Project Design

Breast Cancer

Since global cancer burden estimates were first published in the 1980s, breast cancer has been the cancer most diagnosed among females [1]. Today, it remains the leading cancer diagnosis and cause of cancer-related death in females, and is a major contributor to the cancer burden even when both sexes are combined, representing the second most frequently diagnosed cancer and the fourth most common cause of cancer-related mortality in 2022 [2]. Breast cancer remains a formidable adversary in the landscape of global health challenges, with its intricate pathogenesis and diverse clinical manifestations posing significant obstacles to effective treatment and prevention [2-4]. The application of these emerging diagnostic methods in breast cancer will be discussed in this review. The traditional treatments for breast cancer include surgery, chemotherapy, radiotherapy, endocrine therapy, targeted therapy, and other related approaches [5]. However, constant effort is being made to introduce novel therapies with minimal toxicity. Gene therapy is one of the promising tools, to rectify defective genes and cure various cancers [6]. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) has emerged as a transformative gene-editing tool in various biological fields, including human cancer research and gene therapy. This advancement is largely due to its versatile characteristics, such as high specificity, precision, efficiency, and cost-effectiveness, coupled with a minimal risk profile.

CRISPR/Cas9

CRISPR/Cas system is a phenomenal gene-editing tool and is referred to as genetic scissors. Its potential in precise editing of the DNA revolutionized basic science research. All CRISPR/Cas systems have three principal components: (i) a guide RNA or CrRNA is a unique non-coding RNA, which directs the CRISPR/Cas complex to the target DNA, (ii) auxiliary trans-activating crRNA named tracrRNA. CrRNA and tracrRNA fused to form chimera is termed single-guide RNA (sgRNA), (iii) The Cas protein, an endonuclease that mediates the tailoring of target DNA sequences [7]. Further, the Cas nuclease needs a specific sequence, known as a protospacer adjacent motif (PAM) to cleave the target DNA sequence. To precisely manipulate the DNA sequences, sgRNA plays a fundamental role in ensuring the editing at the desired locus in the target. The sgRNA is composed of two components known as scaffold sequence and ∼ 20 nucleotide spacers. The scaffold sequence is very important for Cas protein binding, and the spacer sequence shares the homology with the target sequence. The seed sequence in the spacer is the first 10–12 nucleotides of sgRNA at the 3′-end close to a PAM sequence that directs the Cas9 nuclease to the target sequence in the genome. The mismatch in seed sequence aborts the interaction between CRISPR/Cas and target DNA sequence, therefore abolishing DNA editing. Hence, sgRNA defines the target and plays a crucial role in the specificity, efficiency, and precision of the CRISPR/Cas-mediated gene manipulations [8]. Structurally, the Cas9 peptide contains the recognition (REC) and nuclease (NUC) lobes [9]. The REC lobe is vital for sgRNA and DNA binding, whereas the NUC lobe is comprised of RuvC and the HNH domains, the HNH and RuvC domains have nuclease activity and nick the complementary and non-complementary target DNA strand, respectively, and create a DNA double-strand break [9].

As in our research, we are targeting the hippo pathway, which plays an essential role in proliferation, migration, invasion, metastasis, and resistance to breast cancer treatment. The upstream factors involved in the Hippo signaling pathway, including mammalian Ste20 kinases 1/2, large tumor suppressor kinases 1/2, and transcription coactivator Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), have been extensively studied as they are considered therapeutic targets for breast cancer, recently, it has been suggested that the transcriptional enhancer factor domain (TEAD) family of transcription factors, particularly TEAD4, plays an important role in breast cancer. TEADs interact with YAP/TAZ to act as transcription factors, in the normal cell, the YAP/TAZ can be phosphorylated, which can be recognized by the cell’s metabolic system to degrade them. In this case, the YAP/TAZ wouldn’t combine with TEAD4 to express a certain gene [10]. When the Hippo pathway functions abnormally, YAP/TAZ proteins are not phosphorylated and consequently evade degradation. This results in an accumulation of YAP/TAZ, which then interacts with TEAD4, leading to the overexpression of specific genes that regulate cell proliferation. This series of reactions can contribute to an increased rate of cancer progression.

Part Design

Utilizing the mechanism of the CRISPR/Cas9 system and TEAD4 mentioned above, our project designs specific guide RNAs (gRNAs) that target the TEAD4 gene and knock out the expression of TEAD4 in Breast cancer cell lines MCF-7 and MDA-MB-231. Theoretically as shown in Figure 1, when the CRISPR/Cas9 were transfected into MCF-7 and MDA-MB-231 cells, the TEAD4 expression was knocked out. TEAD4 expression could affect the downstream Hippo pathway. Therefore, when MCF-7 and MDA-MB-231 cells were treated with CRISPR/Cas9 the proliferation, migration, and invasion ability were inhibited. The tumor cells tend to lose the cancer cell character after CRISPR/Cas9 treatment, which is expected to achieve favorable curative effects in clinical practice. We detected the proliferation ability by cell counting kit-8(CCK-8) test, the migration ability by transwell migration assay, and the internal oxidative stress by Reactive Oxygen Species Assay.

Figure 1 the pipeline of breast cancer therapy.

In summary, we aimed to treat breast cancer using a novel biotherapy involving CRISPR/Cas9 specifically targeting TEAD4. The knockout of the TEAD4 gene has demonstrated itself as a versatile and effective therapeutic strategy, with ongoing advancements in chemistry and pharmaceuticals facilitating the transition of these treatments into clinical settings. While CRISPR/Cas9 shows significant promise in addressing breast cancer due to its stability and cost-effectiveness, potential off-target effects must be carefully considered.

References

1. D. M. Parkin, J.S., and C. S. Muir, Estimates of the worldwide frequency of twelve major cancers. Bull World Health Organ, 1984. 62(2): p. 163.

2. Bray, F., et al., Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2024. 74(3): p. 229-263.

3. Giaquinto, A.N., et al., Breast Cancer Statistics. CA Cancer J Clin, 2022. 72(6): p. 524-541.

4. S. Loibl, P.P., and M. Morrow, Breast cancer. Lancet. 397(10286): p. 1710.

5. Winer, A.G.W.a.E.P., Breast cancer treatment: a review. JAMA, 2019. 321(3): p. 288-300.

6. Karn, V., et al., CRISPR/Cas9 system in breast cancer therapy: advancement, limitations and future scope. Cancer Cell Int, 2022. 22(1): p. 234.

7. Sherwood, B.B.a.R.I., A CRISPR view of gene regulation. Curr Opin Syst Biol, 2017. 1: p. 1-8.

8. X. Chen, J.L., J. M. Janssen, and M. A. F. V Gonçalves, The chromatin structure differentially impacts high-specificity CRISPR-Cas9 nuclease strategies. Mol Ther Nucleic Acids, 2017. 8: p. 558–563.

9. B. Sun, H.C., and X. Gao, Versatile modification of the CRISPR/Cas9 ribonucleoprotein system to facilitate in vivo application. Journal of Controlled Release, 2021. 337: p. 698–717.

10. Y. Wu, M.L., J. Lin, and C. Hu,, Hippo/TEAD4 signaling pathway as a potential target for the treatment of breast cancer. Oncol Lett, 2021. 21(4): p. 313.