Expression Of Stemness-associated Genes Research Articles

LINC00662 Promotes Aggressive Traits by Modulating OCT4 Expression through miR-335-5p in Gallbladder Cancer Cells.

Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133+/CD44+ cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.

Injectable collagen hydrogel combines human umbilical cord mesenchymal stem cells to promote endometrial regeneration in rats with thin endometrium

The regeneration of thin endometrium still remains as a great challenge in the field of reproductive medicine. Stem cells-based therapy has been considered as a promising strategy for the restoration of thin endometrium. However, the low transplantation and retention rate of stem cells and loss of stemness due to in vitro expansion limits the therapeutic efficacy. In our study, we combined collagen hydrogel and human umbilical cord mesenchymal stem cells (uMSCs) for improving the regeneration of thin endometrium, by using the potent pluripotency and low immunogenicity of uMSCs and collagen hydrogel that promotes the anchorage and proliferation of stem cells. Results showed that collagen hydrogel has favorable biocompatibility and the capacity to enhance the cell viability and expression of stemness-associated genes (including organic cation/carnitine transporter4 (Oct-4), Nanog homeobox (Nanog) and SRY-box transcription factor 2 (SOX2)) of uMSCs. The combination of collagen hydrogel and uMSCs prolonged the retention time of the constructs in the uterine cavity and improved endometrial thickness compared with uMSCs alone, leading to increase the fertility of the rats with thin endometrium. These highlighted therapeutic prospects of collagen hydrogel combined with uMSCs for the minimally invasive therapy of thin endometrium in the clinic.

Targeting EZH2 regulates the biological characteristics of glioma stem cells via the Notch1 pathway.

Glioma is the most common malignant brain tumor, and its behavior is closely related to the presence of glioma stem cells (GSCs). We found that the enhancer of zeste homolog 2 (EZH2) is highly expressed in glioma and that its expression is correlated with the prognosis of glioblastoma multiforme (GBM) in two databases: The Cancer Genome Atlas and the Chinese Glioma Genome Atlas. Additionally, EZH2 is known to regulate the stemness-associated gene expression, proliferation, and invasion ability of GSCs, which may be achieved through the activation of the STAT3 and Notch1 pathways. Furthermore, we demonstrated the effect of the EZH2-specific inhibitor GSK126 on GSCs; these results not only corroborate our hypothesis, but also provide a potential novel treatment approach for glioma.

Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.

Abstract 2440: HOTAIR functionality and regulation in ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. We have previously shown that epigenetic changes in OCSCs play a role in post-therapy OCSC persistence and demonstrated the potential to target OCSC using epigenetic therapies. HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in high-grade serous OC (HGSOC). HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and plays a key role in chromatin remodeling. Because HOTAIR is a known epigenetic regulator of differentiation and developmental genes in OC, we hypothesized that HOTAIR regulates OCSCs and epigenetic targeting HOTAIR will prevent tumor relapse. To produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR in a panel high grade serous OC cell lines (Kuramochi, OVCAR3) without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P<0.001) the OCSC population and stemness-related phenotypes, including inhibiting stemness-associated gene expression ALDH1A1, Notch3, Sox9, and PROM1 and in vitro spheroid forming ability under low attachment conditions. Integrated analysis of RNA-seq and ATAC-seq on control and HOTAIR knockout cells revealed HOTAIR altered global chromatin dynamics to change downstream gene expression, particularly, the NF-kB pathway. In xenograft assays using HGSOC cells, combining a HOTAIR inhibitor with an EZH2 inhibitor and chemotherapy significantly decreased in vivo tumor formation and increased mouse survival. In summary, depletion of HOTAIR functionally decreased OCSC phenotypes and malignant potential and reprogrammed HGSOC to a less stem-like phenotype. There is an urgent need to develop new therapeutic strategies to target OCSCs and overcome chemoresistant OC, and the results of this study suggest that targeting HOTAIR in combination with epigenetic therapies may represent a novel therapeutic strategy to prevent tumor relapse in OC. Citation Format: Weini Wang, Yanchi Zhou, Ji Wang, Ali Ozes, Heather M. O’Hagan, Anirban Mitra, Kenneth P. Nephew. HOTAIR functionality and regulation in ovarian cancer stem cells [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 2440.

Indolocarbazole alkaloid Loonamycin A inhibits triple-negative breast cancer cell stemness and Notch signalling.

Enrichment for therapy-resistant cancer stem cells hampers the treatment of triple-negative breast cancer. Targeting these cells via suppression of Notch signalling can be a potential therapeutic strategy. This study aimed to uncover the mode of action of a new indolocarbazole alkaloid loonamycin A against this incurable disease. The anticancer effects were examined in triple-negative breast cancer cells using in vitro methods, including cell viability and proliferation assays, wound-healing assay, flow cytometry and mammosphere formation assay. RNA-seq technology was used to analyse the gene expression profiles in loonamycin A-treated cells. Real-time RT-PCR and western blot were to evaluate the inhibition of Notch signalling. Loonamycin A has stronger cytotoxicity than its structural analog rebeccamycin. Besides inhibiting cell proliferation and migration, loonamycin A reduced CD44high/CD24low/- sub-population, mammosphere formation, as well as the expression of stemness-associated genes. Co-administration of loonamycin A enhanced antitumour effects of paclitaxel by inducing apoptosis. RNA sequencing results showed that loonamycin A treatment caused the inhibition of Notch signalling, accompanied by the decreased expression of Notch1 and its targeted genes. These results reveal a novel bioactivity of indolocarbazole-type alkaloids and provide a promising Notch-inhibiting small molecular candidate for triple-negative breast cancer therapy.

Identification of eIF6 as a prognostic factor that drives tumor progression and predicts arsenic trioxide efficacy in lung adenocarcinoma

BackgroundLung cancer is the leading cause of cancer-related mortality worldwide. Dysregulation of mRNA translation can contribute to the development and progression of cancer whilst also having an impact on the prognosis of different types of malignancies. Eukaryotic translation initiation factors (eIFs) have been reported to serve a key role in the initiation of mRNA translation. However, little was known about the association between eIF6 and lung adenocarcinoma (LUAD) progression. We aimed to elucidate the roles of eIF6 in LUAD tumorigenesis.MethodsBioinformatic analysis was conducted to assess the clinical significance of eIF6 in LUAD. CCK-8, colony formation assays were used to evaluate the biological roles of eIF6. The subcutaneous model was used to assess the in vivo roles of eIF6.ResultsIn the present study, it was found that eIF6 expression was significantly higher in LUAD samples compared with that in normal lung tissues. Higher expression levels of eIF6 were found to be associated with more advanced clinical stages of LUAD and poorer prognoses in patients with LUAD. Subsequently, overexpression of eIF6 was demonstrated to promote LUAD cell proliferation, migration and invasion, which are features of metastasis, in vitro. By contrast, inhibition of eIF6 induced cell cycle arrest and apoptosis in LUAD cells. Further bioinformatics analysis and experimental assays revealed that eIF6 expression positively correlated with the mRNA expression of stemness-associated genes in LUAD cells. Targeting eIF6 suppressed the sphere formation capacity of LUAD cells. In addition, data from the subcutaneous xenograft model in vivo also suggested that eIF6 deficiency could significantly delay tumor growth and improve the prognosis of mice. Targeting eIF6 rendered LUAD cells sensitive to arsenic trioxide treatment.ConclusionThe present study suggest that eIF6 can serve as a prognostic biomarker and a potential therapeutic target for patients with LUAD.

EID3 Promotes Cancer Stem Cell-Like Phenotypes in Osteosarcoma through the Activation of PI3K-AKT Signaling Pathway.

The aim of this study is to elucidate molecular mechanism by which E1A-like inhibitor of differentiation 3 (EID3) promotes cancer stem cell-like phenotypes in osteosarcoma. Overexpression of EID3 in osteosarcoma cells generated more spherical clones, enhanced the expression of stemness-associated genes, and promoted chemoresistance, invasion, and metastasis. Furthermore, osteosarcoma cells overexpressing EID3 had increased ability to grow in suspension as osteospheres with high expression of Sox2 and stem cell marker CD133. In addition, knockdown of EID3 reduced sphere formation and inhibited osteosarcoma cell migration and invasion. RNA sequencing and bioinformatics analysis revealed that PI3K-Akt signaling pathway and MAPK pathway­related genes were enriched in osteosarcoma cells with high expression of EID3. Taken together, EID3 promotes osteosarcoma, and EID3–PI3K-Akt axis is a potential therapeutic target for osteosarcoma treatment.

Abstract 6063: FOXK2 promotes stemness in ovarian cancer by regulating unfolded protein response signaling

Abstract Introduction: Cancer stem cells (CSCs), a rare cell population within tumors that possesses self-renewal and tumor initiation capacity, have been implicated in cancer relapse and resistance to chemotherapy. In ovarian cancer (OC), CSCs are characterized by a high expression of aldehyde dehydrogenase (ALDH). Understanding the regulatory programs promoting stemness could identify new treatments. We identified FOXK2 as a key transcription factor (TF) that promotes stemness in OC. Methods: We generated FOXK2 knockdown OC models by stable transduction of shRNA targeting this TF and measured the population of ALDH+ CSCs, expression of stemness-associated genes, and in vivo tumor initiation capacities. Transcriptomic and ChIP-seq analyses were used to identify key FOXK2 targets. CRISPR-dCas9 genome editing was used to verify the regulatory function of FOXK2. Rescue experiments validated the roles of a newly discovered target gene of FOXK2. Results: Through gene expression analyses in CSCs vs. non-CSCs, we identified FOXK2 as a highly expressed stemness-specific TF in OC. Genetic depletion of FOXK2 diminished stemness features and reduced in vivo tumor initiation capacity. Through ChIP-sequencing we discovered that FOXK2 directly regulates IRE1α (ERN1 gene) expression, a key sensor for the unfolded protein response (UPR). ChIP-sequencing analysis further revealed that FOXK2 binds to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a CRISPR/dCas9 diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 mRNA splicing and activation of stemness pathways, while genetic or pharmacological blockade of IRE1α inhibited CSCs in OC models. Conclusion: Our data establish a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in ovarian CSCs. Citation Format: Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J. Tanner, Mazhar Adli, Daniela E. Matei. FOXK2 promotes stemness in ovarian cancer by regulating unfolded protein response signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6063.

Epiregulin increases stemness-associated genes expression and promotes chemoresistance of non-small cell lung cancer via ERK signaling

BackgroundChemoresistance often causes the failure of treatment and death of patients with advanced non-small-cell lung cancer. However, there is still no resistance genes signature and available enriched signaling derived from a comprehensive RNA-Seq data analysis of lung cancer patients that could act as a therapeutic target to re-sensitize the acquired resistant cancer cells to chemo-drugs. Hence, in this study, we aimed to identify the resistance signature for clinical lung cancer patients and explore the regulatory mechanism.MethodAnalysis of RNA-Seq data from clinical lung cancer patients was conducted in R studio to identify the resistance signature. The resistance signature was validated by survival time of lung cancer patients and qPCR in chemo-resistant cells. Cytokine application, small-interfering RNA and pharmacological inhibition approaches were applied to characterize the function and molecular mechanism of EREG and downstream signaling in chemoresistance regulation via stemness.ResultsThe RTK and vitamin D signaling were enriched among resistance genes, where 6 genes were validated as resistance signature and associated with poor survival in patients. EREG/ERK signaling was activated by chemo-drugs in NSCLC cells. EREG protein promoted the NSCLC resistance to chemo-drugs by increasing stemness genes expression. Additionally, inhibition of EREG/ErbB had downregulated ERK signaling, resulting in decreased expression of stemness-associated genes and subsequently re-sensitized the resistant NSCLC cells and spheres to chemo-drugs.ConclusionsThese findings revealed 6 resistance genes signature and proved that EREG/ErbB regulated the stemness to maintain chemoresistance of NSCLC via ERK signaling. Therefore, targeting EREG/ErbB might significantly and effectively resolve the chemoresistance issue.

SiRNA-mediated silencing of Nanog reduces stemness properties and increases the sensitivity of HepG2 cells to cisplatin

Liver cancer is one of the most lethal cancers having worldwide prevalence. Despite significant progress in cancer therapy, liver cancer-induced mortality is very high. Nanog, as an essential transcription factor modulating cellular multipotency, causes tumor progression, drug resistance, and preserves stemness properties in various tumors such as liver cancer. Thus, this research was conducted to evaluate the impact of combination therapy of Nanog siRNA/cisplatin on the sensitivity of liver cancer cells to this drug. HepG2 cells were transfected with Nanog siRNA and treated with cisplatin, individually and in combination. Then, it was observed that in transfected HepG2 cells, Nanog expression was significantly reduced at mRNA level and also these cells were sensitized to cisplatin. In addition, to assess the impact of Nanog siRNA and cisplatin individually and in combination on cells’ viability, migration capacity, apoptosis, and cell cycle progression, the MTT, wound healing, colony formation assay, Annexin V/PI staining, and flow cytometry assays were applied on HepG2 cells, respectively. Also, the quantitive Real-Time PCR was used to check the expression of stemness-associated genes (CD44, CD133, and Sox2), and apoptosis-related genes (caspase-3, 8, 9, BAX and Bcl2) after combination therapy. It is indicated that the combination of Nanog siRNA and cisplatin significantly reduced proliferation, migration, and colony formation ability, as well as increased apoptosis rate, and cell cycle arrest. Also, it is found that the combination of Nanog siRNA and cisplatin down-regulated the expression of stemness-associated genes and up-regulated apoptosis-related genes in HepG2 cells. Hence, it can be suggested that Nanog inhibition in combination with cisplatin is a potential therapeutic strategy for developing new therapeutic approaches for liver cancer.

Identification of microRNA expression profiles of CD44+ ovarian cancer stem cells.

The aim of our study was to investigate microRNA (miRNA) expression profiles in CD44+ ovarian cancer stem cells (ovarian CSCs). In this study, we enriched CD44+ ovarian CSCs using magnetic activated cell sorting (MACS). A combination of real-time quantitative PCR (qRT-PCR), western blot and sphere formation assays was used to demonstrate stem cell-like properties. RNA sequencing was used to detect the miRNA expression profiles in CD44+ ovarian CSCs. Transient transfection, qRT-PCR, western blot and sphere formation assays were further used to test the function of miR-181a-2-3p. We found that CD44+ ovarian CSCs showed enhanced sphere formation and expression of stemness-associated genes (NANOG, OCT4, SOX2) compared to ovarian cancer cells. The RNA sequencing results showed that the miRNA expression profiles of CD44+ ovarian CSCs were different from those of ovarian cancer cells. GO and KEGG pathway analyses indicated that these miRNAs regulate stem cell-like properties in CD44+ ovarian CSCs. In addition, miR-181a-2-3p negatively regulates the stem cell-like properties of CD44+ ovarian CSCs by targeting EGR1. Our data suggest that miRNAs play important roles in regulating the stem cell-like properties of CD44+ ovarian CSCs.

MUC1-C Dictates JUN and BAF-Mediated Chromatin Remodeling at Enhancer Signatures in Cancer Stem Cells.

These findings show that MUC1-C, which is necessary for the CRPC and TNBC CSC state, activates a novel pathway involving JUN/AP-1 and ARID1A/BAF that regulates chromatin accessibility of stemness-associated gene enhancers.

Tadalafil Reverses the Effect of Three-Dimensional Cell Culture System on Stem Cell Features in A549 and SK-MES-1.

Background: Non-small cell lung cancer is the most common type of lung cancer and is a frequent cause of death. In our research, A549 and SK-MES-1 were used to assess the effect of three-dimensional (3D) culture compared to that of two-dimensional (2D) monolayers in cell proliferation, migration, and invasion, response to chemotherapy, as well as the expression of epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC)-related markers. As tadalafil is a phosphodiesterase type 5 (PDE5) inhibitor with the potential to target CSC maintenance in multiple cancer cell lines, we assessed its function in 3D culture and detected the downstream pathway genes. Methods: We compared the viability and proliferation capacity of A549 and SK-MES-1 cells in 2D and 3D culture by cell counting kit (CCK)-8, foci formation, and Live/Dead double stain (Operetta CLS High content screening). Migration, invasion, and other functions were also assessed. To elucidate the underlying mechanisms, the expression of EMT and CSC markers was analyzed by quantitative real-time PCR (qPCR) and Western blot. Results: A549 and SK-MES-1 cells formed spheroids heterogeneous in shape and size. In our 3D spheroid systems, cells went through EMT, and were also capacitated with higher stemness and chemoresistance. Combination use of tadalafil and cisplatin showed higher chemotherapy efficiency in the 3D model, compared to that of the 2D cell culture. Conclusion: Our research aims at the notable differences between these two cellular systems in terms of cell functions, EMT, and stemness-associated gene expression and chemo-response. We showed that a commonly used drug, tadalafil, showed more pronounced inhibitory effects when cells were cultured in the 3D model. Since the 3D culture system could imitate the in vivo behavior of cancer cells within the tumor, we advocate that this system is superior to traditional 2D culture and should be used in the investigation of new therapeutic compounds.

E2F1 Maintains Gastric Cancer Stemness Properties by Regulating Stemness-Associated Genes.

Purpose To determine the regulatory role of E2F1 in maintaining gastric cancer stemness properties and the clinical significance of E2F1 in gastric cancer. Materials and Methods We conducted a tumor spheroid formation assay to enrich gastric cancer stem-like cells. The protein and mRNA expression levels of genes were measured using Western Blot and qRT-PCR. Lentivirus-mediated overexpression and downregulation of E2F1 were performed to evaluate the effect of E2F1 on the stemness properties of gastric cancer cells. The effect of E2F1 on gastric cancer cell sensitivity of 5-Fu was evaluated using cell viability assay and TdT-mediated dUTP Nick-End Labeling staining. We also analyzed the association between E2F1 expression and clinical characteristics in gastric cancer patients. The KM plotter database was used to analyze the relationship between E2F1 and overall survival in GC patients. Results We found that E2F1 expression was significantly higher in gastric cancer tissues than in the paired adjacent normal tissues (p < 0.05) and was positively correlated with tumor size (p < 0.05), T stage (p < 0.05), and differentiation degree (p < 0.05). KM plotter database demonstrated a close association between higher E2F1 expression level and worse overall survival of gastric cancer patients (p < 0.05). In vitro assay illustrated that E2F1 could regulate the expression of stemness-associated genes, such as BMI1, OCT4, Nanog, and CD44, and maintain the tumor spheroid formation ability of gastric cancer cells. E2F1 enhanced 5-Fu resistance in gastric cancer cells, and the E2F1 expression level was correlated with the prognosis of gastric cancer patients receiving 5-Fu therapy. The expression levels of stemness-associated genes were also significantly higher in gastric cancer tissues than the paired adjacent normal tissues (p < 0.05). A positive correlation was observed between E2F1 and BMI1 (r = 0.422, p < 0.05), CD44 (r = 0.634, p < 0.05), OCT4 (r = 0.456, p < 0.05), and Nanog (r = 0.337, p < 0.05) in gastric cancer tissues. The co-overexpression of E2F1 and stemness-associated genes was associated with worse overall survival. Conclusion E2F1 plays a significant role in gastric cancer progression by maintaining gastric cancer stemness properties through the regulation of stemness-associated genes. The close association between E2F1 and poor prognosis of patients suggests that E2F1 could serve as a prognostic biomarker and a therapeutic target in gastric cancer patients.

The α2δ1 subunit of the voltage-gated calcium channel acts as a potential candidate for breast cancer tumor initial cells biomarker.

The voltage-gated calcium channel subunit alpha 2 delta 1 (α2δ1) is a functional tumor initial cells (TICs) marker for some solid cancer cells. This study aimed to investigate whether α2δ1 can be used as a potential TIC marker for breast cancer cells. α2δ1+ and α2δ1- cells were identified and sorted from the breast cancer cell lines MDA-MB-231, MDA-MB-435s and ZR-75-1 by Immunofluorescence (IF) and Fluorescent-activated cell sorting (FACS) analyses. Spheroid formation in vitro and tumorigenesis in NOD/SCID mice were assessed to determine the self-renewal and serial transplantation abilities of these cells. Using a lentivirus infection system for α2δ1 in breast cancer cell lines, we determined the mRNA levels of stemnessassociated genes by quality real-time PCR (qRT-PCR). Boyden chamber and wounding assays were further performed to detect the migration of α2δ1 overexpression cells. Bioinformatics explored the relationship of molecular classification of breast cancer and drug resistance. α2δ1 presents on the cytomembrane of breast cancer cells, with a positive rate of 1.5-3%. The α2δ1+ cells in breast cancer cell lines have a stronger self-renewal ability and tumor initiating properties in vitro and in vivo. Overexpressing α2δ1 successfully enhanced the sphere-forming efficiency, and upregulated the expression of stemness-associated genes, and increased cell migration. However, seldom significant was available between estrogen receptor +/- (ER+/-), progesterone receptor (PR+/-), and Her2+/-. Breast cancer cells positive for the α2δ1 charactered tumor initiation, and α2δ1 is a potential TIC marker for breast cancer that further promotes the migration.

The MEK1/2 Pathway as a Therapeutic Target in High-Grade Serous Ovarian Carcinoma.

Simple SummaryHigh-grade serous ovarian carcinoma (HGSOC) has poor prognosis for patients due to its high rate of recurrence and acquired resistance to therapy. MEK1/2-ERK1/2 signaling pathway that controls cell proliferation and survival is active in the majority of HGSOC cases, but its functional impact is unclear. We suggest that inhibition of MEK1/2 with specific inhibitor trametinib may exert anti-cancer effects upon HGSOC cells. Here we demonstrate that trametinib treatment of HGSOC cells indeed prominently inhibits cell proliferation and tumor growth, and that cisplatin-resistant cells displaying high MEK1/2 activity are particularly sensitive to trametinib. However, we also discovered that trametinib treatment of HGSOC cells has no cytotoxic effects and promotes cancer stem-like characteristics. We therefore suggest to use MEK1/2 inhibitors with other treatment strategies targeting cancer stem-like cells, like aldehyde dehydrogenase 1 inhibition that might show together strong synergy.High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to its high recurrence rate and acquired chemoresistance. RAS/MEK/ERK pathway activation is linked to cell proliferation and therapeutic resistance, but the role of MEK1/2-ERK1/2 pathway in HGSOC is poorly investigated. We evaluated MEK1/2 pathway activity in clinical HGSOC samples and ovarian cancer cell lines using immunohistochemistry, immunoblotting, and RT-qPCR. HGSOC cell lines were used to assess immediate and lasting effects of MEK1/2 inhibition with trametinib in vitro. Trametinib effect on tumor growth in vivo was investigated using mouse xenografts. MEK1/2 pathway is hyperactivated in HGSOC and is further stimulated by cisplatin treatment. Trametinib treatment causes cell cycle arrest in G1/0-phase and reduces tumor growth rate in vivo but does not induce cell death or reduce fraction of CD133+ stem-like cells, while increasing expression of stemness-associated genes instead. Transient trametinib treatment causes long-term increase in a subpopulation of cells with high aldehyde dehydrogenase (ALDH)1 activity that can survive and grow in non-adherent conditions. We conclude that MEK1/2 inhibition may be a promising approach to suppress ovarian cancer growth as a maintenance therapy. Promotion of stem-like properties upon MEK1/2 inhibition suggests a possible mechanism of resistance, so a combination with CSC-targeting drugs should be considered.

Autologous decellularized extracellular matrix promotes adipogenic differentiation of adipose derived stem cells in low serum culture system by regulating the ERK1/2-PPARγ pathway

ABSTRACT High viability and further adipogenic differentiation of adipose-derived stem cells (ADSCs) are fundamental for engraftment and growth of the transplanted adipose tissue. It has been demonstrated that extracellular matrix (ECM) regulates cell proliferation and differentiation by interacting with ERK1/2 signalling pathway. In this study, we prepared autologous decellularized extracellular matrix (d-ECM) and explored its effect on the proliferation and adipogenic ability of ADSCs in low serum culture. We found that 2% foetal bovine serum (FBS) in growth medium inhibited cell viability and DNA replication, and decreased mRNA and protein levels of PPARγ and C/EPBα compared with 10% FBS. Correspondingly, after 14-days adipogenic induction, cells cultured in 2% FBS possessed lower efficiency of adipogenesis and expressed less adipocyte differentiation markers ADIPOQ and aP2. On the contrary, the d-ECM-coated substrate continuously promoted the expression of PPARγ, and regulated the phosphorylation of ERK1/2 in different manners during differentiation. Pretreatment with ERK1/2 inhibitor PD98059 neutralized the effects of d-ECM, which suggested d-ECM might regulate the adipogenesis of ADSCs through ERK1/2-PPARγ pathway. In addition, d-ECM was revealed to regulate the transcription and expression of stemness-associated genes, such as OCT4, NANOG and SOX2, in the undifferentiated ADSCs, which might be related to the initiation of differentiation.

IKKβ Kinase Promotes Stemness, Migration, and Invasion in KRAS-Driven Lung Adenocarcinoma Cells.

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase β (IKKβ) to promote lung tumourigenesis, we hypothesized that inhibition of IKKβ would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKβ kinase activity. IKKβ targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKβ targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKβ is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease.

Slug is a novel molecular target for head and neck squamous cell carcinoma stem-like cells.

The acquisition of stem-like phenotype is partly attributed to the induction of epithelial-mesenchymal transition (EMT). Thus, the activation of factors involved in EMT can be linked to cancer stem cell genesis. However, the underlying mechanisms in head and neck squamous cell carcinoma (HNSCC) remain largely unknown. Herein, we investigate whether slug, one of the major effectors of EMT, affects the stemness of HNSCC cells. We performed in vitro experiments to determine whether slug gene manipulation can influence the stemness phenotypes, including the capacity for self-renewal, expression of putative stemness markers, chemoresistance, and invasion in HNSCC cells. Further, we identified whether Slug knockout attenuates tumorigenicity of HNSCC cells in vivo. Finally, we examined whether prognosis of HNSCC patients after curative treatment may be affected by the level of slug expression. Overexpression of slug promoted self-renewal of HNSCC cells via activation of sphere formation, the expression of stem cell markers, and induction of chemoresistance to cisplatin. Also, slug overexpression increased the migration and invasion of HNSCC cells in vitro and was mainly observed during the invasion in HNSCC xenograft mouse model. By contrast, slug expression knockdown abrogated their self-renewal capacity, stemness-associated gene expression, and cisplatin chemoresistance. Furthermore, high levels of slug expression correlated with poor prognosis of patients with HNSCC. Inhibition of slug expression may represent a novel therapeutic strategy targeting HNSCC stem-like cells.