spacer
home > ebr > autumn 2010 > welcome interference
PUBLICATIONS
European Biopharmaceutical Review

Welcome Interference

Balasubramanyam Nistla at GBI Research reviews the latest developments in RNA interference technology for cancer care

Ribonucleic acid (RNA) is one of the key components responsible for protein synthesis and cell proliferation. It is made up of a long chain of nucleotide sequences and each nucleotide consists of a nitrogenous base, a ribose sugar and a phosphate. The function of RNA is to encode protein information from the DNA in the nucleus and transfer the information to the amino acids which produce the desired proteins (1). RNA polymerases located in the nucleus transcribe DNA and form RNA. Here, RNA is called messenger RNA (mRNA) since it contains the protein information. mRNA then moves from nucleus to cytoplasm and in the cytoplasm, ribosomes read and translate the protein information from mRNAs.

KEY COMPONENTS OF AN RNA INTERFERENCE (RNAi) PATHWAY

RNA interference is a pathway through which protein synthesis can be blocked. This is carried out either by blocking mRNA from being translated or by degrading mRNA through a cleavage process. RNAi helps to regulate active and inactive genes. Two non-coding RNAs, microRNA (miRNA) and short interfering RNA (siRNA), are the key components in the RNAi pathway (2,3). These RNA molecules can selectively bind to other RNAs and regulate the activity of the genes. These non-coding proteins can perform functions such as blocking mRNA from producing proteins. RNAi has a very important role in safeguarding cells against foreign organisms such as viruses and transposons (4).

The RNA interference pathway becomes activated by an enzyme known as dicer (5,6). When a double-stranded RNA (dsRNA) is introduced into the cell, dicer cleaves dsRNA into small fragments that are approximately 20 nucleotides in length (6). These are known as small interfering RNAs (siRNA). siRNA molecules activate the RNA-induced silencing complex (RISC), which takes up one of the two strands, also known as the guide strand (7). This integration leads to posttranscriptional gene silencing, triggered when the guide strand forms a complementary base pairing with a sequence of mRNA. The complementary base pairing initiates mRNA cleavage.

Various experiments conducted on siRNA molecules have proved that siRNA molecules have the capability to induce 70 to 100 per cent gene suppression. In addition, RNAi technology is more effective than antisense oligonucleotides and ribosomes in specific gene silencing.

THE POTENTIAL OF RNAi TECHNOLOGY IN CANCER CARE IS MULTIFOLD

RNAi technology is highly promising in gene therapy applications intended for the treatment of various cancers. The results of clinical trials on siRNA molecules in the treatment of macular degeneration indicate that siRNA molecules could effectively target vascular endothelial growth factor (VEGF) (8).

The key application of RNAi in oncology is to aptly explain the function of mutant oncogenes. Oncogenes are the genes that help a normal cell to turn into a tumour cell. Upon activation, these genes can help abnormal cells to survive apoptosis and proliferate rapidly. The use of RNAi in understanding the function of these oncogenes can lead to the development of a new class of therapies that can cure cancer. In addition, the effectiveness of the current oncology drugs can also be enhanced using RNAi. Oncology drugs such as imatinib and rapamycin become ineffective against drug-resistant tumours. Resistance to these drugs occurs due to the activation of the resistance-associated gene BCR-ABL (7). 

THE ROLE OF miRNA IN TUMORIGENESIS

miRNAs are non-coding RNAs, which are processed from long precursor molecules encoded by the miRNA gene in the nucleus. The miRNA gene in the nucleus forms a stem-loop primary miRNA (primiRNA). Stem-loops are double-stranded RNA structures which form double-helical structures through imperfect base pairing. A ribonuclease enzyme, Drosha, excises the stem-loop structure to form precursor miRNA (pre-miRNA). The pre-miRNA then moves to the cytoplasm where it gets cleaved by another ribonuclease dicer to form a short RNA duplex (10). The duplex RNA then divides and one of the two strands becomes mature miRNA while the other gets degraded. The mature miRNA then forms a complex with RISC and initiates either mRNA translation repression or mRNA cleavage (11,12).

Cancer is one of the most complex diseases in the world and the exact reason for its occurrences is still unknown. Cancer cells are often associated with mutations, down-regulation, over expression and deletion of tumour suppressor genes. Studies have shown that tumour cells often have defects in noncoding RNAs and normal cells do not have any defective non-coding RNAs. One such defective non-coding RNA found was the H19 gene (13). After the discovery of this important observation, many other defective non-coding RNAs were associated with other tumours and cancers. These non-coding RNAs can be classified into small and large non-coding RNAs, and large non-coding RNAs can generate miRNAs. It has also been found that over expression of miR155 is associated with B-cell lymphoma. miR155 is an miRNA that is produced by non-coding RNA BIC. BIC is often linked with growth control and oncogenesis in cancer cells (13). Similarly, prostate cancer is associated with over expression of non-coding RNA prostate specific gene 1 (PCGEM1). Studies have confirmed that over expression of PCGEM1 promoted cell proliferation and aided in colony formation, suggesting that PCGEM1 has a role to play in prostate tumorigenesis (14).

Some of the non-coding RNAs are also used as markers for the detection of specific tumours. The presence of DD3 non-coding RNA in a urine sample indicates the over expression of the protein and confirmation of prostate cancer. Similarly, many other non-coding RNAs were identified which can be used as markers in various cancers such as breast, bladder and gastrointestinal. For example, the presence of metastasis associated in lung adenocarcinoma transcript 1 (MALAT-1) confirms non-small cell lung cancer (NSCLC). MALAT-1 is currently used as a marker to identify the risk of metastasis in early-stage NSCLC (15).

It has been identified that miRNAs are generally down regulated in tumour cells when compared to the normal cells. Therefore, research on miRNAs reveals vital information about the cancers and their root causes, and clear classification can result in the comprehensive understanding of human cancers and their stages (15).

miRNAs ARE INVOLVED IN THE PATHOGENESIS OF VARIOUS CANCERS

Absence or down-regulation of miRNAs has been observed in many cancers. The first observation on the relation between miRNAs and cancer was studied in chronic lymphocytic leukaemia (CLL). Studies have found that the expression of two miRNA genes, miR-15 and miR-16, was either absent or down-regulated in patients with CLL. Following this observation, many other miRNAs, miR-17-92, and the Myc oncogenic pathway were discovered. Another study elucidated the relationship between let-7 miRNA and the RAS proto-oncogene (16).

Further studies have discovered a cluster of six miRNAs known as miR-17-92 in chromosome 13 – the key chromosome which is amplified in human-B cell lymphomas (17,18). Studies have demonstrated that the miR-17-92 is overexpressed in lymphoma cells and this was confirmed through various laboratory tests. A mouse model was used to confirm the relationship between miR-17-92 with lymphoma. It is a well known fact that mice develop expression of miR-17-92 stepped-up Myc oncogene-induced tumorigenesis in mice (19).

miRNAs HAVE THE POTENTIAL TO SUPPRESS CANCER TUMOURS

A reduction in miRNA levels has been linked to the proliferation of various cancer tumours. Therefore, identification of the absent or down-regulated miRNA will immensely boost the development of therapies targeted at cancer treatment. One such discovery was made in lung cancer, where over-expression of the oncogene was clearly observed. Such RAS oncogene mutations are generally observed in many cancers. Studies have proven that the over expression of the RAS oncogene was due to the down regulation of let-7, a family of miRNAs. Further studies have confirmed that reduced expression of let-7 in lung cancers led to RAS over-expression and, consequently, to tumour proliferation and tumorigenesis (16).

RNAi HAS BECOME THE IDEAL CHOICE FOR SPECIFIC GENE SILENCING

The discovery of new non-coding RNAs has led to the development of new drugs that can target cancer cells. Furthermore, these non-coding RNAs have also become the choice for cancer diagnosis. The success of RNAi in silencing specific genes depends on the effectiveness of siRNA delivery and stability. Currently, the potential of siRNA is studied in gene function characterisation and in the development of therapeutic agents for cancer treatment. The ability of the siRNA to induce specific gene silencing is studied based on its biochemical, pharmacological and histological assays. RNAi technology and the selective gene knock-down mechanism have been used extensively to investigate critical genes and pathways that can be targeted by siRNAs alone or in combination with other drugs.

Oncogenes are potential targets for siRNAs intended for cancer treatment, and are normally genes located inside the cells. Activation of oncogenes results in increased cell growth and tumour formation. Many proteins such as growth factors, signal transducers and transcription factors are generally encoded by oncogenes. These proteins are often used to regulate intracellular functions. For example, down-regulation of the K-RAS protein using RNAi technology in pancreatic cells leads to loss of anchorageindependent growth and tumorigenesis (20). A study was conducted to evaluate the efficacy of siRNA molecules in selective gene silencing in such cancer cells (21,22). Results of the study indicated that siRNA-dependent downregulation of the respective mRNA expression significantly suppressed the proliferation of tumour cells (22,23).

siRNA MOLECULES CAN INHIBIT ANGIOGENESIS & THEREFORE, BLOCK METASTASIS OF TUMOURS

Angiogenesis, the formation of new blood vessels, is one of the critical events required for metastasis. Tumour angiogenesis is the generation of new blood vessels that penetrate into the cancerous cells, thereby supplying nutrients and oxygen. Tumour angiogenesis takes place when cancerous cells release molecules that activate nearby blood vessels. The molecules are generally growth factors such as VEGF, basic fibroblast growth factor (bFGF), matrix metalloproteinase (MMP) and Delta-like ligand 4 (DLL4). Histone deacetylase (HDAC) inhibitors can block angiogenesis. MMPs play a key role in extra-cellular matrix modelling and a membrane-anchored glycoprotein, RECK negatively regulates MMP-9, thereby leading to inhibition of tumour invasion and metastasis. RECK also regulates other MMPs, such as MMP-2 and MT1-MMP, which play a key role in cancer progression. Studies have shown that inhibition of RECK by siRNA in CL-1 lung cancer cells blocked the inhibition of HDAC inhibitors on MMP-2 activation (24). The same study found that CXC chemokine receptor-4 (CXCR4) is over expressed in MDAMB- 231 breast cancer cells. In vitro tests on siRNA expression in the breast cancer cells indicated that siRNA can effectively knock down CXCR4 and suppress breast cancer metastasis.

siRNA have also been used to identify the key proteins and receptors responsible for the metastasis of specific cancers. For example, VEGF, one of the stimulators of angiogenesis, is regulated through Sp protein interactions with several proximal GC-rich motifs in the VEGF promoter. Studies have found that Sp proteins play an important role in tumour progression and metastasis. siRNA molecules were used to investigate the role of Sp proteins in pancreatic cancer cells. Results of the investigation revealed that proximal GC-rich sites were required for VEGF expression in pancreatic cells. Furthermore, sequential knock of the Sp proteins using RNAi technology showed that three proteins, Sp1, Sp3 and Sp4 are involved in VEGF regulation in pancreatic cancer cells. Investigations of the proteins indicated that Sp4 knock down using RNAi decreased the tumour activity by 50 per cent. As a result, it was confirmed that Sp1, Sp3 and Sp4 regulate VEGF expression.

COMBINATION OF RNAi TECHNOLOGY & CHEMOTHERAPY MIGHT PROVE EFFECTIVE IN DRUG-RESISTANT CANCERS

The efficacy of the majority of chemotherapies lies in their ability to induce apoptosis of cancer cells. However, in many cancers, tumour cells develop resistance to the drugs due to the activation of anti-apoptotic factors such as the livin (ML-IAP, KIAP) gene, Bcl-2 and xIAP (22). The livin (ML-IAP, KIAP) gene is an anti-apoptotic factor and suppression of this gene expression in melanoma and cervical carcinoma cells significantly increased the apoptosis rates (22). Similarly, silencing anti-apoptotic factor genes Bcl-2 and xlAP led to improved apoptosis rates in breast cancer cells when treated with drugs such as etoposide and doxorubicin (22). This proved that gene silencing of antiapoptotic factors by siRNA might lead to the development of new therapeutic drugs for cancer treatment.

Table 1: RNA therapeutics: global, siRNA and shRNA delivery options, November 2009

Method

 Type of RNA molecule  Advantages  Disadvantages
 Non-viral delivery      
Lipid    siRNA Systemic delivery, stable Non-selective delivery
 Stable nucleic acid-lipid particles  siRNA Systemic delivery, highly stable Non-selective delivery
 Aptamers  siRNA Receptor-specific delivery Large-scale sequence screening required   
 Nanoparticles  siRNA Receptor-specific, self-assembling Sophisticated preparation required
Viral delivery      
Lentivirus    shRNA Stable expression, transduces non-dividing cells Gene-disruption risk, localised delivery
 Adenovirus   shRNA Episomal, no insertional mutagenesis Immunogenic, dose-dependent hepatotoxicity 
 Adeno-associated virus   shRNA Episomal, low genomic integration  Immunogenic, small  vector capacity

Source: GBI Research, Nature

CONCLUSION

The ability of RNAi to silence diseasecausing genes effectively has placed them as one of the most promising technologies that will drive the future of the pharmaceutical market. The production of synthesised siRNAs is cheaper than the production of proteins or antibody therapies and, due to their favourable pharmacokinetic properties, siRNAs can be delivered to any organ. However, stability and delivery of siRNAs in the blood stream are the major hurdles that are yet to be solved in order to develop effective therapies. siRNAs are very vulnerable to serum nucleases and they degrade rapidly when exposed to ribonucleases (RNAses). They are therefore unstable in cells and biological mediums such as serum.

Synthetic siRNAs have been developed and are currently used widely as reagents for silencing mRNA targets in cells. Several methods were adopted to develop synthetic siRNAs. The 3’-end of the sense strand of siRNA was conjugated with cholesterol through a pyrrolidine linker and this conjugation has significantly improved the pharmacological properties of siRNA. Studies have proved that the conjugated siRNA was resistant to degradation by serum nucleases. Similarly, studies have confirmed that siRNA when conjugated with boranophosphate becomes 10 times more stable and nucleus resistant than naked siRNAs (25).

Drug delivery technologies, both viral and non-viral, that can deliver RNAi therapies to the site of action are now required, and several delivery methods have been developed to deliver these therapies efficiently.

Topical gels have been used to deliver siRNAs to the target site in cervical cancer patients. siRNA was also delivered using intradermal administration via gene guns. siRNA compounds can be delivered using either viral vectors or non-viral vectors for both local and systemic delivery. Some of the siRNA delivery systems include viral delivery, the use of liposomes or nanoparticles, bacterial delivery, and also chemical modification of siRNA to improve stability. siRNA delivery through nanoparticles has been tested for biological activity, and studies have confirmed the accumulation of siRNA compounds near the nuclear membrane. Therefore, in the near future, RNA interference technology will enter and dominate the cancer market.

References

  1. Cooper GC and Hausman RE, The Cell: A Molecular Approach (3rd ed): pp261-276, 297, 339-344, 2004
  2. Holen T, Amarzguioui M, Babaie E and Prydz H, Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway, Nucleic Acids Research 31(9): pp2,401- 2,407, 2003
  3. Meister G and Tuschl T, Mechanisms of gene silencing by double-stranded RNA, Nature 431: pp343-349, 2004
  4. Vaury C and Buchon N, RNAi: a defensive RNA-silencing against viruses and transposable elements, Heredity 96: pp195-202, 2006
  5. Ketting RF et al, Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C elegans, Genes Dev 15: pp2,654-2,659, 2001
  6. Bass BL and Knight SW, A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans, Science 293: pp2,269- 2,271, 2001
  7. Hammond SM, Caudy AA and Hannon GJ, Post-transcriptional gene silencing by double-stranded RNA, Nature Reviews Genetics 2: pp110-119, 2001
  8. Dykxhoorn DM, Palliser D and Lieberman J, The silent treatment: siRNAs as small molecule drugs, Gene Therapy: pp541-552, 2006
  9. Chen J, Wall NR, Kocher K, Duclos N, Fabbro D, Neuberg D, Griffin JD, Shi Y and Gilliland DG, Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin, J Clin Invest 113: pp1,784-1,791, 2004
  10. Bartel DP, MicroRNAs: genomics, biogenesis, mechanism and function, Cell 116: pp281-297, 2004
  11. Caldas C, Eric A, Sassen M and Sassen S, MicroRNA – implications for cancer, Virchows Arch 452: pp1-10, 2008
  12. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S and Kim VN, The nuclear RNase III Drosha initiates microRNA processing, Nature 425: pp415- 419, 2003
  13. Espinosa CES and Slack FJ, The Role of MicroRNAs in Cancer, Yale J Biol Med 79(3-4): pp131-140, 2006
  14. Petrovics G et al, Elevated expression of PCGEM1, a prostatespecific gene with cell growthpromoting function, is associated with high-risk prostate cancer patients, Oncogene 23: pp605-611, 2004
  15. Ji P et al, MALAT-1, a novel noncoding RNA, and thymosin 4 predict metastasis and survival in early-stage non-small cell lung cancer, Oncogene 22: pp8,031- 8,041, 2003
  16. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D and Slack FJ, RAS is regulated by the let-7 microRNA family, Cell 120: pp635-647, 2005
  17. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ and Hammond SM, A microRNA polycistron as a potential human oncogene, Nature 435: pp828-833, 2005
  18. Ota A, Tagawa H, Karnan S, Tsuzuki S, Karpas A, Kira S, Yoshida Y, Seto, Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma, Cancer Res 64: pp3,087-3,095, 2004
  19. O’Donnell KA, Wentzel EA, Zeller KI, Dang CV and Mendell JT, c-Mycregulated microRNAs modulate E2F1 expression, Nature 435: pp839-843, 2005
  20. Garber K, Better Blocker: RNA Interference Dazzles Research Community, Journal of the National Cancer Institute 95(7): pp500-502, 2003
  21. Adachi E et al, The membraneanchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis, PubMed Central 107(6): pp789- 800, 2001
  22. Abudayyeh A, Baker C, Abdelrahim M and Safe S, RNAi and cancer: Implications and applications, Journal of RNAi and Gene Silencing 2(1): pp136-145, 2006
  23. Agami R, Brummelkamp TR and Bernards R, Stable suppression of tumorigenicity by virus-mediated RNA interference, Cancer Cell 2: pp243-247, 2002
  24. Vanderlaag K, Higgins KJ, Yoon K, Abdelrahim M, Metz RP, Liu S, Safe S and Porter W, Vascular Endothelial Growth Factor Receptor-2 Expression Is Induced by 17_- Estradiol in ZR-75 Breast Cancer Cells by Estrogen Receptor _/Sp Proteins, Endocrinology 147(7): pp3,285-3,295, 2006
  25. Turner JJ, Lindsay MA, Gait MJ, Jones SW and Moschos SA, MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse Alike activity, Mol BioSyst 3: pp43-50, 2007

Read full article from PDF >>

Rate this article You must be a member of the site to make a vote.  
Average rating:
0
     

There are no comments in regards to this article.

spacer
Balasubramanyam Nistla has over three years of experience in market research and healthcare consulting. Prior to joining GBI Research, he was a Business Analyst with Evalueserve, India. He is a graduate from BITS, Pilani, with a B Pharma (Hons) degree. He has been with the Healthcare Team at GBI Research since June 2009.
spacer
Balasubramanyam Nistla
spacer
spacer
Print this page
Send to a friend
Privacy statement
News and Press Releases

Arcis Biotechnology and Mirnax Biosens sign exclusive license agreement for Arcis sample prep technology

Daresbury, UK, and Madrid, Spain, 16 July 2019: Arcis Biotechnology (“Arcis”), the nucleic acid sample preparation solution provider, and Mirnax Biosens (“Mirnax”), the developer of microRNA (miRNA) biomarkers for clinical diagnostics, today announced they have signed an exclusive license agreement for use of Arcis sample preparation and preservation technology.
More info >>

White Papers

Is Your Biobank Ready for the Challenge of Biomarker-based Research?

BioFortis

Targeted and personalized studies with well-defined patient segmentation biomarkers are becoming the norm in clinical trials. This increased interest in molecular biomarker studies necessitates a rigor and sophistication in sample management within the clinical trial context that is often not supported either by traditional clinical trial management software (CTMS), or biobanking systems.  Download our Next Generation Biobanking whitepaper and learn about how to overcome the key challenges in clinical trial sample management from working in a distributed network of partners and stakeholder to managing consents and generating scientific insights.
More info >>

 
Industry Events

Nordic Life Science Days 10/12 September 2019

10-12 September 2019, Malmo Sweden

Nordic Life Science Days is the largest Nordic partnering conference for the global Life Science industry. Bringing together the best talents in Life Science, offering amazing networking and partnering opportunities, providing inputs and content on the most recent trends. Nordic Life Science Days attracts leading decision makers from the Life Science sector, not only from biotech, pharma and medtech but also from finances, research, policy and regulatory authorities.
More info >>

 

 

©2000-2011 Samedan Ltd.
Add to favourites

Print this page

Send to a friend
Privacy statement