v
Search
Advanced Search

Publications > Journals > Gene Expression > Article Full Text

  • OPEN ACCESS

Exploring the Advantages and Limitations of CRISPR-Cas in Breast Cancer

  • Nelson Rangel1,
  • Valentina Camargo2,
  • Giovanny Castellanos2,
  • Maribel Forero-Castro2 and
  • Milena Rondón-Lagos2,* 
 Author information
Gene Expression   2024;23(2):116-126

doi: 10.14218/GE.2023.00154

Abstract

Breast cancer (BC) is the type of cancer with the highest incidence and mortality rates in women in the world. In the treatment of this neoplasia, several therapies are applied, including radiotherapy, hormonal therapy, chemotherapy, and biological therapy. Although most patients respond to these types of therapy, some patients over time, develop resistance or eventually relapse. Considering the above, future therapeutic concepts in BC are being directed at individualization of therapy and escalation of treatment based on tumor biology through the use of gene therapy. In this regard, a new genomic engineering technology, called the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9), has acquired great importance in recent years, as a potential gene editing tool, extensively applied in human cancer research and cancer treatment. The aim of this review was to describe the advantages, limitations, and applications of CRISPR gene editing technology in BC treatment. Our review emphasizes the innovative facets and profound importance of CRISPR gene editing technology within the BC treatment landscape. Additionally, it provides valuable information to consider when evaluating the risks associated with the implementation of CRISPR-Cas9 technology in BC therapy.

Keywords

Breast cancer, Gene therapy, Resistance to therapy, Therapeutic targets

Introduction

Breast cancer (BC) is the type of cancer with the highest incidence and mortality rates in women in the world, being therefore, a worldwide concern considering the high rates of incidence (47.8%) and mortality (13.6%) recently reported. Specifically, in 2020, 2,300,000 new cases (11.7%) and 684,996 deaths from this neoplasia were reported. Therefore, it is the type of cancer with the highest incidence and mortality rates in women.1 At the molecular level, BC is a very heterogeneous disease, being classified by molecular subtypes based mainly on the presence of hormone receptors (estrogen-ER, and progesterone receptors-PR), human epidermal growth factor receptor 2 (HER2), and/or BRCA mutations. Depending on the tumor subtypes, treatment strategies have been developed that generally include: endocrine therapy (for ER-positive BC patients), poly(ADP-ribose) polymerase (for BRCA mutation carriers), anti-HER2 therapy (for HER2 positive BC patients), chemotherapy (for triple negative breast cancer—TNBC), and immunotherapy, among others. However, despite the development of several new technologies and the emergence of new classes of anticancer drugs, current clinicopathological, immunohistochemical and molecular markers, leave a significant number of patients at risk of side effects, over-treatment or eventually the development of resistance. Considering the above, future therapeutic concepts in BC are being directed at individualization of therapy and escalation of treatment based on tumor biology through the use of gene therapy. In fact, gene therapy has become a potential tool to correct defective genes and treat various types of cancer.

In recent years, a novel targeted genome editing technology, known as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9), is being extensively applied in human cancer research and cancer treatment. In this review, we describe the advantages, limitations, and applications of CRISPR gene editing technology in BC treatment. By exploring the intricacies of CRISPR-Cas9 technology and its potential implications in BC therapeutics, this review provides valuable insights that are crucial for understanding its scientific significance and clinical relevance. Moreover, it offers a comprehensive analysis of the risks associated with the implementation of CRISPR-Cas9 technology in BC treatment.

CRISPR-Cas9 technology

CRISPR-Cas technology has developed rapidly and is now an efficient alternative for cancer treatment. CRISPR is naturally occurring genome editing systems found in bacteria. The system serves as a genetic memory that helps the cell detect and destroy invading viruses.2 CRISPR-Cas is composed of two components: CRISPR repeat-spacer arrays and a set of CRISPR-associated (Cas) genes, which encode Cas proteins with endonuclease activity. According to the involvement of the different Cas proteins within the CRISPR framework, CRISPR-Cas technology has been classified into 2 classes (Class 1 and Class 2), 6 types (I to VI) and 33 subtypes.3,4 In general, the system works as follows: CRISPR “spacer” sequences, are transcribed into short RNA sequences (single guide RNA- sgRNA) capable of guiding the system towards matching or complementary DNA sequences. When the target DNA is found, Cas9 binds to the DNA and cuts it, generating DNA double-strand breaks (DSBs). Then, DNA double-strand break repair pathway, including homologous recombination (HR) and non-homologous DNA end joining (NHEJ), is activated to repair DSBs.5–7 (Fig. 1). Considering that activation mainly of NHEJ repair pathways is error-prone, chromosomal rearrangements and large deletions, as consequences of target activity, have been reported.8,9

CRISPR mechanism of action and potentials target genes.
Fig. 1  CRISPR mechanism of action and potentials target genes.

CRISPR “spacer” sequences are transcribed into short RNA sequences (single guide RNA-sgRNA) capable of guiding the system toward matching or complementary DNA sequences. When the target DNA is found, Cas9 binds to the DNA and cuts it, generating DNA double-strand breaks (DSBs). Then, the DSB repair pathway, including homologous recombination (HR) and non-homologous DNA end joining (NHEJ), is activated to repair DSBs. The error-prone NHEJ pathway can lead to random indel mutations in the binding site. Indel mutations that occur within the coding region of a gene, can lead to gene knockout. CRISPR-Cas9 has been used in breast cancer research to edit oncogenes and tumor suppressors genes, leading to their inactivation or activation, respectively. ACKR3, Atypical Chemokine Receptor 3; BRCA1, Breast cancer type 1; Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; CXCR4, C-X-C Motif Chemokine Receptor 4; DSBs, DNA double-strand breaks; HR, homologous recombination; HER2, human epidermal growth factor receptor 2; MYC, MYC Proto-Oncogene; NHEJ, non-homologous DNA end joining; OPN, osteopontin gene; PAM, Protospacer Adjacent Motif; PTEN, Phosphatase and Tensin homolog; sgRNA, single guide RNA; TP53, Tumor Protein P53.

Although the apparent advantages of the use of CRISPR-Cas9 have been elucidated, its limitations have also been indicated, including: cancer risk, immunological reactions and ethical concerns, among others.

CRISPR-Cas9 technology in the editing of genes involved in BC

BC is characterized by being a very heterogeneous neoplasm, which is governed by genes that control proliferation, apoptosis, genomic instability, replicative immortality, cell metabolism, invasion and metastasis, among others.10 Despite the development of several new technologies and the emergence of new classes of anticancer drugs, resistance to therapy, side effects, overtreatment or, eventually, the development of resistance, continue to be the biggest concerns for the correct management of BC patients. Therefore, in recent years, a large amount of research has been directed at understanding the biological and molecular mechanisms that lead to resistance, as well as the design of personalized therapies that allow such resistance to be overcome. In fact, the investigations carried out to date, have made it possible to identify the role played by oncogenes and tumor suppressor genes (TSGs), in the resistance to therapy in BC. Taking the above into account, both, oncogenes and TSGs, constitute potential therapeutic targets for the treatment of BC, using CRISPR (Table 1 and Fig. 1).11–23

Table 1

Application of CRISPR-Cas9 in the treatment of Breast Cancer by targeting different genes

Target geneCRISPR-Cas9 approachEffectsReferences
HER2Induction of a frameshift mutation in exons 5, 10 and 12Cell growth inhibition in HER2 positive cell lines11
MYCEpigenetic modifications of MYC regulatory elements. Elimination of the MYC enhancer docking siteCell proliferation reduction and MYC decreased expression levels12
Alteration of MYC binding sites (E-boxes)Alterations in MYC binding to target genes, in target gene expression, in tumor growth in vivo and in cell proliferation in vitro13
CXCR4Gene knockoutCell proliferation and cell invasion reduction14
ACKR3Gene knockoutCell proliferation and cell invasion reduction14
MAP3K11Gene depletionMetastasis reduction15
OPNGene knockoutOPN gene expression reduction. Apoptosis and cell viability decrease16
TP53Reversion of a missense mutationBase substitution in the TP53 gene18
PTENActivation of gene expressionIncreased PTEN expression, and repression of the AKT, mTOR and MAPK signaling pathways.19
BRCA1Decreased DNA methylationTranscriptional up-regulation of BRCA1 gene17
MDR1Gene disruptionIncreased response to doxorubicin and cell death20
PARP1Gene depletionSensitization of TNBC cells to chemotherapeutic drugs: doxorubicin, gemcitabine and docetaxel,21
DSTYKGene deletionActivation of apoptosis in chemoresistant cells in in vitro and in vivo models.22
ATP8B3Gene knockoutIncreased resistance to paclitaxel in TNBC23
FOXR2Gene knockoutIncreased resistance to paclitaxel in TNBC23
FRG2Gene knockoutIncreased resistance to paclitaxel in TNBC23

Targeting oncogenes

Proto-oncogenes are normal genes involved in cell proliferation and differentiation. Among the mechanisms associated with the conversion of proto-oncogenes into oncogenes are amplifications, translocations and mutations. These types of alterations, lead to the permanent activation of oncogenes, and therefore to the alteration of the cellular functions in which they participate,24 promoting tumorigenesis.25 In BC, oncogenes that have often been found to be deregulated include: HER2, MYC, CXCR4, ACKR3, MAP3K11 (MLK3) and OPN, among others. Therefore, CRISPR-Cas9 can be used to target directly these oncogenes, inhibiting cell proliferation and tumorigenicity.

HER2 gene

The HER2 gene is amplified in approximately 15% of primary BC.26HER2 amplification/overexpression, has been identified as oncogenic driver and potential therapeutic target in BC patients.27 In fact, HER2 gene amplification is used for both, the prognosis and guide treatment with trastuzumab in BC patients.28,29 However, although anti-HER2 therapies have dramatically improved the prognosis of cancers that overexpress HER2, some BC patients relapse or develop resistance over time. Therefore, it is necessary to use treatments that allow such resistance to be overcome. In this regard, some studies have been directed to the use of CRISPR-Cas9 technology to target the HER2 gene in HER2 amplified BC cell lines. The results of one of such studies, show that CRISPR-Cas9-mediated HER2 targeting, inhibited cell growth in HER2 positive cell lines. Additional analyzes showed that the inhibition of cell growth of CRISPR-Cas9 on the cell lines, was related to the induction of a frameshift mutation in exon 12 of HER2, which led to the production of a truncated HER2mut protein (Fig. 1). Additionally, it was indicated that the inhibition of cell proliferation was accompanied by the suppression of the MAPK-ERK signaling pathway (Fig. 2).30 This study showed that, targeting exons 5, 10 and 12 of HER2 using CRISPR-Cas9, led to the inhibition of cell growth of two HER2+ BC cell lines (BT474 and SKBR3), but not in HER2- BC cells (MCF7).30 Exons 5, 10, and 12, encode parts of the extracellular domain of the HER2 protein.11 Specifically, in this study, Cas9 together with three gRNAs, were introduced into HER2+ (BT474 and SKBR3) and HER2- BC (MCF7) cell lines, observing suppression of cell growth in the HER2+ cell lines but not in the HER2- cell line. These results indicate that the use of CRISPR-Cas9 to target HER2, results in a decrease in cell growth in HER2+ BC cell lines.30

CRISPR/Cas9 as a gene editing tool in HER2+ Breast Cancer.
Fig. 2  CRISPR/Cas9 as a gene editing tool in HER2+ Breast Cancer.

CRISPR/cas9 induces a frameshift mutation in exons 5, 10, and 12 of HER2, generating a truncated HER2mut protein. The mutated HER2 protein leads to growth inhibition and negative regulation of the endogenous MAPK-ERK and PI3K signaling pathways. These signaling pathways are associated with the activation of target genes related to some hallmarks of cancer. AKT, protein kinase B; BCL2, B-cell lymphoma 2; Cas9, CRISPR associated protein 9; C-MYC, MYC Proto-Oncogene; ERK, extracellular signal-regulated kinase; FOXO1, Fork head Box O1; GLUT4, Glucose transporter type 4; CRISPR, clustered regularly interspaced short palindromic repeat; EGFR, Epidermal Growth Factor Receptor; GSK3A, Glycogen Synthase Kinase 3 alpha; GSK3B, Glycogen Synthase Kinase 3 beta; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; HER4, human epidermal growth factor receptor 4; MEK, mitogen-activated protein kinase kinase; mTOR2, Mechanistic Target Of Rapamycin Kinase; PIP2, Phosphatidylinositol 4,5-bisphosphate; PIP3, Phosphatidylinositol (3,4,5)-trisphosphate; PI3K, phosphatidylinositol 3-kinase; PTEN, Phosphatase and Tensin homolog; RAF, Raf Proto-Oncogene, Serine/Threonine Kinase; RAS, Rat sarcoma virus protein; TSC1/2, TSC Complex Subunit ½; VEGF, Vascular Endothelial Growth Factor.

MYC gene

MYC is an oncogene frequently amplified in BC and associated with apoptosis inhibition and activation of cell proliferation. The application of CRISPR-Cas9 technology in cMYC gene editing, has been directed at the transcriptional deregulation of MYC, either through epigenetic modifications of MYC regulatory elements or through mediated elimination of the MYC enhancer docking site. This leads to reduced cell proliferation, associated with inhibition of transcription factor binding and thus decreased expression levels of the MYC protein (Fig. 1).12 Considering that MYC binds to specific E-box sequences in the genome to regulate gene expression, a recent study showed the potential application of CRISPR-Cas9 in altering MYC binding sites (E-boxes) in the MCF7 BC cell line. The results of this study showed that E-box disruption, in genes essential for tumor cell growth, affected MYC binding, target gene expression, tumor growth in in vivo studies, and cell proliferation in in vitro studies. The authors conclude that this approach could constitute a useful tool for the genome-wide identification of E-boxes that are important for MYC-dependent networks in cancer cells.13

CXCR4 and ACKR3 genes

CXCR4 and ACKR3 genes, encodes a G-protein-coupled seven transmembrane receptor, and are highly expressed and dysregulated in BC. Both, CXCR4 and ACKR3 genes, play important roles in the progression, metastasis and prognosis of BC.31 In fact, higher expression of CXCR4 and CKR3 has been associated with both, poor prognosis and metastasis in TNBC. Further, upregulation of cytoplasmic expression of CXCR4 was also suggested as one of the molecular mechanisms that could facilitate lymph node metastasis in invasive micropapillary breast carcinoma (IMPC).32,33 IMPC is a relatively rare subtype of invasive ductal breast carcinoma, accounting for less than 5% of all BC cases, associated with lymphovascular invasion.34 Considering the high implications of CXCR4 and CKR3 genes in BC, CRISPR-Cas9 technology was used in the TNBC cell line (MDA-MB231), in order to create CXCR4 or CKR3 knockout or co-knockout (Fig. 1). The results of these assays showed a significant reduction in both, cell proliferation and cell invasion.14

MAP3K11 (MLK3) gene

Mitogen-Activated Protein Kinase Kinase Kinase 11 (MAP3K11), encodes for a member of the serine/threonine kinase family. This kinase activates MAPK8/JNK kinase, and functions as a positive regulator of the c-Jun N-terminal kinase (JNK) signaling pathway, being implicated in the metastasis process in TNBC.35 Given its implication in TNBC, previous studies demonstrated that editing of MLK3 gene, using CRISPR-Cas9, led to a significant reduction of TNBC metastasis (Fig. 1), thus revealing the crucial role that MAP3K11 gene plays in this BC tumoral subtype.15

OPN gene

OPN gene, encodes for a member of Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING).36OPN is overexpressed in several diseases,37,38 being associated with poor prognosis, survival, growth and radio resistance in BC.39,40 Given the implications of OPN in BC, this gene has been proposed as a potential prognostic biomarker and also as therapeutic target.41 For instance, a recent study showed that the combined use of the CRISPR-Cas9 system with radiation, in the MDA-MB231 BC cell line, led to a significant reduction in the OPN gene expression, as well as an increase in the rate of apoptosis (Fig. 1) and a greater decrease in cell viability.16 These results suggest that the combination of conventional radiotherapy with OPN gene knockout, could become an effective treatment for the treatment of BC.

Targeting TSGs

TSGs are genes that regulate several cellular functions, such us: Cell cycle regulation, apoptosis induction, DNA repair mechanisms and surveillance of genomic integrity, among others. Inactivation or loss of function of TSGs has been correlated with a high risk of cell growth deregulation, a well-known mechanism for the development and progression of many types of cancers.17 Among the mechanisms associated with the inactivation of TSGs are: chromosomal deletions, mutations and loss of expression due to transcriptional silencing mediated by hypermethylation at the promoter site. Considering the important roles that TSGs play in cancer control, CRISPR-Cas9 technology has acquired great importance in recent years, as a promising tool aimed at activating suppressed TSGs. Among the TSGs that may be potential targets for BC treatment using CRISPR, are: TP53, PTEN and BRCA1, among others.

TP53 gene

The TP53 gene, encodes a tumor suppressor protein with important functions in maintaining cellular integrity. The p53 protein, responds to various cellular stresses to regulate the expression of target genes, activating cell cycle arrest, DNA repair, apoptosis, senescence or alterations in metabolism.42 Mutations in this gene have been associated with a variety of human cancers, with the most frequent mutations occurring within the DNA-binding domain.43 An increase in TP53 mutation burden (predominantly missense mutations) in BC, has been correlated with advanced disease, higher genetic instability and metastatic risk,44 worse overall survival and poor clinical outcome.45,46 Given the high implications of TP53 gene mutations in cancer prognosis, this gene constitutes an attractive therapeutic target for cancer therapy. In fact, several studies have been directed at reversing mutations in TP53 (Fig. 1). For instance, a recent study used CRISPR-Cas9 technology to reverse a TP53 missense mutation (L194F), in the T47D luminal A BC cell line.18 The results of this study showed success in the desired base substitution in the TP53 gene, although the editing efficiencies were lower than expected by the authors. Despite the above, the importance of improving the efficiency of the main edition is highlighted, proposing ways forward that could be beneficial for research in BC.

PTEN gene

Phosphatase and tensin homolog (PTEN), is a TSG that encodes for a negative regulator of the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway.47 Therefore, this gene is involved in the inhibition of cell cycle progression, survival and migration. Deletions, mutations, transcriptional repression and epigenetic silencing due to promoter methylation, are the mechanisms mainly associated with PTEN inactivation.19 Despite the above, deletions together with promoter methylation, are the main causes of the loss of expression of the PTEN protein observed in many BCs.48,49 In addition, loss of PTEN activity has been associated with resistance to therapy, poor outcomes in BC,48,50 and also with more aggressive phenotypes.51 Taking into account both, that the inactivation of PTEN has been associated with the severity of BC,52,53 and that the expression of PTEN can be regulated transcriptionally and epigenetically in the absence of mutations in PTEN,54,55 several studies have focused on transcriptional reactivation of PTEN expression. The goal of these types of studies has been to achieve inhibition of progression and increase drug sensitivity in aggressive PTEN-deficient cancers in which PTEN is not mutated. In this regard, one of these studies activated PTEN expression in TNBC cells with low levels of PTEN expression (Fig. 1),19 by using the dCas9-VPR system. The dCas9-VPR system, consists of a deactived (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). The results of this study showed that the dCas9-VPR system, increased the PTEN expression in TNBC cells, also observing repression of the AKT, mTOR and MAPK oncogenic signaling pathways. The results of this study constitute the basis for the design of potential therapies for the treatment of triple-negative breast tumors, for which there is currently no specific target therapy available.

BRCA1 gene

BRCA1 is another relevant gene in BC, associated with the DNA double-strand break repair and DNA stability. Among the mechanisms associated with genetic silencing of BRCA1, in non-familial BC, including TNBC, is promoter methylation, leading to genetic silencing and conferring poor prognosis.56–59 With the aim of decreasing DNA methylation, a recent study reactivated gene expression and restoring the functional activity of BRCA1 in BC (Fig. 1), by using the CRISPR/deactivated Cas9 (dCas9)-Ten-Eleven Translocation dioxygenase1 catalytic domain (TET1cd) demethylation system.17 The CRISPR/dCas9-TET1cd system has the ability to bind to the target site without cutting the DNA strands. The results of this study showed that CRISPR/dCas9-TET1cd, lead to the transcriptional up-regulation of BRCA1 gene. The results of this study open the possibility of using the CRISPR/dCas9-TET1cd system, as a gene editing tool, for the targeted demethylation of epigenetically silenced TSGs in human cancers.

CRISPR-Cas9 system and treatment resistance in BC

Treatment resistance has been reported as the main cause of high mortality in BC, where more than 90% of failed treatments are due to multidrug resistance (MDR) and acquired resistance.60,61 In fact, it has been indicated that exposure to chemotherapeutic agents can lead to an MDR phenotype, and may also involve various cellular and molecular changes.62

For instance, overexpression of ATP binding cassette (ABC) transporters has been associated with MDR. This is due to the involvement of such transporters in the elimination of drugs from breast tumor cells, before they accumulate in therapeutically active concentrations.63 Given their role in resistance to therapy, the induction of alterations in these membrane transport proteins, could facilitate re-sensitization to existing therapies and reduce the possibility of applying new therapies.20 Established strategies to improve drug therapy include increasing drug efflux by altering the membrane transporter protein, thereby enhancing DNA repair, and reversing MDR.64 Specifically, blocking resistance factors using CRISPR-Cas9, is an attractive strategy to overcome MDR and thus continue using existing anticancer agents. In fact, CRISPR-Cas9 has also been suggested as a potential therapeutic tool to overcome chemoresistance in BC.

Another drug efflux pumps commonly overexpressed in BC and contributing to drug resistance are: P-gp and breast cancer resistance protein (BCRP).65 In addition, Glutathione S-Transferase Pi 1 (GSTP1) gene, has also been associated with chemoresistance in BC.66

P-glycoprotein (P-gp) is encoded by the multidrug resistance gene 1 (MDR1).67 P-gp is a membrane glycoprotein transporter that belongs to the largest superfamily of ATP-binding cassette (ABC) proteins.68 Recently, the CRISPR-Cas9 technology was used to edit the MDR1 gene, and thus overcome doxorubicin resistance in a MDR BC cell model (MCF7/ADR cells). Disruption of MDR1 by CRISPR-Cas9 in MCF7/ADR (adriamycin-resistant cell line) cells, showed an increase in doxorubicin potency in treated cells, which also led to an increase in cell death compared to cells not edited.20 The results of this study suggest that, Cas9-mediated disruption of MDR1 gene, could be considered as a potential tool to overcome MDR in BC cells.

BCRP is encoded by the ATP-binding cassette subfamily G member 2 (ABCG2) gene.65 BCRP is an ABC transporter, associated with MDR in various cancer cells. ABCG2 acts as energy-dependent efflux pumps capable of effluxing out of the cell a wide range of xenobiotics, such us: chemotherapeutics (doxorubicin) and anticancer drugs based on natural products.65,69 Additionally, it has been indicated that the expression of BCRP/ABCG2 in cancer cells, in addition to being associated with drug resistance mechanisms, could be associated with invasiveness, self-renewal and with poor prognosis For example,69 BCRP/ABCG2 has been associated with an MDR phenotype in the MCF7 cell line.70 Taking the above into account, blocking active efflux mediated by BCRP/ABCG2 could constitute a promising therapy to overcome resistance in cancer.

Additionally, the glutathione S-transferase P1 (GSTP1) gene has also been associated with chemoresistance in BC. GSTP1 is a gene that encodes a protein involved in many cellular processes, including: phase II detoxification of xenobiotics, in the metabolism of a variety of carcinogenic compounds and in the protection of cells against DNA damage, among others. Indeed, overexpression of GSTP1, has been associated with cisplatin resistance in BC.71

Taking into account that one of the main causes of therapeutic failure in BC is chemoresistance, especially in TNBC, the CRISPR-Cas9 system is being applied in in vitro and in vivo studies with the aim of sensitizing tumor cells to the chemotherapy. For instance, in this tumoral subtype, mutations in the BRCA1 gene (BRCA1m) have been frequently associated with chemoresistance. In this regard, a recent study, aimed at sensitizing BRCA1m cancer cells to chemotherapy, used CRISPR-Cas9 to generate Poly(ADP-Ribose) Polymerase 1 (PARP1) deficient TNBC cell lines (MDA-MB231 and MDA-MB436). The results of this study showed that CRISPR-Cas9-mediated PARP1 deficiency, sensitized TNBC cells with BRCA1m (MDA-MB436) to chemotherapeutic drugs: doxorubicin, gemcitabine and docetaxel, compared to the wild-type cell line (MDA-MB231).21

In addition, another gene that has recently been associated with promoting chemoresistance in TNBC, is the Dual Serine/Threonine and Tyrosine Protein Kinase (DSTYK). DSTYK gene encodes a dual serine/threonine and tyrosine protein kinase which is thought to function as a regulator of cell death. DSTYK has recently been established as a potential therapeutic target, given its involvement in resistance to chemotherapeutic treatment in TNBC cells. In fact, a recent study demonstrated that deletion of DSTYK by using CRISPR-Cas9, led to apoptosis of chemoresistant cells after drug treatment, both in in vitro and in vivo models. These findings suggest that DSTYK exerts an important and previously unknown role in promoting chemoresistance.22

Additional studies have shown the potential use of CRISPR in identifying cancer vulnerabilities and developing new therapeutic strategies. This is the case in a recent study that identified potential paclitaxel-sensitizing/resistant genes, using a combined in vitro/in vivo genome-wide CRISPR synthetic lethality screening approach in a TNBC cell line.23 The results of this study showed that silencing of the ATP8B3, FOXR2 and FRG2 genes led to increased resistance to paclitaxel in TNBC. Altogether, these results suggest the potential therapeutic value of the ATP8B3, FOXR2 and FRG2 genes for chemotherapy treatments in TNBC.

Overall, the results of all of the above studies suggest the potential use of CRISPR-Cas 9 to restore drug sensitivity and overcome chemotherapy resistance in BC.

CRISPR-Cas9 limitations—induction of chromosomal alterations

As indicated above, CRISPR genome editing has emerged in recent years as a potential tool for the treatment of cancer and other diseases. However, since CRISPR technology is primarily based on creating specific DNA DSBs in almost any part of the genome, has been indicated that gene editing with CRISPR-Cas9, in addition to inducing DSBs, can lead to the induction of a broad spectrum of genomic rearrangements, chromosomal variations and structural chromosomal alterations.72–74 In fact, it has been reported that CRISPR induces structural chromosomal alterations, such as: dicentric chromosomes, chromosomal translocations, micronuclei and chromothripsis.72 Dicentric chromosomes (dic) can be generated because DNA breaks, produced by Cas9, can lead to ligation of the central fragments of sister chromatids cleaved by Cas9.75,76 In regard to chromosomal translocations (t), has been indicated that gene editing protocols that induce more than one on-target DSB, could lead to incorrect joining of DNA ends and, therefore, the induction of chromosomal translocations (Fig. 3) that could persist in treated patients over time.77

Induction of chromosomal alterations by CRISPR/Cas9.
Fig. 3  Induction of chromosomal alterations by CRISPR/Cas9.

CRISPR technology is primarily based on creating specific Double-strand breaks in DNA (DSBs) in almost any part of the genome. These DSBs are subsequently corrected, through the activation of DSBs repair mechanisms, including homologous recombination (HR) and non-homologous end joining repair (NHEJ). In HR repair, the pair of homologous chromosomes are brought together and the region of the undamaged homolog or chromatid is taken as a template to reconstruct the DSB of the affected chromosome. While, Non-homologous end joining (NHEJ) repair, allows the joining of broken ends without requiring a homologous or complementary sequence to guide the repair. These mechanisms are error prone and can leave the ends of the affected chain free, which after erroneous processes of splicing, resection, alignment, invasion and/or replication, lead to structural chromosomal alterations as: chromosomal deletions (del), chromosomal duplications (dup), derivatives chromosomes (der), isochromosomes (i), isodicentric chromosomes (idic), dicentric chromosomes (dic), acentric fragments chromosomes (ace), and chromosomal translocations (t). Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; del, chromosomal deletions; dup, chromosomal duplications; der, derivatives chromosomes; i, isochromosomes; idic, isodicentric chromosomes; dic, dicentric chromosomes; ace, acentric fragments chromosomes; t, chromosomal translocations.

Furthermore, recent findings reveal that CRISPR-Cas9 genome editing can induce the formation of micronuclei (MN) and nucleoplasmic bridges in dividing cells, leading to both numerical and structural chromosomal alterations, including chromothripsis (Fig. 4).72 Chromothripsis is a mutational process, in which up to thousands of massive chromosomal rearrangements occur in a single event and in genomic regions confined to one or a few chromosomes. This phenomenon is involved in cancer and congenital diseases. In fact, it has been indicated that in cancer, chromothripsis leads both, to the amplification of oncogenes, mainly through the formation of double minute chromosomes (dmin), and to the loss of TSGs.78–80

Micronuclei formation and chromothripsis induced by CRISPR/Cas9.
Fig. 4  Micronuclei formation and chromothripsis induced by CRISPR/Cas9.

The main limitation of CRISPR/Cas9 as a therapeutic alternative is the induction of chromosomal alterations. (a) DNA double-strand breaks (DSBs) may not be corrected by (b) DNA DSB repair mechanisms, which may favor the formation of (c) acentric (ace) chromosome fragments. Acentric chromosome fragments that persist until mitosis, fail to align in metaphase (d), or even anchor to the mitotic spindle in (e) anaphase. The above leads to the fact that, while the chromosomes go to the opposite poles of the mitotic spindle, the acentric chromosome fragments remain lagging. In telophase (f), the formation of a new nuclear membrane leads to the emergence of daughter nuclei, each with a copy of the complete genetic material, except for the lagging chromosome fragments, which are (g) surrounded by their own membrane, leading to the formation of micronuclei (MN). MN formation, can favors the acquisition of additional chromosomal alterations (h), including, chromosomal translocations (t), chromosomal insertions (ins), chromosomal deletions (del), dicentric chromosomes (dic), and chromothripsis (i). Chromothripsis is an event of genetic chaos in which one or more chromosomes are fragmented into many segments and then randomly rearranged, losing some regions and promoting the formation of double chromosomes (dmin) and ring chromosomes (r). ace, acentric chromosome fragments; Cas9, CRISPR associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; del, chromosomal deletions; dic, dicentric chromosomes; dmin, double chromosomes; DSBs, DNA double-strand breaks; ins, chromosomal insertions; MN, micronuclei; r, ring chromosomes; t, chromosomal translocations.

For instance, it has been suggested that the mechanisms by which chromothripsis can emerge include, the fragmentation and subsequent reassembly of a single chromatid into small nuclear structures surrounded by membranes called MN and,81,82 the breakage of dicentric chromosomes during telomere crises (Fig. 4).83,84 The above constitutes a limitation for the application of therapeutic genome editing strategies that require the induction of DSBs, since these DSBs, have been associated with the induction of structural chromosomal alterations including chromothripsis. Furthermore, chromothripsis can lead to the acquisition of multiple additional alterations that can promote tumorigenesis in many tissues, including those with cells with intact p53.78,79,85 However, it is important to highlight that to date, the rates of MN formation, nucleoplasmic bridges and chromosomal alterations, including chromothripsis, associated with genome editing therapies in humans are unknown.

Regarding p53, it was recently indicated that in addition to inducing chromosomal alterations, CRISPR-Cas9 technology can also, cause changes in the p53 signaling pathway in many cell lines, leading to an increase in mutations that inactivate p53 and therefore promoting the development of cancer.86,87 Further, recent studies indicated that numerical (chromosomal losses) and structural (chromosomal translocations) chromosomal alterations caused by gene editing, apparently do not disappear over time and, on the contrary, increase in frequency, thus showing a clear random clonal expansion.73 The above could be explained because during the active period of gene editing, changes occur in chromosome segregation and nuclear division, evidencing a probable mechanism for the induction of chromosomal alterations following gene editing.88 Overall, the research carried out to date shows a limitation for the application of DSB-inducing CRISPR therapy in the clinic, so it is important to consider the possibility that extensive chromosomal rearrangements may be induced as a result of the application of this gene editing technology.

Our study presents limitations related to the reduced literature available about the use of CRISPR-Cas9 in in vivo models and in BC patients. This is possibly due to the recent application of this gene editing system in cancer research. Despite the above, CRISPR-Cas9 constitutes a potential tool in the treatment of cancer.

Conclusions

Although, CRISPR-Cas9 genome editing technology has therapeutic potential both, to direct personalized therapy in BC and to overcome drug resistance, it is necessary to consider and monitor the possibility of induction of genomic rearrangements, chromosomal variations and structural chromosomal alterations. This is due to the fact that such genomic rearrangements could have an implication in the prognosis of the disease and in the response to therapy. The studies carried out to date provide important data to take into account when determining the risks associated with the use of CRISPR-Cas9 technology in the clinic. In fact, more studies are necessary to delve into both, the mechanisms related to the safety of CRISPR and the potential risks involved with CRISPR-Cas9 system.

Abbreviations

ABC: 

ATP binding cassette

ACKR3

Atypical Chemokine Receptor 3

AKT: 

protein kinase B

AKT: 

protein kinase B

ATP8B3

ATPase Phospholipid Transporting 8B3

BC: 

breast cancer

BCL2: 

B-cell lymphoma 2

BRCA1

Breast cancer type 1

Cas9: 

CRISPR associated protein-9

C-MYC: 

MYC Proto-Oncogene

CRISPR: 

clustered regularly interspaced short palindromic repeat

CXCR4

C-X-C Motif Chemokine Receptor 4

dCas9: 

deactivated Cas9

DSBs: 

DNA double-strand breaks

DSTYK

Dual Serine/Threonine And Tyrosine Protein Kinase

EGFR: 

Epidermal Growth Factor Receptor

ER: 

estrogen receptor

ERK: 

extracellular signal-regulated kinase

FOXO1: 

Fork head Box O1

FOXR2

Fork head Box R2

FRG2

FSHD Region Gene 2

GLUT4: 

Glucose transporter type 4

GSK3A: 

Glycogen Synthase Kinase 3 alpha

GSK3B: 

Glycogen Synthase Kinase 3 beta

HER2: 

human epidermal growth factor receptor 2

HER3: 

human epidermal growth factor receptor 3

HER4: 

human epidermal growth factor receptor 4

HR: 

homologous recombination

MAP3K11

Mitogen-Activated Protein Kinase Kinase Kinase 11

MAPK: 

mitogen-activated protein kinase

MDR: 

multidrug resistance

MDR1

multidrug resistance protein 1

MEK: 

mitogen-activated protein kinase kinase

MN: 

micronuclei

mTOR: 

Mechanistic Target Of Rapamycin Kinase

mTOR2: 

Mechanistic Target Of Rapamycin Kinase

MYC

MYC Proto-Oncogene

NHEJ: 

non-homologous DNA end joining

OPN

osteopontin gene

PARP1

Poly [ADP-ribose] polymerase 1

PI3K: 

phosphatidylinositol 3-kinase

PIP2: 

Phosphatidylinositol 4,5-bisphosphate

PIP3: 

Phosphatidylinositol (3,4,5)-trisphosphate

PR: 

progesterone receptor

PTEN: 

Phosphatase and Tensin homolog

PTEN: 

Phosphatase and Tensin homolog

RAF: 

Raf Proto-Oncogene, Serine/Threonine Kinase

RAS: 

Rat sarcoma virus protein

TNBC: 

triple negative breast cancer

TNBC: 

triple negative breast cancer

TP53

Tumor Protein P53

TSC1/2: 

TSC Complex Subunit ½

TSGs: 

tumor suppressor genes

VEGF: 

Vascular Endothelial Growth Factor

VPR: 

transactivator VP64-p65-Rta

Declarations

Acknowledgement

None.

Funding

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sector.

Conflict of interest

The authors have no conflict of interests related to this publication.

Authors’ contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by NR, VC, GC, MF-C and MR-L. The first draft of the manuscript was written by MR-L and NR. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71(3):209-249 View Article PubMed/NCBI
  2. Mohammadzadeh I, Qujeq D, Yousefi T, Ferns GA, Maniati M, Vaghari-Tabari M. CRISPR/Cas9 gene editing: A new therapeutic approach in the treatment of infection and autoimmunity. IUBMB Life 2020;72(8):1603-1621 View Article PubMed/NCBI
  3. Hille F, Richter H, Wong SP, Bratovič M, Ressel S, Charpentier E. The Biology of CRISPR-Cas: Backward and Forward. Cell 2018;172(6):1239-1259 View Article PubMed/NCBI
  4. Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 2020;18(2):67-83 View Article PubMed/NCBI
  5. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8(11):2281-2308 View Article PubMed/NCBI
  6. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339(6121):823-826 View Article PubMed/NCBI
  7. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819-823 View Article PubMed/NCBI
  8. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 2018;36(8):765-771 View Article PubMed/NCBI
  9. Cullot G, Boutin J, Toutain J, Prat F, Pennamen P, Rooryck C, et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat Commun 2019;10(1):1136 View Article PubMed/NCBI
  10. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov 2022;12(1):31-46 View Article PubMed/NCBI
  11. Iqbal N, Iqbal N. Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications. Mol Biol Int 2014;2014:852748 View Article PubMed/NCBI
  12. Schuijers J, Manteiga JC, Weintraub AS, Day DS, Zamudio AV, Hnisz D, et al. Transcriptional Dysregulation of MYC Reveals Common Enhancer-Docking Mechanism. Cell Rep 2018;23(2):349-360 View Article PubMed/NCBI
  13. Kazimierska M, Podralska M, Żurawek M, Woźniak T, Kasprzyk ME, Sura W, et al. CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells. Mol Oncol 2023;17(11):2295-2313 View Article PubMed/NCBI
  14. Yang M, Zeng C, Li P, Qian L, Ding B, Huang L, et al. Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells. Onco Targets Ther 2019;12:3849-3858 View Article PubMed/NCBI
  15. Rattanasinchai C, Gallo KA. MLK3 Signaling in Cancer Invasion. Cancers (Basel) 2016;8(5):51 View Article PubMed/NCBI
  16. Behbahani RG, Danyaei A, Teimoori A, Neisi N, Tahmasbi MJ. Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique. Cancer/Radiothérapie 2021;25(3):222-228 View Article PubMed/NCBI
  17. Choudhury SR, Cui Y, Lubecka K, Stefanska B, Irudayaraj J. CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 2016;7(29):46545-46556 View Article PubMed/NCBI
  18. Abuhamad AY, Mohamad Zamberi NN, Sheen L, Naes SM, Mohd Yusuf SNH, Ahmad Tajudin A, et al. Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations. Cells 2022;11(10):1612 View Article PubMed/NCBI
  19. Moses C, Nugent F, Waryah CB, Garcia-Bloj B, Harvey AR, Blancafort P. Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System. Mol Ther Nucleic Acids 2019;14:287-300 View Article PubMed/NCBI
  20. Ha JS, Byun J, Ahn DR. Overcoming doxorubicin resistance of cancer cells by Cas9-mediated gene disruption. Sci Rep 2016;6:22847 View Article PubMed/NCBI
  21. Mintz RL, Lao YH, Chi CW, He S, Li M, Quek CH, et al. CRISPR/Cas9-mediated mutagenesis to validate the synergy between PARP1 inhibition and chemotherapy in BRCA1-mutated breast cancer cells. Bioeng Transl Med 2020;5(1):e10152 View Article PubMed/NCBI
  22. Ogbu SC, Rojas S, Weaver J, Musich PR, Zhang J, Yao ZQ, et al. DSTYK Enhances Chemoresistance in Triple-Negative Breast Cancer Cells. Cells 2021;11(1):97 View Article PubMed/NCBI
  23. Yan G, Dai M, Poulet S, Wang N, Boudreault J, Daliah G, et al. Combined in vitro/in vivo genome-wide CRISPR screens in triple negative breast cancer identify cancer stemness regulators in paclitaxel resistance. Oncogenesis 2023;12(1):51 View Article PubMed/NCBI
  24. Torry DS, Cooper GM. Proto-oncogenes in development and cancer. Am J Reprod Immunol 1991;25(3):129-132 View Article PubMed/NCBI
  25. Osborne C, Wilson P, Tripathy D. Oncogenes and tumor suppressor genes in breast cancer: Potential diagnostic and therapeutic applications. Oncologist 2004;9(4):361-377 View Article PubMed/NCBI
  26. Kurebayashi J, Miyoshi Y, Ishikawa T, Saji S, Sugie T, Suzuki T, et al. Clinicopathological characteristics of breast cancer and trends in the management of breast cancer patients in Japan: Based on the Breast Cancer Registry of the Japanese Breast Cancer Society between 2004 and 2011. Breast Cancer 2015;22(3):235-244 View Article PubMed/NCBI
  27. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244(4905):707-712 View Article PubMed/NCBI
  28. Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L. Treatment of HER2-positive breast cancer: current status and future perspectives. Nat Rev Clin Oncol 2011;9(1):16-32 View Article PubMed/NCBI
  29. Baselga J, Bradbury I, Eidtmann H, Di Cosimo S, de Azambuja E, Aura C, et al. Lapatinib with trastuzumab for HER2-positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial. Lancet 2012;379(9816):633-640 View Article PubMed/NCBI
  30. Wang H, Sun W. CRISPR-mediated targeting of HER2 inhibits cell proliferation through a dominant negative mutation. Cancer Lett 2017;385:137-143 View Article PubMed/NCBI
  31. Wu W, Qian L, Chen X, Ding B. Prognostic significance of CXCL12, CXCR4, and CXCR7 in patients with breast cancer. Int J Clin Exp Pathol 2015;8(10):13217-24 PubMed/NCBI
  32. Liu F, Lang R, Wei J, Fan Y, Cui L, Gu F, et al. Increased expression of SDF-1/CXCR4 is associated with lymph node metastasis of invasive micropapillary carcinoma of the breast. Histopathology 2009;54(6):741-750 View Article PubMed/NCBI
  33. Verras GI, Tchabashvili L, Mulita F, Grypari IM, Sourouni S, Panagodimou E, et al. Micropapillary Breast Carcinoma: From Molecular Pathogenesis to Prognosis. Breast Cancer 2022;14:41-61 View Article PubMed/NCBI
  34. Nangong J, Cheng Z, Yu L, Zheng X, Ding G. Invasive micropapillary breast carcinoma: A retrospective study on the clinical imaging features and pathologic findings. Front Surg 2022;9:1011773 View Article PubMed/NCBI
  35. Cronan MR, Nakamura K, Johnson NL, Granger DA, Cuevas BD, Wang JG, et al. Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis. Oncogene 2012;31(34):3889-3900 View Article PubMed/NCBI
  36. Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS. Flexible Structures of SIBLING Proteins, Bone Sialoprotein, and Osteopontin. Biochem Biophys Res Commun 2001;280(2):460-465 View Article PubMed/NCBI
  37. Zhao JJ, Yang L, Zhao FQ, Shi SM, Tan P. Osteopontin Levels are Elevated in Patients with Asthma. J Int Med Res 2011;39(4):1402-1407 View Article PubMed/NCBI
  38. Dombai B, Ivancsó I, Bikov A, Oroszi D, Bohács A, Müller V, et al. Circulating Clusterin and Osteopontin Levels in Asthma and Asthmatic Pregnancy. Can Respir J 2017;2017:1602039 View Article PubMed/NCBI
  39. Bellahcène A, Castronovo V. Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer. Am J Pathol 1995;146(1):95-100 PubMed/NCBI
  40. Bramwell VH, Doig GS, Tuck AB, Wilson SM, Tonkin KS, Tomiak A, et al. Serial plasma osteopontin levels have prognostic value in metastatic breast cancer. Clin Cancer Res 2006;12:3337-3343 View Article PubMed/NCBI
  41. Saleh S, Thompson DE, McConkey J, Murray P, Moorehead RA. Osteopontin regulates proliferation, apoptosis, and migration of murine claudin-low mammary tumor cells. BMC Cancer 2016;16:359 View Article PubMed/NCBI
  42. Wang H, Guo M, Wei H, Chen Y. Targeting p53 pathways: mechanisms, structures, and advances in therapy. Signal Transduct Target Ther 2023;8(1):92 View Article PubMed/NCBI
  43. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012;490(7418):61-70 View Article PubMed/NCBI
  44. Pont M, Marqués M, Sorolla MA, Parisi E, Urdanibia I, Morales S, et al. Applications of CRISPR Technology to Breast Cancer and Triple Negative Breast Cancer Research. Cancers (Basel) 2023;15(17):4364 View Article PubMed/NCBI
  45. Børresen-Dale AL. TP53 and breast cancer. Hum Mutat 2003;21(3):292-300 View Article
  46. Bai H, Yu J, Jia S, Liu X, Liang X, Li H. Prognostic Value of the TP53 Mutation Location in Metastatic Breast Cancer as Detected by Next-Generation Sequencing. Cancer Manag Res 2021;13:3303-3316 View Article PubMed/NCBI
  47. Di Cristofano A, Pandolfi PP. The multiple roles of PTEN in tumor suppression. Cell 2000;100(4):387-90 View Article PubMed/NCBI
  48. Li S, Shen Y, Wang M, Yang J, Lv M, Li P, et al. Loss of PTEN expression in breast cancer: association with clinicopathological characteristics and prognosis. Oncotarget 2017;8(19):32043-32054 View Article PubMed/NCBI
  49. Shetty PJ, Pasupuleti N, Chava S, Nasaruddin K, Hasan Q. Altered transcription and expression of PTEN in breast tumors: Is it regulated by hypermethylation?. Breast Dis 2011;33(1):27-33 View Article PubMed/NCBI
  50. Saal LH, Johansson P, Holm K, Gruvberger-Saal SK, She QB, Maurer M, et al. Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity. Proc Natl Acad Sci USA 2007;104(18):7564-7569 View Article PubMed/NCBI
  51. Luo S, Chen J, Mo X. The association of PTEN hypermethylation and breast cancer: a meta-analysis. Onco Targets Ther 2016;9:5643-5650 View Article PubMed/NCBI
  52. Carracedo A, Alimonti A, Pandolfi PP. PTEN level in tumor suppression: how much is too little?. Cancer Res 2011;71(3):629-633 View Article PubMed/NCBI
  53. Alimonti A, Carracedo A, Clohessy JG, Trotman LC, Nardella C, Egia A, et al. Subtle variations in Pten dose determine cancer susceptibility. Nat Genet 2010;42(5):454-458 View Article PubMed/NCBI
  54. Khan S, Kumagai T, Vora J, Bose N, Sehgal I, Koeffler PH. PTEN promoter is methylated in a proportion of invasive breast cancers. Int J Cancer 2004;112(3):407-10 View Article PubMed/NCBI
  55. García JM, Silva J, Peña C, Garcia V, Rodríguez R, Cruz MA, et al. Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosomes Cancer 2004;41(2):117-124 View Article PubMed/NCBI
  56. Esteller M, Silva JM, Dominguez G, Bonilla F, Matias-Guiu X, Lerma E, et al. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 2000;92(7):564-569 View Article PubMed/NCBI
  57. Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001;61(8):3225-3229 PubMed/NCBI
  58. Marqués M, Sorolla MA, Urdanibia I, Parisi E, Hidalgo I, Morales S, et al. Are Transcription Factors Plausible Oncotargets for Triple Negative Breast Cancers?. Cancers (Basel) 2022;14(5):1101 View Article PubMed/NCBI
  59. Sporikova Z, Koudelakova V, Trojanec R, Hajduch M. Genetic Markers in Triple-Negative Breast Cancer. Clin Breast Cancer 2018;18(5):e841-e850 View Article PubMed/NCBI
  60. Alamolhodaei N, Behravan J, Mosaffa F, Karimi G. MiR 221/222 as New Players in Tamoxifen Resistance. Curr Pharm Des 2017;22(46):6946-6955 View Article PubMed/NCBI
  61. Marquette C, Nabell L. Chemotherapy-Resistant Metastatic Breast Cancer. Curr Treat Options Oncol 2012;13(2):263-275 View Article PubMed/NCBI
  62. Simon SM, Schindler M. Cell biological mechanisms of multidrug resistance in tumors. Proc Natl Acad Sci USA 1994;91(9):3497-3504 View Article PubMed/NCBI
  63. Padayachee J, Singh M. Therapeutic applications of CRISPR/Cas9 in breast cancer and delivery potential of gold nanomaterials. Nanobiomedicine (Rij) 2020;7:1849543520983196 View Article PubMed/NCBI
  64. Wu Q, Yang Z, Nie Y, Shi Y, Fan D. Multi-drug resistance in cancer chemotherapeutics: Mechanisms and lab approaches. Cancer Lett 2014;347(2):159-166 View Article PubMed/NCBI
  65. Leonessa F, Clarke R. ATP binding cassette transporters and drug resistance in breast cancer. Endocr Relat Cancer 2003;10(1):43-73 View Article PubMed/NCBI
  66. Mao Q, Unadkat JD. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport—an update. AAPS J 2015;17(1):65-82 View Article PubMed/NCBI
  67. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 1993;62(1):385-427 View Article PubMed/NCBI
  68. Dean M, Hamon Y, Chimini G. The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 2001;42(7):1007-1017 View Article PubMed/NCBI
  69. Nakanishi T, Ross DD. Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression. Chin J Cancer 2012;31(2):73-99 View Article PubMed/NCBI
  70. Austin Doyle L, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003;22(47):7340-7358 View Article PubMed/NCBI
  71. Dong X, Sun R, Wang J, Yu S, Cui J, Guo Z, et al. Glutathione S-transferases P1-mediated interleukin-6 in tumor-associated macrophages augments drug-resistance in MCF-7 breast cancer. Biochem Pharmacol 2020;182:114289 View Article PubMed/NCBI
  72. Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Sun L, Yao Y, et al. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nat Genet 2021;53(6):895-905 View Article PubMed/NCBI
  73. Wu J, Zou Z, Liu Y, Liu X, Zhangding Z, Xu M, et al. CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo. Nucleic Acids Res 2022;50(19):11128-11137 View Article PubMed/NCBI
  74. Höijer I, Emmanouilidou A, Östlund R, van Schendel R, Bozorgpana S, Tijsterman M, et al. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat Commun 2022;13(1):627 View Article PubMed/NCBI
  75. Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genome instability. Nat Rev Mol Cell Biol 2017;18(3):175-186 View Article PubMed/NCBI
  76. Umbreit NT, Zhang CZ, Lynch LD, Blaine LJ, Cheng AM, Tourdot R, et al. Mechanisms generating cancer genome complexity from a single cell division error. Science 2020;368(6488):eaba0712 View Article PubMed/NCBI
  77. Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, et al. CRISPR-engineered T cells in patients with refractory cancer. Science 2020;367(6481):eaba7365 View Article PubMed/NCBI
  78. Cortés-Ciriano I, Lee JJ, Xi R, Jain D, Jung YL, Yang L, et al. PCAWG Structural Variation Working Group, PCAWG Consortium. Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing. Nat Genet 2020;52(3):331-341 View Article PubMed/NCBI
  79. Stephens PJ, Greenman CD, Fu B, Yang F, Bignell GR, Mudie LJ, et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 2011;144(1):27-40 View Article PubMed/NCBI
  80. Ly P, Brunner SF, Shoshani O, Kim DH, Lan W, Pyntikova T, et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat Genet 2019;51(4):705-715 View Article PubMed/NCBI
  81. Zhang CZ, Spektor A, Cornils H, Francis JM, Jackson EK, Liu S, et al. Chromothripsis from DNA damage in micronuclei. Nature 2015;522(7555):179-184 View Article PubMed/NCBI
  82. Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y, et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature 2012;482(7383):53-58 View Article PubMed/NCBI
  83. Maciejowski J, Li Y, Bosco N, Campbell PJ, de Lange T. Chromothripsis and Kataegis Induced by Telomere Crisis. Cell 2015;163(7):1641-1654 View Article PubMed/NCBI
  84. Mardin BR, Drainas AP, Waszak SM, Weischenfeldt J, Isokane M, Stütz AM, et al. A cell-based model system links chromothripsis with hyperploidy. Mol Syst Biol 2015;11(9):828 View Article PubMed/NCBI
  85. Leibowitz ML, Zhang CZ, Pellman D. Chromothripsis: A New Mechanism for Rapid Karyotype Evolution. Annu Rev Genet 2015;49(1):183-211 View Article PubMed/NCBI
  86. Enache OM, Rendo V, Abdusamad M, Lam D, Davison D, Pal S, et al. Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat Genet 2020;52(7):662-668 View Article PubMed/NCBI
  87. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 2018;24(7):927-930 View Article PubMed/NCBI
  88. Yang L, Li H, Han Y, Song Y, Wei M, Fang M, et al. CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation. Genes (Basel) 2023;14(4):806 View Article PubMed/NCBI
  • Gene Expression
  • pISSN 1052-2166
  • eISSN 1555-3884
Back to Top

Exploring the Advantages and Limitations of CRISPR-Cas in Breast Cancer

Nelson Rangel, Valentina Camargo, Giovanny Castellanos, Maribel Forero-Castro, Milena Rondón-Lagos
  • Reset Zoom
  • Download TIFF