The Hippo pathway in chemotherapeutic drug resistance

Chemotherapy is one of the major treatments for cancer patients. Although chemotherapeutic drugs can sometimes effectively suppress tumor growth in cancer patients, a significant proportion of patients are either intrinsically resistant or later develop resistance to primary chemotherapy, leading to disease relapse and patient mortality. The best way to conquer the resistance is the better understanding of the molecular network in cancer cells in response to drugs. Therefore, identification of signaling pathways and molecules involved in drug resistance is essential for successful treatment of cancers. The Hippo pathway is an emerging signaling pathway that plays important roles in tumorigenesis, stem cell self‐renewal and differentiation, organ size control as well as many other biological processes. Therefore, exploring novel roles of the Hippo pathway in various biological functions has become one of the cutting‐edge research areas in cancer and other biomedical research. Recently, we and others have provided new evidence that the Hippo pathway is involved in the resistance of different types of cancer cells to various chemotherapeutic drugs. In this review, we will discuss the specific roles of the Hippo pathway in chemotherapy, potential applications for studying this network in response to drugs as well as the future direction in identification of therapeutic targets.     

微环境乏氧对胰腺癌PC-2细胞生长及形态的影响

目的：观察体外培养的胰腺癌PC-2细胞在化学乏氧剂CoCl2作用后形态学及生长曲线的变化,为进一步探讨乏氧对肿瘤细胞的影响寻求直观依据。方法：用CoCl2处理PC-2细胞模拟肿瘤体内乏氧模型,应用倒置显微镜、透射电镜观察细胞形态学变化,MTT法检测不同浓度、不同时间CoCl2处理后的PC-2细胞生长情况。结果：化学乏氧剂CoCl2作用后的胰腺癌PC-2细胞形态发生改变,与正常细胞相比,乏氧后的细胞体积变小、伪足少而短,易分辩个体,但并不可见脱落细胞增多或减少;电镜下可见线粒体肿胀,染色质边集,少见凋亡小体。不同浓度CoCl2对胰腺癌PC-2细胞生长曲线有不同程度的改变,对数生长期和平台期提前,对数期缩短。随着CoCl2浓度增加,曲线越趋于低平。结论：化学乏氧剂CoCl2对胰腺癌PC-2细胞具有一定的凋亡诱导作用,同时也缩短了细胞正常的生长期,细胞形态发生相应改变。     

缺氧调控端粒酶活性及抑制鼻咽癌细胞增殖实验研究

目的探讨缺氧及siRNA沉默缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)后对鼻咽癌细胞中端粒酶催化亚单位(telomerase catalytic subunit,hTERT)、细胞周期和化疗耐药的影响。方法采用三气培养箱对鼻咽癌细胞5-8F和CNE2进行缺氧处理(1%O2),蛋白质印迹法检测不同乏氧时相(0~72h)HIF-1α和hTERT蛋白的表达。将HIF-1α基因特异性siRNA分别转染鼻咽癌细胞株5-8F和CNE2,筛选出沉默效率最高的siRNA,实验分为未处理组(常氧)、未处理组(缺氧)、Negative-siRNA(缺氧)和HIF-1α-siRNA(缺氧),荧光定量PCR及蛋白质印迹检测瞬时转染后hTERT及HIF-1α的表达。流式细胞术(flow cytometry,FCM)分析缺氧或沉默HIF-1α后对细胞周期的影响。MTT法检测缺氧或沉默HIF-1α后,鼻咽癌细胞对顺铂(DDP)和5-氟尿嘧啶(5-FU)的化疗敏感性。结果缺氧处理0~72h后鼻咽癌5-8F细胞HIF-1α(F=37.147,P<0.001)和hTERT(F=70.069,P<0.001)蛋白的表达上调,差异有统计学意义。HIF-1α-siRNA对5-8F细胞瞬时转染率>98%。HIF-1α-siRNA组hTERT mRNA表达量为0.37±0.05,显著低于未处理组(缺氧)的1.00±0.00和Negative-siRNA(缺氧)的0.95±0.01,F=360.339,P<0.001;hTERT蛋白表达量为(0.27±0.05),显著低于未处理组(缺氧)0.54±0.00和Negative-siRNA组(缺氧)0.53±0.01,F=24.010,P<0.001。未处理组(缺氧)G0/G1期细胞比例明显增加(45.63±2.01)%,显著高于未处理组(常氧)的(26.75±1.28)%,P<0.001。5-8F细胞未处理组(常氧)对5-FU的IC50分别为(17.30±3.31)μg/mL,未处理组(缺氧)为(32.04±12.75)μg/mL,Negative-siRNA组为(33.90±0.87)μg/mL,HIF-1α-siRNA组为(13.72±2.36)μg/mL,F=3.704,P<0.001。5-8F细胞缺氧组对DDP的化疗敏感性也降低,沉默HIF-1α后,5-8F细胞对DDP的化疗敏感性明显提高。除细胞周期外,CNE2与5-8F的结果均一致。结论缺氧促使鼻咽癌细胞发生G1/S阻滞及化疗耐药,可能与上调hTERT表达,进而上调端粒酶活性有关;沉默HIF-1α显著逆转缺氧诱导的肿瘤耐药并下调端粒酶活性。     

Mislocalization of death receptors correlates with cellular resistance to their cognate ligands in human breast cancer cells

Multiple clinical trials are ongoing to evaluate the potential antitumor activity of human TNF variants, Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL) and its agonistic antibodies. These drug products act through the death receptors (DRs) TNF receptor 1 (TNFR1), Fas/CD95, DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2), respectively. Therefore, characterization of the level and localization of DR expression in cancer cells is important for DR-targeted therapy. In this study, we examined the subcellular distribution of the four DRs in a panel of 10 human breast cancer cell lines by western blots and flow cytometry and 50 human breast tumors by immunohistochemistry. Despite their total protein expressions, the DRs were found to be absent on the surface of some cell lines. Consistent with this result, all four DRs were found to be mostly expressed in the cytoplasm and/or the nucleus of primary breast tumors (n=50). We further determined the growth inhibition activity (GI50) of the death ligands, recombinant human TNFα, FasL and TRAIL, and found a correlation with the subcellular localization of the corresponding DRs. These results demonstrate an aberrant expression of the death receptors in breast cancer cells, and suggest that the lack of surface DRs appears to be predictive of tumor resistance to DR-targeted therapies.     

缺氧微环境诱导胶质母细胞瘤耐药机制研究进展

胶质母细胞瘤（glioblastoma,GBM）的耐药性是导致其临床治疗失败的主要原因。目前,诸多研究认为肿瘤内部缺氧的微环境能够诱导GBM对放化疗的抵抗。GBM的生长极为迅速,贫瘠的氧供不能满足其对氧气的需求,缺氧的微环境由此形成。缺氧可通过不同机制影响GBM的耐药性。本文将根据缺氧微环境的形成与缺氧诱导GBM耐药的机制进行综述。      

Androgen deprivation results in time‐dependent hypoxia in LNCaP prostate tumours: Informed scheduling of the bioreductive drug AQ4N improves treatment response

Androgen withdrawal induces hypoxia in androgen‐sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time‐dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT‐induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti‐androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide‐treated and vehicle‐only‐treated tumours were re‐established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 ± 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide‐treated tumours were more malignant than vehicle‐treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide‐induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.     

A new superinvasive in vitro phenotype induced by selection of human breast carcinoma cells with the chemotherapeutic drugs paclitaxel and doxorubicin

Doxorubicin- and paclitaxel-selected variants of an in vitro invasive clonal population of the human breast cancer cell line, MDA-MB-435S, were established by pulse selection, and exhibited a novel 'superinvasive' phenotype. This phenotype is characterised by an ability to relocate to another surface following invasion through matrigel and membrane pores, by decreased adhesion to extracellular matrix proteins and by increased motility. This may represent an in vitro model of a step in the metastatic process occurring subsequent to invasion. The paclitaxel-resistant variants, MDA-MB-435S-F/Taxol-10p and MDA-MB-435S-F/Taxol-10p4p were resistant to paclitaxel, vincristine and docetaxel, but not to doxorubicin, carboplatin, etoposide or 5-fluorouracil. The doxorubicin-selected variants MDA-MB-435S-F/Adr-10p and MDA-MB-435S-F/Adr-10p10p, in contrast, exhibited only small increases in resistance to doxorubicin, although they were slightly resistant to VP-16 and docetaxel, and exhibited increased sensitivity to paclitaxel, carboplatin and 5-fluorouracil.     

MicroRNA-17-5p promotes chemotherapeutic drug resistance and tumour metastasis of colorectal cancer by repressing PTEN expression

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide, especially in Western countries. Although chemotherapy is used as an adjuvant or as a palliative treatment, drug resistance poses a great challenge. This study intended to identify biomarkers as predictive factors for chemotherapy.Patients and methods: By microarray analysis, we studied miRNAs expression profiles in CRC patient, comparing chemoresistant and chemosensitive groups. The miRNAs of interest were validated and the impact on clinical outcomes was assessed in a cohort of 295 patients. To search for potential targets of these miRNAs, tissue samples were subject to in situ hybridization and immunohistochemistry analysis. Colorectal adenocarcinoma cells were also used for in vitro experimentation, where cellular invasiveness and drug resistance were examined in miRNA-transfected cells.Results: The expression level of miRNA-17-5p was found increased in chemoresistant patients. Significantly higher expression levels of miR-17-5p were found in CRC patients with distant metastases and higher clinical stages. Kaplan-Meier analysis showed that CRC patients with higher levels of miR-17-5p had reduced survival, especially in patients who had previously received chemotherapy. Overexpression of miR-17-5p promoted COLO205 cell invasiveness. We found that PTEN was a target of miR-17-5p in the colon cancer cells, and their context-specific interactions were responsible for multiple drug-resistance. Chemotherapy was found to increase the expression levels of miR-17-5p, which further repressed PTEN levels, contributing to the development of chemo-resistance.Conclusions: MiR-17-5p is a predictive factor for chemotherapy response and a prognostic factor for overall survival in CRC, which is due to its regulation of PTEN expression.     

自噬与肺癌化疗和靶向药物耐药关系的研究进展

肺癌是全球癌症相关死因第一位的恶性肿瘤,目前临床上治疗中晚期肺癌主要以化疗和分子靶向治疗为主。肿瘤耐药是临床上化疗和靶向治疗失败的最主要原因之一。自噬相关分子参与机体生理病理众多过程而且调控机制非常复杂。本文主要综述在肺癌化疗和靶向药物耐药的过程中自噬相关分子发生的改变,旨在阐明自噬与肺癌耐药的关系。     

In vitro cellular drug resistance adds prognostic information to other known risk-factors in childhood acute lymphoblastic leukemia

Leukemic cells from 230 children with newly diagnosed B-cell precursor ALL were tested for in vitro drug resistance to a panel of anti-cancer drugs. Minimal residual disease (MRD) was measured by RQ-PCR. During follow-up, 24 relapses occurred in the 159 children with MRD <0.1% day 29. The risk of any relapse was correlated to vincristine and doxorubicin resistance, with a relative risk of 3.7 (95% CI 1.3-10.5; p = 0.016) for patients resistant to both drugs. There was a significant correlation also for the subgroup with extra-medullary relapses. Our findings indicate that analysis of drug resistance can add prognostic information to other known risk-factors including MRD.     

活体细胞凋亡检测筛选肝癌敏感化疗药物的实验研究

目的:通过动物实验探讨99 Tcm-HYNIC-Annexin V活体细胞凋亡检测筛选肝癌敏感化疗药物的可行性。方法:20只VX-2荷瘤免随机分为4组,通过治疗剂量的紫杉醇(PAC,n=6),5-氟尿嘧啶(5-FU,n=5),表柔比星(EPI,n=5)分组干预治疗,同时建立对照组(C,n=4)。分别予化疗前2h、化疗后46h静脉注射99 Tcm-HYN-IC-Annexin V,2h后再以单光子发射计算体层摄影(SPECT)成像,选取肿瘤部位放射性计数(T)及腿部非肿瘤区部位放射性计数(NT),计算其比值(T/NT),进行半定量分析,确定敏感化疗药物。取肿瘤组织,分别予免疫组化(Tunel)和流式细胞仪(FCM)分析组织细胞凋亡率,放射性核素聚集显像与肿瘤细胞凋亡进行相关性分析。实验数据以SPSS 17.0软件分析。结果:99 Tcm-HY-NIC-Annexin V放射显像显示,化疗前各组肿瘤部位均无明显显像(F=0.025,P=0.980),放射性核素分布主要聚集在肾脏及膀胱区。药物干预48h后,对照组仍无显像,而药物干预组显像清楚,放射性计数显著高于对照组(F=16.52,P<0.001)及化疗前(P<0.01)。药物干预组中,PAC组肿瘤组织放射性显像显著高于5-FU组和EPI组(P值分别为0.042和0.003),而5-FU组与EPI组间放射性显像差异无统计学意义(P=0.224)9。9 Tcm-HYNIC-Annexin V活体细胞凋亡检测结果与免疫组化及流式细胞检测结果一致(R2=0.537 7,P=0.002;R2=0.695 4,P<0.001)。结论9:9 Tcm-HYNIC-Annexin V核素显像检测能够反映活体组织细胞凋亡情况,该方法能够在活体状态下筛选敏感化疗药物。     

Transport of glutathione conjugates and chemotherapeutic drugs by RLIP76 (RALBP1): a novel link between G-protein and tyrosine kinase signaling and drug resistance

Our studies have shown that RLIP76 (RALBP1), a 76 kDa Ral-binding, Rho/Rac-GAP and Ral effector protein, is a novel multispecific transporter of xenobiotics as well as GS-Es. Like previously characterized ABC transporters, it mediates ATP-dependent transport of structurally unrelated amphiphilic xenobiotics and displays inherent ATPase activity, which is stimulated by its substrate allocrites. It does not have significant sequence homology with ABC transporters and differs from the ABC transporters in several other important aspects, including (i) lack of any close homologs in humans, (ii) lack of a classical Walker domain, (iii) integral membrane association without clearly defined transmembrane domains and (iv) its role as a direct link to Ras/Ral/Rho and EGF-R signaling through its multifunctional nature, including GAP activity, regulation of exocytosis as well as clathrin-coated pit-mediated receptor endocytosis. Its multifunctional nature derives from the presence of multiple motifs, including a Rho/Rac GAP domain, a Ral effector domain binding motif, 2 distinct ATP-binding domains, a H(+)-ATPase domain, PKC and tyrosine kinase phosphorylation sites and the ability to undergo fragmentation into multiple smaller peptides which participate as components of macromolecular functional complexes. One of the physiologic functions of RLIP76 is regulation of intracellular concentration of the electrophilic intermediates of oxidative lipid metabolism by mediating efflux of GS-E formed from oxidative degradation of arachidonic acid, including leukotrienes and the 4HNE-GSH conjugate. RLIP76-mediated transport of amphiphilic chemotherapeutic agents such as anthracyclines and vinca alkaloids as well as GS-E produced during oxidative metabolism places this multifunctional protein in a central role as a resistance mechanism for preventing apoptosis caused by chemotherapeutic agents and a variety of external/internal stressors, including oxidative stress, heat shock and radiation.     

Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia

Bone marrow and peripheral blood samples from 362 patients with acute lymphoblastic leukaemia (ALL) proliferating cell and 90 patients with acute myeloid leukaemia (AML) were analysed for S-phase fractions, Ki67 antigen, and proliferating cell nuclear antigen expression. The S-phase fractions were correlated with in vitro drug resistance to 15 different anticancer agents. Leukaemia cells isolated from bone marrow had higher S-phase fractions than leukaemia cells isolated from peripheral blood (in initial ALL, median values resp. 6.9 and 2.7%, in initial AML resp. 5.3 and 1.3%; both P<0.01). Relapse ALL samples derived from bone marrow showed increased S-phase fractions (median 9.9%) compared with initial ALL samples (median 6.9%; P<0.01). ALL samples obtained at initial diagnosis showed higher S-phase fractions (median 6.9%) and higher Ki67 expression (median 30%) than initial AML samples (median resp. 5.3 and 14%; both P<0.05). The S-phase fractions were not related to white blood cell count, age, or gender. Within initial ALL, the S-phase fraction correlated significantly but modestly strong (rho=0.3-0.5; P<0.05) with sensitivity to antimetabolites (cytarabine, mercaptopurine, thioguanine), L-asparaginase, teniposide, and vincristine. Similar results were found within subgroups of initial ALL (nonhyperdiploid and common/precursor-B-lineage ALL). In relapsed ALL and AML such correlations were not found. In conclusion, cell proliferation differs between leukaemia subgroups and increased proliferation is associated with increased in vitro sensitivity to several anticancer agents in initial ALL.     

药物联合逆转肿瘤细胞耐药的机制及研究进展

针对肿瘤细胞的特异性目前开发了各种治疗药物,但由于耐药机制的存在,最终限制了抗肿瘤药物的疗效。因此采用新颖的联合疗法,了解联合疗法的相互作用,通过这些相互作用来干扰肿瘤细胞生存或生长途径和它们之间建立的串扰,来逆转肿瘤细胞的耐药机制,是未来解决肿瘤细胞耐药性问题的关键点之一。本文就目前联合用药抗肿瘤细胞耐药性的研究进展作出系统回顾。     

Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia

Purpose A high-rate glycolysis is a fundamental property of solid tumors and is associated with an over-expression of glucose transporters and glycolytic enzymes. We hypothesize that over-expression of glucose transporters in tumors prevents apoptosis, promotes cancer cell survival, and confers drug resistance. Inhibition of glucose transporter will preferentially sensitize the anticancer effects of chemotherapeutic drugs to overcome drug resistance in hypoxia. Methods Glucose transporter expressions were detected in cancer tissues and NCI 60 cancer cells with immunostaining and DNA microarray. Glucose uptake was measured with ³H-2-deoxy-glucose. Cytotoxicity of daunorubicin (DNR) in combination of glucose inhibitor was detected by MTS assay under hypoxic condition. Early stage apoptosis was monitored with Annexin V-FITC staining. Results Immunostaining showed that GLUT1 was significantly increased in hypoxic regions of the human colon and breast tumors. The expression profiles of all glucose transporters in NCI 60 cancer cells exhibited distinct expression patterns. Phloretin exhibited more than 60% glucose uptake inhibition. Hypoxia conferred two to fivefold higher drug resistance in SW620 and K562 to DNR. Inhibition of glucose uptake by phloretin sensitized cancer cells to DNR for its anticancer activity and apoptosis to overcome drug resistance only under hypoxia. Conclusion Cancer cells heavily rely on glucose transporters for glucose uptake to facilitate a high-rate glycolysis under hypoxia for their survival and drug resistance. Combination of glucose transporter inhibitors and chemotherapeutic drugs may provide a preferential novel therapeutic strategy to overcome drug resistance in hypoxia.