RNAi can be introduced into cells by either the "expressed" or the "delivered" route, depending on whether the RNAi molecules are expressed in cells or whether they are delivered exogenously. Benitec´s patented DNA-directed RNA interference (ddRNAi) technology uses the "expressed" pathway. It involves inserting a DNA construct into a cell, triggering the production of double stranded RNA (dsRNA) that is immediately cleaved into small interfering RNA (siRNA) by Dicer, a specific type of RNAse III, as part of the RNAi process. The siRNA then enters the cellular RNAi pathway and causes the destruction of the rogue gene or viral genomes.
Consequently both the expressed and the delivered strategies operate using a similar mechanism however each application has unique advantages and thus different therapeutic applications. Benitec’s “expressed” strategy is ideal for targeting infectious diseases and cancer, and Benitec has collaborative research therapeutic programs underway in three key areas:
Scientists at Benitec Limited in collaboration with researchers at the City of Hope National Medical Center in Duarte, California, are investigating a novel method to fight HIV, the virus that causes AIDS, using Benitec’s RNAi technology in two separate studies. One is targeting patients’ blood stem cells and the second is targeting their T cells. Both strategies use specially engineered, HIV-fighting genes that are inserted into patients´ own cells. The treatment uses a three-pronged target strategy, aimed at preventing the HIV from entering the patient’s T cells, and at preventing those viruses that may enter from being able to reproduce within the cell. This is done using a harmless virus (called a lentivirus) to carry the therapeutic molecules into the stem cells. Technically, the lentivirus vector encodes three forms of anti-HIV RNA: RNAi in the form of a short hairpin RNA (shRNA) targeted to an exon in HIV-1 tat/rev (shI), a decoy for the HIV TAT-reactive element (TAR), and a ribozyme that targets the host cell CCR5 chemokine receptor (CCR5RZ) that acts as a key protein used by the virus to enter the cell.
The advantage of using a multi-target approach is that it is likely to reduce the chances that HIV will develop resistance to the therapy.
HIV Stem Cell Project
A Phase I clinical trial entitled, A pilot study of the safety and feasibility of stem cell therapy for AIDS lymphoma using stem cells treated with a lentiviral vector-encoding multiple anti-HIV RNAs," commenced in 2008 at the City of Hope. The rationale behind modifying stem cells is that the treatment could allow patients´ bodies to produce HIV-resistant white blood cells indefinitely.
A primary objective of this study was to determine the safety and feasibility of Benitec´s RNAi technology in treating these patients. After 12 months, the interim clinical trial results suggest this approach is safe and feasible, with the following outcomes reported by the City of Hope researchers:
Benitec plans to continue this research with a second Phase I clinical trial at the City of Hope, using improved methods to increase the level of gene modification within the stem cells.
HIV T Cell Project
Benitec’s collaboration with the City of Hope National Medical Center has extended to conducting a second research study using the same lentiviral delivered gene constructs to target HIV patients’ CD4+ T cells. Entitled A pilot study of safety and feasibility of T cell immunotherapy using lentivirus vector-expressed RNAi in autologous T cells of HIV-1 infected patients who have failed anti-retroviral therapy, the trial represents a product-development-step toward commercialisation by building on the current AIDS/lymphoma pilot study. The trial will provide additional and valuable pre-clinical information regarding the development of RNAi as a therapeutic strategy for HIV in a clinical setting other than lymphoma and stem cell transplantation.
Subjects must be HIV-infected adults aged 18-60 who have been on HAART therapy for at least one year and have significant levels of HIV in their blood. They must also have reasonable numbers of CD4+ T cells. Similarly to the stem cell study, the T cells will be removed from the patients’ blood, grown in the laboratory, transfected with the multi-gene construct and then returned to the same patient.
We expect this program to be in the clinic in 2010.
HIV and AIDS Lymphoma
Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), has infected over 40 million people worldwide and caused 3 million deaths in 2003. AIDS-related lymphoma is the occurrence of lymphatic cancer in AIDS patients. AIDS-related lymphoma usually grows and spreads faster to other parts of the body more often than lymphoma that is not related to AIDS. About 10% of HIV infections lead to AIDS-related lymphoma (about 4000 patients in the USA).
Current Therapy
Although there is no cure for HIV/AIDS, there are a number of medical treatments available to help keep HIV in check and prevent progression to AIDS. Highly Active Anti Retroviral Therapy (HAART) is the standard of care for HIV infection. HAART has made AIDS more manageable, although it has severe side effects and patients remain infectious. Furthermore, the high mutation rate of HIV makes multiple drug resistance a continuing and challenging problem.
Treatment of AIDS lymphoma is extremely difficult. Autologous bone marrow stem cell (BMSC) transplantation after chemotherapy is the only therapy currently available, but HIV rapidly re-establishes in the newly derived blood cells, helped by the fact that HAART is temporarily stopped during the procedure.
References and Further Reading
Lentiviral vectors are able to transduce non-dividing cells and maintain sustained long-term expression of the transgenes. Many cell types including brain, liver, muscle, and hematopoietic stem cells have been successfully transduced with lentiviral vectors carrying a variety of genes. These properties make lentiviral vectors attractive vehicles for delivering small interfering RNA (siRNA) genes into mammalian cells. RNA polymerase III (Pol III) promoters are most commonly used for expressing siRNAs from lentiviral vectors. Pol III promoters are relatively small, have high activity, and use simple termination signals of short stretches of U. It is possible to include several Pol III expression cassettes in a single lentiviral vector backbone to express different siRNAs or to combine siRNAs with other transgenes. This chapter describes the delivery of Pol III promoted siRNAs by HIV-based lentiviral vectors and covers vector design, production, and verification of siRNA expression and function. This chapter should be useful for establishing a lentiviral vector-based delivery of siRNAs in experiments that require long-term gene knockdown or developing siRNA-based approaches for gene therapy applications.
HIV infection is a lifelong problem requiring continual medication for suppressing viral replication. Current strategies of antiretroviral drug combinations have proven effective in prolonging the time from infection to the symptoms of AIDS. Nevertheless, chemotherapy is not without its problems, which include toxicities and eventual emergence of virus mutants that are resistant to current antiretrovirals. Gene therapy refers to the introduction of effector oligonucleotides to transiently or stably alter gene expression or the delivery and expression of an exogenous gene within a specific target cell. A number of studies have demonstrated effective silencing/inhibition of HIV-1 replication by using RNA-based effector oligonucleotides for RNA interference. In this study, we have taken advantage of lentiviral vector-mediated delivery of anti-HIV short hairpin RNA for the treatment of HIV infection in hematopoietic cells.
Since the first description of RNA interference (RNAi) in animals less than a decade ago, there has been rapid progress towards its use as a therapeutic modality against human diseases. Advances in our understanding of the mechanisms of RNAi and studies of RNAi in vivo indicate that RNAi-based therapies might soon provide a powerful new arsenal against pathogens and diseases for which treatment options are currently limited. Recent findings have highlighted both promise and challenges in using RNAi for therapeutic applications. Design and delivery strategies for RNAi effector molecules must be carefully considered to address safety concerns and to ensure effective, successful treatment of human diseases.
RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) operates to specifically and potently downregulate the expression of a target gene. This downregulation has been thought to predominantly function at the level of mRNA, as post-transcriptional gene silencing. The discovery that siRNAs can suppress gene expression at the level of transcription, that is, transcriptional gene silencing, has created a major paradigm shift in mammalian RNAi. These findings significantly broaden the role that RNA, specifically siRNA and potentially microRNA, plays in the regulation of gene expression, as well as the breadth of potential siRNA target sites. Indeed, the specificity and simplicity of design makes the use of siRNAs to target and suppress virtually any gene of interest a realized technology. Furthermore, since siRNAs are small nucleic acid reagents, they are unlikely to elicit an immune response, theoretically making them good therapeutics. The development, delivery and potential therapeutic use of antiviral siRNAs in treating viral infections and emerging viral threats are reviewed.
RNA interference (RNAi) is a natural mechanism by which small interfering RNAs (siRNAs) operate to specifically and potently downregulate the expression of a target gene. This downregulation has been demonstrated by targeting siRNAs to the mRNA (posttranscriptional gene silencing) as well as to the gene promoter, regulating gene expression epigenetically by transcriptional gene silencing. These observations significantly broaden the role RNA plays in the cell and suggest that siRNAs could prove to be a potent future therapeutic for the treatment of diseases such as human immunodeficiency virus type 1 (HIV-1) infection. The specificity and simplicity of design and the ability to express siRNAs from mammalian promoters make the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a soon-to-be-realized technology. However, the delivery and stable expression of siRNAs to target cells remain an enigma that could be surmounted, at least regarding the treatment of HIV-1 infection, by the application of lentiviral vectors to deliver and express anti-HIV-1 siRNAs in target cells. This review focuses on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating HIV-1.
We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.
Snove, O., Jr. and Rossi, J.J. (2006) Expressing short hairpin RNAs in vivo. Nature Methods 3: 689-695. Promoter-based expression of short hairpin RNAs (shRNAs) may in principle provide stable silencing of genes in any tissue. As for all approaches that require transgene expression, safe delivery is the biggest obstacle, but toxicity can also occur via expression of the sequence itself. Innate immunity mechanisms can be triggered by expressed hairpin RNAs, critical cellular factors can be saturated, and genes other than the intended target can be silenced. Nevertheless, shRNAs constitute a valuable tool for in vivo research and have great therapeutic potential if the challenges with delivery and side effects are appropriately addressed.
RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs). In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter-expressed shRNAs in primary CD34+ progenitor-derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNalpha and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.
Combinatorial therapies for the treatment of HIV infection have changed the course of the AIDS epidemic in developed nations where the antiviral drug combinations are readily available. Despite this progress, there are many problems associated with chemotherapy for AIDS including toxicities and emergence of viral mutants resistant to the drugs. Our goal has been the development of a hematopoietic gene therapy treatment for HIV infection. Like chemotherapy, gene therapy for treatment of HIV infection should be used combinatorially. We have thus combined three different inhibitory genes for treatment of HIV infection into a single lentiviral vector backbone. The inhibitory agents engage RNAi via a short hairpin RNA targeting HIV tat/rev mRNAs, a nucleolar localizing decoy that binds and sequesters the HIV Tat protein, and a ribozyme that cleaves and downregulates the CCR5 chemokine receptor used by HIV for cellular entry. This triple combination has proven to be highly effective for inhibiting HIV replication in primary hematopoietic cells, and is currently on track for human clinical application.
Combinatorial therapies for the treatment of HIV-1 infection have proven to be effective in reducing patient viral loads and slowing the progression to AIDS. We have developed a series of RNA-based inhibitors for use in a gene therapy-based treatment for HIV-1 infection. The transcriptional units have been inserted into the backbone of a replication-defective lentiviral vector capable of transducing a wide array of cell types, including CD34+ hematopoietic progenitor cells. The combinatorial therapeutic RNA vector harbors a U6 Pol III promoter-driven short hairpin RNA (shRNA) targeting the rev and tat mRNAs of HIV-1, a U6 transcribed nucleolar-localizing TAR RNA decoy, and a VA1-derived Pol III cassette that expresses an anti-CCR5 ribozyme. Each of these therapeutic RNAs targets a different gene product and blocks HIV infection by a distinct mechanism. Our results demonstrate that the combinatorial vector suppresses HIV replication long term in a more-than-additive fashion relative to the single shRNA or double shRNA/ribozyme or decoy combinations. Our data demonstrate the validity and efficacy of a combinatorial RNA-based gene therapy for the treatment of HIV-1 infection.
Here we demonstrate that an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. Our strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and hematopoietic stem cell-derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection.
In September 2009, Benitec entered into a binding agreement with China-based Biomics Biotechnologies Co. Ltd. to collaborate on a DNA directed (ddRNAi) or vector expressed RNAi for the treatment of chronic hepatitis B virus (HBV) infection.
Current therapies for chronic HBV infection (see below) have only limited inhibitory effects on viral gene expression and replication in the majority of chronically infected patients.
The application of ddRNAi technology to HBV infection has the potential to revolutionise the treatment of chronic hepatitis B. The aim of the collaborative research project is to directly target the activity of a specific HBV gene with minimum off-target effects on human genes. This could provide a unique strategy to address the current unmet clinical treatment needs for HBV infection.
Although numerous ddRNAi targets on the HBV genome could be chosen the Benitec/Biomics research team has selected a gene which has been shown to be critical for HBV survival based on pre-existing studies of the mode of action of a common drug used for treatment of this condition.
As a unique and foreign viral gene sequence within the infected liver cell any ddRNAi targeted to this gene is unlikely to trigger off-target events.
The project commenced in September 2009. The first step is to identify suitable target sequences on the gene. Researchers at Biomics have extensive experience with cloning and sequencing of siRNA constructs. Resulting sequences will be tested in vitro for their anti-viral efficacy. Once one or more targets are validated they will undergo preclinical testing using in vitro and in vivo models of chronic HBV disease. Should these studies be successful, Benitec plans to take the ddRNAi technology to early stage clinical trials.
About Hepatitis B
Hepatitis is a general term meaning ‘inflammation of the liver’ and can be caused by a variety of different viruses including hepatitis A, B, C, D and E. Of the many viral causes of human hepatitis few are of greater global importance than Hepatitis B virus (HBV). HBV is a serious and common infectious disease of the liver, affecting millions of people throughout the world.
More than 2,000 million people alive today have been infected with HBV at some time in their lives and of these about 350 million remain chronically infected and become carriers of the virus. In the USA alone there are over 1.25 million people living with the consequences of chronic active HBV, and over 60,000 new cases per year.
The severe pathological consequences of persistent HBV infections include the development of chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma. Every year about 25% of the over 4 million acute clinical cases (i.e. 1 million people worldwide) die from chronic active hepatitis, cirrhosis or HBV-induced liver cancer.
Persons with chronic HBV infection ("carriers" - worldwide about 350-400 million people) have a 12 to 300 times higher risk of developing hepatocellular carcinoma than non-carriers and globally HBV causes 60-80% of the world’s primary liver cancers. HBV carriers can transmit the disease for many years.
Worldwide Prevalence of HBV
High [carrier rate >8%] endemicity areas include south-east Asia and the Pacific Basin (excluding Japan, Australia, and New Zealand), sub-Saharan Africa, the Amazon Basin, parts of the Middle East, the central Asian Republics, and some countries in Eastern Europe. In these areas, about 70-90% of the population becomes infected with HBV before the age of 40, and 8-20% of people are HBV carriers. In countries such as China, Senegal, and Thailand infection rates are very high in infants, and continue through early childhood. In other countries such as Panama, Papua New Guinea, Solomon Islands, Greenland, and in populations such as Alaskan Indians, infection rates in infants are relatively low and increase rapidly during early childhood.
Low [<2%] endemicity areas include North America, Western and Northern Europe, Australia, and parts of South America. The carrier rate in these areas is less than 2%, and less than 20% of the population is infected with HBV. The rest of the world falls into the intermediate range of HBV prevalence, with 2 to 8% of a given population being carriers.
Current Therapy
Currently, although there is a prophylactic vaccine available there is no treatment available for acute HBV infection. There are two main classes of treatment for chronic HBV infection:
Currently, chronic HBV is treated with partial success using interferon-based drugs or an oral 2',3'-dideoxy cytosine analogue (lamivudine). A major limitation of chronic lamivudine therapy, however, is the development of viral resistance. Resistance to lamivudine typically develops after 6 months of treatment and is associated with mutations in the highly conserved catalytic region of the HBV polymerase gene.
References and Further Reading
Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-alpha and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection.
RNA interference (RNAi) is an ancient defensive mechanism in eukaryotes to control gene expressing and defend their genomes from foreign invaders. It refers to the phenomenon that double-stranded RNA results in the sequence-specific silencing of target gene expression. Although it was documented in a relatively short time ago, intensive research has facilitated making its mechanism clear. Researchers have found that it was a powerful tool for analyzing the functions of genes and treating tumors, infectious diseases and genetic abnormalities that are associated with a dominant gene defect. However, delivery in vivo, low blood stability and poor intracellular uptake present significant challenges for the development of RNAi reagents in clinical use. Thus, long-term inducible RNAi was designed. There are hundreds of millions of hepatitis B virus (HBV) carriers in the world at present, a portion of whom will lose their lives after several years due to chronic complications such as cirrhosis, hepatocellular carcinomas or both. Although a preventive vaccine is now available, the present therapeutic options for chronically infected patients are limited and of low efficiency. Admittedly, to date most RNAi experiments have been done in vitro, but it is hoped that they may be developed into a therapeutic strategy for HBV in the near future. In this article the principles and construction of long-term RNA are discussed. Its therapeutic potentiality and attention to the potential hazards will also outlined. We conclude that this ancient defensive mechanism can be recruited as a powerful weapon in the fight against HBV.
For chronic hepatitis B virus (HBV) infection the effects of current therapies are limited. Recently, RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we studied the effects of HBV-specific 21-bp short hairpin RNAs (shRNAs) targeted to the surface antigen (HBsAg) region and the core antigen (HBcAg) region both in a cell culture system and in a mouse model for HBV replication. METHODS: HBsAg and hepatitis B e antigen (HBeAg) in the media of the cells and in the sera of the mice were analyzed by time-resolved immunofluorometric assay, intracellular HBcAg by immunofluorescence assay, HBsAg and HBcAg in the livers of the mice by immunohistochemical assay, HBV DNA by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and HBV mRNA by semi-quantitative reverse transcriptase PCR (RT-PCR). RESULTS: Transfection with the shRNAs induced an RNAi response. Secreted HBsAg was reduced by >80% in cell culture and >90% in mouse serum, and HBeAg was also significantly inhibited. Immunofluorescence detection of intracellular HBcAg revealed 76% reduction. In the liver tissues by immunohistochemical detection, there were no HBsAg-positive cells and >70% reduction of HBcAg-positive cells for shRNA-1. And for shRNA-2 the detection of HBsAg and HBcAg also revealed substantial reduction. The shRNAs caused a significant inhibition in the levels of viral mRNA relative to the controls. HBV DNA was reduced by >40% for shRNA-1 and >60% for shRNA-2. CONCLUSIONS: RNAi is capable of inhibiting HBV replication and expression in vitro and in vivo and thus may constitute a new therapeutic strategy for HBV infection.
Benitec is collaborating with researchers at the University of New South Wales on a project aimed at developing a DNA-directed RNA interference (ddRNAi) therapeutic for lung cancer using the beta III ( β III) isoform of tubulin as a target for knock down.
The principal research scientist, A/Professor Maria Kavallaris, Head of Pharmacoproteomics Program at UNSW’s Children’s Cancer Institute Australia has long been interested in the role of the tubulin/microtubule system in mediating tumour resistance to chemotherapy. Two of the most clinically valuable classes of anti-cancer chemotherapeutic agents target the tubulin system: vinca alkaloids and taxanes. Build-up of resistance to these classes of drugs in cancer patients is associated with aberrant expression of β -tubulin isotypes in tumours, lending weight to the hypothesis that β -tubulin isotype expression has prognostic and theranostic value in cancer treatment.
The research team showed that targeting specific sequences on the β III-tubulin gene using vector expressed RNAi increases the sensitivity of lung cells to the anti-cancer effects of both tubulin-binding agents and DNA-damaging agents. This finding significantly extends the potential of the RNAi approach to act as an adjuvant therapy for a range of chemotherapeutic drugs in non small cell lung cancer, a disease with a dismal prognosis (see below). Benitec expects that should the project reproduce this result in pre-clinical and clinical studies, it would be of significant interest to potential pharmaceutical company collaborators
In 2008, the CCIA team found that targeting β III tubulin alone via vector expressed RNA interference mechanisms inhibited tumorigenesis in mice bearing H460 human non-small cell lung cancer (NSCLC) clones, while targeting β II- or β IV-tubulin alone did not affect tumour growth.
In October 2009, Benitec signed a term sheet with NewSouth Innovations Pty Limited (NSi), the commercial arm of the university, in securing an option to licence patent applications arising from the Children’s Cancer Institute Australia for Medical Research (CCIA). Under the agreement, the CCIA team and Benitec will carry out further studies using target sequences with proven knock down efficacy. The studies are aimed at optimising the effect in vitro utilising Benitec’s patented vector expressed RNAi technology and delivering the constructs to lung cancer tissue in vivo, with the aim of taking this to a Phase 1 clinical trial as soon as possible.
About Lung Cancer
Lung cancer is the leading form of cancer worldwide in terms of incidence and mortality. NSCLC accounts for more than 80% of all lung cancers. First line therapy for NSCLC includes a combination of a tubulin-binding agent (TBA) (taxanes, vinca alkaloids removed epothilones as not yet approved in NSCLC) and DNA-damaging agents (platinums - cisplatin, carboplatin; doxorubicin; etoposide). The prognosis for patients with advanced NSCLC however remains dismal as the tumours rapidly become resistant to these drugs. Upregulation of bIII-tubulin is associated with clinical resistance to these drugs, which is what makes the Benitec-CCIA approach so promising.
References and Further Reading
First line therapy for non-small cell lung carcinoma (NSCLC) commonly includes combination therapy with a tubulin-binding agent (TBA) and a DNA-damaging agent. TBAs suppress microtubule dynamics by binding to the beta-tubulin subunit of alpha/beta-tubulin, inducing mitotic arrest and apoptosis. Up-regulation of class III beta-tubulin (betaIII-tubulin) has been implicated in clinical resistance in NSCLC, ovarian and breast tumors treated in combination with a TBA and DNA-damaging agent. To investigate the functional significance of betaIII-tubulin in resistance to both these classes of agents, small interfering RNA (siRNA) was used to silence the expression of this isotype in two NSCLC cell lines, NCI-H460 and Calu-6. Reverse transcription-PCR and immunoblotting showed that betaIII-siRNA potently inhibited the expression of beta III-tubulin, without affecting the expression of other major beta-tubulin isotypes. Clonogenic assays showed that betaIII-siRNA cells were significantly more sensitive to TBAs, paclitaxel, vincristine, and vinorelbine, and for the first time, DNA-damaging agents, cisplatin, doxorubicin, and etoposide compared with controls. Cell cycle analysis of H460 betaIII-siRNA cells showed reduced accumulation at the G(2)-M boundary and an increase in the sub-G(1) population in response to TBA treatment compared with control cells. Importantly, betaIII-siRNA cells displayed a significant dose-dependent increase in Annexin V staining when treated with either paclitaxel or cisplatin, compared with controls. These findings have revealed a novel role for betaIII-tubulin in mediating response to both TBA and DNA-damaging agent therapy and may have important implications for improving the targeting and treatment of drug-refractory NSCLC.
OBJECTIVE: To assess the relationship between in vitro chemosensitivity evaluated by the histoculture drug response assay (HDRA) and the expression of beta-tubulin isotypes in tumors of patients with completely resected NSCLC in order to determine the predictive value of beta-tubulin in chemotherapy for NSCLC. METHODS: Expression of beta-tubulin isotypes was immunohistochemically analyzed in a series of 58 tumor samples from patients with completely resected NSCLC. The sensitivity of individual tumors to anticancer agents was evaluated by HDRA. RESULTS: Class III beta-tubulin expression by tumor cells was significantly correlated with resistance to docetaxel (p=0.0250), but not related with resistance to gemcitabine. Patient characteristics (age, gender, histology, and stage) were not associated with class III beta-tubulin expression. CONCLUSION: An abundance of class III beta-tubulin in tumor cells could be a biomarker for resistance to docetaxel in patients with completely resected NSCLC.
