NFIB promotes cell survival by directly suppressing p21 transcription in TP53-mutated triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options and poor prognosis. There is an urgent need to identify and understand the key factors and signalling pathways driving TNBC tumour progression, relapse, and treatment resistance. In this study, we report that gene copy numbers and expression levels of nuclear factor IB (NFIB), a recently identified oncogene in small cell lung cancer, are preferentially increased in TNBC compared to other breast cancer subtypes. Furthermore, increased levels of NFIB are significantly associated with high tumour grade, poor prognosis, and reduced chemotherapy response. Concurrent TP53 mutations and NFIB overexpression (z-scores > 0) were observed in 77.9% of TNBCs, in contrast to 28.5% in non-TNBCs. Depletion of NFIB in TP53-mutated TNBC cell lines promotes cell death, cell cycle arrest, and enhances sensitivity to docetaxel, a first-line chemotherapeutic drug in breast cancer treatment. Importantly, these alterations in growth properties were accompanied by induction of CDKN1A, the gene encoding p21, a downstream effector of p53. We show that NFIB directly interacts with the CDKN1A promoter in TNBC cells. Furthermore, knockdown of combined p21 and NFIB reverses the docetaxel-induced cell growth inhibition observed upon NFIB knockdown, indicating that NFIB's effect on chemotherapeutic drug response is mediated through p21. Our results indicate that NFIB is an important TNBC factor that drives tumour cell growth and drug resistance, leading to poor clinical outcomes. Thus, targeting NFIB in TP53-mutated TNBC may reverse oncogenic properties associated with mutant p53 by restoring p21 activity. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes.Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC.Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care.     

Effect of anthracycline and taxane on the expression of programmed cell death ligand-1 and galectin-9 in triple-negative breast cancer

This study identified chemotherapeutic agents that up-regulate programmed cell death ligand-1 (PD-L1) and galectin-9 (Gal-9) in breast cancer cells. Immunohistochemical (IHC) staining was used to evaluate changes in PD-L1 and Gal-9 expression in the tumor tissue of triple-negative breast cancer (TNBC) patients who received anthracycline- and taxane-based neoadjuvant chemotherapy. To determine whether PD-L1 and Gal-9 expression changes were attributable directly to chemotherapeutics, MDA-MB-231 cells and HS578T cells were treated with different concentrations of anthracycline and taxane. Expression levels of PD-L1 and Gal-9 were evaluated and the activation status of NFκB in MDA-MB-231 and HS578T cells was determined to identify the PD-L1 and Gal-9 up-regulation mechanism. Three cases of increased PD-L1 expression and two of increased Gal-9 expression were observed among the TNBC patients. PD-L1 and Gal-9 expression were up-regulated by anthracycline and taxane in MDA-MB-231 cells, but not in HS578T cells. Increased nuclear levels of NFκB were observed in MDA-MB-231 cells treated with 0.5?μM epirubicin. Anthracycline and taxane up-regulated PD-L1 and Gal-9 expression in some subtypes of TNBC. This study provides useful reference data for clinical trials investigating combination treatments with immune checkpoint inhibitors and chemotherapy.     

TFPI‐2 Downregulates Multidrug Resistance Protein in 5‐FU‐Resistant Human Hepatocellular Carcinoma BEL‐7402/5‐FU Cells

Tissue factor pathway inhibitor‐2 (TFPI‐2) is known to induce apoptosis and to suppress tumor metastasis in several types of cancer cells. However, there is little known about its reversal effect on chemoresistant tumor cells. This study investigated the effect of TFPI‐2 in 5‐fluorouracil (5‐FU)‐resistant human hepatocellular cancer BEL‐7402/5‐FU cells in vitro. We constructed TFPI‐2 overexpression BEL‐7402/5‐FU cell lines and explored resistance index (RI) of 5‐FU, function of the P‐glycoprotein (P‐gp) efflux pump, and the mRNA and protein expression of drug resistance gene, including multidrug resistance gene (MDR1), lung‐resistance protein (LRP), multidrug resistance‐associated protein (MRP1), glutathione‐S‐transferase‐π (GST‐π), excision repair cross‐complementing gene 1 (ERCC1), and p38 phosphorylation. We found that TFPI‐2 improved the RI of 5‐FU and inhibited P‐gp function. Western blotting and real‐time PCR revealed that TFPI‐2 also decreased mRNA and protein expression of MDR1, LRP, MRP1, GST‐π, and ERCC1, whereas p38 phosphorylation was increased. We considered that TFPI‐2 reduces 5‐FU resistance in BEL‐7402/5‐FU cells, and the mechanism appears to involve p38‐mediated downregulation of drug resistance gene expression such as MDR1, LRP, MRP1, GST‐π, and ERCC1. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple‐negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three‐dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6‐depleted, triple‐negative MDA‐MB‐231 cells rearranged the actin cytoskeleton and restored epidermal growth factor‐mediated invasion. In patients with localized, lymph node‐negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse‐free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6–fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Expression of multidrug resistance 1 gene in B‐cell lymphomas: association with follicular dendritic cells

Aims: Multidrug resistance (MDR) in B‐cell lymphomas still constitutes a major obstacle to the effectiveness of chemotherapy even in the anti‐CD20 antibody therapy era. The aim of this study was to investigate the expression of MDR‐associated molecules in reactive lymphadenopathy (RL), follicular lymphoma (FL), and diffuse large B‐cell lymphoma (DLBCL). Methods and results: The expression of mRNA for ABC‐transporter family genes was determined by real‐time RT‐PCR in lymph nodes from RL, FL, and DLBCL cases. MDR1 exhibited significantly stronger expression in RL, FL, and DLBCL than Raji B‐cell lymphoma cells. RL and FL showed significantly higher expression than DLBCL. Immunohistochemically, MDR1 positive cells were localized in the germinal centers of RL and center of the nodular lesions of FL showing associations with CD21 positive follicular dendritic cells (FDCs). Raji cells were co‐cultured with FDC sarcoma‐derived cells and the expression of MDR1 and drug resistance were analyzed. The co‐culture of Raji cells with FDCs induced strong expression of MDR1 and introduced resistance to doxorubicin‐induced apoptosis. Conclusions: These results suggest that FDCs induce MDR1 expression in reactive as well as neoplastic B‐cells. Inhibition of the interaction of FDCs with B‐cells may provide a novel strategy for treating the chemotherapy resistant fraction.     

胃癌干细胞对氟尿嘧啶敏感性及化疗药物耐受细胞的生物学机制

背景：氟尿嘧啶是胃癌化疗的基础药物,临床上耐药现象较为常见。肿瘤干细胞对化疗药敏感性较低,可能是导致化疗后肿瘤复发进展的重要原因。 目的：探讨胃癌干细胞对氟尿嘧啶敏感性,从细胞生物学角度分析胃癌的化疗耐药机制。 方法：利用免疫组织化学染色法检测69例胃癌组织内干细胞标志物CD44和耐药蛋白胸苷酸合成酶表达情况;基于克隆形态的分选策略,从AGS胃癌细胞系内分离胃癌干细胞克隆,检测CD44和胸苷酸合成酶的表达和自我更新能力;利用CCK-8法检测不同AGS细胞克隆的5-氟尿嘧啶半数抑制浓度(IC50)。 结果与结论：69例胃癌组织标本中,胸苷酸合成酶和CD44的表达阳性率分别为57%(39/69)和61%(42/69),CD44与胸苷酸合成酶的表达之间呈正相关关系(Kappa=0.41,χ²=11.59,P < 0.05)。AGS细胞系的克隆形成率为39%(29/69),其中,副克隆、次克隆、全克隆所占比例分别为 17%(5/29)、69%(20/29)、14%(4/29)。二代克隆形成后,采用胰酶消化克隆并再次低密度接种传代,副克隆均无法传代,次克隆仅少数可连续传代,全克隆均可连续传代。不同浓度5-氟尿嘧啶作用之后,全克隆的生长抑制率均显著低于次克隆和AGS细胞,经比较差异均有显著性意义(P < 0.05)。以上结果表明胃癌干细胞对5-氟尿嘧啶敏感性较低,其对化疗药物耐受,可能是临床胃癌化疗耐药的产生机制之一。     

Gene therapy to overcome drug resistance in cancer: targeting key regulators of the apoptotic pathway

A better understanding of the molecular events responsible for the development of drug resistance in cancer cells has emerged in recent years. It is now established that tumor cells can acquire drug resistance by alterations of pathways involved in the regulation of apoptosis and that failure to activate this pathway in cancer cells may confer resistance to chemotherapy. This resistance to drug-induced apoptosis is likely to play an important role in tumors that are refractory to chemotherapy. The identification of points in the apoptotic pathway at which dysregulation occurs opens up new therapeutic opportunities in situations where conventional cytotoxic chemotherapy approaches fail. Although these gene therapy-based strategies are still in their infancy they will likely lead to more effective treatments for human cancers. This review will focus on gene therapy strategies developed to specifically target the apoptotic pathway and how these strategies can affect the sensitivity of tumor cells to chemotherapy.     

Molecular mechanisms of drug resistance

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Tumours may be intrinsically drug-resistant or develop resistance to chemotherapy during treatment. Acquired resistance is a particular problem, as tumours not only become resistant to the drugs originally used to treat them, but may also become cross-resistant to other drugs with different mechanisms of action. Resistance to chemotherapy is believed to cause treatment failure in over 90% of patients with metastatic cancer, and resistant micrometastic tumour cells may also reduce the effectiveness of chemotherapy in the adjuvant setting. Clearly, if drug resistance could be overcome, the impact on survival would be highly significant. This review focuses on molecular mechanisms of drug resistance that operate to reduce drug sensitivity in cancer cells. Drug resistance can occur at many levels, including increased drug efflux, drug inactivation, alterations in drug target, processing of drug-induced damage, and evasion of apoptosis. Advances in DNA microarray and proteomic technology, and the ongoing development of new targeted therapies have opened up new opportunities to combat drug resistance. We are now able to characterize the signalling pathways involved in regulating tumour cell response to chemotherapy more completely than ever before. This will facilitate the future development of rational combined chemotherapy regimens, in which the newer targeted therapies are used in combination with cytotoxic drugs to enhance chemotherapy activity. The ability to predict response to chemotherapy and to modulate this response with targeted therapies will permit selection of the best treatment for individual patients.     

Epigenetic silencing of miR‐200c in breast cancer is associated with aggressiveness and is modulated by ZEB1

Loss of expression of miR‐200 family members has been implicated in cellular plasticity, a phenomenon that accounts for epithelial‐to‐mesenchymal transition (EMT) and stem‐like features of many carcinomas and is considered a major cause of tumor aggressiveness and drug resistance. Nevertheless, the mechanisms of miR‐200 downregulation in breast cancer are still largely unknown. Here we show that miR‐200c expression inversely correlates with miR‐200c/miR‐141 locus methylation in triple‐negative breast tumors (TNBC). Importantly, low levels of miR‐200c expression and high levels of miR‐200c/miR‐141 locus methylation associated with lymph node metastasis. Moreover, miR‐200c/miR‐141 locus methylation was significantly related to high expression of ZEB1 in two independent TNBC series. Silencing of ZEB1 in vitro reduced miR‐200c/miR‐141 DNA methylation and, concurrently, decreased histone H3K9 trimethylation. This chromatin modifications were paralleled by an increase in the expression of both miR‐200c and E‐cadherin. Similar effects were achieved by treatment with a demethylating agent. Our data suggest that gene methylation is an important element in the regulation of the miR‐200c/ZEB1 axis and that chromatin remodeling of the miR‐200c/miR‐141 locus is affected by ZEB1 and, thus, contributes to ZEB1‐induced cellular plasticity. © 2016 Wiley Periodicals, Inc.     

Establishment of pancreatic cancer stem cells by flow cytometry and their biological characteristics

To investigate the method of separating human pancreatic cancer stem cells by Hoechst 33342 labeled flow cytometry and to analyze the biological properties of pancreatic cancer stem cells. The human pancreatic cancer cell line PC-3 was divided into SP and non-SP cells by flow cytometry. The number of two cell clone spheres and nude mice tumor formation rates were compared by cultivating in serum-free medium; The expression of CD133, Nestin mRNA and protein was analyzed by real-time fluorescence quantitative PCR and Western blot; The expression of two cell drug resistance genes (MDR1, ABCG2, ABCA2 and MRP1) was analyzed by real time fluorescent quantitative PCR. The number of the cloned spheres in SP cells in serum-free medium was significantly higher than that of non-SP cells (P<0.05). The incidence of SP cells in the tumor of immunodeficiency nude mice was significantly higher than that of non-SP cells, and the difference was statistically significant (P<0.05). Real-time fluorescence quantitative PCR analysis showed that the expression of CD133 and Nestin mRNA in SP cells was significantly higher than those of non-SP cells, and the expression of CD133 and Nestin protein in SP cells was also significantly higher than those of non-SP cells (P<0.05). In conclusion, SP side population pancreatic cancer cells by Hoechst 33342 separation have the stem cell characteristics, higher tumor formation rate and higher drug resistance, which may be related to chemotherapy resistance.     

Comparison of E-selectin expression at mRNA and protein levels in murine models of inflammation

Background:Drug targeting to activated endothelial cells via E-selectin is currently being explored as a new approach to treat chronic inflammatory disorders. This approach uses E-selectin directed antibodies as carrier molecules to selectively deliver anti-inflammatory drugs into activated endothelial cells, thereby theoretically decreasing drug-associated side-effects. Therapeutic effects of developed drug targeting constructs will have to be tested in animal models of inflammation, in which E-selectin is expressed during the course of the disease. In this study several murine models of inflammation were investigated regarding expression of E-selectin.Methods:E-selectin expression was determined both at the mRNA level using RT-PCR and at the protein level by immunohistochemistry using two monoclonal antibodies (10E9.6 and MES-1). The models studied included delayed type hypersensitivity induced skin inflammation, dextran sodium sulphate induced colitis, kidney ischemia/reperfusion injury, atherosclerosis in ApoE knockout mice, and collagen induced arthritis.Results:In all animal models E-selectin mRNA expression was detected, although to a different extent. In contrast, only the delayed type hypersensitivity model and, to a minor extent, the collagen induced arthritis model showed E-selectin protein expression.Conclusion:These results stress the need to determine E-selectin protein expression and not only mRNA expression, when choosing an animal model for testing E-selectin directed drug targeting preparations. In addition, in the arthritis model, E-selectin protein detection was dependent on the particular anti-E-selectin antibody used. This finding may not only have implications for the development and/or choice of homing devices to be used in E-selectin directed drug targeting preparations, but also for inflammation research in general.     

Zinc finger E-box-binding homeobox 2 (ZEB2) regulated by miR-200b contributes to multi-drug resistance of small cell lung cancer

Zinc finger E-box-binding homeobox 2 (ZEB2) was closely related to the oncogenesis, development and response to chemotherapy of cancer. However, its biological functions in small cell lung cancer (SCLC) remain unknown. The aim of this study is to investigate the roles of ZEB2 in chemoresistance of SCLC and its possible molecular mechanism. Expression of ZEB2 was examined in sixty-eight cases of SCLC tissues by immunohistochemistry. Knockdown of ZEB2 was carried out in SCLC multidrug resistant cells (H69AR) to assess its influence on chemoresistance. The results showed that ZEB2 was expressed in 23.5% (16/68) of SCLC. Overexpression of ZEB2 was associated with the poor pathologic stage of SCLC (P < 0.001 by the Fisher's Exact Test) and the shorter survival time (by the Kaplan–Meier method). Inhibition of ZEB2 expression using small interfering RNA in H69AR cells sensitized cancer cells to chemotherapeutic drugs through increasing drug-induced cell apoptosis accompanied with S phase arrest. In silico analysis demonstrated that there are complementary binding sites between miR-200b and ZEB2 3′-UTR, and identified miR-200b as a potential regulator of ZEB2. We found that miR-200b was down-regulated in the resistant cells and enforced expression of miR-200b by miRNA mimics increased cell sensitivity. Overexpression of miR-200b led to the downregulation of ZEB2 at protein level. Luciferase reporter gene assay showed that 3′UTR ZEB2 activity was regulated by miR-200b. Our results suggest that ZEB2 modulates drug resistance and is regulated by miR-200b. All findings provide insight into the ZEB2 signaling mechanism and ZEB2 may be a potentially novel target for multi-drug resistance in SCLC.     

维生素D通过下调谷氨酰胺合成酶抑制乳腺癌细胞增殖

目的 探究维生素D对非三阴性乳腺癌(non-triple negative breast cancer,Non-TNBC)和TNBC癌细胞增殖的影响及分子机制.方法 收集TNBC和Non-TNBC患者乳腺组织,原代培养TNBC和Non-TNBC患者乳腺细胞;免疫荧光检测乳腺细胞雌激素受体(ER)、孕激素受体(PR)和H...     

Adrenaline induces chemoresistance in HT-29 colon adenocarcinoma cells

Psychological distress and its ensuing chronic elevation of plasma catecholamines (adrenaline and noradrenaline) lead to poor response of tumors to chemotherapy, and constitute a poor prognostic factor for survival. Colorectal cancer patients suffer from various forms of psychological stress reflected in elevated plasma catecholamines, and their cancer cells express adrenergic receptors. Our objective was to investigate whether adrenergic activation contributes to the chemoresistance of colon cancers, and to explore the signal transduction pathway involved in the activation. The mRNA expression of the ABCB1 gene (previously MDR1) in human colon carcinoma HT-29 cell line was measured after treatment with an adrenergic receptor agonist (adrenaline) and various antagonists (propranolol, prazosin, and yohimbine). The function of P-glycoprotein, the protein product of the ABCB1 gene, was assessed by rhodamine 123 (Rh123)–retention assay, and chemosensitivity was determined by evaluating the cytotoxicity of 5-fluorouracil (5-FU) on the tumor cells. Increased ABCB1 mRNA expression and P-glycoprotein function levels in HT-29 cells by adrenaline was dose-dependent. This was accompanied by promotion of Rh123 efflux, and resistance to the growth-inhibiting effect of 5-FU in the tumor cells. The α₂-adrenergic receptor antagonist yohimbine completely abolished the induction of ABCB1 mRNA, the stimulatory effect of adrenaline on Rh123 efflux, and the growth-inhibiting effect of 5-FU. The α₁-adrenergic receptor and β-adrenergic receptor antagonists did not inhibit the induction of ABCB1. The stimulating effects were coupled with extracellular receptor kinase 1/2 (Erk1/2) phosphorylation, but were not associated with protein kinase A activity. We conclude that adrenaline induces multidrug resistance in colon cancer cells by upregulating ABCB1 gene expression via α₂-adrenergic receptors, and such effects were associated with the mitogen activated protein kinase (MAPK) pathway.     

内源性IL-8诱导卵巢癌细胞对顺铂与紫杉醇产生耐药的机制研究

目的探讨内源性IL-8诱导卵巢癌细胞对顺铂和紫杉醇产生耐药的机制及相关信号转导通路。方法在原有工作基础上,以2种人卵巢癌细胞系A2780(不分泌IL-8,对顺铂、紫杉醇敏感)和SKOV-3(高分泌IL-8,对顺铂、紫杉醇耐药)为研究模型,分别将正义(sense,ss)IL-8基因或反义(antisense,as)IL-8基因稳定转染至A2780细胞或SKOV3细胞,应用MTT法、Caspase-3活性测定、RT-PCR及Western blot技术等观察内源性IL-8是否影响卵巢癌细胞对顺铂和紫杉醇的敏感性,并对其作用的机制和可能的信号传导通路进行研究。结果 1)内源性过表达IL-8可诱导A2780细胞对顺铂和紫杉醇产生耐药,而抑制IL-8表达可恢复SKOV3细胞对顺铂和紫杉醇的敏感性,IL-8诱导的卵巢癌细胞化疗耐药是通过降低Caspase-3活性来实现的;2)内源性过表达IL-8可上调A2780细胞的耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达,而抑制IL-8表达可使上述基因的表达明显降低;3)Wortmannin(PI3K抑制剂)和PD98059(MEK1/2抑制剂)能分别阻断IL-8诱导下卵巢癌细胞的Akt和ERK活化及化疗耐药作用。结论 IL-8诱导的卵巢癌细胞化疗耐药可能与其上调耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达以及活化Raf/MEK/ERK和PI3K/Akt信号通路相关,提示调节IL-8表达或其相关信号通路可能是治疗耐药性卵巢癌的一种良好策略。     

LINE-1 amplification accompanies explosive genome repatterning in rodents

Transposable elements (TEs) sometimes induce karyotypic changes following recombination, breakage and rearrangement. We used FISH and Southern blot analyses to investigate the amount and distribution of LINE-1 retrotransposons in rodents (genus Taterillus, Muridae, Gerbillinae) that have recently undergone an important genome repatterning. Our results were interpreted in a known phylogenetic framework and clearly showed that LINE-1 elements were greatly amplified and non-randomly distributed in the most rearranged karyotypes. A comparison between FISH and conventional banding patterns provided evidence that LINE-1 insertion sites and chromosome breakpoints were not strongly correlated, thus suggesting that LINE-1 amplification subsequently accompanied Taterillus chromosome evolution. Similar patterns are observed in some cases of genomic stresses (hybrid genomes, cancer and DNA-damaged cells) and usually associated with DNA hypomethylation. We propose that intensively repatterned genomes face transient stress phases during which some epigenetic features, such as DNA methylation, are relaxed, thus allowing TE amplification.