The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas.     

新型miRNA表达框架下调端粒酶活性并抑制视网膜母细胞瘤细胞生长

目的 本研究致力于设计一种新的miRNA表达框架（MEC）,其以hTERT和hTR的特异性序列为靶点。该框架能够有效改善传统RNAi方法中miRNA易降解和细胞毒性问题,为miRNA的合成提供一种简便的方法。方法 采用重叠PCR方法构建hTERT和hTR特异的miRNA表达框架。采用TRAP银染色和TRAP实时荧光定量PCR检测端粒酶活性。实时荧光定量PCR法测定端粒长度,MTT法测定细胞活力。annexin V/PI双染色法检测细胞凋亡,PI单染色法检测细胞周期,流式细胞仪检测细胞凋亡。结果 成功构建了靶向hTERT/ hTR特异性miRNA表达框架。不同MEC对端粒酶活性的抑制程度不同。端粒酶的沉默可引起视网膜母细胞瘤（RB）细胞G0/G1期生长阻滞,导致细胞凋亡。结论 miRNA介导的端粒酶沉默是一种有效抑制RB细胞生长的策略,开发一个强大的系统来充分探索miRNA的作用是必要的。本文构建的MEC显示了强大的RNAi效应,可成为筛选用于RB基因治疗的RNAi靶向序列的有效工具。     

Bilirubin Inhibits Tumor Cell Growth via Activation of ERK

Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.     

MicroRNA-126 Inhibits Tumor Cell Growth and Its Expression Level Correlates with Poor Survival in Non-Small Cell Lung Cancer Patients

Background

It is controversial whether microRNA-126 is a tumor suppressive or oncogenic miRNA. More experiments are needed to determine whether microRNA-126 is associated with non-small cell lung cancer risk and prognosis.

Methods

Over-expression of microRNA-126 was performed to evaluate the cell invasion and tumor growth in non-small cell lung cancer (NSCLC) cell lines and nude mouse xenograft model. Gain-of-function experiments and luciferase assays were performed to reveal the relationship between microRNA-126 and PI3K-Akt signal pathway in A549 cells. We analyzed the associations of the microRNA-126 expression between genetic variants within microRNA-126 and clinical information including smoking status, sex, age, and histological type and the tumor stage.

Results

Over-expression of microRNA-126 in NSCLC cell lines decreased cell proliferation in vitro and tumor growth in the nude mouse xenograft model. And microRNA-126 repressed the activity of PI3K-Akt pathway by targeting binding sites in the 3′-untranslated region of PI3KR2 mRNA. The expression level of microRNA-126 was decreased in NSCLC lines and tumor tissues. The patients with low microRNA-126 expression had significantly poorer survival time than those with high microRNA-126 expression (means for survival time (month): 24.392±1.055 vs. 29.282±1.140, P = 0.005). However, there was no significant difference in the genotype and allele frequencies of the microRNA-126 variant (G>A, rs4636297) between cases and controls (P = 0.366). In addition, there was no association between SNP rs4636297 and survival time in NSCLC patients (P = 0.992). And microRNA-126 expression had no significant difference among the three genotype groups (P = 0.972).

Conclusions

Our data indicate that microRNA-126 is a tumor-suppressor gene in NSCLC and low microRNA-126 expression is a unfavorable prognostic factor in NSCLC patients. However, the regulatory mechanism of microRNA-126 remains to be elucidated in different normal and malignant tissues. Therefore, further research is needed to explore the tumor suppressive functions of microRNA-126 in NSCLC.     

siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model

Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers.     

Blockade of the SNARE Protein Syntaxin 1 Inhibits Glioblastoma Tumor Growth

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM.     

Cyclic Phosphatidic Acid Stimulates cAMP Production and Inhibits Growth in Human Colon Cancer Cells

Colon cancer is a malignancy that develops in colon and rectal tissues. The prognosis for metastatic colon cancer remains poor, and novel therapeutic options are required to reduce colon cancer mortality. Recently, intracellular cAMP levels have been suggested to influence the behavior of cancer cells. Intriguingly, cyclic phosphatidic acid (cPA) and its structural analogs inhibit growth in many cancer cell lines, and our previous work has suggested that cPA increases cAMP production. Phosphodiesterase (PDE) type 3 isoforms PDE3A and PDE3B are expressed mainly in cardiovascular tissue and adipose tissue, respectively. Moreover, increase in intracellular cAMP levels has been associated with the inhibition of growth in colon cancer cells. These findings suggest that cPA could be used in colon cancer therapy. In this study, we found that cPA inhibited the growth of HT-29 cells, which express high levels of PDE3B, but not the growth of DLD-1 cells, which express low levels of PDE3B. Furthermore, cPA inhibited the phosphorylation of Akt in HT-29 cells in a dose-dependent fashion. Our results suggest that PDE3B expression and intracellular cAMP levels are correlated with the proliferation of colon cancer cells. These findings demonstrate for the first time that cPA may serve as a useful a molecule in targeted therapy for colon cancer.     

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.     

一种新型肿瘤细胞抑制蛋白的分离纯化

介绍了从健康牛骨髓中分离到一种具有抑制肿瘤活性的糖蛋白,并对该糖蛋白进行了纯化,获得了电泳纯和层析纯的纯品．测得该糖蛋白N末端为两氨酸残基．用SDS-PAGE法测得该溏蛋白的表现分子量为65kD．氨基酸组成分析显示其为一种富含丝氨酸的蛋白质．生物活性测定表明,该糖蛋白纯品对小白鼠白血病P388细胞和人慢粒白血病HL-60细胞株的增殖均有抑制作用．有关该糖蛋白的糖含量的定量测定及蛋白质一级结构分析正在进一步的研究中．     

Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors

Cancer stem cells (CSCs) are considered to be responsible for the dismal prognosis of cancer patients. However, little is known about the molecular mechanisms underlying the acquisition and maintenance of CSC properties in cancer cells because of their rarity in clinical samples. We herein induced CSC properties in cancer cells using defined factors. We retrovirally introduced a set of defined factors (OCT3/4, SOX2 and KLF4) into human colon cancer cells, followed by culture with conventional serum-containing medium, not human embryonic stem cell medium. We then evaluated the CSC properties in the cells. The colon cancer cells transduced with the three factors showed significantly enhanced CSC properties in terms of the marker gene expression, sphere formation, chemoresistance and tumorigenicity. We designated the cells with CSC properties induced by the factors, a subset of the transduced cells, as induced CSCs (iCSCs). Moreover, we established a novel technology to isolate and collect the iCSCs based on the differences in the degree of the dye-effluxing activity enhancement. The xenografts derived from our iCSCs were not teratomas. Notably, in contrast to the tumors from the parental cancer cells, the iCSC-based tumors mimicked actual human colon cancer tissues in terms of their immunohistological findings, which showed colonic lineage differentiation. In addition, we confirmed that the phenotypes of our iCSCs were reproducible in serial transplantation experiments. By introducing defined factors, we generated iCSCs with lineage specificity directly from cancer cells, not via an induced pluripotent stem cell state. The novel method enables us to obtain abundant materials of CSCs that not only have enhanced tumorigenicity, but also the ability to differentiate to recapitulate a specific type of cancer tissues. Our method can be of great value to fully understand CSCs and develop new therapies targeting CSCs.     

槲皮素对结肠癌细胞生长的影响

通过MTT试验观察槲皮素对结肠癌细胞系RKO生长的影响;通过流式细胞仪技术观察槲皮素对细胞周期和细胞凋亡的影响;采用RT-PCR和western印迹技术,确定槲皮素对p21、p27和p53表达的影响.MTT法显示槲皮素加药组生长抑制作用明显,且具有剂量依赖性;流式细胞仪分析结果显示,经5μmol/L槲皮素作用后细胞周期明显阻滞在G0/G1期;5、10、20μmol/L3个剂量组的细胞凋亡率分别为23.4%、24.2%、47.9%,而对照组为13.2%;p21和p27的mRNA及蛋白质表达水平上调,促凋亡蛋白p53表达水平上调.因此,槲皮素对RKO生长有明显的抑制作用,槲皮素可能通过上调p21和p27表达使RKO细胞周期阻滞于G0/G1期,可能通过上调促凋亡蛋白p53表达诱导RKO细胞发生凋亡.     

Contribution of Soft Substrates to Malignancy and Tumor Suppression during Colon Cancer Cell Division

In colon cancer, a highly aggressive disease, progression through the malignant sequence is accompanied by increasingly numerous chromosomal rearrangements. To colonize target organs, invasive cells cross several tissues of various elastic moduli. Whether soft tissue increases malignancy or in contrast limits invasive colon cell spreading remains an open question. Using polyelectrolyte multilayer films mimicking microenvironments of various elastic moduli, we revealed that human SW480 colon cancer cells displayed increasing frequency in chromosomal segregation abnormalities when cultured on substrates with decreasing stiffness. Our results show that, although decreasing stiffness correlates with increased cell lethality, a significant proportion of SW480 cancer cells did escape from the very soft substrates, even when bearing abnormal chromosome segregation, achieve mitosis and undergo a new cycle of replication in contrast to human colonic HCoEpiC cells which died on soft substrates. This observation opens the possibility that the ability of cancer cells to overcome defects in chromosome segregation on very soft substrates could contribute to increasing chromosomal rearrangements and tumor cell aggressiveness.     

Growth Characteristics and Substrate Specificity of Aerobic Photosynthetic Bacterium, Erythrobacter Sp. (OCh 114)

Optimal growth conditions and substrate specificity of the aerobicphotosynthetic bacterium, Erythrobacter sp. (OCh 114), wereinvestigated. Erythrobacter utilized 19 out of 26 substratestested, including several sugars and amino acids. Glycerol andlactate were the most effective as electron donors or carbonsources. Maximum growth was obtained at a salinity of about25, pH 8–9 and temperature 28°C in a glycerol-enrichedmedium. A suitable growth medium for Erythrobacter sp. (OCh114) is proposed. (Received October 18, 1985; Accepted January 10, 1986)