Introduction: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. TNBC stem cells, identified by the cell surface marker CD44, have been implicated in tumor initiation, metastasis, and therapy resistance. The ATP-binding cassette sub-family G member 2 (ABCG2) gene, which encodes a drug efflux pump, has been associated with stemness and chemoresistance in various cancer types, including TNBC. In this study, we aimed to investigate the impacts of ABCG2 gene knockout using high-fidelity CRISPR-Cas9 technology in CD44+ TNBC stem cells, utilizing adeno-associated virus (AAV) vectors for efficient gene delivery.
Methods: The study design involved the generation of AAV vectors carrying the CRISPR-Cas9 system targeting the ABCG2 gene. These vectors were then used to transduce CD44+ TNBC stem cells. The efficiency of gene knockout was assessed using various molecular techniques, such as polymerase chain reaction (PCR), Western blotting, and flow cytometry. Additionally, functional assays were performed to evaluate the impact of ABCG2 gene knockout on stemness properties, chemoresistance, and tumorigenic potential of CD44+ TNBC stem cells. AAV vectors are chosen for their numerous advantages in gene therapy applications. These include their non-pathogenic nature, high transduction efficiency, ability to infect both dividing and non-dividing cells, long-term gene expression capability, and low immunogenicity. By utilizing AAV vectors, we aimed to deliver the CRISPR-Cas9 system targeting the ABCG2 gene to CD44+ TNBC stem cells.
The ABCG2 gene, also known as the ATP-binding cassette sub-family G member 2, encodes a protein called breast cancer resistance protein (BCRP). This protein is a member of the ATP-binding cassette (ABC) transporter superfamily and is involved in drug efflux, which can contribute to multidrug resistance in cancer cells. While all exons of the ABCG2 gene are important, specific mutations or variants within certain exons can affect the function or expression of the protein. For example, a well-studied genetic variant in the ABCG2 gene is the Q141K polymorphism, which is located in exon 5. This variant has been associated with altered drug transport activity and drug resistance in certain cancers.
In order to achieve this, we can employ the high fidelity Crispr-Cas9 system to specifically target and knockout exon number 5 of the ABCG2 gene in CD44+ TNBC stem cells. We should confirm successful gene editing through genomic DNA sequencing and verified the loss of ABCG2 protein expression using immunoblotting. Subsequently, evaluating the effects of ABCG2 knockout on tumor growth inhibition in CD44+ TNBC stem cells using in vitro and in vivo models is vital.
Preliminary results demonstrated that ABCG2 knockout significantly inhibited the growth and proliferation of CD44+ TNBC stem cells compared to control cells. Moreover, ABCG2 knockout does sensitize CD44+ TNBC stem cells to conventional chemotherapeutic agents commonly used in TNBC treatment. Mechanistic investigations reveals that ABCG2 knockout designing appropriate gRNA (via CHOPCHOP) led to increased intracellular drug accumulation and enhanced apoptosis induction in CD44+ TNBC stem cells.
Results: The results of this study will provide valuable insights into the role of ABCG2 in TNBC stem cells and its potential as a therapeutic target. Furthermore, the use of AAV vectors for efficient gene delivery in CD44+ TNBC stem cells will contribute to the advancement of gene therapy strategies for TNBC treatment. Ultimately, this research may lead to the development of novel therapeutic approaches that specifically target TNBC stem cells, improving patient outcomes and reducing the risk of relapse and metastasis. This study contributes to the growing body of knowledge on TNBC stem cell biology and provides a foundation for further research aimed at developing targeted therapies for this challenging disease.
Conclusion: Consequently, the goal of this research is to integrate gene therapy with stem cells which considering that important role in the recurrence on cancer and its formation, by targeting the key gene in drug resistance in them, it can be at the same time, inhibiting drug resistance, metastasis and cancer invasion.
Keywords: TNBC, ABCG2, breast cancer stem cell (CSC), HF-Crispr-Cas9, gene editing