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Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Shenzhen University School of Medicine

Reprogramming of intracellular metabolism is common in liver cancer cells. Understanding the mechanisms of cell metabolic reprogramming may present a new basis for liver cancer treatment. In our previous study, we reported that a novel oncogene eukaryotic translation initiation factor 5A2 (EIF5A2) promotes tumorigenesis under hypoxic condition. Here, we aim to investigate the role of EIF5A2 in cell metabolic reprogramming during hepatocellular carcinoma (HCC) development. In this study, we reported that the messenger RNA (mRNA) level of EIF5A2 was upregulated in 59 of 105 (56.2%) HCC clinical samples (P = 0.015), and EIF5A2 overexpression was significantly associated with shorter survival time of patients with HCC (P = 0.021). Ectopic expression of EIF5A2 in HCC cell lines significantly promoted cell growth and accelerated glucose utilization and lipogenesis rates. The high rates of glucose uptake and lactate secretion conferred by EIF5A2 revealed an abnormal activity of aerobic glycolysis in HCC cells. Several key enzymes involved in glycolysis including glucose transporter type 1 and 2, hexokinase 2, phosphofructokinase liver type, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase M2 isoform, phosphoglycerate mutase 1 and lactate dehydrogenase A were upregulated by overexpression of EIF5A2. Moreover, EIF5A2 showed positive correlations with FASN and ACSS2, two key enzymes involved in the fatty acid de novo biosynthetic pathway, at both protein and mRNA levels in HCC. These results indicated that EIF5A2 may regulate fatty acid de novo biosynthesis by increasing the uptake of acetate. In conclusion, our findings demonstrate that EIF5A2 has a critical role in HCC cell metabolic reprogramming and may serve as a prominent novel therapeutic target for liver cancer treatment.

Eukaryotic translation initiation factor 5A2 promotes metabolic reprogramming in hepatocellular carcinoma cells.

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer.

The high-risk (HR) human papillomaviruses (HPV) are causative agents of anogenital tract dysplasia and cancers and a fraction of head and neck cancers. The HR HPV E6 oncoprotein possesses canonical oncogenic functions, such as p53 degradation and telomerase activation. It is also capable of stimulating expression of several oncogenes, but the molecular mechanism underlying these events is poorly understood. Here, we provide evidence that HPV16 E6 physically interacts with histone H3K4 demethylase KDM5C, resulting in its degradation in an E3 ligase E6AP- and proteasome-dependent manner. Moreover, we found that HPV16-positive cancer cell lines exhibited lower KDM5C protein levels than HPV-negative cancer cell lines. Restoration of KDM5C significantly suppressed the tumorigenicity of CaSki cells, an HPV16-positive cervical cancer cell line. Whole genome ChIP-seq and RNA-seq results revealed that CaSki cells contained super-enhancers in the proto-oncogenes  and  Ectopic KDM5C dampened these super-enhancers and reduced the expression of proto-oncogenes. This effect was likely mediated by modulating H3K4me3/H3K4me1 dynamics and decreasing bidirectional enhancer RNA transcription. Depletion of KDM5C or HPV16 E6 expression activated these two super-enhancers. These results illuminate a pivotal relationship between the oncogenic E6 proteins expressed by HR HPV isotypes and epigenetic activation of super-enhancers in the genome that drive expression of key oncogenes like  and  This study suggests a novel explanation for why infections with certain HPV isotypes are associated with elevated cancer risk by identifying an epigenetic mechanism through which E6 proteins expressed by those isotypes can drive expression of key oncogenes. .

E6 Protein Expressed by High-Risk HPV Activates Super-Enhancers of the  and  Oncogenes by Destabilizing the Histone Demethylase KDM5C.

Frizzled receptors (FZDs) are a family of seven-span transmembrane receptors with hallmarks of G protein-coupled receptors (GPCRs) that serve as receptors for secreted Wingless-type (WNT) ligands in the WNT signaling pathway. Functionally, FZDs play crucial roles in regulating cell polarity, embryonic development, cell proliferation, formation of neural synapses, and many other processes in developing and adult organisms. In this review, we will introduce the basic structural features and review the biological function and mechanism of FZDs in the progression of human cancers, followed by an analysis of clinical relevance and therapeutic potential of FZDs. We will focus on the development of antibody-based and small molecule inhibitor-based therapeutic strategies by targeting FZDs for human cancers.

Frizzled Receptors as Potential Therapeutic Targets in Human Cancers.

Angiogenesis is essential in the early stage of solid tumor recurrence, but how a suspensive tumor is reactivated before angiogenesis is mostly unknown. Herein, we stumble across an interesting phenomenon that s.c. xenografting human lung cancer tissues can awaken the s.c. suspensive tumor in nude mice. We further found that a high level of insulin-like growth factor 1 (IGF1) was mainly responsible for triggering the transition from suspensive tumor to progressive tumor in this model. The s.c. suspensive tumor is characterized with growth arrest, avascularity, and a steady-state level of proliferating and apoptotic cells. Intriguingly, CD133 lung cancer stem cells (LCSCs) are highly enriched in suspensive tumor compared with progressive tumor. Mechanistically, high IGF1 initiates LCSCs self-renewal from asymmetry to symmetry via the activation of a PI3K/Akt/β-catenin axis. Next, the expansion of LCSC pool promotes angiogenesis by increasing the production of CXCL1 and PlGF in CD133 LCSCs, which results in lung cancer recurrence. Clinically, a high level of serum IGF1 in lung cancer patients after orthotopic lung cancer resection as an unfavorable factor is strongly correlated with the high rate of recurrence and indicates an adverse progression-free survival. Vice versa, blocking IGF1 or CXCL1/PlGF with neutralizing antibodies can prevent the reactivation of a suspensive tumor induced by IGF1 stimulation in the mouse model. Collectively, the expansion of LCSC pool before angiogenesis induced by IGF1 is a key checkpoint during the initiation of cancer relapse, and targeting serum IGF1 may be a promising treatment for preventing recurrence in lung cancer patients.

Expansion of cancer stem cell pool initiates lung cancer recurrence before angiogenesis.

Carboxyl-containing metabolites (CCMs) widely exist in living systems and are the essential components for life. Global characteristics of CCMs in biological samples are critical for the understanding of physiological processes and the discovery for the onset of relevant diseases. However, their determination represents a challenge due to enormous polarity differences, structural diversity, high structural similarity, and poor ionization efficiency in mass spectrometry. Herein, 5-(diisopropylamino)amylamine (DIAAA) derivatization coupled with liquid chromatography-mass spectrometry (LC-MS) was developed for mapping the CCMs. With this methodology, the sensitivity was significantly enhanced. More importantly, the hydrophobicity of polar CCMs, amino acids, TCA cycle intermediates, and short-chain fatty acids and the hydrophilicity of low-polar CCMs, long-chain fatty acids, and bile acids were significantly increased, resulting in a remarkable separation efficiency for which 68 CCMs can be simultaneously determined. Furthermore, the polarity-tuning effect was confirmed to be induced by the different impacts of aliphatic chains and nitrogen atom in DIAAA, the latter existing as a cation in the acidic mobile phase, using different derivatization reagents. Finally, this derivatization method was utilized to hunt for the potential biomarkers in colorectal cancer (CRC) patients and 52 CCMs, related with several key metabolic pathways, including amino acids metabolism, TCA cycle, fatty acid metabolism, pyruvate metabolism, and gut flora metabolism were identified. This innovative polarity-tuning derivatization-LC-MS approach was proved to be a valuable tool for probing global metabolome with high separation efficiency and sensitivity in various biological samples.

Polarity-Tuning Derivatization-LC-MS Approach for Probing Global Carboxyl-Containing Metabolites in Colorectal Cancer.

Esophageal squamous cell carcinoma (ESCC) occurs with the highest frequency in China, especially in the high-risk Northern Chinese. Recent studies have reported that  is significantly downregulated in non-tumor (NT) esophageal tissues from familial ESCC patients compared with those from sporadic ESCC. However, the mechanism of how  regulates familial ESCC remains unknown. In this study, post hoc genome-wide association studies (GWAS) in 496 cases with a family history of upper gastrointestinal tract cancers and 1056 controls were performed and the results revealed that  is a novel susceptibility gene for familial ESCC. Reduced expression of  in NT esophageal tissues from familial ESCC patients significantly correlates with its promoter hypermethylation. Moreover, case-control study of Chinese descendants from different risk areas of China revealed that the methylation of the  gene in peripheral blood leukocyte (PBL) DNA samples could be a risk factor for developing ESCC in this high-risk population. Functional studies showed that  is a novel antioxidant gene, and deregulation of  facilitates heat stress-induced oxidative DNA damage and formation of γ-H2AX foci in normal esophageal epithelial cells. Collectively, we show that epigenetic alterations of  predispose susceptible individuals to increased risk of esophageal cancer.

Epigenetic alterations of a novel antioxidant gene  predispose susceptible individuals to increased risk of esophageal cancer.

The histone demethylase KDM4B is frequently overexpressed in various cancer types, and previous studies have indicated that the primary oncogenic function of KDM4B is its ability to demethylate H3K9me3 in different tumors, resulting in altered gene expression and genome instability. A genome-wide analysis to evaluate the effect of KDM4B on the global or local H3K9me3 level has not been performed. In this study, we assess whole-genome H3K9me3 distribution in cancer cells and find that H3K9me3 is largely enriched in long interspersed nuclear element-1 (LINE-1). A significant proportion of KDM4B-dependent H3K9me3 was located in evolutionarily young LINE-1 elements, which likely retain retrotransposition activity. Ectopic expression of KDM4B promoted LINE-1 expression, while depletion of KDM4B reduced it. Furthermore, KDM4B overexpression enhanced LINE-1 retrotransposition efficacy, copy number, and associated DNA damage, presumably via the histone demethylase activity of KDM4B. Breast cancer cell lines expressing high levels of KDM4B also exhibited increased LINE-1 expression and copy number compared with other cell lines. Pharmacologic inhibition of KDM4B significantly reduced LINE-1 expression and DNA damage in breast cancer cells with excessive KDM4B. Our study not only identifies KDM4B as a novel regulator of LINE-1, but it also suggests an unexpected oncogenic role for KDM4B overexpression in tumorigenesis, providing clues for the development of new cancer prevention strategies and therapies. SIGNIFICANCE: The histone demethylase KDM4B promotes tumorigenesis by inducing retrotransposition and DNA damage.

Histone Demethylase KDM4B Promotes DNA Damage by Activating Long Interspersed Nuclear Element-1.

Deletion of Chromosome 3p is one of the most frequently detected genetic alterations in nasopharyngeal carcinoma (NPC). We reported the role of a novel 3p26.3 tumor suppressor gene (TSG)  in NPC. Down-regulation of  was detected in 4/6 of NPC cell lines and 71/95 (74.7%) in clinical tissues. Ectopic expressions of CHL1 in NPC cells significantly inhibit colony formation and cell motility in functional study. By up-regulating epithelial markers and down-regulating mesenchymal markers  could induce mesenchymal-epithelial transition (MET), a key step in preventing tumor invasion and metastasis.  could also cause the inactivation of RhoA/Rac1/Cdc42 signaling pathway and inhibit the formation of stress fiber, lamellipodia, and filopodia.  could co-localize with adhesion molecule Integrin-β1, the expression of  was positively correlated with Integrin-β1 and another known tumor suppressor gene (TSG) Merlin. Down-regulation of Integrin-β1 or Merlin was significantly correlated with the poor survival rate of NPC patients. Further mechanistic studies showed that  could directly interact with integrin-β1 and link to Merlin, leading to the inactivation of integrin β1-AKT pathway. In conclusion,  is a vital tumor suppressor in the carcinogenesis of NPC.

 suppresses tumor growth and metastasis in nasopharyngeal carcinoma by repressing PI3K/AKT signaling pathway via interaction with Integrin β1 and Merlin.

Cancer-associated fibroblasts (CAFs) play critical roles in cancer progression and treatment failure. CAFs display extreme phenotypic heterogeneity and functional diversity. Some subpopulations of CAFs have the ability to reconstitute cancer stemness by promoting the expansion of cancer stem cells (CSCs) or by inducing the generation of CSCs from differentiated cancer cells. CAFs regulate cancer stemness in different types of solid tumors by activating a wide array of CSC-related signaling by secreting proteins and exosomes. As feedback, the CSCs can also induce the proliferation and further activation of CAFs to promote their CSC-supporting activities, thus completing the loop of CAF-CSC crosstalk. Current research on targeting CAF-CSC crosstalk could be classified into (i) specific depletion of CAF subpopulations that have CSC-supporting activities and (ii) targeting molecular signaling in CAF-CSC crosstalk, such as the IL6/STAT3, TGF-β/SDF-1/PI3K, WNT/β-catenin, HGF/cMET and SHH/Hh pathways. Strategies targeting CAF-CSC crosstalk may open new avenues for overcoming cancer progression and therapeutic resistance.

Therapeutic targeting of the crosstalk between cancer-associated fibroblasts and cancer stem cells.

Esophageal cancer (EC) seriously threatens human health, and a promising new avenue for EC treatment involves cancer immunotherapy. To improve the efficacy of EC immunotherapy and to develop novel strategies for EC prognosis prediction or clinical treatment, understanding the immune landscapes in EC is required. EC cells harbor abundant tumor antigens, including tumor-associated antigens and neoantigens, which have the ability to initiate dendritic cell-mediated tumor-killing cytotoxic T lymphocytes in the early stage of cancer development. As EC cells battle the immune system, they obtain an ability to suppress antitumor immunity through immune checkpoints, secreted factors, and negative regulatory immune cells. Cancer-associated fibroblasts also contribute to the immune evasion of EC cells. Some factors of the immune landscape in EC tumor microenvironment are associated with cancer development, patient survival, or treatment response. Based on the immune landscape, peptide vaccines, adoptive T cell therapy, and immune checkpoint blockade can be used for EC immunotherapy. Combined strategies are required for better clinical outcome in EC. This review provides directions to design novel and effective strategies for prognosis prediction and immunotherapy in EC.

The immune landscape of esophageal cancer.

Drug resistance is a major challenge for chemotherapy in treating human gastric cancer (GC), as the underlying molecular mechanism of chemoresistance in GC remains unknown. Caveolin-1 (Cav-1) is a scaffold protein of plasma membrane caveolae that acts as a tumor modulator by interacting with several cell signals. In this research, we showed that the survival rate of GC cells to cisplatin (CDDP) increased in the presence of Cav-1. Moreover, Cav-1 overexpression inhibited cisplatin-induced apoptosis and improved the survival rate of GC cells. Cav-1 overexpression and knock-down experiments indicated that Cav-1 expression stimulated wingless-type MMTV integration site (WNTs) pathway through the phosphorylation of LRP6 and dephosphorylation of β-catenin. Cav-1 was positively associated with the increase of WNT downstream target gene Met, which led to the activation of HER2 signaling. Moreover, our results demonstrated that the expression of Cav-1 and Met were positively associated with the resistance of GC cells to cisplatin. Collectively, Cav-1 enhances the cisplatin-resistance of GC cells by activating the WNT signaling pathway and Met-HER2 crosstalk. Understanding the role of Cav-1 in the chemoresistance of GC would help to develop novel therapies for a better treatment outcome of GC patients.

Li Fu

Shenzhen University School of Medicine

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells