencing to assess the impact of PPP2R2A knockdown using two shRNAs in a NSCLC cell line. Gene set enrichment analysis (GSEA) of the RNA-sequencing data revealed significant enrichment of the epithelial-mesenchymal transition (EMT) pathway, with SNAI2 (the gene encoding Slug) emerging as one of the top candidates. Our findings demonstrate that PP2A B55α suppresses EMT, as PPP2R2A deficiency through knockdown or homozygous or hemizygous depletion promotes EMT and metastatic behavior in NSCLC cells, as evidenced by changes in EMT biomarkers, invasion and migration abilities, as well as metastasis in a tail vein assay. Mechanistically, PP2A B55α inhibits EMT by downregulating SNAI2 expression via the GSK3β-β-catenin pathway. Importantly, PPP2R2A deficiency also slows cell proliferation by disrupting DNA replication, particularly in PPP2R2A−/− cells. Furthermore, PPP2R2A deficiency, especially PPP2R2A−/− cells, leads to an increase in the cancer stem cell population, which correlates with enhanced resistance to chemotherapy. Overall, the decrease in PP2A B55α levels due to hemizygous/homozygous depletion heightens EMT and the metastatic or stemness/drug resistance potential of NSCLC cells despite their proliferation disadvantage. Our study highlights the significance of PP2A B55α in EMT and metastasis and suggests that targeting EMT/stemness could be a potential therapeutic strategy for treating PPP2R2A-deficient NSCLC.

Suppress the cell growth of cancer stem-like cells (NTERA-2) using Sox2-Oct4 decoy oligodeoxynucleotide−encapsulated niosomes-zinc hybrid nanocarriers under X-irradiation

Sox2 and Oct4 dysregulations could significantly increase in the cancer stem cell (CSC) population in some cancer cells and resistance to common treatments. In this study, the synergistic effects of Sox2-Oct4 decoy oligodeoxynucleotides-encapsulated Niosomes-zinc hybrid nanocarriers along with X‐irradiation conditions as a combinational therapy tool were investigated in the treatment of cancer-like stem cells (NTERA-2). The NTERA-2 cell line known as a cancer-like stem cell line was used in this investigation. Sox2-Oct4 decoy oligodeoxynucleotides were designed based on the sequence of the Sox2 promoter and synthesized. Physicochemical characteristics of ODNs-encapsulated niosomes-zinc hybrid nanocarriers (NISM@BSA-DEC-Zn) investigated with FT-IR, DLS, FESEM, and ODNs release kinetic estimation assays. Further investigations such as hemolysis, uptake, cell viability, apoptosis, cell cycle, and scratch repair tests were performed. All the above assays were completed with and without X-ray exposure conditions (fractionated 2Gy). Physicochemical characteristics results showed that the Niosomes-Zn nanocarriers were successfully synthesized. NISM@BSA-DEC-Zn was efficiently taken up by NTERA-2 cells and significantly inhibited cell growth, increased apoptosis, and reduced cell migration in both conditions (with and without X-ray exposure). Furthermore, NISM@BSA-DEC-Zn treatment resulted in G1 and G2/M cell cycle arrest without and with X-irradiation, respectively. The prepared nanocarrier system can be a promising tool for drug delivery in cancer treatment. Decoy ODN strategy along with zinc nanoparticles could increase the sensitivity of cancer cells toward irradiation, which has the potential for combinational cancer therapies.

Atypical cell cycle regulation promotes mammary stem cell expansion during mammary development and tumourigenesis

BackgroundThe cell cycle of mammary stem cells must be tightly regulated to ensure normal homeostasis of the mammary gland to prevent abnormal proliferation and susceptibility to tumorigenesis. The atypical cell cycle regulator, Spy1 can override cell cycle checkpoints, including those activated by the tumour suppressor p53 which mediates mammary stem cell homeostasis. Spy1 has also been shown to promote expansion of select stem cell populations in other developmental systems. Spy1 protein is elevated during proliferative stages of mammary gland development, is found at higher levels in human breast cancers, and promotes susceptibility to mammary tumourigenesis when combined with loss of p53. We hypothesized that Spy1 cooperates with loss of p53 to increase susceptibility to tumour initiation due to changes in susceptible mammary stem cell populations during development and drives the formation of more aggressive stem like tumours.MethodsUsing a transgenic mouse model driving expression of Spy1 within the mammary gland, mammary development and stemness were assessed. These mice were intercrossed with p53 null mice to study the tumourigenic properties of Spy1 driven p53 null tumours, as well as global changes in signaling via RNA sequencing analysis.ResultsWe show that elevated levels of Spy1 leads to expansion of mammary stem cells, even in the presence of p53, and an increase in mammary tumour formation. Spy1-driven tumours have an increased cancer stem cell population, decreased checkpoint signaling, and demonstrate an increase in therapy resistance. Loss of Spy1 decreases tumor onset and reduces the cancer stem cell population.ConclusionsThis data demonstrates the potential of Spy1 to expand mammary stem cell populations and contribute to the initiation and progression of aggressive, breast cancers with increased cancer stem cell populations.