Clustered regularly interspaced short palindromic repeats (CRISPR) is a third-generation genome editing technique that has revolutionized the world with its high-throughput results. It has been used to treat various biological diseases and infections. Various bacteria and other prokaryotes (such as archaea) also have CRISPR/Cas9 systems to defend against phages. It has been reported that CRISPR/Cas9-based strategies can inhibit the growth and progression of triple negative breast cancer (TNBC) by targeting potentially altered resistance genes, transcription, and epigenetic regulation. These therapeutic interventions may help address challenging problems such as drug resistance observed even in TNBC. Currently, various methods are used to deliver CRISPR/Cas9 to target cells, such as physical (microinjection, electroporation and hydrodynamic modes), viral (adeno-associated virus and lentivirus) and non-viral (liposomes and lipid nanoparticles). )-particles). Although various models have been developed to study the molecular causes of TNBC, the lack of sensitive and targeted methods for delivering genome editing tools in vivo limits their clinical application. In summary, this review comprehensively examines the progress, challenges, limitations, and prospects of CRISPR/Cas9 treatment for TNBC based on existing evidence. We also highlight how the combination of artificial intelligence and machine learning can improve CRISPR/Cas9 strategies in the treatment of TNBC.
Cancer is characterized by uncontrolled cell division, disruption of cell cycle checkpoints, and mutations in tumor suppressor genes (TSGs) (Matthews et al., 2022). Among the different types of cancer, breast cancer is the most common type of cancer in women and has a high mortality rate worldwide (Waks and Winer, 2019). Breast cancer is a heterogeneous disease with diverse characteristics, such as histological and biological features, clinical presentation and behavior, and response to treatment (Weigelt et al., 2010). Breast cancer classification provides precise insights for breast cancer diagnosis and tumor prognosis. The use of common biomarkers and clinicopathological features has been the main criterion for the classification of breast cancer (Tsang and Tse, 2019). The prognosis and response to treatment of breast cancer is influenced by numerous factors, including the presence of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), and histological grade, tumor type, and grade. size and lymph node metastasis (Al-Tubaiti, 2020). Five intrinsic molecular subtypes of breast cancer have been identified, including luminal A, luminal B, HER2-enriched, basal-like, and claudin-low (Prat et al., 2015). TNBC is a molecular subtype of cancer that does not express all ER, PR, and HER2 ( Yin et al., 2020 ). These pathological features are associated with TNBC, confirming its rapid progression and more aggressive nature than any other type of breast cancer (Feng et al., 2018). Additionally, Peru et al (2000) used microarray technology to reclassify breast cancer and identify five intrinsic subtypes of breast cancer (Cadenas, 2012). Basal-like breast cancer is a subtype of breast cancer that has a triple-negative phenotype and is associated with rapid progression. It should be noted that all basal-like breast cancers are commonly misdiagnosed as TNBC, but only 77% are TNBC; In contrast, 71–91% of TNBC are basal-like, indicating that these two types of breast cancer overlap and represent different classifications (Wang D.-Y. et al., 2019). This creates a need to characterize the heterogeneity of TNBC to elucidate prognosis and identify potential responses to current and future treatments. Additionally, TNBC accounts for 15–20% of all breast cancer cases and is more common in women under 50 years of age. BRCA1 or BRCA2 mutations have been reported in approximately 20% of TNBC cases (Xie et al., 2017; Tzikas et al., 2017). , 2020). Studies have also shown that TNBC has a distinct immune microenvironment that includes high levels of vascular endothelial growth factors, tumor-associated macrophages (TAMs), tumor-infiltrating lymphocytes (TILs), and other molecules involved in tumor growth and migration. Therefore, understanding the microenvironment of TNBC is critical for its prognosis and treatment (Fan and He, 2022).
To assess the prognosis of TNBC and ensure effective treatment, accurate diagnosis, mainly based on immunohistochemistry (IHC) to detect ER, PR and HER2, and mammography to detect breast neoplasms, is important. However, mammography cannot adequately depict intratumoral features such as necrosis and fibrosis (Deepak Singh et al., 2021). Since poor prognosis and diagnosis prevent doctors from prescribing the right medications (Chaudhary, 2020), various strategies are used to improve care for patients with TNBC. Currently, two drugs, doxorubicin and cyclophosphamide, are used in patients with TNBC and have shown good results. In addition, other platinum drugs are used, such as carboplatin and cisplatin (Sikov et al., 2015). In addition, PARP inhibitors such as Olaparib, Velaparib, and PF-01367338 have been used as potential chemotherapy drugs for the treatment of TNBC (Ishino et al., 2018). Since Wnt/b-Catenin, NOTCH, and Hedgehog signaling pathways have been reported to be involved in the occurrence and progression of TNBC, targeting drugs to these pathways may be an important strategy (Aysola et al., 2013). Although surgery, radiation therapy and chemotherapy remain the mainstay of treatment for TNBC today, significant progress has been made in developing new treatments including targeted therapy, immunotherapy, various CRISPR-related gene editing tools such as Cas9n, dCas9. , CRISPR/Cas12, prime editing, and CRISPR/Cas9-targeted gene therapy. Here we will focus on various CRISPR-related gene editing tools used in the treatment of TNBC. Among them, CRISPR/Cas9 has received widespread attention.
Cas9n, also known as Cas9 nickase, is a genetically engineered variant of the Cas9 protein derived from the CRISPR/Cas9 genome editing system (Gupta et al., 2019). In its original form, the Cas9 protein contains two nuclease domains: RuvC and HNH, the main function of which is the cleavage of both DNA strands. However, in Cas9n, one of the nuclease domains, HNH, is genetically mutated and becomes inactive. Therefore, the HNH domain of Cas9n remains nonfunctional. Only the RuvC domain remains active, allowing Cas9n to cut or create breaks on single DNA strands (Trevino and Zhang, 2014). Compared with Cas9, Cas9n can effectively reduce off-target effects and accurately improve cell repair mechanisms. Cas9n can be used to solve various problems, such as creating breaks at specific locations in double-stranded DNA. For this purpose, two Cas9n molecules were combined and used together (Yee, 2016). Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase that serves as a key regulator during TNBC metastasis (Cronan et al., 2012).
MLK3 can activate multiple signaling pathways leading to TNBC metastasis. For example, the c-Jun N-terminal kinase (JNK) pathway regulates cell motility and extracellular matrix degradation, and MLK3 activates the JNK pathway, thereby enhancing cell motility and conferring invasive properties (Rattanasinchai and Gallo, 2016). MLK3 also regulates EMT. To do this, it activates downstream transcription factors such as Snail, Slug and Twist. These factors inhibit the expression of epithelial markers and promote the expression of mesenchymal markers, leading to the acquisition of a metastatic phenotype (Casalino et al., 2023). MLK3 has been observed to play a role in ECM remodeling and activation of proteases such as matrix metalloproteinases (MMPs) that degrade the ECM. This facilitates tumor cell invasion and spread to distant sites (Katari et al., 2019). Thus, previous studies have shown that MLK3 plays a critical role in TNBC. Rattanasinchai and Gallo used TNBC models to study the role of MLK3 and found that it promotes cancer development through specific signaling pathways. They used CRISPR/Cas9n to edit MLK3 and observed a significant reduction in TNBC metastasis (Rattanasinchai and Gallo, 2016).
dCas9, often called inactive Cas9, is a modified derivative form of the Cas9 protein. Unlike active Cas9, dCas9 lacks endonuclease activity, making it unable to induce DNA double-strand breaks. Therefore, dCas9 can be used to precisely target specific regions of the genome without any changes or modifications to the DNA sequence ( Wang et al., 2016 ). In dCas9, two endonuclease proteins, RuvC and HNH, are inactivated by suppressing important amino acid residues. (Richter et al., 2016). Despite its lack of DNA cleavage activity, dCas9 plays a number of important roles in genetic research and biotechnology. For example, it allows visualization of specific regions of the genome, facilitates transcriptional regulation, and promotes epigenetic modifications (Brocken et al., 2018).
The transcription factor ZEB1 (zinc finger E-box binding homeobox 1) plays a specific and critical role in mediating epithelial-mesenchymal transition (EMT), a cellular process that occurs during embryonic development, tissue repair, and cancer progression. It involves the transformation of epithelial cells into mesenchymal cells, resulting in changes in cell morphology, motility, invasiveness, etc. (Wu et al., 2020). ZEB1 inhibits multiple epithelial markers, such as E-cadherin and Occludin, which are responsible for maintaining cell-cell adhesion and polarity of epithelial cells in TNBC (Moreno-Bueno et al., 2008). In addition, ZEB1 activates certain markers such as N-cadherin, vimentin and fibronectin (Konradi et al., 2014) and also regulates genes involved in cytoskeletal remodeling such as Rho GTPases and matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), in addition, it also affects various signaling pathways such as transforming growth factor-β (TGF-β), Wnt signaling, etc., thereby promoting tumor invasion and metastasis in TNBC (Chen et al. , 2016). ). Numerous studies and scientific evidence indicate that ZEB1 has great potential as a valuable agent for the detection and therapeutic intervention of TNBC. In a recent study, Waryah et al. used a TNBC model and achieved complete suppression of ZEB1 by dCas9. Thus, they observed very high specificity and almost complete inhibition of ZEB1 in in vivo conditions (Waryah et al., 2023).
CRISPR/Cas12 is a gene editing tool called CRISPR/Cpf1. It is derived from the CRISPR/Cas system, where the term Cas12 refers to CRISPR-associated protein 12 (Bharathkumar et al., 2022), which is completely similar to Cas9. The only difference is that it contains the Cas12 protein. Compared with Cas9, Cas12 has some unique functions such as generating sticky ends during the gene editing process, whereas Cas9 generates blunt ends (Wang et al., 2021). This property of Cas12 contributes to its specific DNA manipulation technology. As a protein capable of precisely recognizing and cutting target DNA, it has extraordinary versatility, making it an effective tool for a variety of applications, including gene editing (Pickar-Oliver and Gersbach, 2019).
Similar to other CRISPR variants, CRISPR/Cas12 is also capable of knocking out or activating genes in TNBC, targeting genes that play an important role in its pathogenesis or response to treatment (Yang and Zhang, 2023). To accomplish this process, a guide RNA (gRNA) was developed that directs Cas12 to the target gene. Once Cas12 binds to its target, it introduces double-strand breaks in DNA and activates DNA repair mechanisms. During the repair process, incorrect nucleotides may be incorporated, leading to gene mutations and loss of function (Zhang et al., 2021a). In addition, an improved version of the Cas12 enzyme (called dCas12) was also used to activate genes. By combining it with a transcription activator, it is possible to induce certain genes, thereby activating them. This technology has great potential in promoting the activation of tumor suppressor genes (Sultan et al., 2022).
Prime editing is a newly developed and extremely superior genome editing technology. It is capable of modifying the DNA of an organism very precisely (Chen and Liu, 2023). Prime editing is achieved through the integration of two main components: a modified CRISPR/Cas9 enzyme and reverse transcriptase. The role of the CRISPR/Cas9 enzyme is to selectively target precise sites in the genome, while reverse transcriptase helps to thoroughly modify the DNA at the target location (Hassan et al., 2021). In the primer editing mechanism, the first step involves the creation of a primer editing guide RNA (pegRNA) (Standage-Beier et al., 2021). It contains the target sequence and RNA template corresponding to the desired editing site in the target DNA. The pegRNA is then introduced into target cells along with a primer editing nuclease (PE2). PE2 is a fusion protein consisting of a Cas9 enzyme, a reverse transcriptase, and a primer editing adapter (Martín-Alonso et al., 2021). Inside the cell, pegRNA and PE2 complexes search for the specific DNA they want to modify. The Cas9 enzyme cuts DNA and creates a template consisting of single strands (Choi et al., 2022). Reverse transcriptase uses this template to prepare the DNA for editing. Along with copying DNA, instructions for editing the RNA template are also included. Finally, the newly created DNA strand is used as a template to repair the break in the DNA, resulting in an altered DNA sequence containing the desired modification (Ochoa-Sanchez et al., 2021). Primer editing mechanisms can lead to widespread mutations in genes. It can target point mutations, insertions, deletions, and even gene replacements ( Anzalone et al., 2019 ; Chen and Liu, 2023 ). Prime editing offers several advantages over previous genome editing technologies. These include increased accuracy, reduced off-target effects, and the ability to edit DNA without relying on DNA double-strand breaks ( Anzalone et al., 2020 ).
CRISPR/Cas9 technology was originally developed to protect bacteria from plasmid transfer and phage infection and was later repurposed as an effective RNA-based DNA targeting tool for genome editing (Jiang and Doudna, 2017). In addition, the CRISPR/Cas9 system has been reported to be present in 50% and 87% of bacterial and archaeal genomes, respectively (Ishino et al., 2018). CRISPR/Cas9 is a potential tool for deletion, insertion and correction of any abnormal genetic sequence using in vivo and in vitro modes ( Sabit et al., 2021 ). Moreover, CRISPR/Cas9 has been shown to be part of the adaptive immune system due to its specificity for target genes of interest (Chen and Zhang, 2018).
CRISPR/Cas9 consists of Cas9 and a single guide RNA (sgRNA). Cas9 is an endonuclease composed of multiple protein components. In addition, Cas9 has unique structural and conformational properties (Pacesa et al., 2022) as it consists of two lobes: recognition (REC) and nuclease (NUC). The REC lobe is further divided into three regions: REC1, REC2, and bridge helices (Cromwell et al., 2018). The NUC consists of three lobes, namely RuvC (RuvC I, RuvC II, RuvC III), HNH and the protospacer adjacent motif (PAM) interaction domain (Fig. 1) (Song et al., 2016). sgRNA consists of two components, including CRISPR RNA (crRNA) and transcoding small RNA (tracer RNA) (Figure 1). The sgRNA connects the Cas9 protein to connexins to form an active complex, also known as the effector complex (Richter et al., 2012). crRNA is an 18–20 nucleotide base pair that plays a critical role in recognizing target DNA sequences. In addition, crRNA pairs with the target DNA, and tracrRNA acts as a scaffold for the Cas9 nuclease to bind the target DNA (Manghwar et al., 2019). On the other hand, the PAM sequence has 3 nucleotides, which confirms, specifies and mediates the binding of the effector complex to DNA. The Cas9 protein complex subunits RuvC and HNH have catalytic activity ( Richter et al., 2012 ; Asmamaw and Zawdie, 2021 ). The HNH nuclease domain of the Cas9 subunit cleaves the DNA strand associated with the crRNA. The RuvC nuclease domain cleaves other DNA strands and generates double-strand breaks (DSBs), after which two different DNA break repair mechanisms are activated (Jiang and Doudna, 2017) (Figure 1).
Figure 1. Overview of CRISPR/Cas9 (A). Components of the CRISPR/Cas9 system: (i). Cas9 endonuclease is responsible for cutting the target DNA sequence, (ii) a single guide (sg) RNA resulting from the fusion of crRNA and tra-crRNA chimeras. (two). Cas9 includes several components such as Rec I, Rec II, NUC lobe (HNH and Ruv C are subcomponents) and PAM interaction domain (C) with corresponding functions. The CRISPR/Cas9 protein complex cleaves DNA sequences into non-complementary and complementary forms (D). CRISPR/Cas9 edits the genome in three stages: recognition, cutting and repair. The designed sg-RNA guides Cas9 and recognizes the desired sequence through the complementary component crRNA. Cas9 recognizes the PAM sequence at 5′-NGG-3′ and melts the DNA, forming a DNA-RNA hybrid and activating cleavage. The HNH domain of Cas9 cleaves the complementary strand, and the RuvC domain cleaves the non-complementary strand. CRISPR/Cas9 repairs dsDNA by breaking it in two ways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ repairs double-stranded DNA in the absence of foreign homologous DNA through an enzymatic process, an error-prone mechanism that can insert or remove random DNA sequences. HDR is very specific and requires homologous DNA templates.
CRISPR/Cas9-mediated repair pathways include non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. NHEJ is an error-prone path because it involves insertion or deletion and does not require any pattern during the repair mechanism. It uses random nucleotides to generate standard proteins (Abbasi et al., 2021). This repair system consists of four complexes such as the KU complex, the cross-complementing protein complex type 4 (XRCC-4), the DNA end processing enzyme, and the protein kinase DNA-PKcs ( Abbasi et al., 2021 ). The KU complex protein has two subunits, Ku 70 and Ku 80, and plays an important role in the NHEJ mechanism, since the repair mechanism is initiated by the binding of two subunits (Ku 70 and Ku 80) to the blunt or nearly blunt ends of the target DNA (Abbasi et al., 2021 ), serving as a scaffold to recruit other NHEJ-related factors to the site of injury (Yang et al., 2020). XRCC-4 and DNA ligase are composed of 334 and 911 amino acids, respectively, and the XRCC4-DNA ligase IV complex stimulates ligation of DNA ends ( Chatterjee et al., 2015 ). DNA end processing enzyme, also known as polynucleotide kinase 3′-phosphate, is a DNA end processing enzyme. It can remove the 3′P group in DNA and phosphorylate the 5′OH group during DSB repair. It also involves repair of single-strand breaks (SSBs) using the SSB repair pathway (Chatterjee et al., 2015). Protein kinase DNA-PKcs is a DNA-dependent protein kinase that consists of a catalytic subunit of the PIKKs (phosphatidylinositol 3-kinase-related kinases) and ataxia telangiectasia mutated (ATM) family, as well as ATM and Rad3-related ATRs. strand breaks (DSBs) and single-strand breaks ( Yue et al., 2020 ; Peng et al., 2016 ).
HDR is a more accurate and suitable repair mechanism because the information is copied using the intact form of the homologous DNA duplex, although it requires the presence of sister chromatids. This occurs during the S/G2 phase of the mammalian cell cycle. HDR occurs mainly in yeast species, but NHEJ is critical in mammals ( Burma et al., 2006 ; Abbasi et al., 2021 ). The complete repair mechanism is shown in Figure 1.
Since TNBC is caused by genetic and epigenetic abnormalities, correction of malignant genomic/epigenomic abnormalities using CRISPR/Cas9 may be a reasonable therapeutic approach (Chen et al., 2019). In addition, some transcription factors involved in cell-specific transcriptional regulation may exhibit unique characteristics of cancer cells, suggesting that transcriptional regulation may be an excellent approach for cancer treatment (Drost et al., 2017). Exploiting these molecular features of tumors, such as genetic, epigenetic and transcriptional defects, for drug development can improve clinical outcomes and reduce screening costs. CRISPR is a potential gene editing tool that can not only detect and identify cancer-causing genomic targets, but can also be used to edit, suppress, and epigenetically modify oncogenes in human cells (Table 1) (Ahmed et al., 2021). Several CRISPR screens have been conducted to look for genes associated with tumor suppressors, oncogenes, and drug resistance. The use of CRISPR/Cas9 to edit various TNBC oncogenes is shown in Figure 2.
Figure 2. Gene editing of various TNBC oncogenes using CRISPR/Cas9 resulting in reduced tumor growth and metastasis. These oncogenes include CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 and CXCR7, Crypto1, ROR1 and ST8SIA, which are involved in the occurrence and metastasis of TNBC.
Cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT) are associated with ITGA9. Previous studies have shown that ITGA9 is a key player in the Notch pathway and plays an interesting role in rhabdomyosarcoma metastasis (Molist et al., 2020). In addition, ITGA9 was found to be closely associated with patient prognosis in several tumor types, including breast cancer (Wang Z. et al., 2019). In addition, bioinformatics analysis of ITGA9 showed that its expression in TNBC was significantly higher than in other breast cancer subtypes. Elevated ITGA9 levels are associated with tumor metastasis and relapse in TNBC patients. Knockout of ITGA9 by CRISPR/Cas9 resulted in cancer stem cell (CSC)-like properties, tumor angiogenesis, tumor growth, and reduced metastasis by promoting β-catenin degradation in TNBC (Wang Z. et al., 2019).
Crypto-1 is a member of the TGF-β family and is critical for early embryogenesis, stem cell maintenance, and cancer metastasis (Ishii et al., 2021). Also known as Tdgf-1, it is an oncogenic GPI-anchored signaling protein involved in the regulation of the formation of the primitive streak, mesoderm and endoderm, as well as the establishment of left/right asymmetry in the development of body organs during embryogenesis (Zhang et al., 2021b). ). Additionally, Cripto-1 has been shown to be involved in epithelial-mesenchymal transition (EMT) as a stem cell marker ( Zhang et al., 2021b ). EMT is critical not only for various processes such as embryonic development, fibrosis and wound healing, but also for cancer invasion and metastasis. Additionally, Cripto-1 has been shown to interact with four Notch receptors and enhance their post-translational maturation (Brandstadter and Maillard, 2019). The Notch signaling pathway is known to be involved in the maintenance of human breast cancer cells. Studies have shown that CRISPR/Cas9-mediated knockout of Crypto-1 suppresses cancer growth and metastasis. Therefore, Crypto-1 may become an important therapeutic target for TNBC (Castro et al., 2015).
XCL12 protein and its chemokine receptor CXC (CXCR4 and CXCR7) play various roles in cancer cell proliferation, growth, migration and invasion (Wu et al., 2015). These chemoreceptors have been associated with the development of TNBC through multiple signaling pathways in both in vivo and in vitro models (Wu et al., 2015). In addition, activation of CXCR4 and CXCR7 is associated with greater susceptibility to metastasis and poor prognosis in TNBC (Karn et al., 2022). Therefore, knockout of CXCR4 and CXCR7 may become effective drug target genes for the treatment of breast cancer, including TNBC. Study by Yang et al. Yang et al (2019) used CRISPR/Cas9 to co-knock out the CXCR4 and CXCR7 genes and found that TNBC proliferation, growth, migration and invasion were significantly inhibited (Yang et al., 2019).
Increased levels of miR-3662, a TNBC oncogene, were observed in breast cancer tissues (Yi et al., 2022). Knockdown of miR-3662 has been shown to inhibit breast cancer tumor growth and metastasis both in vivo and in vitro (Agarwal and Gupta, 2021). HBP-1 is a potent inhibitor of Wnt/-catenin signaling and is likely responsible for miR-3662-mediated growth of TNBC cells. Recently, Yi et al. found that the miR-3662-HBP1 axis regulates the Wnt/-catenin signaling pathway in TNBC cells (Yi et al., 2022). Due to its tumor-specific expression, miR-3662 may be a potential therapeutic target for TNBC. Thus, CRISPR/Cas9-mediated knockdown of miR-3662 may be an excellent approach for developing new drugs for the treatment of TNBC.
UBR5 is a 300 kDa nucleophosphoprotein that has been identified as a key regulator of tumorigenesis, metastasis, and immune response in various cancers ( Shearer et al., 2015 ; Fu et al., 2023 ). UBR5 has been reported to be highly upregulated in TNBC samples and stimulates ERα function by inducing proliferation through its ubiquitin ligase activity ( Bolt et al., 2015 ). Whole exome sequencing studies of primary TNBC samples also showed increased expression of UBR5, suggesting its role in the development of TNBC. Additionally, CRISPR/Cas9-driven deletion of UBR5 showed significant inhibition of TNBC metastasis and growth in experimental mouse models. Moreover, inclusion of UBR5 in a wild-type mouse model restored its full functionality, whereas this effect was not observed in inactivated mutant strains (Liao et al., 2017). Lack of UBR5 is associated with increased apoptosis, necrosis, and inhibition of tumor growth in TNBC due to poor angiogenesis. Due to the loss of UBR5, tumor spread to distant organs is reduced, and UBR5 induces abnormal EMT mainly by reducing E-cadherin expression (Zhang and Weinberg, 2018). Recently, UBR5 was shown to be a very important factor in IFN-γ-induced PDL1 transcription in TNBC due to its lack of E3 ubiquitination activity. RNA transcriptome analysis revealed that UBR5 may have systemic effects on genes associated with the IFN-γ pathway and promote PDL1 transactivation by increasing activation levels of protein kinase RNA (PKR) and its signal transducers and activators. transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1). However, CRISPR/Cas9-mediated combined ablation of UBR5 and PD-L1 expression has a synergistic therapeutic effect than either blockade alone, with profound effects on the tumor microenvironment (Wu et al., 2022). Thus, CRISPR/Cas9 may be an important tool to inhibit UBR5 function, thereby suppressing TNBC metastasis and tumor growth.
ROR1 is a type I transmembrane protein that is expressed during cancer and embryonic development and has been identified as an oncofetal protein (Nicholas Borcherding, 2014). The invasive behavior of various human cancers is associated with upregulation of ROR1. Promising results have been obtained from in vivo and in vitro studies involving therapeutic compounds targeting ROR1 (Chien et al., 2016). Elevated levels of ROR1 mRNA in breast tissue biopsies have also been associated with aggressive basal-like breast (BL) tumors and their migration to other parts of the body. Moreover, overexpression of ROR1 has been identified as a prognostic marker for the development of TNBC (Chien et al., 2016). However, silencing ROR1 using CRISPR/Cas9 to suppress TNBC growth and metastasis would be an effective strategy.
The sialylation process involves the addition of sialic acid to glycoconjugates, which is catalyzed by sialyltransferases (STs). ST8SIA1 belongs to the ST family and plays an important role in the pathogenesis of various diseases such as lymphocytic leukemia and colorectal cancer ( Chang et al., 2018 ). RNA sequence analysis shows that ST8SIA1 is highly expressed in breast tissue of TNBC patients and is positively correlated with mutations in the tumor suppressor gene p53, which may contribute to the pathogenesis of TNBC (Battula et al., 2017). In addition, ST8SIA1 is involved in the metastasis and recurrence of TNBC, suggesting that it plays an important role in the occurrence and development of TNBC. In an in vitro model of TNBC, knockdown of ST8SIA1 by CRIPSR/Cas9 was shown to block growth and metastasis (Battula et al., 2017). This suggests that CRISPR/Cas9 may be an important tool for inhibiting the function of the ST8SIA1 oncogene in the treatment of TNBC.
NAT1 is a metabolic enzyme that catalyzes the formation of phase II xenobiotic compounds and is expressed in almost all human tissues. NAT1 can use the cofactor folate to target acetyl-CoA (acetyl-CoA) even in the absence of the aromatic amine substrate ( Stepp et al., 2015 ; Laurieri et al., 2014 ). Studies have shown that NAT1 regulates matrix metalloproteinase 9 (MMP9) function in breast cancer cell models and protects against reactive oxygen species (ROS) during glucose starvation (Wang et al., 2018). Deletion of NAT1 has been shown to inhibit the pyruvate dehydrogenase complex, leading to mitochondrial dysfunction (Wang L. et al., 2019). Additionally, various other reports have shown that NAT1 inhibition using small molecules and siRNA silencing can reduce the invasiveness and proliferation of breast cancer cells (Stepp et al., 2018). Recently, CRISPR/Cas9 was used to suppress NAT1 in the breast cancer cell line MDA-MB-231, affecting cellular metabolism depending on its expression level. This study also showed that NAT-1 is critical for the progression and metastasis of TNBC (Carlisle et al., 2020).
Coordinated transcription of oncogenes is regulated by super enhancers, transcription factors and cofactors (Hnisz et al., 2015). In addition, a group of cyclin-dependent kinases (CDKs), such as CDK7, CDK8, CDK9, CDK12, and CDK13, are required for transcriptional regulation. Among them, CDK7 is involved in the phosphorylation of RNA polymerase II, which is very important for the initiation and expansion of oncogene transcription in the pathogenesis of TNBC. In a seminal study, deletion of CDK7 by CRISPR/Cas9 inhibited TNBC, indicating that the pathogenesis of TNBC is CDK7 dependent (Wang Y. et al., 2015).
Studies have shown that mutations in MYC and RBP (RNA binding protein) can lead to apoptosis, while single gene mutations in MYC or RBP do not affect the growth of cancer cells (Einstein et al., 2021). More than 1000 RBPs in the human genome have been screened using a CRISPR/Cas9-based library. Among them, 57 RBPs were found to be critical for the growth of cancer cells with highly elevated MYC levels (Wheeler et al., 2020). In addition, YTHDF2 is important for maintaining TNBC cell growth and reduces the amount of methylated transcripts during high-level transcription and translation in cancer cells with higher MYC expression. Additionally, YTHDF2 is not essential for cancer cells, which are less dependent on elevated MYC levels to prolong survival of TNBC patients (Einstein et al., 2021), suggesting that it may be a potential therapeutic target for drugs that can overcome TNBC .
Zinc finger proteins (ZNFs) make up approximately 1% of the total human genome. Studies have shown that ZNF can regulate cell proliferation in various cancers, such as liver cancer, breast cancer and colorectal cancer (Zhang W. et al., 2021; Li et al., 2017). Libraries generated by CRISPR knockout have been used to screen various tumor suppressor genes (TSGs) in breast cancer cells (Shalem et al., 2014). Since then, a transcriptomic study of CRISPR/Cas9 ZNF319-deleted breast cancer cells has shown that ZNF319 is a tumor suppressor gene that reduces breast cancer proliferation and is therefore involved in various potential signaling mechanisms and other biological functions (Wang L. et al., 2022).
Ferroptosis is a type of programmed cell death that is dependent on the presence of iron. It is known that the development of ferroptosis is influenced by the presence of lipid peroxides. In addition, PKCβII has been reported to exhibit early lipid peroxidation, and increased lipid peroxidation is associated with ferroptosis. Screening a library of CRISPR/Cas9-mediated kinase inhibitors revealed that PKCβII is involved in the process of lipid peroxidation, which is critical for ferroptosis in MDA-MB-231 cells (Zhang et al., 2022). Therefore, this suggests that knockout of PKCβII by CRISPR/Cas9 may be a potential tumor suppressor gene target for the treatment of ferroptosis-related diseases (Zhang et al., 2022).
Drug resistance is believed to be responsible for approximately 90% of deaths in cancer patients and is one of the major challenges in cancer treatment (Bukowski et al., 2020). The results indicate that a sufficient number of genes related to drug efflux, DNA repair, apoptosis and various cell signaling pathways are associated with drug resistance (Haider et al., 2020). Among them, several genes have been targeted by CRISPR/Cas9 tools and have shown promising results in reducing drug resistance and increasing the effectiveness of anti-cancer treatments (Vaghari-Tabari et al., 2022). In addition, high-throughput CRISPR/Cas9 gene knockout screening libraries have been implicated in modifying the function of potential drug resistance gene targets (Shalem et al., 2015). To identify paclitaxel resistance genes, RNA sequencing coupled with genome-wide sgRNA library screening was used to identify eight candidate genes, including histone deacetylase 9 (HDAC9), that are associated with drug resistance in patients with recurrent TNBC (B et al., 2020 ). ). In another study, increased expression of diserine/threonine and tyrosine protein kinase (DSTYK) was observed during survival of TNBC patients treated with anticancer drugs. Moreover, CRISPR/Cas9-mediated knockdown of DSTYK significantly enhanced apoptosis of drug-resistant cancer cells in vitro (SUM102PT cells and MDA-MB-468 cells) and an in vivo TNBC model ( Ogbu et al., 2021 ).
Although TNBC has abnormal activation of the MAPK pathway, the clinical effect of MEK-targeted therapies is poor. A CRISPR/Cas9 genomic library screen revealed that inhibition of PSMG2 (proteome assembly chaperone 2) sensitizes TNBC BT549 and MB468 cells to the MEK inhibitor AZD6244. CRISPR/Cas9 knockdown of PSMG2 altered normal proteasome function, leading to autophagy-mediated disassembly of PDPK1, thereby further improving tumor cells in TNBC mouse models induced by AZD6244 (MEK inhibitor) and MG132 (proteasome inhibitor). Thus, proteasome and MAP kinase (MEK) inhibitors can be administered synergistically to reduce tumor cell proliferation (Wang X. et al., 2022).
CRISPR/Cas9 has also been used to screen for loss of gene function leading to TNBC resistance ( Shu et al., 2020 ). Ge et al explained the mechanism of drug resistance of cancer cells treated with JQ1, a BET bromodomain inhibitor (BBDI), in TNBC. Using CRISPR/Cas9, they found that deleting the rb1 gene caused TNBC to become resistant to the anti-cancer drug JQ1. Therefore, rb1 function is important in the response to JQ1 drugs in TNBC. They also reported that paclitaxel is a CDK4/6 kinase/microtubule inhibitor, and its combination with BBDIs such as JQ1 may provide promising therapeutic responses to drug-resistant TNBC (Ge et al., 2020).
The transcriptional landscape of long noncoding RNA (lncRNA) has been reported to be responsible for resistance to neoadjuvant therapy in TNBC. This study shows that five different MALAT1 lncRNA transcripts are highly expressed in TNBC. Additionally, CRISPR/Cas9-mediated deletion of MALAT1 increased the sensitivity of TNBC BT-549 cells to paclitaxel and doxorubicin, suggesting a potential role for MALAT1 resistance in TNBC (Shaath et al., 2021).
Mutations in BRCA1 (BRCA1m) are heterogeneous and therefore difficult to detect. Targeting PARP1 (poly(ADP-ribose) polymerase), the synthetic lethal partner of BRCA1, may increase TNBC drug chemosensitivity. Using CRISPR/Cas9, deletion of PARP1 increased the sensitivity of anticancer drugs such as doxorubicin, gemcitabine and docetaxel to mBRCA1 mutant TNBC cells, suggesting that PARP1 is also involved in drug resistance in TNBC (Vaghari-Tabari et al., 2022) . . Mutations in the tumor suppressor genes BRCA1 or BRCA2 are known to increase the likelihood of developing breast cancer in people. Treatment of these patients requires the use of PARP inhibitors. Additionally, it has been demonstrated that knockout of the nucleotide salvage factor DNPH1 using CRISPR/Cas9 can remove the toxic nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate, thereby enhancing the response of BRCA-deficient cells to sensitivity to PARP inhibitors (Fugger). et al., 2021). Thus, CRISPR/Cas9 and PARP1 inhibitors may become an important treatment strategy for TNBC.
The penetrant glycoprotein (P-gp) gene is a multidrug resistance gene that is generally upregulated in approximately 41% of all TNBC ( Sun et al., 2020 ). P-gp-induced drug efflux has been identified as a key regulator of drug resistance in breast cancer. P-gp inhibitors have demonstrated increased sensitivity to anticancer drugs in breast cancer ( Famta et al., 2021 ). Thus, CRISPR/Cas9-mediated ablation or suppression of P-gp and P-gp inhibitors may be very important methods to overcome drug resistance in TNBC.
ATP-binding cassette transporter G2 (ABCG2) is known to cause drug resistance in TNBC (Palasuberniam et al., 2015), although there are currently no reports on the relationship between CRISPR/Cas9 and ABCG2 silencing and tumor suppressor gene inactivation. PTEN May enhance ABCG2 activation (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Therefore, using CRISP/Ca9 to remove ABCG2 and combining it with an ABCG2 inhibitor may be a suitable treatment to overcome drug resistance in TNBC. Table 2 mentions CRISPR/Cas9 targeting all resistance genes.
Table 2. CRISPR/Cas9 targets various TNBC resistance genes to sensitize cells to anticancer drugs.
CRISPR/Cas9 libraries are potential tools for screening gene mutations associated with cancer pathogenesis (Chan et al., 2022). This screening method involves four steps such as (a) library construction, (b) lentiviral transduction, (c) phenotypic screening, and (d) target gene analysis. Although TNBC has abnormal activation of the MAPK pathway, the clinical prognosis of MEK targeted therapy for TNBC patients carrying mutations in tumor suppressor genes such as PTEN, RB1, and TP53 is very poor.
Additionally, a gene knockout screen using CRISPR/Cas9 tested the most potent and selective molecule, dehydrocatrol, which is abundant in Guatemalan flower and leaf extracts, to understand the effect of dehydrocatrol on MDA-MB-231. Mechanisms of selective cell cytotoxicity. Mesenchymal stemness characteristics of TNBC subtypes. This CRISPR/Cas9-based screen also revealed that HSD17B11, a gene encoding 17β-hydroxysteroid dehydrogenase type 11, is highly expressed in MDA-MB-231 cells and results in dehydrofalcarinol specific for MDA-MB-231 cells (Grant et. al., 2020). Thus, this suggests that CRISPR/Cas9 genomic screening has great potential in identifying the mechanisms underlying the anticancer properties of potential natural compounds.
In addition, the vulnerability of TNBC to cancer was studied using an unbiased genome-wide in vivo CRISPR/Cas9 screen, which showed a link between oncogenic and tumor suppressor pathways. Key components of the mTOR and Hippo pathways have been reported to play important roles in tumor regulation in TNBC. Additionally, studies have shown that pharmacological inhibition of mTORC1/2 and YAP oncoprotein effectively inhibits the pathogenicity of TNBC using an in vitro drug-matrix synergy model and in vivo patient-derived xenografts. Moreover, Torin-1-mediated mTORC1/2 inhibition enhances macropinocytosis, whereas verteporfin-induced YAP inhibition leads to TNBC cell death. Taken together, these results highlight the power and robustness of genome-wide in vivo CRISPR screening to identify novel and effective treatments for TNBC ( Dai et al., 2021 ).
Dysregulation of the immune system is an important factor in tumorigenesis. Cancer cells evade immune clearance by bypassing defense mechanisms, including interfering with the activity of immune cells in the tumor microenvironment and compromising the immune system. Therefore, developing an improved immune system may be a key approach to fighting tumors. The use of CRISPR/Cas9-based gene modification addresses several issues related to immune dysfunction from different perspectives. CRISPR/Cas9 has been used to improve antitumor immunity against breast cancer through the following pathways (Figure 3).
Figure 3. CRISPR/Cas9 immunotherapy targets TNBC cells. Loss of CDK5 and knockdown of PDL1 and CD155 enhance the immune system. Likewise, loss of A2AR and knockdown of TAAs such as HER2, mucin 1 and TEM8 increased the efficiency of CAR-T cells in killing cancer cells. CRISPR/Cas9-driven T cell screening has shown that disruption of p38 kinase enhances T cell antitumor activity. T cells modified by CRISPR/Cas9 increase the expression of TCR (T cell receptor), resulting in T cells having antitumor activity.
The goal of immunotherapy is to stimulate the immune system to attack cancer cells. Overexpression of immune checkpoint proteins usually prevents autoimmune reactions but may help cancers avoid them (Topalian et al., 2015). These proteins prevent immune reactions by binding to receptors on the surface of immune cells. Multiple checkpoint proteins (such as CD155 and PD-L1) target the PD-1 receptor on immune cells and are expressed in breast cancer, especially TNBC (Li Y.-C. et al., 2020). Knockdown of PD-L1 or its receptor using CRISPR/Cas9 can stimulate the immune system to attack TNBC tumors (Yahata et al., 2019). Additionally, downregulation of PD-L1 expression through CRISPR/Cas9-mediated deletion of CDK5 has been shown to inhibit tumor growth in vitro and in vivo (Deng et al., 2020). Growth inhibition in in vitro and in vivo models of breast cancer suggests that shRNA-mediated reduction of CD155 may have a therapeutic effect on breast cancer (Gao et al., 2018).
CAR T cells express CARs that recognize tumor-associated antigens (TAAs) and can use knockdown of checkpoint proteins to enhance their activity (Li C. et al., 2020). There are various potential targets for CAR T cell therapy in breast cancer, including several TAAs such as HER2, mucin1, and TEM8 (Bajgain et al., 2018). There is evidence that CAR T cells targeting mesothelin (overexpressed in TNBC BT-459 cells) are more effective against cancer when PD-1 is knocked out using CRISPR/Cas9 (Hu et al., 2019). However, isolating T cells from patients and then editing them ex vivo is a labor-intensive and time-consuming process. Although universal T cells can reduce isolation requirements, donor T cells expressing human leukocyte antigen (HLA) class I and T cell receptor (TCR) should be removed to prevent graft-versus-host abnormalities (Ren et al ., 2017). , 2017a). Using CRISPR/Cas9, HDR can simultaneously eliminate TCR and HLA and knock out the gene encoding CAR. Studies have shown that CRISPR/Cas9 can be used to introduce anti-CD19 CARs into the TCR locus, thereby efficiently producing CARs without depleting T cells (Dimitri et al., 2022). Multiplex technologies have been developed to generate allogeneic CAR T cells by simultaneously eliminating TCR, beta-2-microglobulin (B2M), HLA-I subunit and other proteins such as PD-1 and CTLA-4, which may have greater anti-cancer properties . against TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Using CRISPR/Cas9 could improve the effectiveness of CAR-T cells. Adenosine is known to have immunosuppressive properties and can reduce anticancer immunity by blocking T cell function and activating adenosine A2A receptors (A2AR) (Vigano et al., 2019). Silencing A2AR using CRISPR/Cas9 significantly improved the efficacy of CAR-T cells in vivo (Giuffrida et al., 2021). T cells are known to exhibit various phenotypic characteristics such as cellular expansion, differentiation, oxidative stress and genomic stress. CRISPR/Cas9-based T cell screening revealed disruption of 25 different T cell receptor kinases. Among them, deletion of p38 kinase has been shown to enhance the antitumor activity of T cells, indicating that p38 kinase is a key regulator of CAR-T cell regulation ( Gurusamy et al., 2020 ).
T cells can be genetically modified using CRISPR/Cas9 to produce highly expressed TCRs. Transferring genetically modified T cells to patients has shown stronger anticancer activity than endogenous T cells. Therefore, endogenous TCRs may compete with genetically altered TCRs in patients, which may impact the potential of cancer immunotherapy. To overcome this problem, using CRISPR/Cas9 to remove endogenous TCR-β in recipient cells and subsequently transfer TCR-β T cells to cancer patients may exhibit better anti-cancer immune responses without the need for endogenous TCR sexual competition (Fan et al. 2018). Thus, with the exception of CRISPR-modified T cells, TCRs are a thousand times more sensitive to tumor antigens than normal TCR-transduced T cells. Additionally, in various leukemias, altered T cells generated by γδ TCR+ CRISPR exhibit higher expression of CD4+ and CD8+ T cells than standard TCR transfer (Legut et al., 2018). Thus, modified T cells generated by CRISPR/Cas9 may be an effective immunotherapy method to overcome TNBC.
Integrins are cell adhesion molecules that are present in cellular transmembranes and promote the binding of cells to the extracellular matrix (ECM) (Hamidi and Ivaska, 2018). Dysregulation of integrins is associated with cancer development and migration by altering the extracellular matrix, leading to cancer cell survival in the circulation (Hamidi and Ivaska, 2018). CRISPR/Cas9-mediated integrin knockdown slows tumor progression, metastasis, and colonization in TNBC. Knockdown of integrin a5 (ITGA5) has been reported to reduce cell migration and progression in other cancers such as lung cancer (Ju et al., 2017), suggesting that integrin a5 may also be a key factor in the pathogenesis of TNBC.
Generating knockout mice using traditional embryonic stem (ES) cell methods is a labor-intensive, time-consuming, and inefficient process. It takes months and years to target ES cells through homologous recombination, breed chimeric mice, and then cross them with heterozygous mice to produce homozygous offspring. Complex crossbreeding is required to create mice with multiple genetic mutations. However, many problems have arisen when using mice generated by ablation of ES cells using CRISPR/Cas9 or by microinjecting CRISPR/Cas9 components into single-cell fertilized eggs. For example, introduction of changes often occurs at biallelic loci and is not gene specific. It was recently reported that ES cells using CRISPR/Cas9 technology can simultaneously insert biallelic mutations in up to 5 genes (Nishizono et al., 2021). For this purpose, Cas9 mRNA and five gene-specific gRNAs were simultaneously transfected into ES cells. This highlights the promise and effectiveness of this procedure, although these mutations may have been passed on when founder lines were bred to produce quintuple knockout mice. The study also reports a surprising discovery: ES cells are no longer required to create genetically modified mice. Instead, to remove specific gene products, Cas9 mRNA and gRNA were injected into single-cell stage embryos. As a result, knockout founder lines have been created that can theoretically be used to study the consequences of gene deletion in mice (Qin et al., 2016). Thus, CRISPR/Cas9 allows the creation of transgenic mice to treat TNBC at a lower cost than traditional genetic engineering. Using the CRISPR/Cas system, a new knockout mouse model has been developed that can successfully introduce point mutations in one or more endogenous genes in TNBC. Based on this concept, animal models of TNBC can also be developed using CRISPR/Cas9-mediated deletion of BRCA1 and p53, resulting in loss of HR repair, genomic instability, and mutant phenotypes ( Annunziato et al., 2020 ).
Currently, various methods such as mammography, magnetic resonance imaging (MRI) and ultrasound are used to diagnose TNBC. However, these methods have some limitations. Mammography is used to diagnose local breast tissue rather than metastases, where cancer cells have migrated to other organs. Ultrasonography is not a reliable method for diagnosing TNBC (Chen and Lee-Felker, 2023). MRI has higher sensitivity than ultrasound and mammography, but its diagnostic accuracy is limited (Sha and Chen, 2022). Tissue biopsy is an invasive way to identify cancer cells. However, in some cases, a biopsy may miss cancerous tissue if the needle strays away from the area of interest. In addition, it is very expensive and can be traumatic for the patient. TNBC is a heterogeneous type of cancer, so a biopsy may not provide enough information about the type of cancer. Therefore, a new diagnostically reliable method for detecting TNBC is urgently needed. CRISPR/Cas9 may serve as a highly sensitive and minimally invasive alternative for breast cancer diagnosis. To this end, CRISPR/Cas9-based strategies can be used to improve PCR methods for the diagnosis of TNBC. First, the Cas9 and cpf1 proteins of the CRISPR system are used to remove non-specific DNA, and then these two proteins (Cas9 and cpf1) can recognize the PAM sequence before binding to the target DNA (Deepak Singh et al., 2021). . Thus, PCR can identify mutations associated with cancer development. Several studies have adopted this CRISPR-based strategy to detect different mutations in different cancers ( Safari et al., 2019 ). Thus, CRISPR-based PCR methods may reduce the dependence of TNBC diagnosis on invasive methods such as biopsy-based immunohistochemistry.
Genotypic changes in hormone receptor regulation have also been demonstrated in TNBC (Chen and Russo, 2009). CRISPR/Cas9-based PCR strategies using microarrays for point-of-care diagnostics can effectively monitor these mutations (Hajian et al., 2019). It could also provide healthcare providers with relevant information about specific mutations in TNBC patients, which could help improve their treatment course. However, this combined technique has limitations. Therefore, extensive research efforts are required before this CRISPR/Cas-PCR method can be used in healthcare for the diagnosis of TNBC (Yang et al., 2019).
Post time: Oct-14-2024