Introduction

RNA interference (RNAi) is a post-transcriptional mechanism of gene silencing through chromatin remodeling, inhibition of protein translation or direct mRNA degradation, which is ubiquitous in eukaryotic cells (for review, see Caplen,¹ Dorsett and Tuschl² and Shankar et al.³). RNAi regulates the expression of key genes that determine cell fate and differentiation, and considerable progress has been made in understanding how RNAi mediates gene silencing. It was first found that the introduction of foreign double-stranded RNAs (dsRNA) can initiate a potent cascade of sequence-specific degradation of endogenous mRNAs that bear homology to the dsRNA trigger.⁴ This phenomenon was referred to as RNAi and has since been associated with a number of previously described silencing phenomena, such as post-transcriptional gene silencing in plants and quelling in fungi. Mechanistically, when dsRNAs are introduced into the cytoplasm, they are processed by the RNase-III enzyme Dicer, which cleaves the long dsRNAs into short 21–28 nucleotide duplexes which have symmetric 2–3 nucleotide 3' overhangs and 5' phosphate and 3' hydroxyl groups.^{5, 6} These RNA duplexes are referred to as short interfering RNAs (siRNA), and these duplexes associate with a multiprotein RNA-inducing silencing complex (RISC), guiding RISC to a homologous target mRNA and triggering its endonucleolytic cleavage by Slicer (Argonaute-2), an enzyme residing within the RISC complex. The target mRNA is cleaved at a single site in the center of the duplex region between the guide siRNA and the target mRNA, 10 nucleotides from the 5' end of the siRNA, resulting in gene silencing. This silencing process is highly sequence specific, and also very efficient because the antisense strand of the dsRNA is protected within the RISC complex and therefore is preserved as a catalyst to degrade additional copies of the target mRNA. Although it was initially believed that effective RNAi required almost complete sequence homology throughout the length of the mRNA, it now appears that as few as seven contiguous complementary base pairs can direct RNAi-mediated silencing.⁷

With the emerging technology of high-throughput gene expression profiling of cancer cells (for review, see Ameyar-Zazoua et al.⁸ and Leung and Whittaker⁹), genes that are dysregulated in cancers are discovered almost every day. RNAi technology has been applied in this setting to silence the expression of dominant mutant oncogenes, gene amplifications, translocations and viral oncogenes in order to elucidate their function and their interaction with other genes in a number of critical cellular pathways. The understanding gleaned from the study of these various gene interactions has already facilitated a systematic search for new drug targets, and has also improved the efficacy of existing chemotherapeutic agents by specifically targeting and silencing resistance-associated genes.

In this review, we will highlight the utility of RNAi to target cancer-associated gene products in a number of important cellular pathways, discuss the application of RNAi therapy for targeting genes that play a role in tumor–host interactions, and consider the use of RNAi in silencing genes that are important for the resistance of cancers to chemotherapy or radiotherapy, in an effort to provide an understanding of the current status of RNAi therapy for cancer, explore its current limitations and propose more novel therapeutic applications for its future use. We hope to demonstrate that the initial utility of RNAi as a tool to help elucidate the role of genes in the development of carcinogenesis has evolved into the clinical application of RNAi to cancer gene therapy.

RNAi targeting of molecules involved in carcinogenesis

The advantage of RNAi technology is that it can be used to target a large number of different genes involving a number of distinct cellular pathways. This is particularly important for a disease as complex as cancer. The major cellular pathways altered in cancer include the receptor protein tyrosine kinase (PTK) pathway, adenomatous polyposis coli (APC) pathway, glioma-associated oncogene (GLI) pathway, phosphoinositide 3-kinase (PIK3) pathway, SMAD pathway, hypoxia-inducible transcription factor (HIF) pathway, retinoblastoma (Rb) pathway, p53 pathway, and apoptosis (APOP) pathway (for review, see Vogelstein and Kinzler¹⁰). Most of the RNAi candidate cancer gene targets are involved in pathways that contribute to net tumor growth (either through increased tumor-cell proliferation or reduced tumor-cell death, or both). While mRNAs expressed from mutated cancer oncogenes can be directly targeted for RNAi intervention, RNAi can also be used to target and silence gene products that negatively regulate the function of endogenous tumor suppressor genes. Other gene products that can be targeted by RNAi include proteins involved in cellular senescence, or protein stability and degradation. Although these additional targets are not directly involved in the oncogenesis pathway, they can indirectly contribute to net tumor growth, and therefore represent potential candidates for RNAi intervention. Table 1 summarizes some of the genes involved in oncogenesis, cell-cycle regulation, apoptosis, cellular senescence, and protein stability and degradation, which have been targeted by RNAi.^{11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77}

Table 1.

Full table

RNAi targeting of gene products involved in oncogenesis pathways

RNAi targeting of genes in the receptor PTK pathway

The receptor PTKs are important regulators of intracellular signal transduction, and represent some of the most frequently occurring mutations found in human malignancies.⁷⁸ They are a subclass of transmembrane-spanning receptors that have intrinsic, ligand-stimulatable PTK activity. This pathway is normally tightly regulated. When mutated or altered structurally, PTKs can become potent oncoproteins, causing cellular transformation from enhanced or constitutive kinase activity, with quantitatively or qualitatively altered downstream signaling (for review, see Blume-Jensen and Hunter⁷⁹). Such transformation can occur as a result of genomic rearrangements such as chromosomal translocations, which produce oncogenic fusion proteins that include a PTK catalytic domain and an unrelated protein that provides constitutive activity to the tyrosine kinase. An example is the Bcr-Abl fusion protein, which is commonly observed in chronic myeloid leukemia. Several groups have designed siRNAs specific to the fusion sequence of Bcr-Abl and have demonstrated that these RNAis are capable of decreasing Bcr-Abl protein expression and protein-dependent cell growth.²⁸ Additionally, this Bcr-Abl knockdown is accompanied by a strong induction of apoptotic cell death,⁸⁰ and the introduction of Bcr-Abl siRNA increases the sensitivity of Bcr-Abl overexpressing cells to the chemotherapeutic agent imatinib.²⁸

In addition to genomic rearrangements, oncogenic transformation can also result from gain-of-function mutations in PTKs. For example, some non-small-cell lung cancers have mutations within the epidermal growth factor receptor's (EGFR) catalytic kinase domain, which can lead to constitutive activation. The oncogenic activity of EGFR reflects the activation of signals that promote both cell proliferation and cell survival. RNAi-mediated inactivation of the mutant EGFR in vitro results in rapid and massive apoptosis. Sequence-specific siRNAs target specifically the mRNA of the mutant EGFR allele and have no effect on the expression of the wild-type EGFR. This study suggests that the expression of the mutant EGFR is essential for the oncogenicity in lung cancers harboring these mutations, and that the RNAi-mediated knockdown of the mutant EGFR results in specific and extensive apoptosis of NSCLC cells.^{11, 81}

PTK overexpression can also result from gene amplification, as demonstrated in several common human cancers. An example of PTK overexpression leading to constitutive kinase activation is the Neu/ErbB2 pathway, which is often amplified in breast and ovarian carcinomas. RNAi technology has been used to silence the expression of Her-2/neu in Her2/neu-expressing human breast and ovarian cancer cell lines. The introduction of the siRNA into the Her2/neu-positive cell lines dramatically reduces the cell surface expression of the Her2/neu protein, resulting in growth inhibition, increased apoptosis, increased G0/G1 arrest, and decreased tumor growth. Moreover, knockdown of Her-2/neu expression by siRNA has also been found to be associated with increased expression of the antiangiogenic factor thrombospondin-1 and decreased expression of the proangiogenic vascular endothelial growth factor (VEGF),¹² suggesting that Her-2/neu may stimulate tumor growth in part by regulating angiogenesis. These findings demonstrate how RNAi-mediated gene silencing of Her-2/neu may be a useful therapeutic strategy for abrogating the effects of Her-2/neu-mediated carcinogenesis in Her2/neu-expressing breast or ovarian cancers.^{12, 82}

Other molecules in the PTK pathway that have been targeted by RNAi technology are listed in Table 1. In summary, the functional blockade of these various molecules in the PTK-signaling pathway through gene silencing can affect cellular growth, apoptosis or angiogenesis, and might have great potential for cancer therapy.

RNAi targeting of genes involved in HIF-1, APC, GLI, PI3K and SMAD oncogenesis pathways

Other oncogenesis pathways include the HIF-1 pathway, APC pathway, GLI pathway, PI3K pathway and the SMAD pathway. Table 1 lists examples of RNAi targeting of molecules related to these oncogenesis pathways. Although a discussion of each gene is beyond the scope of this review, the silencing of these various genes results in the inhibition of cellular proliferation and/or enhanced apoptosis of cancer cells, which again emphasizes the potential of RNAi as a therapeutic modality to treat human cancers.

RNAi targeting of molecules related to cell-cycle regulation

Several molecules involved in cell-cycle regulation have been targeted for RNAi intervention in an effort to suppress cancer cell growth. Two cell-cycle regulators, Rb tumor suppressor protein (pRb) and p53, are of special importance in cancer therapy and are worthy of discussion.

Rb pathway

The pRb is an important cell-cycle regulator. One of its functions is to bind to the transcription factor E2F and prevent it from activating the expression of cyclins E and A, thus serving as a cell-cycle checkpoint (for review, see Kaelin⁸³). Alterations in the various genes involved in the pRb pathway result in the loss or delay of cellular senescence and have been found to play a role in a number of human cancers.

The Human Papillomavirus (HPV) oncogenic protein E7, which is expressed only and uniquely in virus-infected cells, represents an ideal target within the Rb pathway for RNAi intervention. E7 binds and inactivates pRb and its subsequent downstream signaling, thereby contributing to the malignant transformation of HPV-infected cells.⁸⁴ HPV-16 E7 silencing by siRNA induces apoptotic cell death in HPV-16-associated cervical cancer cell lines, while HPV-negative cells are unaffected by the siRNAs,⁴⁶ again demonstrating the specificity of RNAi technology. Other molecules involved in the Rb pathway, such as E2F4, have also been targeted by siRNA. For example, it has been found that the disruption of E2F4 by siRNA prevents p130/E2F4 complex formation and sensitizes cells to irradiation-induced apoptosis.⁴⁷

p53 pathway

The tumor suppressor protein p53 is inactivated in approximately one-half of all human cancers. The p53 pathway is composed of a network of genes and their products that are designed to respond to a variety of intrinsic and extrinsic stress signals that impact the cellular homeostatic mechanisms that monitor DNA replication, chromosome segregation and cell division. In response to a stress signal, the p53 protein is activated by post-translational modification, which leads to a variety of processes, such as cell-cycle arrest, cellular senescence and apoptosis. In addition to these responses to cellular stress at the single-cell level, the p53 pathway within a cell communicates with neighboring cells by secreting a series of proteins that may alter the local microenvironment (extracellular matrix (ECM)) or initiate local angiogenic signaling. Thus, the p53 pathway interacts with a large number of other signal transduction pathways in the cell, and a number of positive and negative autoregulatory feedback loops act upon the p53 response (for review, see Harris and Levine⁸⁵).

Several molecules involved in the p53 pathway have been studied using RNAi technology and some of these may serve as potential therapeutic targets for RNAi intervention. For example, Hdmx, which is a key p53 negative regulator, has been found to be overexpressed in many human tumors and amplified in 5% of primary breast tumors, all of which have wild-type p53 alleles.⁵⁰ RNAi-mediated reduction of Hdmx markedly inhibits the growth potential of transfected cells in a p53-dependent manner.⁵⁰ Similarly, downregulation of Notch-1, Delta-like-1 or Jagged-1 within the p53 pathway by RNAi also induces apoptosis and inhibits proliferation in multiple glioma cell lines transfected with siRNAs.⁵¹ Another example of a viral oncogene that may be targeted for gene-specific silencing is the HPV E6 protein, which interacts with the p53 pathway. As with the HPV E7 protein, HPV E6 is also constitutively expressed in HPV-associated neoplasms and plays an important role in the malignant transformation of HPV-associated epithelial cells. HPV E6 binds, inactivates, and promotes the degradation of p53.⁸⁴ HPV-16-positive cells transfected with siRNAs targeting HPV E6 undergo massive apoptotic cell death.⁴⁸ Other genes in the p53 pathway that may be potential targets for RNAi gene silencing are listed in Table 1.

Other molecules involved in cell-cycle regulation that have been targeted by RNAi technology include cyclin B1/cdc2,^{52, 53} cyclin D1,⁵⁴ and Checkpoint kinase 1(Chk1)⁵⁵ (see Table 1). For example, cyclin B1 represents the regulatory subunit of the M-phase promoting factor, and proper regulation of cyclin B1 is important for the initiation of mitosis. Suppression of cyclin B1 by siRNA has been found to inhibit proliferation and induce apoptosis in human tumor cells.⁵² Likewise, Chk1 has been found to be the major mediator in the activation of cell-cycle checkpoints (S or G2/M) in response to a variety of chemotherapeutic agents. Inhibition of Chk1 potentiates the toxicity of conventional DNA-damaging agents and enhances the toxicity of antimetabolites in cancer cell lines.⁵⁵

RNAi targeting of genes in the APOP pathway

It is now clear that many cancers express antiapoptotic proteins. Apoptosis defects are recognized as an important complement to proto-oncogene activation, as many deregulated oncoproteins that drive aberrant cell division also trigger apoptosis. As apoptosis plays a pivotal role in the cytotoxic activity of most chemotherapeutic drugs as well as radiation therapy, the antiapoptotic proteins expressed by neoplastic cells may provide a basis for chemo- and/or radioresistance. Therefore, the restoration of apoptosis by using RNAi to target key antiapoptotic proteins expressed by cancer cells would have important therapeutic implications. Several antiapoptotic proteins are expressed in cancer cells and can serve as targets for RNAi. These antiapoptotic proteins include the Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory protein (FLIP),⁵⁶ Bcl-2,^{57, 58, 59, 60} Bcl-xL,^{57, 61, 62} Mcl-1,⁸⁶ survivin,^{57, 63, 64, 65, 66, 67, 68} and X chromosome-linked IAP (XIAP).^{60, 69} Figure 1 depicts a simplified schematic of the key antiapoptotic proteins within the extrinsic and intrinsic apoptotic pathways that may serve as effective targets for RNAi.

Figure 1.

A simplified schematic of key antiapoptotic proteins within the extrinsic and intrinsic apoptotic pathways that can be targeted by siRNA. There are two major apoptotic pathways: the extrinsic apoptotic pathway and the intrinsic apoptotic pathway. In general, death domain-containing receptors (such as CD95 (APO-1/Fas)) can sense an external signal (such as Fas ligand) and activate the extrinsic apoptosis pathway through the Fas-associated death domain. This pathway is mediated by recruitment and activation of caspase-8, an initiator caspase, in the death-inducing signaling complex (DISC) followed by direct cleavage of downstream effector caspases. Thus, caspase 8 is an important proapoptotic protein for the extrinsic apoptotic pathway. In comparison, the intrinsic apoptosis pathway initiates from within the cell. For example, it can be activated through intracellular changes, such as DNA damage, resulting in the release of a number of proapoptotic factors from mitochondria, such as cytochrome c. The release of these factors leads to the activation of another initiator caspase, caspase-9, and ultimately results in the activation of effector caspases in a protein complex called the apoptosome. The release of these proapoptotic factors from the mitochondria is tightly controlled by the proteins Bak and Bax. Thus Bak, Bax, and caspase 9 are important proapoptotic proteins for the intrinsic apoptotic pathway. Effector-caspases, such as caspase 3, eventually lead to proteolysis of a panel of death substrates, resulting in apoptotic cell death. Thus, caspase 3 is also an important proapoptotic protein. FLIP, Bcl-2, Bcl-xL, Mcl, survivin and IAP are the key antiapoptotic proteins within the extrinsic and intrinsic apoptotic pathways that may serve as effective targets for RNA interference.

Full figure and legend (140K)

Other molecules with antiapoptotic function, though not directly related to the APOP pathway, can also serve as targets for RNAi technology. For example, clusterin is a cytoprotective protein that is commonly upregulated in many cancers in the presence of a variety of apoptotic triggers such as chemotherapy. The overexpression of clusterin confers chemoresistance to the cancer cells. However, cancer cells transfected with siRNA targeting clusterin demonstrate significantly enhanced chemosensitivity in vitro.⁸⁷ Thus, clusterin may serve as a therapeutic target for RNAi technology in strategies employing novel multimodality therapy for advanced cancer. Additional candidate molecules that regulate death receptor-mediated apoptosis are stem cell antigen-2,⁸⁸ glycogen synthase kinase-3beta⁸⁹ (which regulates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis), and protein kinase casein kinase II⁹⁰ (which protects cancer cells from TRAIL-induced apoptosis). RNAi targeting of these molecules results in augmented apoptotic responses.^{88, 89, 90}

RNAi targeting of molecules related to cellular senescence

The terminal ends of linear chromosomes are organized into telomeres to prevent their recognition as DNA breaks. Telomeres are composed of proteins bound to double-stranded specific DNA repeats and to single-stranded DNA tails, the G-tails.⁷¹ During successive cycles of DNA replication, telomere repeats are continuously shortened, and the synthesis of new repeats by telomerase is required to prevent cellular senescence. Although telomerase levels are usually very low in normal, slowly dividing cells, telomere length in rapidly dividing human cancers is maintained by elevated levels of telomerase. Telomerase contains an intrinsic templating RNA moiety (human telomerase RNA; hTER) and a core protein (human telomerase reverse transcriptase). It has been shown that silencing the hTER template region by siRNA can cause p53-independent cell growth inhibition and apoptosis.⁷⁰

Other molecules associated with cellular senescence may also serve as targets for RNAi cancer therapy. The mammalian heterogeneous nuclear ribonucleoparticule A1 and A2 proteins bind to the G-tails of telomeres with high affinity. siRNA-mediated reduction in A1/A2 protein expression in many human cancer cell lines, such as those derived from cervical, colon, breast, ovarian and brain cancers, induces specific and rapid apoptosis.⁷¹ Moreover, Id1, a member of the Id family of helix–loop–helix transcriptional regulatory proteins, can suppress p16 (INK4a) expression and is also implicated in cellular senescence. Suppression of Id1 expression in young cells by siRNA results in an increased p16 (INK4a) level and premature cellular senescence.⁷²

RNAi targeting of genes influencing protein stability and degradation

Other molecules that are indirectly related to cancer gene expression and the aforementioned pathways can also serve as targets for RNAi technology. For example, molecules related to proteosome-dependent pathways such as Cks-1,⁷³ Skp-2^{74, 75} and E3-ubiquitin ligase receptor subunit betaTRCP1⁷⁶ have been used as targets for RNAi intervention. Suppression of these proteins by siRNA may influence the degradation of cell-cycle regulators such as p27Kip1 and p21, resulting in the inhibition of tumor cell growth in experimental models.^{73, 74} Another protein that may influence cell-cycle regulation is cathepsin L, which interacts with p21/WAF1. Suppression of cathepsin L by siRNA results in p21/WAF1 stabilization and its subsequent intracellular accumulation, leading to cellular senescence and drug sensitivity in neoplastic cells.⁷⁷

RNAi therapy targeting gene products involved in tumor–host interactions

Neoplastic cells grow within the context of the host environment, and must respond to numerous physical, chemical and cellular challenges. Therefore, neoplastic cells develop multiple strategies with which to take control of the tumor–host interaction. In order for a neoplasm to grow and spread, it needs to obtain sufficient oxygen and nutrients to break down the ECM in order to invade surrounding tissues and metastasize, and to evade the host immune response. Figure 2 summarizes the use of RNAi technology to target the molecules involved in angiogenesis, invasion/metastasis, and immune evasion.

Figure 2.

Schematic diagram to illustrate siRNA targets important for tumor-host interaction. siRNA technology can be used to target molecules that are important for tumor angiogenesis, invasion, metastasis and immune evasion. Metastasis and invasion of the surrounding tissue by tumor cells require several important steps, including the abnormal expression of adhesion molecules and degradation of the cellular matrix. siRNA targeting these molecules can potentially inhibit these processes and lead to control of invasion and metastasis. Furthermore, molecules important for tumor evasion from immune attack can potentially be targeted by siRNA in order to control tumors. These molecules can potentially be expressed by the tumor cells or cells located in the tumor microenvironment. The biological significance as well as examples of silencing by siRNA of these molecular targets shown in this figure are described in the text.

Full figure and legend (219K)

RNAi targeting of gene products important for tumor angiogenesis

Continuous tumor growth eventually results in a hypoxic environment where tumor cells outstrip their surrounding blood supply. As the tumor must respond to this altered environment through the generation of angiogenic vessels, RNAi inhibiting proangiogenic genes can serve as a potential target for cancer therapy. Table 2 summarizes some of the proangiogenic molecules that are important for neovascularization.⁹¹

Table 2.

Full table

RNAi technology may be used to block the angiogenesis activators listed in Table 2. For example, VEGF plays a critical role in the pathological angiogenesis that occurs in a number of cancers. siRNAs targeting human VEGF have been found to inhibit the secretion of VEGF in the human prostate cancer cell line PC-3.⁹² In addition, administration of VEGF siRNA directly into PC-3 tumors significantly suppresses tumor angiogenesis and tumor growth in a xenograft model.⁹² Furthermore, systemic administration of crude anti-VEGF siRNA in vivo has also been found to reduce the growth of tumor cells resistant to the antiangiogenic effects of thrombospondin-1.⁹³ siRNAs targeting VEGF receptor 2 have also been reported.⁹⁴ The intravenous administration of these siRNAs into tumor-bearing mice leads to a decrease in the expression of VEGF receptor 2 within the tumors, as well as a reduction in both tumor angiogenesis and tumor growth.⁹⁴

Another example of a molecule that can be targeted by RNAi therapy is cell adhesion molecule-1 (CEACAM1). CEACAM1-overexpressing microvascular endothelial cells survive longer and have increased tubule formation when stimulated with VEGF, whereas silencing of CEACAM1 via siRNA blocks these effects.⁹⁵ Such CEACAM1 silencing also abrogates the VEGF-induced morphogenetic effects seen during capillary formation. Strategies targeting the endothelial upregulation of CEACAM1 can potentially be used for antiangiogenic cancer therapy.

RNAi targeting of molecules related to ECM degradation

Tumor progression and angiogenesis require the degradation of the ECM and the basement membrane. The enzymes that are primarily responsible for ECM degradation in vivo are cysteine protease, serine protease and matrix metalloprotease (MMP), all of which may serve as targets for RNAi-mediated cancer therapy. For example, the serine protease urokinase-type plasminogen activator (u-PA) is thought to be involved in tumor invasion. u-PA mRNA has been found to be upregulated in many human hepatocellular carcinomas (HCC), and its level of expression is inversely correlated with patient survival. HCC cells transfected with siRNA targeting u-PA demonstrate reduced migration, invasion and proliferation in vitro.⁹⁶ Moreoever, the simultaneous targeting of multiple molecules involved in ECM degradation yield even more promising results. For instance, RNAi targeting cathepsin B (a cysteine protease) and urokinase plasminogen activator receptor (uPAR) results in decreased cell invasion, angiogenesis and tumor growth in a xenogenic tumor model.⁹⁷ In addition, human glioma cells transfected with DNA encoding siRNA targeting uPAR and MMP-9 have reduced formation of capillary-like structures in in vitro and in vivo models of angiogenesis,⁹⁸ and blocking the expression of these genes results in the total regression of pre-established intracerebral tumors in a preclinical model.⁹⁸

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the ECM and basement membrane. Expression of the heparanase gene is thought to account for the invasive, angiogenic and metastatic potential of several malignant cell lines. RNAi targeting of heparanase leads to a decrease in tumor invasiveness and prolonged survival.⁹⁹ In mice, tumors derived from cells transfected with siRNA-targeting heparanase are less vascularized and less metastatic than tumors derived from cells transfected with control vectors, and mice injected with cells transfected with these siRNAs live longer than mice injected with control cells.⁹⁹

RNAi targeting of molecules related to invasion or metastasis

Tumor invasion and metastasis rely on the mobility of tumor cells and their interaction with the host microenvironment. Thus, RNAi targeting of molecules important for the mobility of neoplastic cells or molecules important for tumor invasion or metastasis may have therapeutic potential. For example, the small GTPase RhoA has been associated with the regulation of cell morphology, motility and transformation, and the RhoA protein has been found to be frequently overexpressed in gastric cancers. siRNAs targeting RhoA significantly inhibit the proliferation and tumorigenicity of cancer cells, and enhance their chemosensitivity to adriamycin and 5-fluorouracil.¹⁰⁰ Another example is the 64 integrin, which is a laminin adhesion receptor. The 64 integrin has been implicated to play an important role in the invasive phenotype of many carcinomas. siRNAs targeting the 64 integrin reduce the cell-surface expression of this integrin and result in decreased invasiveness of a breast carcinoma cell line in an in vitro assay.¹⁰¹

CXC chemokine receptor-4 (CXCR4) gene expression in breast cancer may be important for breast cancer metastasis. Knockdown of endogenous CXCR4 gene expression by siRNA in breast cancer cells significantly inhibits breast cancer cell migration in vitro.¹⁰² More importantly, direct injection of a pool of naked siRNA targeting CXCR4 has been found to prevent tumorigenesis of a breast cancer cell line in an animal model.¹⁰³ Thus, siRNA targeting CXCR4 may represent a potentially novel preventive and therapeutic strategy for cancer management.

Other molecules that are important for tumor invasion and metastasis include insulin-like growth factor-binding protein 2 (IGFBP2) and EphA2 receptor tyrosine kinase. IGFBP2 is frequently overexpressed in ovarian carcinomas, particularly in serous carcinoma.¹⁰⁴ An in vitro Matrigel invasion assay found that these IGFBP2-overexpressing cells are more invasive than control cells. Furthermore, silencing of IGFBP2 expression by RNAi technology in an ovarian cancer cell line (PA-1) decreases its invasiveness. EphA2 is also overexpressed in a variety of human cancers. For example, the overexpression of EphA2 in pancreatic adenocarcinoma cells has been associated with increased cellular invasiveness. Suppression of EphA2 expression by siRNA leads to reduced cellular invasiveness in vitro and slows tumor growth and inhibits metastasis in vivo.²³ Thus, EphA2 may be a determinant of malignant cellular behavior and EphA2-specific siRNA may serve as a potential therapeutic adjunct for the treatment of pancreatic adenocarcinoma.

RNAi targeting of cell adhesion molecules

The regulation of cell-to-cell adhesion among neoplastic cells is important for tumor invasion, metastasis and angiogenesis. Molecules that are involved in the modulation of cell-to-cell adhesion may represent good targets for RNAi-mediated gene silencing to influence tumor invasion and/or metastasis. For example, epithelial cell adhesion molecule (EpCAM) is a molecule involved in cell–cell adhesion and is known to be highly expressed in colon, breast and other epithelial carcinomas.¹⁰⁵ Silencing EpCAM gene expression with siRNA decreases cell proliferation in several different breast cancer cell lines and significantly decreases migration and invasiveness of a breast cancer cell line in vitro. Furthermore, EpCAM siRNA treatment also increases the expression of the cell adhesion molecule E-cadherin.¹⁰⁵ Thus, EpCAM may serve as a regulator of cell adhesion, and modification of EpCAM expression by siRNA may enhance E-cadherin-mediated cell-to-cell adhesion and inhibit cell migration, invasion or proliferation.

RNAi targeting of molecules related to tumor immune evasion

Despite the existence of tumor-specific antigens and the demonstrated presence of tumor-specific immune cells, the majority of tumors manage to avoid immune-mediated destruction. Tumor escape from immune surveillance and immune attack is one of the most important determinants of tumor survival in the host (for reviews, see Dunn et al.¹⁰⁶ and Khong and Restifo¹⁰⁷). Various mechanisms have been suggested for tumor evasion from the immune response. One such mechanism is mediated by transforming growth factor-beta (TGF-), an immunosuppressive cytokine expressed by most tumors. It has been found that the T-cell-specific blockade of TGF- signaling through modification of adopted T cells can generate an immune response capable of eradicating tumors in mice.¹⁰⁸ Alternatively, direct suppression of TGF- secretion by tumors through RNAi may overcome cancer immune evasion. At least one report found that RNAi targeting TGF- leads to suppression of TGF- protein expression and cell proliferation.¹⁰⁹ Another important immunosuppressive cytokine, interleukin-10 (IL-10), has been found to be secreted by some cancers and to interfere with T-cell function.¹¹⁰ IL-10 also has antiapoptotic properties, and RNAi targeting IL-10 has efficiently induced apoptosis in transfected cells.¹¹¹ Thus RNAi targeting immunosuppressive cytokines represents a plausible approach for addressing tumor immune evasion.

The neoplasm's microenvironment is an impediment to successful antitumor immune responses. Several molecules that are expressed by neoplastic cells confer resistance to effective T cell killing, thus forming a microenvironment that allows the neoplastic cells to escape immune attack. These molecules include: B7-H1, indoleamine 2,3-dioxygenase (IDO) enzyme, galectin-1, and secreted forms of MHC class I chain-related gene A (MIC-A) and MIC-B. B7-H1, a member of the B7 family of costimulatory molecules, can interact with activated T and B cells through the PD-1 receptor and negatively regulate immune responses. Expression of B7-H1 in mouse tumors increases apoptosis of activated tumor-reactive T cells and allows highly immunogenic B7-1-positive tumors to grow in vivo.¹¹² IDO is an endogenous immunosuppressive molecule. Expression of IDO by tumors leads to the degradation of the essential amino acid tryptophan around the tumor, resulting in the suppression of immune effector cells (for review, see Munn and Mellor¹¹³). Galectin-1 is a negative regulator of T-cell activation and survival. It plays a critical role in promoting the escape from T-cell-dependent immunity. It has been found that the blockade of galectin-1 in tumor cells promotes tumor rejection and stimulates the generation of an antitumor T-cell-mediated responses in mice.¹¹⁴ MIC-A and MIC-B molecules, the major histocompatibility complex class I homologues, are the ligands of NKG2D, which is expressed on most natural killer cells and CD8+ T cells. These molecules are absent from most cells but are frequently expressed in epithelial tumors. MIC-A/MIC-B engagement of NKG2D can activate natural killer cells and costimulate antigen-specific effector T cells. It has been found that tumor-derived soluble MIC-A may cause the downregulation of NKG2D and lead to significant impairment of the responsiveness of tumor-antigen-specific CD8⁺ T cells.¹¹⁵ It is conceivable that the silencing of these immunosuppressive molecules by using RNAi technology may improve antitumor immune responses.

RNAi targeting of gene products important for tumor resistance to chemotherapy and/or radiotherapy

Many of the genes described above may contribute to the resistance to chemotherapy or irradiation. For example, the expression of antiapoptotic proteins by cancer cells is an important mechanism by which cancer cells resist chemotherapy or irradiation. Using RNAi to target antiapoptotic proteins may represent a promising strategy to be used in conjunction with chemotherapy and radiotherapy for cancer treatment (see section on RNAi targeting of genes in the APOP pathway). There are several additional mechanisms that also contribute to the resistance to chemotherapy or irradiation, and molecules related to these mechanisms might provide opportunities for RNAi intervention.

RNAi targeting of multidrug resistance (MDR) proteins

The expression of MDR genes in cancer cells represents an important mechanism by which cancer cells resist chemotherapy. RNAi technology has been used to silence the expression of several MDR genes in cancer cells such as ABCB1 (MDR 1),^{116, 117, 118, 119, 120} ABCB4 (MDR 3),¹¹⁹ and ABCB5.¹²¹ In general, RNAi targeting of these MDR genes can sensitize several chemoresistant cancer cells in vitro and suggests that RNAi treatment may represent a novel approach for the treatment of MDR gene-mediated drug resistance.

RNAi targeting of molecules related to DNA repair mechanisms

DNA repair mechanisms are crucial for the maintenance of genomic stability and are emerging as potential therapeutic targets for cancer. In the stress of chemo- or radiotherapy, many cancer cells overexpress proteins related to DNA repair mechanisms in order to restore therapy-induced DNA damage. RNAi technology has been used in this setting to downregulate DNA repair genes and enhance the sensitivity of cancer cells to chemotherapeutic agents or irradiation. These molecules include the excision repair cross-complementing 1 (ERCC1) gene product,^{122, 123} DNA double-stranded break repair protein endo-exonuclease,¹²⁴ ribonucleotide reductase,¹²⁵ double-strand break signaling/repair proteins ATM¹²⁶ and DNA-dependent protein kinase catalytic subunit.¹²⁶ In general, transfection of siRNAs targeting proteins related to DNA repair mechanisms has been found to suppress these DNA repair proteins and render cancer cells sensitive to chemotherapeutic agents or irradiation. Thus, these molecules also represent potential targets for RNAi intervention in conjunction with existing chemotherapy or radiotherapy.

Current limitations of RNAi therapy and future directions

Issues related to in vivo delivery, efficacy and selectivity

The primary obstacle for translating RNAi technology from an effective research tool into a feasible therapeutic strategy remains the efficient delivery of these small molecules to the targeted cell type in vivo. The RNAi used for gene silencing in cancers can be derived from dsRNA, DNA vectors or viral vectors. The efficacy of direct introduction of chemically synthesized siRNAs into cells is limited by the short-lived nature of their transient gene silencing effects as well as their relative instability. It has been reported that the extracellular degradation of siRNAs peaks around 36–48 h after their introduction and begins to decrease after 96 h, and that the levels of silencing are not absolute because of the significant interanimal variation, as well as differences in the efficiency with which the siRNAs are taken up by target cells and tissues (for review, see Ryther et al.¹²⁷). Furthermore, whereas the duration of gene silencing in differentiated or slowly dividing cells is relatively long (on the order of several weeks), in more rapidly dividing cells, the effect of siRNAi is short lived, peaking at around 3 days and lasting for approximately 1 week. This may be due to the increasing dilution of the siRNA with repeated cell division, as well as ongoing cellular enzymatic degradation.

It is possible to chemically modify siRNAs to make them more resistant to serum RNases without sacrificing biological activity. siRNAs can be coupled with fusogenic peptides, linked to antibodies to cell surface receptor ligands for cell-specific delivery, or encased in lipid complexes, cationic liposomes or other types of particles. These strategies may potentially generate advantages such as deliver into specific cell types and evasion of filtration by the kidney (for review, see Shankar et al.³). Other strategies to target siRNA specifically to neoplastic cells employ conditionally replicating viruses that only express siRNA in neoplastic cells,¹²⁸ use the highly tissue-specific RNA polymerase II promoter to express siRNA rather than the ubiquitous RNA polymerase III,¹²⁹ or use ligand targeted, sterically stabilized nanoparticles to deliver siRNA to particular cell types.⁹⁴ More research is needed to improve the efficiency and specificity of deliverying siRNA to tumor cells in vivo.

In order to obtain a more sustained inhibitory effect, several groups have developed a variety of viral vectors to deliver siRNAs to target cells. Viral vectors can stably integrate into the genome and mediate the long-term knockdown of endogenous transcripts in cell culture and in vivo. The viral vectors that have been used to date as gene delivery systems include retroviral,^{18, 20, 130} lentiviral,^{24, 74, 88, 131} adenoviral,^{17, 65, 74} and adeno-associated viral¹³² vectors. Other recombinant viral vectors such as the hemagglutinating virus of Japan (also called Sendai virus) have also been used to delivery siRNA for gene silencing.¹³³ However, significant limitations of the viral vectors include a general lack of specificity, low efficiency of gene delivery to the target cell of interest, as well as the potential toxicity of the viral vectors themselves.

Plasmid-based expression systems using RNA polymerase III (pol III) promoters that produce short RNA species have been developed by several groups. Two pol III promoters that have been used include the U6 promoter and the H1 promoter.⁴⁰ Several groups have recently described tandem U6 promoters that express the sense and antisense strands from separate transcription units (for review, see Sui and Shi¹³⁴). Other groups designed an H1 RNA-pol-III-based shRNA expression vector to produce a hairpin RNA with a 19-nucleotide stem and a short loop.¹³⁵ Subsequently, other groups developed similar plasmid-based shRNA expression systems that differ in their stem and loop length and composition. Although most expression systems use either the U6 or H1 promoter, Kawasaki and Taira¹³⁶ described an expression system that uses the transfer RNA (tRNA) promoter. Despite these advances, efficient uptake and long-term stability of siRNAs still represent significant obstacles in establishing RNAi as a therapeutic approach, and considerable effort is being devoted to the development of more selective and efficient delivery mechanisms.

Issues related to nonspecific immune stimulation

While there is a high degree of specificity associated with RNAi, some effects have been observed that are independent of the specific gene targeted for silencing. In general, 21 base-pair or longer dsRNAs can lead to a sequence-independent interferon response.¹³⁷ In addition, it has been reported that high concentrations of synthetic or vector-based siRNA can trigger the interferon response in sensitive cell lines.^{137, 138} Interferon triggers the degradation of mRNA by inducing 2–5' oligoadenylate synthase, which in turn activates RNAse L.¹³⁹ Interferon can also activate the dsRNA-dependent protein kinase (PKR), which phosphorylates and subsequently inactivates the translation factor eIF2, leading to a global inhibition of mRNA translation. The length of the initiating dsRNA (siRNA or shRNA) clearly has some role in triggering the interferon response, with more recent data suggesting that sequences shorter than 19 nucleotides are more likely to escape the interferon antiviral response. It is anticipated that the judicious selection of siRNA/shRNA sequences together with a greater understanding of their interactions with any given target gene will resolve this issue.

Similarly, evidence exists that siRNAs and shRNAs can activate dendritic cells and other cells of the immune system through a much more specific and restricted class of receptors, the toll-like receptors, that can recognize foreign nucleic acids including dsRNAs and when activated can send a danger signal to trigger a proinflammatory response.¹⁴⁰ These findings raise the possibility that RNAi reagents may trigger unforeseen immune responses, including autoimmune diseases, in vivo.

Off target interference

Nucleic acid-based gene-silencing molecules may also have effects on genes that are not considered targets, the so-called off-target effects, due to similarities in nucleic acid sequences (for review, see Scherr and Eder¹⁴¹). The degree of the off-target effect is dependent upon the mode of silencing and the stability of the nucleic acid hybrid. If siRNAs are not carefully selected, siRNAs having partial complementarity to an mRNA target can repress translation or subject unintended mRNAs to degradation. A study that compared the gene-expression profiles created by different siRNAs targeted against the same transcript revealed that in extreme cases, as little as seven nucleotide complementarity between the 5' end of either siRNA strand to an mRNA can cause a reproducible reduction in transcript levels.^{7, 142} Interestingly, it has been found from studies in primitive organisms that off-target effects are not observed when complete dsRNAs are introduced instead of synthetic siRNAs. This may be explained by the fact that the siRNAs derived endogenously from the cleavage of dsRNAs are generated and selected by Dicer and the RISC complex, which may have a proofreading mechanism that protects against the generation of siRNA sequences that might result in the silencing of endogenous genes. Therefore, it is possible that mammalian siRNAs generated from dsRNA precursors through the action of Dicer and the RISC complex may be less prone to induce off-target effects than synthetically designed siRNAs. Several algorithms and software are available to select siRNA target sequences with reduced off-target effects (for review, see Boese¹⁴³), and it will be important to select siRNA targets with relatively sophisticated sequence comparison tools to minimize potential off-target effects.

Resistance

Many mechanisms for the development of resistance to RNAi therapy are conceivable. In general, the specificity of RNAi-mediated degradation of homologous mRNA makes this therapeutic strategy highly prone to the development of resistance, as simple changes in target sequences may make previously effective siRNA triggers impotent.^{144, 145} Alternatively, some sequences may be inaccessible to RNAi therapy as a result of physical hindrance by RNA-binding proteins or by complex secondary structures.¹⁴⁵ Therefore, RNAi therapy for cancer should consider multiple sequences per target and multiple targets per cell. Moreover, the targeting of proteins with a long half-life may result in therapeutic failure despite successful gene silencing, since silencing at the transcript level does not affect pre-existing proteins. The maximal effect of RNAi technology is observed in proteins with rapid turnover. Finally, the genes involved in the machinery of RNAi, such as the RISC complex, may become mutated and thereby limit the efficacy of RNA silencing.

Conclusion

RNAi has evolved from a powerful laboratory tool utilized to elucidate the function of novel genes, to a potential therapeutic modality in the diverse armamentarium of cancer therapy. Although a number of important oncogenic targets within many critical cellular pathways have been identified, and RNAi-mediated gene silencing of these targets has produced favorable effects in vitro and in preclinical animal models, the translation of such findings to the more complex clinical arena remains a foreboding challenge. RNAi therapy can potentially be used in conjunction with chemotherapy, radiotherapy and/or immunotherapy. As with these treatment modalities, RNAi therapy will likely require multiple dosing regimens to accomplish the desired effects. As we begin to more clearly understand the mechanisms by which RNAi regulates gene expression, we can hope to overcome the many obstacles that currently exist in this system and exploit this powerful tool as an adjunct in the multimodality therapy of malignancies.

References

1. Caplen NJ. Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference. Gene Therapy 2004; 11: 1241–1248. | Article | PubMed | ISI | ChemPort |
2. Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 2004; 3: 318–329. | Article | PubMed | ISI | ChemPort |
3. Shankar P, Manjunath N, Lieberman J. The prospect of silencing disease using RNA interference. Jama 2005; 293: 1367–1373. | Article | PubMed | ISI | ChemPort |
4. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806–811. | Article | PubMed | ISI | ChemPort |
5. Tuschl T, Zamore PD, Lehmann R, Bartel DP, Sharp PA. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 1999; 13: 3191–3197. | Article | PubMed | ISI | ChemPort |
6. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999; 286: 950–952. | Article | PubMed | ISI | ChemPort |
7. Jackson AL, Linsley PS. Noise amidst the silence: off-target effects of siRNAs? Trends Genet 2004; 20: 521–524. | Article | PubMed | ISI | ChemPort |
8. Ameyar-Zazoua M, Guasconi V, Ait-Si-Ali S. siRNA as a route to new cancer therapies. Expert Opin Biol Ther 2005; 5: 221–224. | Article | PubMed | ISI | ChemPort |
9. Leung RK, Whittaker PA. RNA interference: from gene silencing to gene-specific therapeutics. Pharmacol Ther 2005; 107: 222–239. | Article | PubMed | ISI | ChemPort |
10. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004; 10: 789–799. | Article | PubMed | ISI | ChemPort |
11. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004; 305: 1163–1167. | Article | PubMed | ISI | ChemPort |
12. Yang G, Cai KQ, Thompson-Lanza JA, Bast Jr RC, Liu J. Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against Her-2/neu gene expression. J Biol Chem 2004; 279: 4339–4345. | Article | PubMed | ISI | ChemPort |
13. Sithanandam G, Fornwald LW, Fields J, Anderson LM. Inactivation of ErbB3 by siRNA promotes apoptosis and attenuates growth and invasiveness of human lung adenocarcinoma cell line A549. Oncogene 2005; 24: 1847–1859. | Article | PubMed | ISI | ChemPort |
14. Surmacz E. Growth factor receptors as therapeutic targets: strategies to inhibit the insulin-like growth factor I receptor. Oncogene 2003; 22: 6589–6597. | Article | PubMed | ISI | ChemPort |
15. Macaulay VM. The IGF receptor as anticancer treatment target. Novartis Found Symp 2004; 262: 235–243. | PubMed | ChemPort |
16. Panta GR, Nwariaku F, Kim LT. RET signals through focal adhesion kinase in medullary thyroid cancer cells. Surgery 2004; 136: 1212–1217. | Article | PubMed | ISI |
17. Chen LM, Le HY, Qin RY, Kumar M, Du ZY, Xia RJ et al. Reversal of the phenotype by K-rasval12 silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1. World J Gastroenterol 2005; 11: 831–838. | PubMed | ChemPort |
18. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002; 2: 243–247. | Article | PubMed | ISI | ChemPort |
19. Aharinejad S, Paulus P, Sioud M, Hofmann M, Zins K, Schafer R et al. Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice. Cancer Res 2004; 64: 5378–5384. | Article | PubMed | ISI | ChemPort |
20. Chen J, Wall NR, Kocher K, Duclos N, Fabbro D, Neuberg D et al. Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin. J Clin Invest 2004; 113: 1784–1791. | Article | PubMed | ISI | ChemPort |
21. Walters DK, Stoffregen EP, Heinrich MC, Deininger MW, Druker BJ. RNAi-induced down-regulation of FLT3 expression in AML cell lines increases sensitivity to MLN518. Blood 2005; 105: 2952–2954. | Article | PubMed | ISI | ChemPort |
22. Ma PC, Jagadeeswaran R, Jagadeesh S, Tretiakova MS, Nallasura V, Fox EA et al. Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res 2005; 65: 1479–1488. | Article | PubMed | ISI | ChemPort |
23. Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE. EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma. Oncogene 2004; 23: 1448–1456. | Article | PubMed | ISI | ChemPort |
24. Sumimoto H, Miyagishi M, Miyoshi H, Yamagata S, Shimizu A, Taira K et al. Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference. Oncogene 2004; 23: 6031–6039. | Article | PubMed | ISI | ChemPort |
25. Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F et al. B-RAF is a therapeutic target in melanoma. Oncogene 2004; 23: 6292–6298. | Article | PubMed | ISI | ChemPort |
26. Pal A, Ahmad A, Khan S, Sakabe I, Zhang C, Kasid UN et al. Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer. Int J Oncol 2005; 26: 1087–1091. | PubMed | ISI | ChemPort |
27. Zhang SQ, Du QY, Ying Y, Ji ZZ, Wang SQ. Polymerase synthesis and potential interference of a small-interfering RNA targeting hPim-2. World J Gastroenterol 2004; 10: 2657–2660. | PubMed | ChemPort |
28. Wohlbold L, van der Kuip H, Miething C, Vornlocher HP, Knabbe C, Duyster J et al. Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571). Blood 2003; 102: 2236–2239. | Article | PubMed | ISI | ChemPort |
29. Lefevre G, Glotin AL, Calipel A, Mouriaux F, Tran T, Kherrouche Z et al. Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis. J Biol Chem 2004; 279: 31769–31779. | Article | PubMed | ISI | ChemPort |
30. Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE. siRNA directed against c-Src enhances pancreatic adenocarcinoma cell gemcitabine chemosensitivity. J Am Coll Surg 2004; 198: 953–959. | Article | PubMed | ISI |
31. Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB. Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res 2003; 9: 1291–1300. | PubMed | ISI | ChemPort |
32. van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep 2003; 4: 609–615. | Article | PubMed | ISI | ChemPort |
33. Wang YH, Liu S, Zhang G, Zhou CQ, Zhu HX, Zhou XB et al. Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo. Breast Cancer Res 2005; 7: R220–R228. | Article | PubMed | ISI | ChemPort |
34. Valsesia-Wittmann S, Magdeleine M, Dupasquier S, Garin E, Jallas AC, Combaret V et al. Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells. Cancer Cell 2004; 6: 625–630. | Article | PubMed | ISI | ChemPort |
35. Bienvenu F, Barre B, Giraud S, Avril S, Coqueret O. Transcriptional regulation by a DNA-associated form of cyclin D1. Mol Biol Cell 2005; 16: 1850–1858. | Article | PubMed | ISI | ChemPort |
36. Zhang L, Yang N, Liang S, Barchetti A, Vezzani C, Huang J et al. RNA interference: a potential strategy for isoform-specific phosphatidylinositol 3-kinase targeted therapy in ovarian cancer. Cancer Biol Ther 2004; 3: 1283–1289. | PubMed | ISI | ChemPort |
37. Gagnon V, Mathieu I, Sexton E, Leblanc K, Asselin E. AKT involvement in cisplatin chemoresistance of human uterine cancer cells. Gynecol Oncol 2004; 94: 785–795. | Article | PubMed | ISI | ChemPort |
38. Vandermoere F, El Yazidi-Belkoura I, Adriaenssens E, Lemoine J, Hondermarck H. The antiapoptotic effect of fibroblast growth factor-2 is mediated through nuclear factor-kappaB activation induced via interaction between Akt and IkappaB kinase-beta in breast cancer cells. Oncogene 2005; 24: 5482–5491. | Article | PubMed | ISI | ChemPort |
39. Guo J, Verma UN, Gaynor RB, Frenkel EP, Becerra CR. Enhanced chemosensitivity to irinotecan by RNA interference-mediated down-regulation of the nuclear factor-kappaB p65 subunit. Clin Cancer Res 2004; 10: 3333–3341. | Article | PubMed | ISI | ChemPort |
40. Zheng L, Liu J, Batalov S, Zhou D, Orth A, Ding S et al. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc Natl Acad Sci USA 2004; 101: 135–140. | Article | PubMed | ChemPort |
41. Dohjima T, Lee NS, Li H, Ohno T, Rossi JJ. Small interfering RNAs expressed from a Pol III promoter suppress the EWS/Fli-1 transcript in an Ewing sarcoma cell line. Mol Ther 2003; 7: 811–816. | Article | PubMed | ISI | ChemPort |
42. Chansky HA, Barahmand-Pour F, Mei Q, Kahn-Farooqi W, Zielinska-Kwiatkowska A, Blackburn M et al. Targeting of EWS/FLI-1 by RNA interference attenuates the tumor phenotype of Ewing's sarcoma cells in vitro. J Orthop Res 2004; 22: 910–917. | Article | PubMed | ISI | ChemPort |
43. Sowter HM, Raval RR, Moore JW, Ratcliffe PJ, Harris AL. Predominant role of hypoxia-inducible transcription factor (Hif)-1alpha versus Hif-2alpha in regulation of the transcriptional response to hypoxia. Cancer Res 2003; 63: 6130–6134. | PubMed | ISI | ChemPort |
44. Zhang X, Kon T, Wang H, Li F, Huang Q, Rabbani ZN et al. Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1alpha. Cancer Res 2004; 64: 8139–8142. | Article | PubMed | ISI | ChemPort |
45. Romero-Ramirez L, Cao H, Nelson D, Hammond E, Lee AH, Yoshida H et al. XBP1 is essential for survival under hypoxic conditions and is required for tumor growth. Cancer Res 2004; 64: 5943–5947. | Article | PubMed | ISI | ChemPort |
46. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 2002; 21: 6041–6048. | Article | PubMed | ISI | ChemPort |
47. DuPree EL, Mazumder S, Almasan A. Genotoxic stress induces expression of E2F4, leading to its association with p130 in prostate carcinoma cells. Cancer Res 2004; 64: 4390–4393. | Article | PubMed | ISI | ChemPort |
48. Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M, Hoppe-Seyler F. siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells. Oncogene 2003; 22: 5938–5945. | Article | PubMed | ISI | ChemPort |
49. Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T, Ikeda Y et al. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther 2003; 8: 762–768. | Article | PubMed | ISI | ChemPort |
50. Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, Frenk R et al. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol Cell Biol 2004; 24: 5835–5843. | Article | PubMed | ISI | ChemPort |
51. Purow BW, Haque RM, Noel MW, Su Q, Burdick MJ, Lee J et al. Expression of Notch-1 and its ligands, Delta-like-1 and Jagged-1, is critical for glioma cell survival and proliferation. Cancer Res 2005; 65: 2353–2363. | Article | PubMed | ISI | ChemPort |
52. Yuan J, Yan R, Kramer A, Eckerdt F, Roller M, Kaufmann M et al. Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene 2004; 23: 5843–5852. | Article | PubMed | ISI | ChemPort |
53. Roberson RS, Kussick SJ, Vallieres E, Chen SY, Wu DY. Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers. Cancer Res 2005; 65: 2795–2803. | Article | PubMed | ISI | ChemPort |
54. Zen Y, Harada K, Sasaki M, Chen TC, Chen MF, Yeh TS et al. Intrahepatic cholangiocarcinoma escapes from growth inhibitory effect of transforming growth factor-beta1 by overexpression of cyclin D1. Lab Invest 2005; 85: 572–581. | Article | PubMed | ISI | ChemPort |
55. Xiao Z, Xue J, Sowin TJ, Rosenberg SH, Zhang H. A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation. Oncogene 2005; 24: 1403–1411. | Article | PubMed | ISI | ChemPort |
56. Abedini MR, Qiu Q, Yan X, Tsang BK. Possible role of FLICE-like inhibitory protein (FLIP) in chemoresistant ovarian cancer cells in vitro. Oncogene 2004; 23: 6997–7004. | Article | PubMed | ISI | ChemPort |
57. Zangemeister-Wittke U. Antisense to apoptosis inhibitors facilitates chemotherapy and TRAIL-induced death signaling. Ann NY Acad Sci 2003; 1002: 90–94. | Article | PubMed | ChemPort |
58. Yano J, Hirabayashi K, Nakagawa S, Yamaguchi T, Nogawa M, Kashimori I et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin Cancer Res 2004; 10: 7721–7726. | Article | PubMed | ISI | ChemPort |
59. Holle L, Hicks L, Song W, Holle E, Wagner T, Yu X. Bcl-2 targeting siRNA expressed by a T7 vector system inhibits human tumor cell growth in vitro. Int J Oncol 2004; 24: 615–621. | PubMed | ISI | ChemPort |
60. Lima RT, Martins LM, Guimaraes JE, Sambade C, Vasconcelos MH. Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells. Cancer Gene Ther 2004; 11: 309–316. | Article | PubMed | ISI | ChemPort |
61. Tran NL, McDonough WS, Savitch BA, Sawyer TF, Winkles JA, Berens ME. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling system regulates glioma cell survival via NFkappaB pathway activation and BCL-XL/BCL-W expression. J Biol Chem 2005; 280: 3483–3492. | Article | PubMed | ISI | ChemPort |
62. Zhu H, Guo W, Zhang L, Davis JJ, Teraishi F, Wu S et al. Bcl-XL small interfering RNA suppresses the proliferation of 5-fluorouracil-resistant human colon cancer cells. Mol Cancer Ther 2005; 4: 451–456. | PubMed | ISI | ChemPort |
63. Ning S, Fuessel S, Kotzsch M, Kraemer K, Kappler M, Schmidt U et al. siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth. Int J Oncol 2004; 25: 1065–1071. | PubMed | ISI | ChemPort |
64. Kappler M, Bache M, Bartel F, Kotzsch M, Panian M, Wurl P et al. Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53. Cancer Gene Ther 2004; 11: 186–193. | Article | PubMed | ISI | ChemPort |
65. Uchida H, Tanaka T, Sasaki K, Kato K, Dehari H, Ito Y et al. Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo. Mol Ther 2004; 10: 162–171. | Article | PubMed | ISI | ChemPort |
66. Caldas H, Jiang Y, Holloway MP, Fangusaro J, Mahotka C, Conway EM et al. Survivin splice variants regulate the balance between proliferation and cell death. Oncogene 2005; 24: 1994–2007. | Article | PubMed | ISI | ChemPort |
67. Cheng SQ, Wang WL, Yan W, Li QL, Wang L, Wang WY. Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721. World J Gastroenterol 2005; 11: 756–759. | PubMed | ChemPort |
68. Kappler M, Taubert H, Bartel F, Blumke K, Panian M, Schmidt H et al. Radiosensitization, after a combined treatment of survivin siRNA and irradiation, is correlated with the activation of caspases 3 and 7 in a wt-p53 sarcoma cell line, but not in a mt-p53 sarcoma cell line. Oncol Rep 2005; 13: 167–172. | PubMed | ISI | ChemPort |
69. Hatano M, Mizuno M, Yoshida J. Enhancement of C2-ceramide antitumor activity by small interfering RNA on X chromosome-linked inhibitor of apoptosis protein in resistant human glioma cells. J Neurosurg 2004; 101: 119–127. | PubMed | ISI | ChemPort |
70. Li S, Rosenberg JE, Donjacour AA, Botchkina IL, Hom YK, Cunha GR et al. Rapid inhibition of cancer cell growth induced by lentiviral delivery and expression of mutant-template telomerase RNA and anti-telomerase short-interfering RNA. Cancer Res 2004; 64: 4833–4840. | Article | PubMed | ISI | ChemPort |
71. Patry C, Bouchard L, Labrecque P, Gendron D, Lemieux B, Toutant J et al. Small interfering RNA-mediated reduction in heterogeneous nuclear ribonucleoparticule A1/A2 proteins induces apoptosis in human cancer cells but not in normal mortal cell lines. Cancer Res 2003; 63: 7679–7688. | PubMed | ISI | ChemPort |
72. Zheng W, Wang H, Xue L, Zhang Z, Tong T. Regulation of cellular senescence and p16(INK4a) expression by Id1 and E47 proteins in human diploid fibroblast. J Biol Chem 2004; 279: 31524–31532. | Article | PubMed | ISI | ChemPort |
73. Kitajima S, Kudo Y, Ogawa I, Bashir T, Kitagawa M, Miyauchi M et al. Role of Cks1 overexpression in oral squamous cell carcinomas: cooperation with Skp2 in promoting p27 degradation. Am J Pathol 2004; 165: 2147–2155. | PubMed | ISI | ChemPort |
74. Sumimoto H, Yamagata S, Shimizu A, Miyoshi H, Mizuguchi H, Hayakawa T et al. Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference. Gene Therapy 2005; 12: 95–100. | Article | PubMed | ISI | ChemPort |
75. Kudo Y, Kitajima S, Ogawa I, Kitagawa M, Miyauchi M, Takata T. Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell growth of oral cancer cells by inhibiting p27 degradation. Mol Cancer Ther 2005; 4: 471–476. | PubMed | ISI | ChemPort |
76. Muerkoster S, Arlt A, Sipos B, Witt M, Grossmann M, Kloppel G et al. Increased expression of the E3-ubiquitin ligase receptor subunit betaTRCP1 relates to constitutive nuclear factor-kappaB activation and chemoresistance in pancreatic carcinoma cells. Cancer Res 2005; 65: 1316–1324. | Article | PubMed | ISI |
77. Zheng X, Chou PM, Mirkin BL, Rebbaa A. Senescence-initiated reversal of drug resistance: specific role of cathepsin L. Cancer Res 2004; 64: 1773–1780. | Article | PubMed | ISI | ChemPort |
78. Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R et al. A census of human cancer genes. Nat Rev Cancer 2004; 4: 177–183. | Article | PubMed | ISI | ChemPort |
79. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001; 411: 355–365. | Article | PubMed | ISI | ChemPort |
80. Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002; 21: 5716–5724. | Article | PubMed | ISI | ChemPort |
81. Zhang M, Zhang X, Bai CX, Chen J, Wei MQ. Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells. Acta Pharmacol Sin 2004; 25: 61–67. | PubMed | ISI | ChemPort |
82. Choudhury A, Charo J, Parapuram SK, Hunt RC, Hunt DM, Seliger B et al. Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines. Int J Cancer 2004; 108: 71–77. | Article | PubMed | ISI | ChemPort |
83. Kaelin Jr WG. Functions of the retinoblastoma protein. Bioessays 1999; 21: 950–958. | Article | PubMed | ISI |
84. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002; 2: 342–350. | Article | PubMed | ISI | ChemPort |
85. Harris SL, Levine AJ. The p53 pathway: positive and negative feedback loops. Oncogene 2005; 24: 2899–2908. | Article | PubMed | ISI | ChemPort |
86. Taniai M, Grambihler A, Higuchi H, Werneburg N, Bronk SF, Farrugia DJ et al. Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells. Cancer Res 2004; 64: 3517–3524. | Article | PubMed | ISI | ChemPort |
87. July LV, Beraldi E, So A, Fazli L, Evans K, English JC et al. Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma cells both in vitro and in vivo. Mol Cancer Ther 2004; 3: 223–232. | PubMed | ISI | ChemPort |
88. He J, Chang LJ. Functional characterization of hepatoma-specific stem cell antigen-2. Mol Carcinog 2004; 40: 90–103. | Article | PubMed | ISI | ChemPort |
89. Liao X, Zhang L, Thrasher JB, Du J, Li B. Glycogen synthase kinase-3beta suppression eliminates tumor necrosis factor-related apoptosis-inducing ligand resistance in prostate cancer. Mol Cancer Ther 2003; 2: 1215–1222. | PubMed | ISI | ChemPort |
90. Izeradjene K, Douglas L, Delaney A, Houghton JA. Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells. Clin Cancer Res 2004; 10: 6650–6660. | Article | PubMed | ISI | ChemPort |
91. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol 2001; 61: 253–270. | Article | PubMed | ISI | ChemPort |
92. Takei Y, Kadomatsu K, Yuzawa Y, Matsuo S, Muramatsu T. A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics. Cancer Res 2004; 64: 3365–3370. | Article | PubMed | ISI | ChemPort |
93. Filleur S, Courtin A, Ait-Si-Ali S, Guglielmi J, Merle C, Harel-Bellan A et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res 2003; 63: 3919–3922. | PubMed | ISI | ChemPort |
94. Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res 2004; 32: e149. | Article | PubMed |
95. Kilic N, Oliveira-Ferrer L, Wurmbach JH, Loges S, Chalajour F, Neshat-Vahid S et al. Pro-angiogenic signaling by the endothelial presence of CEACAM1. J Biol Chem 2005; 280: 2361–2369. | Article | PubMed | ISI | ChemPort |
96. Salvi A, Arici B, De Petro G, Barlati S. Small interfering RNA urokinase silencing inhibits invasion and migration of human hepatocellular carcinoma cells. Mol Cancer Ther 2004; 3: 671–678. | PubMed | ISI | ChemPort |
97. Gondi CS, Lakka SS, Dinh DH, Olivero WC, Gujrati M, Rao JS. RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas. Oncogene 2004; 23: 8486–8496. | Article | PubMed | ISI | ChemPort |
98. Lakka SS, Gondi CS, Dinh DH, Olivero WC, Gujrati M, Rao VH et al. Specific interference of uPAR and MMP-9 gene expression induced by double-stranded RNA results in decreased invasion, tumor growth and angiogenesis in gliomas. J Biol Chem 2005; 280: 21882–21892. | Article | PubMed | ISI | ChemPort |
99. Edovitsky E, Elkin M, Zcharia E, Peretz T, Vlodavsky I. Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis. J Natl Cancer Inst 2004; 96: 1219–1230. | PubMed | ChemPort |
100. Liu N, Bi F, Pan Y, Sun L, Xue Y, Shi Y et al. Reversal of the malignant phenotype of gastric cancer cells by inhibition of RhoA expression and activity. Clin Cancer Res 2004; 10: 6239–6247. | Article | PubMed | ISI | ChemPort |
101. Lipscomb EA, Dugan AS, Rabinovitz I, Mercurio AM. Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells. Clin Exp Metastasis 2003; 20: 569–576. | Article | PubMed | ISI | ChemPort |
102. Chen Y, Stamatoyannopoulos G, Song CZ. Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro. Cancer Res 2003; 63: 4801–4804. | PubMed | ISI | ChemPort |
103. Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L, Shim H. Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res 2005; 65: 967–971. | PubMed | ISI | ChemPort |
104. Lee EJ, Mircean C, Shmulevich I, Wang H, Liu J, Niemisto A et al. Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion. Mol Cancer 2005; 4: 7. | Article | PubMed | ChemPort |
105. Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA et al. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 2004; 64: 5818–5824. | Article | PubMed | ISI | ChemPort |
106. Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Annu Rev Immunol 2004; 22: 329–360. | Article | PubMed | ISI | ChemPort |
107. Khong HT, Restifo NP. Natural selection of tumor variants in the generation of 'tumor escape' phenotypes. Nat Immunol 2002; 3: 999–1005. | Article | PubMed | ISI | ChemPort |
108. Gorelik L, Flavell RA. Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells. Nat Med 2001; 7: 1118–1122. | Article | PubMed | ISI | ChemPort |
109. Yin JQ, Gao J, Shao R, Tian WN, Wang J, Wan Y. siRNA agents inhibit oncogene expression and attenuate human tumor cell growth. J Exp Ther Oncol 2003; 3: 194–204. | Article | PubMed | ChemPort |
110. Yue FY, Dummer R, Geertsen R, Hofbauer G, Laine E, Manolio S et al. Interleukin-10 is a growth factor for human melanoma cells and down-regulates HLA class-I, HLA class-II and ICAM-1 molecules. Int J Cancer 1997; 71: 630–637. | Article | PubMed | ISI | ChemPort |
111. McCarthy BA, Mansour A, Lin YC, Kotenko S, Raveche E. RNA interference of IL-10 in leukemic B-1 cells. Cancer Immun 2004; 4: 6. | PubMed |
112. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002; 8: 793–800. | Article | PubMed | ISI | ChemPort |
113. Munn DH, Mellor AL. IDO and tolerance to tumors. Trends Mol Med 2004; 10: 15–18. | Article | PubMed | ISI | ChemPort |
114. Rubinstein N, Alvarez M, Zwirner NW, Toscano MA, Ilarregui JM, Bravo A et al. Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection: a potential mechanism of tumor-immune privilege. Cancer Cell 2004; 5: 241–251. | Article | PubMed | ISI | ChemPort |
115. Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 2002; 419: 734–738. | Article | PubMed | ISI | ChemPort |
116. Wu H, Hait WN, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells. Cancer Res 2003; 63: 1515–1519. | PubMed | ISI | ChemPort |
117. Nieth C, Priebsch A, Stege A, Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi). FEBS Lett 2003; 545: 144–150. | Article | PubMed | ISI | ChemPort |
118. Yague E, Higgins CF, Raguz S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1. Gene Therapy 2004; 11: 1170–1174. | Article | PubMed | ISI | ChemPort |
119. Duan Z, Brakora KA, Seiden MV. Inhibition of ABCB1 (MDR1) and ABCB4 (MDR3) expression by small interfering RNA and reversal of paclitaxel resistance in human ovarian cancer cells. Mol Cancer Ther 2004; 3: 833–838. | PubMed | ISI | ChemPort |
120. Peng Z, Xiao Z, Wang Y, Liu P, Cai Y, Lu S et al. Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells. Cancer Gene Ther 2004; 11: 707–712. | Article | PubMed | ISI | ChemPort |
121. Huang Y, Anderle P, Bussey KJ, Barbacioru C, Shankavaram U, Dai Z et al. Membrane transporters and channels: role of the transportome in cancer chemosensitivity and chemoresistance. Cancer Res 2004; 64: 4294–4301. | Article | PubMed | ISI | ChemPort |
122. Chang IY, Kim MH, Kim HB, Lee do Y, Kim SH, Kim HY et al. Small interfering RNA-induced suppression of ERCC1 enhances sensitivity of human cancer cells to cisplatin. Biochem Biophys Res Commun 2005; 327: 225–233. | Article | PubMed | ISI | ChemPort |
123. Youn CK, Kim MH, Cho HJ, Kim HB, Chang IY, Chung MH et al. Oncogenic H-Ras up-regulates expression of ERCC1 to protect cells from platinum-based anticancer agents. Cancer Res 2004; 64: 4849–4857. | Article | PubMed | ISI | ChemPort |
124. Chow TY, Alaoui-Jamali MA, Yeh C, Yuen L, Griller D. The DNA double-stranded break repair protein endo-exonuclease as a therapeutic target for cancer. Mol Cancer Ther 2004; 3: 911–919. | PubMed | ISI | ChemPort |
125. Lin ZP, Belcourt MF, Cory JG, Sartorelli AC. Stable suppression of the R2 subunit of ribonucleotide reductase by R2-targeted short interference RNA sensitizes p53(-/-) HCT-116 colon cancer cells to DNA-damaging agents and ribonucleotide reductase inhibitors. J Biol Chem 2004; 279: 27030–27038. | Article | PubMed | ISI | ChemPort |
126. Collis SJ, Swartz MJ, Nelson WG, DeWeese TL. Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. Cancer Res 2003; 63: 1550–1554. | PubMed | ISI | ChemPort |
127. Ryther RC, Flynt AS, Phillips III JA, Patton JG. siRNA therapeutics: big potential from small RNAs. Gene Therapy 2005; 12: 5–11. | Article | PubMed | ISI | ChemPort |
128. Carette JE, Overmeer RM, Schagen FH, Alemany R, Barski OA, Gerritsen WR et al. Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells. Cancer Res 2004; 64: 2663–2667. | Article | PubMed | ISI | ChemPort |
129. Song J, Pang S, Lu Y, Yokoyama KK, Zheng JY, Chiu R. Gene silencing in androgen-responsive prostate cancer cells from the tissue-specific prostate-specific antigen promoter. Cancer Res 2004; 64: 7661–7663. | Article | PubMed | ISI | ChemPort |
130. Duxbury MS, Ito H, Benoit E, Zinner MJ, Ashley SW, Whang EE. Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: a novel therapeutic strategy in pancreatic cancer. Surgery 2004; 136: 261–269. | Article | PubMed | ISI |
131. Razorenova OV, Ivanov AV, Budanov AV, Chumakov PM. Virus-based reporter systems for monitoring transcriptional activity of hypoxia-inducible factor 1. Gene 2005; 350: 89–98. | Article | PubMed | ISI | ChemPort |
132. Xu D, McCarty D, Fernandes A, Fisher M, Samulski RJ, Juliano RL. Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector. Mol Ther 2005; 11: 523–530. | Article | PubMed | ISI | ChemPort |
133. Ito M, Yamamoto S, Nimura K, Hiraoka K, Tamai K, Kaneda Y. Rad51 siRNA delivered by HVJ envelope vector enhances the anti-cancer effect of cisplatin. J Gene Med 2005; 7: 1044–1052. | Article | PubMed | ISI | ChemPort |
134. Sui G, Shi Y. Gene silencing by a DNA vector-based RNAi technology. Methods Mol Biol 2005; 309: 205–218. | PubMed | ChemPort |
135. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–553. | Article | PubMed | ISI | ChemPort |
136. Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucl Acids Res 2003; 31: 700–707. | Article | PubMed | ISI | ChemPort |
137. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 2003; 5: 834–839. | Article | PubMed | ISI | ChemPort |
138. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003; 34: 263–264. | Article | PubMed | ISI | ChemPort |
139. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem 1998; 67: 227–264. | Article | PubMed | ISI | ChemPort |
140. Kariko K, Bhuyan P, Capodici J, Weissman D. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J Immunol 2004; 172: 6545–6549. | PubMed | ISI | ChemPort |
141. Scherr M, Eder M. RNAi in functional genomics. Curr Opin Mol Ther 2004; 6: 129–135. | PubMed | ISI | ChemPort |
142. Saxena S, Jonsson ZO, Dutta A. Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J Biol Chem 2003; 278: 44312–44319. | Article | PubMed | ISI | ChemPort |
143. Boese Q, Leake D, Reynolds A, Read S, Scaringe SA, Marshall WS et al. Mechanistic insights aid computational short interfering RNA design. Methods Enzymol 2005; 392: 73–96. | Article | PubMed | ISI | ChemPort |
144. Wu HL, Huang LR, Huang CC, Lai HL, Liu CJ, Huang YT et al. RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant. Gastroenterology 2005; 128: 708–716. | Article | PubMed | ISI | ChemPort |
145. Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucl Acids Res 2005; 33: 796–804. | Article | PubMed | ISI | ChemPort |

Acknowledgements

This review is not intended to be an encyclopedic one, and we apologize to any authors not cited. We thank Drs Richard Roden and Ralph Hruban for their critical review of the manuscript.