Potential applications of RNA interference technology in the treatment of cancer

Inhibition of growth and progression of cancer cells is a challenge with major potential impact. RNA interference (RNAi) technology has been rapidly developed as a laboratory tool for the downregulation of the expression of a gene of interest. Moreover, RNAi offers a new potential for gene therapy of particular neoplasms by the specific inhibition of a cancer-associated target. This article will briefly describe the mechanism and application possibilities of RNAi, and illustrate the therapeutic potential in cancer gene therapy. The utilization of RNAi technology as a potential therapeutic tool for the treatment of cancer will be discussed in detail for two specific targets; the Bcr-Abl tyrosine kinase and the multidrug transporter MDR1/P-glycoprotein.     

Mutational Targets in Colorectal Cancer Cells with Microsatellite Instability

Cancers arise from the sequential acquisition of genetic alterations in specific genes. The high number of mutations in cancer cells led to the hypothesis that an early step in tumor progression is the generation of a genetic instability. The potent role of genetic instability in initiation and progression of colorectal cancers has been well defined in hereditary nonpolyposis colon cancer (HNPCC) syndrome. HNPCC is a common hereditary disorder caused by germline mutations of DNA mismatch repair (MMR) genes. Somatic loss of the normal allele of the predisposition gene leads to a strong “mutator phenotype”, characterized by a high rate of mutations in repetitive sequences. Nevertheless, the observation of frequent alterations of key growth regulatory genes in MMR-deficient cells such as NF1, APC, p53, K-Ras, with no significant excess of frameshift mutations and changes at short coding repeats, suggest that even in the presence of an inherited tendency to genomic instability, tumor progression is mainly driven by a process of natural selection.     

RNAi-mediated knockdown of target genes: a promising strategy for pancreatic cancer research

Pancreatic cancer is one of the most aggressive malignancies with a very poor prognosis, partially due to its very low accessibility to resection and resistance to chemoradiotherapy. As such, it is reasonable to find more effective, specific therapies and the related therapeutic targets. The identification of certain genes contributing to the tumorigenesis and poor prognosis provides the specific targets for efficient silencing by RNA interference (RNAi). As a powerful tool to suppress gene expression in mammalian cells, RNAi can be directed against pancreatic cancer through various pathways, including the inhibition of overexpressed oncogenes, suppression of tumor growth, metastasis and enhancement of apoptosis. In combination with chemoradiotherapy agents, RNAi can also attenuate the chemoradiation resistance of pancreatic cancer. In addition, RNAi has been used to define the 'loss of function' of endogenous genes in pancreatic cancer. This review provides a brief introduction to recent developments of RNAi applications in pancreatic cancer studies and suggestions for further exploration. It substantially demonstrates that RNAi holds a promising therapeutic potential as a future treatment for pancreatic cancer.     

Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro

RNA interference (RNAi) is a powerful tool for studying gene function. Here, we describe an inducible small interfering RNA expression system that allows a tight control of the specific gene silencing by RNAi. Using this system, we demonstrated the inducible RNAi effect on the gene expression in mammalian cells. We further showed that inducible knockdown of endogenous CXC chemokine receptor-4 (CXCR4) gene expression in breast cancer cells resulted in significant inhibition of breast cancer cell migration in vitro. This system should be useful for both basic researches on gene function and therapeutic applications of RNAi.     

Cancer: More of polygenic disease and less of multiple mutations? A quantitative viewpoint

The focus of cancer research is on cancer‐specific mutations, with most clinical trials involving targeted drugs. Huge numbers of DNA lesions and tumor resistance events, in each of the >10¹³ cells of a human individual, form a striking contrast to the low, and also very narrow, cancer incidence window (10^?1‐10⁰). A detailed consideration of these quantitative observations seems to question the present paradigm, while suggesting that a systemic regulatory network mechanism is a stronger determinant for overt cancer disease, as compared with cancer‐specific gene products. If we shall ever achieve major improvements in survival, we must gain understanding of this systemic network, rather than targeting therapy to a limited set of molecules or mutations. This may give us new opportunities for development of highly potent therapeutic tools. Cancer 2011. © 2010 American Cancer Society.     

Viral gene therapy

Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses. The ability for the delivery of therapeutic genes, made them desirable for engineering virus vector systems. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits gene expressionin vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many non-dividing and dividing cell types, but has a limited DNA capacity. This review discusses current and emerging virus-based genetic engineering strategies for the delivery of therapeutic molecules or several approaches for cancer treatment. Supported by an unrestricted educational grant from AstraZeneca.     

Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene

Increased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific antiapoptotic factors may provide a rational basis for the development of novel therapeutic strategies. We investigated here whether the RNA interference (RNAi) technology could be used to increase the apoptotic susceptibility of cancer cells. As a molecular target, we chose the antiapoptotic livin (ML-IAP, KIAP) gene, which is expressed in a subset of human tumors. We identified vector-borne small interfering (si)RNAs, which could efficiently block endogenous livin gene expression. Silencing of livin was associated with caspase-3 activation and a strongly increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin, UV-irradiation, or TNFalpha. The effects were specific for Livin-expressing tumor cells. Our results (i) provide direct evidence that the intracellular interference with livin gene expression resensitizes human tumor cells to apoptosis, (ii) define the livin gene as a promising molecular target for therapeutic inhibition, and (iii) show that the livin gene is susceptible to efficient and specific silencing by the siRNA technology.     

Epigenetic reduction of DNA repair in progression to gastrointestinal cancer

Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.     

腺病毒介导Her2／neu siRNA对卵巢癌细胞的生长抑制作用

目的观察腺病毒介导的针对Her2／neu基因RNAi表达载体对Her2／neu的抑制及抑制后对卵巢癌SKOV-3细胞的生长的影响。方法通过构建的针对Her2neu的siRNA重组腺病毒（滴度为1．6×10^8PFU／mL）感染卵巢癌细胞SKOV-3后，采用Western—blot法观察Her2／neu基因的沉默效果，流式细胞仪分析细胞周期变化，cell Proliferation法检查细胞体外增殖能力。结果构建的重组腺病毒Adeno—Her2siRNA感染卵巢癌细胞SKOV-3后Her2／neu的蛋白表达降低；S期细胞较对照组未感染细胞比例增加；肿瘤细胞胞增殖速度缓慢（P〈0．05）。结论成功构建的重组腺病毒Adeno-Her2siRNA感染后可有效降低Her2／neu蛋白水平表达，阻滞细胞感染于S期，造成感染细胞生长速度减慢，有望为卵巢癌的基因治疗提供一种选择手段。     

全外显子测序在乳腺癌发病机制及诊疗中的研究进展

 全外显子测序是利用序列捕获技术将全基因组外显子区域DNA捕捉并富集后进行高通量测序的基因组分析方法，该技术已经应用到各种复杂疾病的基因诊疗中。目前，乳腺癌的发病机制尚未完全阐明，而体细胞基因突变造成的癌基因激活与抑癌基因失活在乳腺癌发生发展的过程中扮演着至关重要的作用。近年来，众多研究小组开展了大量的全外显子测序研究，发现并鉴定了许多与复杂疾病/性状相关联的遗传变异，为复杂疾病包括乳腺癌的发病机制研究提供了重要线索。本文就全外显子测序在乳腺癌的发病机制及其诊治研究进行综述。     

甲基化抗肿瘤药物治疗中的DNA修复:治疗效果与健康结局

在甲基化抗肿瘤药物治疗中,DNA修复是决定治疗效果与不良生物效应（如突变、癌变和致畸）的一个关键机制。本文主要对甲基化抗肿瘤药物在DNA修复过程的作用进行综述。尽管这些抗肿瘤药物无选择性地靶向作用于癌细胞和正常细胞的DNA,但因其削弱了癌细胞内某些特异的DNA修复活动,从而更多地杀死癌细胞。单功能的烷化剂显示出甲基化改变特性（丙卡巴肼、达卡巴嗪、链脲佐菌素、替莫唑胺）,或者氯乙基化形成单加合物并在下一步反应中引起DNA链内交联（洛莫司汀、尼莫地平、卡莫司汀、福莫司汀）。癌细胞对抗肿瘤药物的一个主要机制是通过自杀酶O⁶-甲基鸟嘌呤-DNA甲基转移酶（MGMT）直接逆转DNA损伤,形成O⁶-甲基鸟嘌呤和O⁶-氯化鸟嘌呤。由于MGMT对恶性肿瘤治疗的结局有显著影响,它被认为是一个耐药的重要标志,特别是在高级恶性胶质瘤。MGMT也被认为是甲基化抗肿瘤药物有效性的预测标志,许多临床试验正在分析MGMT抑制对治疗效果的影响。其他涉及甲基化抗肿瘤药物耐药的DNA修复因素包括错配修复、通过同源重组和DNA双链断裂（DSB）信号启动的相关修复。碱基切除修复和alkB同源蛋白（如ABH2）也可能与烷化类药物耐药有关,该现象在高表达MGMT的细胞株异常明显。对于这些机制的进一步了解,将有助于设计更为有效的治疗方案,同时减少副作用。     

癌症靶向治疗的新趋势

靶向性是癌症治疗的关键所在,新的治疗方案必须在特异性作用于肿瘤的同时降低对正常细胞的损伤,目前已取得了许多成果:1)肿瘤的靶向基因-病毒治疗结合了基因治疗和病毒治疗的各自优势。所用的病毒载体具有特异性靶向肿瘤的作用并在肿瘤细胞中大量复制和高效表达抗癌基因,如果用2个特异性启动子严格控制病毒只在肿瘤中表达,并加上2个有协同效应的抗癌基因即可达到极好的抗癌效果。2)在抗体治疗方面,运用第1代腺病毒高效表达Her-ceptin或是Rituxan抗体基因已取得成功,它可以大大地降低目前昂贵的抗体治疗费用。3)RNA干扰技术可以沉默致病基因,选用质粒或是病毒做载体可以达到长期及高效的基因沉默效果。4)具有自我更新能力的肿瘤干细胞是肿瘤复发的关键。5)人们也逐渐意识到肿瘤的形成过程是癌细胞与基质细胞相互作用的结果,以肿瘤基质为靶标的药物也显示出了良好的效果。6)针对蛋白酪氨酸激酶或是抗凋亡蛋白而研制的小分子靶向药物也在肿瘤分子治疗上取得了成功。7)纳米技术正以其独特性而引起广泛关注。总之,在众多新型治疗方案研发的同时,我们必须认识到仅仅靠一种方法是难以成功根治肿瘤的,各种靶向治疗方案之间的相互结合将成为肿瘤治疗的主攻方向。     

Significance of multiple mutations in cancer

There is increasing evidence that in eukaryotic cells, DNA undergoes continuous damage, repair and resynthesis. A homeostatic equilibrium exists in which extensive DNA damage is counterbalanced by multiple pathways for DNA repair. In normal cells, most DNA damage is repaired without error. However, in tumor cells this equilibrium may be skewed, resulting in the accumulation of multiple mutations. Among genes mutated are those that function in guaranteeing the stability of the genome. Loss of this stability results in a mutator phenotype. Evidence for a mutator phenotype in human cancers includes the frequent occurrence of gene amplification, microsatellite instability, chromosomal aberrations and aneuploidy. Current experiments have centered on two mechanisms for the generation of genomic instability, one focused on mutations in mismatch repair genes resulting in microsatellite instability, and one focused on mutations in genes that are required for chromosomal segregation resulting in chromosomal aberrations. This dichotomy may reflect only the ease by which these manifestations can be identified. Underlying both pathways may be a more general phenomenon involving the selection for mutator genes during tumor progression. During carcinogenesis there is selection for cells harboring mutations that can overcome adverse conditions that limit tumor growth. These mutations are produced by direct DNA damage as well as secondarily as a result of mutations in genes that cause a mutator phenotype. Thus, as tumor progression selects for cells with specific mutations, it also selects for cancer cells harboring mutations in genes that normally function in maintaining genetic instability.     

Apoptosis of estrogen-receptor negative breast cancer and colon cancer cell lines by PTP alpha and src RNAi

We show that siRNA-mediated suppression of protein tyrosine phosphatase alpha (PTP alpha) reduces Src activity 2 to 4-fold in breast, colon and other human cancer cell lines. Src and PTP alpha RNAi induced apoptosis in estrogen receptor (ER)-negative breast cancer and colon cancer cells, but not in immortalized noncancerous breast cells, ER-positive breast cancer cells or other cancer cell types tested. RNAi of other Src family members (Fyn and Yes) or of PTP1B, a phosphatase previously suggested to be an activator of Src in breast cancer, had no effect. Although further tests with primary tumor tissues are required, the unexpected correlation between ER status and Src/PTP alpha dependence in breast cancer cell lines may be important for planning therapeutic strategies, and the insensitivity of normal breast cells to the RNAi highlights the potential of PTP alpha, which may be easier to target than Src, as a therapeutic target in ER-negative breast cancer.     

Non-genetic cancer cell plasticity and therapy-induced stemness in tumour relapse: ‘What does not kill me strengthens me'

Therapy resistance and tumour relapse after drug therapy are commonly explained by Darwinian selection of pre-existing drug-resistant, often stem-like cancer cells resulting from random mutations. However, the ubiquitous non-genetic heterogeneity and plasticity of tumour cell phenotype raises the question: are mutations really necessary and sufficient to promote cell phenotype changes during tumour progression? Cancer therapy inevitably spares some cancer cells, even in the absence of resistant mutants. Accumulating observations suggest that the non-killed, residual tumour cells actively acquire a new phenotype simply by exploiting their developmental potential. These surviving cells are stressed by the cytotoxic treatment, and owing to phenotype plasticity, exhibit a variety of responses. Some are pushed into nearby, latent attractor states of the gene regulatory network which resemble evolutionary ancient or early developmental gene expression programs that confer stemness and resilience. By entering such stem-like, stress-response states, the surviving cells strengthen their capacity to cope with future noxious agents. Considering non-genetic cell state dynamics and the relative ease with which surviving but stressed cells can be tipped into latent attractors provides a foundation for exploring new therapeutic approaches that seek not only to kill cancer cells but also to avoid promoting resistance and relapse that are inherently linked to the attempts to kill them.     

Evolving insights: how DNA repair pathways impact cancer evolution

Viewing cancer as a large, evolving population of heterogeneous cells is a common perspective. Because genomic instability is one of the fundamental features of cancer, this intrinsic tendency of genomic variation leads to striking intratumor heterogeneity and functions during the process of cancer formation, development, metastasis, and relapse. With the increased mutation rate and abundant diversity of the gene pool, this heterogeneity leads to cancer evolution, which is the major obstacle in the clinical treatment of cancer. Cells rely on the integrity of DNA repair machineries to maintain genomic stability, but these machineries often do not function properly in cancer cells. The deficiency of DNA repair could contribute to the generation of cancer genomic instability, and ultimately promote cancer evolution. With the rapid advance of new technologies, such as single-cell sequencing in recent years, we have the opportunity to better understand the specific processes and mechanisms of cancer evolution, and its relationship with DNA repair. Here, we review recent findings on how DNA repair affects cancer evolution, and discuss how these mechanisms provide the basis for critical clinical challenges and therapeutic applications.     

Genome-wide RNAi screens in Caenorhabditis elegans: impact on cancer research

Genes linked to human cancers often function in evolutionary conserved pathways, and research in C. elegans has been instrumental in dissecting some of the pathways affected, such as apoptosis and Ras signalling. The advent of RNA interference (RNAi) technology has allowed high-throughput loss-of-function analyses of C. elegans gene functions. Here we review some of the most recent genome-wide RNAi screens that have been conducted and discuss their impact on cancer research and possibilities for future screens. We also show that genes causally implicated in human cancers are significantly more likely to have a C. elegans homologue than average, validating the use of C. elegans as a cancer gene discovery platform. We foresee that genome-wide RNAi screens in C. elegans will continue to be productive in identifying new cancer gene candidates and will provide further insights into cancer gene functions.