Stem-like Cancer Cells Are Inducible by Increasing Genomic Instability in Cancer Cells

The existence of cancer stem cells (CSCs) or stem-like cancer cells (SLCCs) is regarded as the cause of tumor formation and recurrence. However, the origin of such cells remains controversial with two competing hypotheses: CSCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Compelling evidence has determined the chromosomal aneuploidy to be one of the hallmarks of cancer cells, indicating genome instability plays an important role in tumorigenesis, for which CSCs are believed to be the initiator. To gain direct evidence that genomic instability is involved in the induction of SLCCs, we utilized multiple approaches to enhance genomic instability and monitored the percentage of SLCC in cultured cancer cells. Using side population (SP) cells as a marker for SLCC in human nasopharyngeal carcinoma (NPC) and CD133 for human neuroblastoma cells, we found that DNA damage inducers, UV and mitomycin C were capable of increasing SP cells in NPC CNE-2 and neuroblastoma SKN-SH cells. Likewise, either overexpression of a key regulator of cell cycle, Mad2, or knock down of Aurora B, an important kinase in mitosis, or Cdh1, a key E3 ligase in cell cycle, resulted in a significant increase of SP cells in CNE-2. More interestingly, enrichment of SP cells was observed in recurrent tumor tissues as compared with the primary tumor in the same NPC patients. Our study thus suggested that, beside transformation of tissue stem cells leading to CSC generation, genomic instability could be another potential mechanism resulting in SLCC formation, especially at tumor recurrence stage.     

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.     

Induction of Cancerous Stem Cells during Embryonic Stem Cell Differentiation

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.     

肿瘤干细胞研究进展

肿瘤干细胞（cancer stem cell, CSC）假说是近年来提出的关于肿瘤发生的新理论,在所有的肿瘤细胞中,可能只有一小部分细胞具有产生肿瘤并维持肿瘤生长和异质性的能力,目前已经在白血病、乳腺癌、脑癌等肿瘤组织中成功分离出了肿瘤干细胞,深入了解肿瘤干细胞的生物学特性、发展相应的鉴别方法以及特殊的治疗手段对癌症的临床治疗有着重要的意义。主要从肿瘤干细胞的概念、起源、鉴定分离方法、与正常干细胞的比较、比率以及与肿瘤转移的关系等方面进行了综述。     

Artemin,a Member of the Glial Cell Line-derived Neurotrophic Factor Family of Ligands,Is HER2-regulated and Mediates Acquired Trastuzumab Resistance by Promoting Cancer Stem Cell-like Behavior in Mammary Carcinoma Cells

Previous studies have demonstrated that Artemin (ARTN) functions as a cancer stem cell (CSC) and metastatic factor in mammary carcinoma. Herein, we report that ARTN mediates acquired resistance to trastuzumab in HER2-positive mammary carcinoma cells. Ligands that increase HER2 activity increased ARTN expression in HER2-positive mammary carcinoma cells, whereas trastuzumab inhibited ARTN expression. Forced expression of ARTN decreased the sensitivity of HER2-positive mammary carcinoma cells to trastuzumab both in vitro and in vivo. Conversely, siRNA-mediated depletion of ARTN enhanced trastuzumab efficacy. Cells with acquired resistance to trastuzumab exhibited increased ARTN expression, the depletion of which restored trastuzumab sensitivity. Trastuzumab resistance produced an increased CSC population concomitant with enhanced mammospheric growth. ARTN mediated the enhancement of the CSC population by increased BCL-2 expression, and the CSC population in trastuzumab-resistant cells was abrogated upon inhibition of BCL-2. Hence, we conclude that ARTN is one mediator of acquired resistance to trastuzumab in HER2-positive mammary carcinoma cells.     

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc^min/+ mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc^min/+ mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.     

Artemin Stimulates Radio- and Chemo-resistance by Promoting TWIST1-BCL-2-dependent Cancer Stem Cell-like Behavior in Mammary Carcinoma Cells

Artemin (ARTN) has been reported to promote a TWIST1-dependent epithelial to mesenchymal transition of estrogen receptor negative mammary carcinoma (ER-MC) cells associated with metastasis and poor survival outcome. We therefore examined a potential role of ARTN in the promotion of the cancer stem cell (CSC)-like phenotype in mammary carcinoma cells. Acquired resistance of ER-MC cells to either ionizing radiation (IR) or paclitaxel was accompanied by increased ARTN expression. Small interfering RNA (siRNA)-mediated depletion of ARTN in either IR- or paclitaxel-resistant ER-MC cells restored cell sensitivity to IR or paclitaxel. Expression of ARTN was enriched in ER-MC cells grown in mammospheric compared with monolayer culture and was also enriched along with BMI1, TWIST1, and DVL1 in mammospheric and ALDH1+ populations. ARTN promoted mammospheric growth and self-renewal of ER-MC cells and increased the ALDH1+ population, whereas siRNA-mediated depletion of ARTN diminished these CSC-like cell behaviors. Furthermore, increased ARTN expression was significantly correlated with ALDH1 expression in a cohort of ER-MC patients. Forced expression of ARTN also dramatically enhanced tumor initiating capacity of ER-MC cells in xenograft models at low inoculum. ARTN promotion of the CSC-like cell phenotype was mediated by TWIST1 regulation of BCL-2 expression. ARTN also enhanced mammosphere formation and the ALDH1+ population in estrogen receptor-positive mammary carcinoma (ER+MC) cells. Increased expression of ARTN and the functional consequences thereof may be one common adaptive mechanism used by mammary carcinoma cells to promote cell survival and renewal in hostile tumor microenvironments.     

乳腺癌肿瘤干细胞的研究进展

干细胞(stem cell,SC)是一类具有自我更新、多向分化潜能的特殊细胞群体.而肿瘤干细胞(cancer stem cell,CSC)不仅具备了干细胞方面的特征,同时还有其自身特殊性.乳腺癌中肿瘤干细胞的发现为乳腺癌的治疗提供了创新性策略.近年来,有关乳腺癌肿瘤干细胞的筛选标记和方法以及信号转导通路方面的研究颇多,但其中也不乏争议.就乳腺癌干细胞研究的最新进展作一端述.     

Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer

Introduction

Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer;

Methods

We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected;

Results

DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERα-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Erα was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited.

Conclusions

In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERα-positive breast cancer cells.     

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal''s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.     

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin−c-Kit+ and Lin−Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin−Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.     

人乳腺癌干细胞异种移植动物模型的制备和应用

越来越多的证据显示乳腺癌干细胞是导致乳腺癌发生、发展、复发和转移的根源。因此,模拟出与人乳腺癌发病机制相似的动物模型将对乳腺癌的治疗起着至关重要的作用。本文旨在介绍乳腺癌干细胞异种移植动物模型的制备方法、应用以及近年来的研究进展。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

糖基化突变体细胞引发疾病的研究进展

糖基化是细胞表面最常见也是最重要的一种修饰方法,异常的糖基化会影响糖基化产物的结构,改变细胞表面的化学特性;利用基因技术和化学小分子,调控糖基化酶的活性,可以人为地影响糖基化反应进程,揭示糖基化产物对生物体健康的影响.展示糖基化突变体与疾病的关联.     

Synthesis, Shedding, and Intercellular Transfer of Human Medulloblastoma Gangliosides: Abrogation by a New Inhibitor of Glucosylceramide Synthase

Abstract: The biological activity of human medulloblastoma tumor gangliosides very likely involves the interaction of these molecules with host cells in the tumor microenvironment. To trace the hypothesized intercellular transfer of shed medulloblastoma gangliosides, we used an in vitro dual-chamber culture system in which the tumor cells, the shed gangliosides, and the target cells to which they might bind would not be perturbed during the transfer process. We observed that under these unmanipulated conditions, gangliosides were shed by the Daoy medulloblastoma cell line (∼300 pmol/10⁸ cells/h), traversed the chamber membrane, and stably bound to the target fibroblasts at the very high density of 10⁷ molecules per cell within 48 h. To determine if this substantial intercellular transfer of shed gangliosides, with its potential of modifying target cell function, could be blocked, we evaluated a new inhibitor of glucosylceramide synthase, dl - threo -1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). PPPP (1.0 µ M ) reduced (90%) Daoy cell ganglioside content strikingly, without causing toxicity or inhibiting cell proliferation. Subsequently, ganglioside shedding by the medulloblastoma cells was diminished significantly (to ∼50 pmol/10⁸/h), and binding of radiolabeled shed medulloblastoma gangliosides to target fibroblasts was consequently almost completely abrogated. We conclude that the shedding and transfer of potentially biologically active human medulloblastoma gangliosides can be diminished effectively by PPPP.     

NAAGS基因修饰神经干细胞在创伤性颅脑损伤治疗中的研究

目的：探讨移植NAAG合酶（NAAG synthetase,NAAGS）基因修饰的神经干细胞（Neural Stem Cells,NSCs）能否促进创伤性颅脑损伤大鼠神经功能的恢复。方法：利用电穿孔转染大鼠NSCs,通过脑立体定向仪分别将PBS（模型组）、NSCs（NSCs组）、转基因NSCs（NAAGS＋NSCs组）移植到创伤性颅脑损伤（Traumatic Brain Injury,TBI）大鼠局部损伤灶边缘,通过NSS评分评价移植后大鼠神经功能的变化以及用TUNEL法检测NSCs的凋亡情况,并采用放射免疫法分析脑组织中促炎因子水平。结果：Nss评分结果显示NAAGs＋NSCs组和NSCs组在第7、14、21天神经功能评分均低于模型组（P〈0．05）;NAAGS＋NSCs组在第14和21天神经功能评分低于NSCs组（P〈0．05）;在各时间点细胞移植组比模型组的神经细胞凋亡数明显减少;转基因NSCs移植能明显降低TBI脑组织中促炎因子水平。结论：转基因NSCs移植后可以合成NAAGS促进TBI大鼠神经功能的恢复。     

5-Fluorouracil Chemotherapy of Gastric Cancer Generates Residual Cells with Properties of Cancer Stem Cells

Background: 5-Fluorouracil (5Fu) chemotherapy is the first treatment of choice for advanced gastric cancer (GC), but its effectiveness is limited by drug resistance. Emerging evidence suggests that the existence of cancer stem cells (CSCs) contributes to chemoresistance. The aim of the present study was to determine whether 5Fu chemotherapy generates residual cells with CSC-like properties in GC. Methods: Human GC cell lines, SGC7901 and AGS, were exposed to increasing 5Fu concentrations. The residual cells were assessed for both chemosensitivity and CSC-like properties. B lymphoma Mo-MLV insertion region 1 (BMI1), a putative CSC protein, was analyzed by immunohistochemical staining and subjected to pairwise comparison in GC tissues treated with or without neoadjuvant 5Fu-based chemotherapy. The correlation between BMI1 expression and recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy was then examined. Results: The residual cells exhibited 5Fu chemoresistance. These 5Fu-resistant cells displayed some CSC features, such as a high percentage of quiescent cells, increased self-renewal ability and tumorigenicity. The 5Fu-resistant cells were also enriched with cells expressing cluster of differentiation (CD)133⁺, CD326⁺ and CD44⁺CD24^-. Moreover, the BMI1 gene was overexpressed in 5Fu-resistant cells, and BMI1 knockdown effectively reversed chemoresistance. The BMI1 protein was highly expressed consistently in the remaining GC tissues after 5Fu-based neoadjuvant chemotherapy, and BMI1 levels were correlated positively with recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy. Conclusions: Our data provided molecular evidence illustrating that 5Fu chemotherapy in GC resulted in acquisition of CSC-like properties. Moreover, enhanced BMI1 expression contributed to 5Fu resistance and may serve as a potential therapeutic target to reverse chemoresistance in GC patients.     

High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O₂) conditions. Treatment with a high O₂ condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O₂ condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O₂ condition activated Wnt signaling. Optimal stage-specific treatment with a high O₂ condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O₂ condition at a specific stage is useful for the efficient generation of insulin-producing cells.     

Pancreatic Cancer Stem-like Cells Display Aggressive Behavior Mediated via Activation of FoxQ1

Subpopulations of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) have been identified from most tumors, including pancreatic cancer (PC), and the existence of these cells is clinically relevant. Emerging evidence suggests that CSLCs participate in cell growth/proliferation, migration/invasion, metastasis, and chemo-radiotherapy resistance, ultimately contributing to poor clinical outcome. However, the pathogenesis and biological significance of CSLCs in PC has not been well characterized. In the present study, we found that isolated triple-marker-positive (CD44⁺/CD133⁺/EpCAM⁺) cells of human PC MiaPaCa-2 and L3.6pl cells behave as CSLCs. These CSLCs exhibit aggressive behavior, such as increased cell growth, migration, clonogenicity, and self-renewal capacity. The mRNA expression profiling analysis showed that CSLCs (CD44⁺/CD133⁺/EpCAM⁺) exhibit differential expression of more than 1,600 mRNAs, including FoxQ1, compared with the triple-marker-negative (CD44⁻/CD133⁻/EpCAM⁻) cells. The knockdown of FoxQ1 by its siRNA in CSLCs resulted in the inhibition of aggressive behavior, consistent with the inhibition of EpCAM and Snail expression. Mouse xenograft tumor studies showed that CSLCs have a 100-fold higher potential for tumor formation and rapid tumor growth, consistent with overexpression of CSC-associated markers/mediators, including FoxQ1, compared with its parental MiaPaCa-2 cells. The inhibition of FoxQ1 attenuated tumor formation and growth, and expression of CSC markers in the xenograft tumor derived from CSLCs of MiaPaCa-2 cells. These data clearly suggest the role of differentially expressed genes in the regulation of CSLC characteristics, further suggesting that targeting some of these genes could be important for the development of novel therapies for achieving better treatment outcome of PC.