Project Description

Introduction:

Globally, breast cancer is the most common cancer to be diagnosed, and it is also the primary cause of cancer-related fatalities. Affecting both males and females. Molecular diversity is a hallmark of the complex disease known as breast cancer, with multiple subgroups identified by gene expression investigations. Differences in clinical outcomes are linked to these categories[1, 2]. The molecular categorization scheme is based on the histology and immunochemistry of essential proteins such as ER, PR, (and HER2) which are estrogen receptors, progesterone receptors, Human Epidermal Growth factor receptors, & Ki67 [3]respectively.

Figure 1: The molecular classification of Breast Cancer.

Classification of breast cancer:

The hormone receptor proteins allow for categorizing breast cancer into different groups, namely Basal Like, Luminal A, HER2 positive, Luminal B, and Claudin-low TNBC, as shown in Figure 1.  The presence of ER-, PR-, and HER- hormone receptor proteins in a configuration results in Triple Negative Breast Cancer (TNBC) with an unfavorable prognosis. The Ki67 index is highly negative in TNBC and Invasive breast cancer of no specific type (NST) histology. On the other hand, a configuration with ER-, PR-, and HER2 + leads to the development of HER2-positive cancers, which exhibit overexpression of non-luminal HER2. Furthermore, a configuration including ER + and PR + but HER2 leads to the emergence of breast cancer of the Luminal types. Conversely, when ER and PR are present along with HER2 + , it leads to the formation of Luminal B-type breast carcinoma, characterized by higher Ki67 values.

TNBC is most frequent in women under 40 years of age, accounting for around 15%–20% of all cases of breast cancer who have not yet reached menopause. Triple-negative breast cancer (TNBC) is known for its aggressiveness, which is linked to poor clinical outcomes, limited therapy choices, increased invasiveness, and more heterogeneity. The lack of Claudin and terminal expression markers is linked to a poor prognosis for TNBC malignancy, resulting in reduced survival time for patients. TNBC patients have been reported to have brain, liver, and lung metastases[4].

TNBC lacks ER/PR & HER2 receptors, which makes it resistant to hormonal therapies. Furthermore, the effectiveness of chemotherapy diminishes once TNBC has metastasized[5]. TNBC accounts for around 15%–25% of all incidences of breast cancer, thereby representing a substantial proportion of breast cancer diagnoses. Prevalence is higher among younger women and individuals with a familial predisposition to the condition. TNBC exhibits a higher incidence among specific racial and ethnic groups, particularly African American women, who have a greater likelihood of receiving a TNBC diagnosis and frequently encounter inferior prognoses.

Genome editing techniques:

With the use of sophisticated genome editing technology, DNA sequences may be precisely altered, down to the level of a single nucleotide, while avoiding any unintended off-target effects. The transformation of basic knowledge into customized medicine is founded upon this principle[6, 7]. Genome editing has been crucial in the identification and treatment of breast cancer. A variety of methods, including insertion, deletion, knockouts, protein targeting within cells, transcriptional activation/repression, and modification of the epigenetic state within cells, are included in genome editing.

Genome editing has been facilitated by the use of targeted nucleases, which grants researchers the capability to change nearly any sequence in the genome. It allows for easy development of genetically identical cell lines and animal models for studying human diseases. There are three primary techniques used for gene editing in Breast carcinoma: Zinc finger nucleases (ZFNs) [Table 1], transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR associated protein 9 (Cas9).

Table 1 Comparison between the three main gene editing tools

	ZFN	TALEN	CRISPR
Creation	ZNF functions as dimers, with each monomer recognizing a specific half-site sequence, typically 18 bps of DNA via zinc finger DNA binding domain	Engineered to recognize 12–20 bps of DNA with more bases leading to higher genome editing specificity	Target site recognition is mediated by gRNA, Thus there is no need to Engineer new protein to Target new sites
Size of DNA sequence recognition site	18 base pairs (bps)	12–20 bps	Cas 12A- 20–24 nt Cas9 – 100 nt Type –VC Cas1- entire site integration of 18 bps DNA
Efficiency to hit target	High	Moderate	Low
Efficacy	Moderate	Moderate	High
Nuclease – D/M	Fokl – dimer	Fokl – dimer	Cas9 – monomer
Mode of delivery to target	Two sets of ZNF monomers to hybridize around target sequence	Two TALEN to hybridize around the target sequence	With gRNA, Cas9 can reach the target DNA sequence and generate double-strand breaks
Immunosuppressive	Moderate	Low	Low
Benefit–cost analysis	Difficult to construct ZNF arrays, time-consuming	No selection or directed evolution is necessary to engineer TALEN arrays, which are easy to construct, time-consuming	No need to engineer new proteins to target new sites. Cas9 nuclease and a single guide RNA (gRNA) is only required. Affordable and time-efficient

ZFN

ZFN is a gene editing method that utilizes Zinc Finger Nucleases. These nucleases consist of DNA-Binding domains that are specific to certain sequences of DNA, as well as non-specific DNA-cleaving domains derived from the Fokl restriction endonucleases. ZFN allows for extensive gene editing by inducing a double-strand break in DNA. ZFNs are synthetic structures created by a combinatorial method that combines restriction endonucleases with zinc-finger-binding domain proteins. Biotechnology is limited in its ability to bind to three codons on both sides of the DNA strand. Due to its simplicity and specificity, the technique has gained a lot of traction in recent years and is currently being employed in clinical practice for particular disorders [8, 9].

TALEN

DNA binding domains from TALE proteins are combined with the fokI cleavage domain to generate transcription activator-like effector (TALE) nucleases. TALE consists of many domains, each containing 33–35 amino acids that are repeated. Each of these domains can detect a single base pair. Similar to ZFNs, TALEN also consists of targeted double-stranded breaks (DSBs) that initiate DNA damage response pathways and facilitate customized modifications. TALENs and ZFNs differ in their targeting capabilities. TALENs can simultaneously target three nucleotides, while ZFNs can only address one nucleotide. This makes TALENs more site-specific and reduces the occurrence of off-target effects, as supported by reference[10].

CRISPR/Cas9

CRISPR, which stands for Clustered Regulatory Interspaced Short Palindromic repetitions, refers to certain DNA sequences that consist of several short direct repetitions[11]. These sequences play a crucial role in providing bacteria and archaea with acquired immunity. CRISPR systems utilize crRNA and tracrRNA to selectively silence foreign DNA by targeting certain sequences. CRISPR/Cas systems can be divided into three different categories: Cas9 operates in type II systems as an RNA-guided DNA endonuclease, cleaving DNA upon target sequence recognition via crRNA-tracrRNA identification. The CRISPR Cas system exhibits numerous advantages compared to ZFN and TALEN, particularly in terms of its simplicity and flexibility[12]. The primary difference is that the CRISPR system relies on RNA–DNA recognition rather than a protein–DNA binding mechanism. Hence, it is more feasible and simpler to fabricate a personalized CRISPR/Cas9 complex by only modifying the gRNA sequence rather than undertaking protein engineering. Table 1 provides a comparative evaluation of ZFN, TALEN, and CRISPR.

CRISPR is a revolutionary gene-editing technology that was first discovered in the late 1980s. It plays a crucial role in modifying DNA sequences with high precision and efficiency.

CRISPR technology is an innovative gene-editing device that enables accurate alterations to the genetic code of cells. The adaptability and versatility of this instrument have rendered it a valuable tool for scientific inquiry and possible therapeutic interventions. CRISPR technology holds promise in the realm of breast cancer and breast carcinoma, since it has the capacity to tackle multiple facets of the disease. This includes comprehending its genetic foundations, creating precise treatments, and enhancing methods for detecting the disease at an early stage.

A single, readily modifiable guide RNA sequence makes up the CRISPR system, and it is linked to the nuclease CRISPR-associated endonuclease (Cas9). When employed in CRISPR investigations, the sgRNA and Cas9 are amalgamated into a ribonucleoprotein complex.

Bacteria and archaea i.e. prokaryotic species in particular have an inbuilt adaptive immune system called CRISPR. It offers protection to bacteria against foreign DNA invaders like viruses and plasmids. A set of brief, repeating nucleotide sequences (24–28 base pairs) that are encircled by DNA fragments known as spacers are essential to this defense mechanism.

In every previous infection, the PAM spacer regions were incorporated into the bacterial genome [13].  CRISPR genes are connected to CRISPR-associated (Cas) genes and show a high level of conservation. The Cas protein catalyzes the processing of the CRISPR sequence to generate the CRISPR RNA (crRNA), which is then utilized to target and remove the DNA molecule of the invading organism. The molecules exhibit complementarity, facilitating the hybridization of RNA sequences and impeding the invader's activity. The Cas nucleus destroys the duplex, so halting the virulence process and providing protection to the cell. Moreover, the Cas protein is responsible for attaching the PAM sequence after infection to establish the immune "memory."

Thus, when an external entity impacts the bacteria, the system becomes active and the entity might be rendered ineffective[14].

Three primary subgroups comprise the CRISPR-Cas system: Three different CRISPR-casses: Type I, Type II, and Type III. Type I and II encompass specialized Cas endonucleases responsible for crRNA processing[15]. When a crRNA reaches maturity, it joins forces with numerous Cas proteins to form a massive complex that can recognize and cut complementary nucleic acids. On the other hand, Type II CRISPR Cas necessitates the presence of a trans-activation CRISPR activating CRISPR RNA (tracer RNA) that is complementary to the repeating sequence of crRNA. Ribonucleic acid (RNA) enzyme III, in the presence of the Cas9 protein, carries out the processing of the CRISPR RNA (crRNA). Here, crRNA-guided silencing is not exclusively the result of the Cas enzyme.

Therapeutic potential of CRISPR in breast cancer:

The potential therapeutic application of CRISPR in the treatment of breast cancer is being explored.

A potent tool for studying and treating various diseases, including breast tumors (BCs), is CRISPR/Cas9. Tumor suppressor genes (TSGs) are involved in DNA repair and control of cell growth; when mutated or inactivated, they result in genomic instability and uncontrolled cell proliferation. Oncogenes, on the other hand, promote cell growth and proliferation and, when mutated or activated, drive tumor development. Gene expression can become distorted or inhibited as a result of genetic changes that happen at the nucleotide, transcriptional, and epigenetic levels. The versatility of CRISPR/Cas9 and its derivatives enables them to specifically target mutations across several levels, making them highly promising for the treatment of BCs.

Targeting tumor-suppressing genes and oncogenes:

Cancer can be caused by genes that encode transcription factors (TFs), signal transducers, growth factors and their receptors, and chromatin remodeling proteins. One can use the CRISPR/Cas9 technology to target these oncogenes specifically, leading to their eradication and the inhibition of cancer cell growth by several mechanisms. The methodology has been effective in deactivating viral and cellular oncogenes in a range of cancer types, such as prostate cancer [16], endometrial cancer [17], leukemia[18], and cervical cancer [17].

BCs have been discovered to possess mutations in several tumor suppressor genes (TSGs), such as PTEN, BRCA1, and BRCA2. The TSGs play a vital role in maintaining the stability of the genome. They oversee the restoration of double-strand breaks (DSBs) using non-homologous end joining (NHEJ) and homologous recombination (HR) methods. Moreover, they guarantee the seamless progression of replication forks and the resumption of halted forks. Restoring the functionality of a tumor suppressor gene (TSG) is a more difficult undertaking in comparison to deactivating an oncogene. The dysregulation of transcription factors or hypermethylation of promoters can decrease the production of tumor suppressor genes (TSGs). However, CRISPR variants can be employed to improve the expression of TSGs. The PTEN tumor suppressor gene (TSG), known to be associated with heightened aggressiveness of breast cancer (BC) when its function is disrupted, has been induced in TNBC SUM159 cells through the utilization of a CRISPR a approach[19]. This included merging dCas9 with the VPR domain, which encompasses the transcriptional activators VP64, p65, and Rta. The process of hypermethylation represses the expression of Tumor Suppressor Genes (TSGs), such as PTEN and BRCA1.

References

1. Cheang, M.C., et al., Responsiveness of intrinsic subtypes to adjuvant anthracycline substitution in the NCIC.CTG MA.5 randomized trial. Clin Cancer Res, 2012. 18(8): p. 2402-12.

2. Hu, Z., et al., The molecular portraits of breast tumors are conserved across microarray platforms. BMC Genomics, 2006. 7: p. 96.

3. Nielsen, T.O., Parker, J. S., Leung, S., Voduc, D., Ebbert, M., Vickery, T., Davies, S. R., Snider, J., Stijleman, I. J., & Reed, J. , A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor–positive breast cancer. Clinical Cancer Research, 2010. 16(21): p. 5222–5232.

4. Havas, K.M., et al., Metabolic shifts in residual breast cancer drive tumor recurrence. J Clin Invest, 2017. 127(6): p. 2091-2105.

5. Chew, W.L., Immunity to CRISPR Cas9 and Cas12a therapeutics. Wiley Interdiscip Rev Syst Biol Med, 2018. 10(1).

6. Goodwin, S., J.D. McPherson, and W.R. McCombie, Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet, 2016. 17(6): p. 333-51.

7. Levy, S.E. and R.M. Myers, Advancements in Next-Generation Sequencing. Annu Rev Genomics Hum Genet, 2016. 17: p. 95-115.

8. Ochiai, H. and T. Yamamoto, Construction and Evaluation of Zinc Finger Nucleases. Methods Mol Biol, 2023. 2637: p. 1-25.

9. Ji, H., et al., Zinc-Finger Nucleases Induced by HIV-1 Tat Excise HIV-1 from the Host Genome in Infected and Latently Infected Cells. Mol Ther Nucleic Acids, 2018. 12: p. 67-74.

10. Chandrasegaran, S. and D. Carroll, Origins of Programmable Nucleases for Genome Engineering. J Mol Biol, 2016. 428(5 Pt B): p. 963-89.

11. Chehelgerdi, M., et al., Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy. Mol Cancer, 2024. 23(1): p. 9.

12. Norris, A.D., X. Gracida, and J.A. Calarco, CRISPR-mediated genetic interaction profiling identifies RNA binding proteins controlling metazoan fitness. Elife, 2017. 6.

13. Hamidi, H. and J. Ivaska, Every step of the way: integrins in cancer progression and metastasis. Nat Rev Cancer, 2018. 18(9): p. 533-548.

14. Horvath, P. and R. Barrangou, CRISPR/Cas, the immune system of bacteria and archaea. Science, 2010. 327(5962): p. 167-70.

15. Pont, M., et al., Applications of CRISPR Technology to Breast Cancer and Triple Negative Breast Cancer Research. Cancers (Basel), 2023. 15(17).

16. Cai, J., et al., Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells. J Int Med Res, 2019. 47(5): p. 2199-2206.

17. Hu, Z., Yu, L., Zhu, D., Ding, W., Wang, X., Zhang, C., Wang, L., Jiang, X., Shen, H., & He, D. , Disruption of HPV16‐E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. BioMed Research International, , 2014. 1: p. 612823.

18. Narimani, M., M. Sharifi, and A. Jalili, Knockout Of BIRC5 Gene By CRISPR/Cas9 Induces Apoptosis And Inhibits Cell Proliferation In Leukemic Cell Lines, HL60 And KG1. Blood Lymphat Cancer, 2019. 9: p. 53-61.

19. Yedier-Bayram, O., et al., EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities. Cell Death Dis, 2022. 13(8): p. 710.