|Year : 2017 | Volume
| Issue : 2 | Page : 44-49
Application of clustered regularly interspersed short palindromic repeats/cas9 editing technologies in breast cancer research
Biskup Ewelina1, Lu Cai2, Xiaoyan Lin2, Craig Kingston3, Fengfeng Cai2
1 Department of Internal Medicine, University Hospital of Basel, Basel, Switzerland; Basic Medical College, Shanghai University of Medicine and Health Sciences, Shanghai, China
2 Department of Breast Surgery, Yangpu Hospital, Tongji University School of Medicine, Shanghai, China
3 Department of Internal Medicine, University Hospital of Basel, Basel, Switzerland
|Date of Submission||19-Jan-2017|
|Date of Acceptance||29-Mar-2017|
|Date of Web Publication||22-Jun-2017|
Department of Breast Surgery, Yangpu Hospital, Tongji University School of Medicine, Shanghai 200090
Source of Support: None, Conflict of Interest: None
Breast cancer (BC) is one of the most heterogeneous diseases. The specific clinical and pathological features of each patient's BC diagnosis determine the response to specific agents and thus the prognosis. There is a great need for personalized management approaches to avoid over- or under-treatment. Recently, a novel method revolutionized the world of cancer research: the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9, an RNA-programmable system. The advantages of CRISPR Cas9 system mean simple and effective. It is increasingly being used in the field of BC research, including evaluation of the established therapies.
Keywords: Breast cancer, clustered regularly interspersed short palindromic repeats/Cas9, somatic gene editing
|How to cite this article:|
Ewelina B, Cai L, Lin X, Kingston C, Cai F. Application of clustered regularly interspersed short palindromic repeats/cas9 editing technologies in breast cancer research. Transl Surg 2017;2:44-9
|How to cite this URL:|
Ewelina B, Cai L, Lin X, Kingston C, Cai F. Application of clustered regularly interspersed short palindromic repeats/cas9 editing technologies in breast cancer research. Transl Surg [serial online] 2017 [cited 2017 Nov 23];2:44-9. Available from: http://www.translsurg.com/text.asp?2017/2/2/44/208867
| Introduction|| |
Breast cancer (BC) is the most common noncutaneous cancer, accounting for 30% of all female cancers and is the leading cause of cancer deaths among women (15%)., It is also one of the most heterogeneous diseases. Despite great therapeutic and diagnostic progress, the understanding and implementation of individualized screening and management of BC are still in their infancy.,,,,, Oncologists aim to identify high-risk individuals, detect cancer at an early stage, predict outcome, monitor treatment, and screen for disease recurrence.,,,,, Tumor/node/metastasis classification of malignant tumors features such as tumor size, lymph node stage, histological grade, type, and lymphovascular invasion are still the dominant variables in predicting the course of the disease. However, the specific clinical and pathological features of each patient's BC diagnosis determine the response to specific agents and thus the prognosis. Some therapeutic targets (e.g., estrogen receptor [ER], progesterone receptor or human epidermal receptor 2) are also known predictive factors. Prognostic factors, however, are still not sufficiently elaborated.,,,,,, In addition, as the tumor progresses and/or metastasizes, cancer's molecular features change, making marker proteins not always suitable for follow-up.,,,,,,,, There is a tremendous need for personalized management approaches to avoid an over- or under-treatment. Accordingly, gene-expression analyses are evolving. A number of methods for an effective and efficient quantification of specific gene expression status have been established.,,,, Still, proving a statistical correlation between the gene expression and the overall disease-free-survival is merely the first hurdle in finding clinical application for this system.,,,,,
Recently, a novel method revolutionized the world of cancer research: the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9, an RNA-programmable system,,,,,,, which allows rapid generation of any desired modification to the genome in cellular and animal models at low cost.
| Physiogenetic Background|| |
CRISPR are found in most bacteria and archaea. They are segments of DNA with short, repetitive base sequences, divided by short segments of spacer DNA from foreign DNA (e.g., viruses or plasmids).,, CRISPR-associated system (Cas) is found in small clusters, next to CRISPR sequences. CRISPR/Cas is believed to be distinctive for the genome of most prokaryotic DNA and play an important role in the prokaryotic immune system, particularly in providing resistance to bacteriophages. The system allows bacteria to acquire immunity against viruses and develop resistance to them based on a specific spacer, which has been integrated during a viral challenge. A repeated attack of the virus activates the Cas system, which recognizes the exogenous DNA based on the specific RNA in the spacer sequence. Cas proteins cut foreign RNA and protect bacteria from viral replication.,,
| New Development of Clustered Regularly Interspersed Short Palindromic Repeats/cas9 System|| |
More recent developments have brought CRISPR to a new, even more simplified level with only two components: a single synthetic single guide RNA (sgRNA, from fusing trRNA and crRNA).,,, In addition, further Cas9 nucleases have been modified and made more adaptable for targeted gene alterations. The prototype was the wild-type Cas9 that cleaves the DNA at specific sites, which are then repaired by double-strand break repair machinery. A newer model is Cas9D10A, a nickase, which cleaves only one DNA strand, and repairs are conducted in a more precise way.,,,,,,, Another option is a nuclease-deficient Cas9 (dCas9), which does not have a cleavage function, but leads to a sequence-specific gene silencing or activation.,,,,,, More strikingly, by fusing dCas9 with an enhanced green fluorescent protein (EGFP), dCas9 has been used for visualization of repetitive DNA sequences or nonrepetitive loci.,, Most recently, the CRISPR-Cas9 system is increasingly used for repression, activation, and loci imaging.,, This is offering new options for biomedical, therapeutic, industrial, and biotechnological application.
New Implications for Cancer Research
During the development and progression of cancers, there are numerous mutations taking place. Until now, it was practically impossible to track the mutations occurring (and shaping) the cancer. Precise models, for example, homologous recombination, are limited, time-consuming, involve multiple steps, and are vulnerable to human factor bias. With CRISPR-Cas9, multiple alterations of genes are possible in one generation, whereas multigenerative processes would be necessary to create a vaguely similar result. In addition, CRISPR is advantageous in terms of speed: using the very specific zinc finger nuclease or transcription activator-like effector nucleases approach requires a laborious design, which is more complex than creating RNA-sequences.,,,
Known esterases usually recognize a short sequence, slicing specifically at their locations all along the genome. Cas9 can recognize a sequence of ca. 20 bases. Therefore, CRISPS-Cas9 has provided researchers with a new tool to create highly specific chromosomal rearrangements.,, Some of them have been reconstructed in cancers, for example, CD74-ROS1 translocation event and the EML4-ALK and KIF5B-RET inversion events in lung cancer.,,,,, Similar reports have been made for bladder cancer, where circuits against cellular functional genes, including hBAX, p21, and E-cadherin have been constructed, effectively inhibiting cancer cell growth, inducing apoptosis and decreasing cell motility. In animals models, the CRISPR/Cas9 system was used to develop hepatocellular carcinoma in mice tail veins. Constructs have also been achieved for brain cancer and pancreatic cancer.,,
Significant implications were made in leukemia research: using the CRISPR/Cas9 system, BRD4 protein domain inhibitors were identified as one of the major antitumor agents. Further, screens in mice with acute myeloid leukemia (AML) identified 25 domains, which influence survival-6 known therapeutic domains and 19 new targets.,
Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 in Breast Cancer Research
The advantages of CRISPR Cas9 system, such as its simplicity and efficacy, could lead to wider scientific application of this system in modern-day research. Considering the current array of information on the variety of BC-driving mutations and their impact on cancer characteristics, CRISPR could provide crucial insights, in turn, leading to tailored therapeutic approaches.,,
Annunziato et al. developed a method to model invasive lobular breast carcinoma. They used intraductal injection of creencoding lentiviral vectors to mammary glands, which lead to multifocal tumorigenesis in mice carrying Cdh1/Akt-E17K mutation or conditional Pten alleles. Similarly, injection of lentiviruses with sgRNA targeting Pten resulted in ILC formation without an immune response. Wang et al. aimed to investigate whether CRISPR/Cas9-mediated interruption of transcription in triple-negative BC (TNBC) would have a strong impact on cancer development. Since TNBC is the most aggressive type of BC, bearing the most multifold mutation structure, it is quite stable in the transcriptional program. Disruption of transcription processes could, therefore, lead to a significant instability of the cancer cells. It was shown that TNBC cells were indeed dependent on CDK7 and undergo apoptosis when this transcription kinase is being inhibited. This observation opens a new frontier of therapeutic options for TNBC, potentially with a CRISPR/Cas9-CDK7-cluster.
As in any other CRISPR/Cas9 approach, the initial step is to design sgRNAs for specific cancer cells CRISPR/Cas9 cassette.,,, Scientists now have the ability to generate a number of potential sgRNAs, which have to be evaluated in designed in vitro experiments.In vivo trials would, of course, be the ultimate objective.
Research into the epigenetic identity of estrogen positive BC leads to the discovery that noncoding mutations and inherited single-nucleotide variants outside of genes in ER-positive breast tumors are dominant culprits and promote ESR1 expression. Moreover, the researchers were able to alter the functional regulatory components.
CRISPR/Cas9 has also been applied to investigate whether BCL2-associated X protein (BAX), a proapoptotic protein, could be inserted into human BC cells.In vitro experiments have been conducted, which monitored cell growth and protein expression rate in cells at regular intervals., Scientists assume that adding the BAX gene into the mitochondrial COI gene would decrease ATP production in the cells and increase the BAX protein quantitatively and lead to an increased apoptosis or at least a significant loss of function. The results are still pending but if they turn out to be positive, it will open a new frontier of BC therapies, allowing a cost-effective and specific approach, with potentially far less side effects in comparison to current therapies. The proposed reason for fewer side effects is based on the system's mechanism: Cas9 activity requires a guide RNA (gRNA) to mediate the cleavage specificity. The amount of how much Cas9 will show an off-target activity is dependent on the gRNA substrate. The crucial step in this regard will be the development of cancer cell-specific delivery systems.
Another important step in its development was using CRISPR/Cas9 to study genetic constellation and driver mutations in TNBC in African-American and Caucasian women. BRCA1 or BRCA2 DNA repair genes and R248Q (TP53) wild type were introduced in TNBC on these two distinct genetic backgrounds, anticipating BRCA1/2, and R248Q (TP53) mutations as manifest characteristics of TNBC. TNBC cells were then exposed to radiation and bleomycin. Results of the study are still pending, but it will almost certainly provide significant insights into novel, personalized TNBC treatment options.
Buchholz's team used the expression of Cas9 together with the cancer-specific guide (g) RNAs. In that way, they were able to identify driver mutations of cell growth and viability in BC. The scientists screened over 500,000 reported BC mutations. More than 80% could theoretically be targeted and specifically cleaved with CRISPR/Cas9, without significantly targeting the healthy, wildtype alleles.
CRISPR/Cas9 also has implications for the investigation of some of the established therapies of BC, for example, taxol (paclitaxel)., It is frequently and broadly used, with mediocre results. However, more than half of the patients face developing resistance to this chemotherapeutic agent.
Since BC is a heterogeneous neoplasia, new developments of CRISPR/Cas9 aiming to target multiple genes are most promising in the search for new therapeutic approaches. In addition, the epigenetic implication of CRISPR/Cas9 is very promising, as it will prompt further investigation of the epigenetic profile of various BC types to allow their characteristic expression states to be specifically targeted. When a specific combination of mutations in a particular BC type is known, the CRISPR/Cas9 system can also assist in cancer diagnostics, before an individualized therapy could be initiated.
| Conclusion|| |
CRISPR-Cas9 system is a great leap forward in the era of cancer genetics, providing simple and effective genomic manipulation with possible use in personalized medicine and future therapies. Since it can potentially be applied to any cancer cell, it is associated with an enthusiasm rarely seen in oncological research, aiming at modeling cancer, finding tumor suppressor genes or oncogenes, and exploring therapeutic strategies. In the era of whole genome sequencing, we have gained insights into tumorigenetic mutation. However, CRISPR/Cas9 has allowed a functional ratification of tumor mutations. Translating this new knowledge into usable clinical information requires the development of precise, practical tests for clinical diagnostics, monitoring and management of cancer patients.
In simpler models, such as leukemia, the system has already been widely used and was essential in discovering new driver mutations in AML. In solid tumors, especially in extremely heterogeneous ones, such as BC, the research is progressing somewhat slower and faces more challenges. The main obstacle, which is also the main research objective in the CRISPR/Cas9 field, is the delivery of Cas9 into in vivo models. Another main point is the safety aspect-studies needed to make the system applicable to human trials.
The ultimate goal is to use CRISPR-Cas9 as a treatment in cancer by cutting out malignant mutations and replacing them with normal DNA sequences. In the meantime, important knowledge will be gained en route about the pathophysiology and biology of cancers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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