Antisense BCL-2 and HER-2 Oligonucleotide Therapy in Breast Cancer

 

HER2 Positive Breast Cancer

Human Epidermal Growth Factor Receptor 2 (HER2) is involved in the growth & proliferation of breast cancer cells. The patients with breast cancer that are tested positive for HER2 are called HER2-positive breast cancer patients. They have extra copies of those genes that are encoding the HER2 protein receptor. 

HER2-positive breast cancer is the most aggressive form of breast cancer but the good point about this cancer type is that it can be diagnosed easily. About 15 to 20% of breast cancer patients tested positive for HER2. Targeting HER2 in these patients as a treatment for breast cancer can be effective (Loibl & Gianni, 2017). 

BCL-2 and Its Role in Cancer

The BCL-2 family of proteins plays a crucial role in programmed cell death PCD or apoptosis in all cells. It makes the outer mitochondrial membrane permeable for different apoptotic factors like cytochrome C that can enter cytosol from the mitochondria and regulate intrinsic apoptosis in the cell. The BCL-2 family has different proteins, some are proapoptotic proteins while others are anti-apoptotic. Anti-apoptotic proteins control cell death pathways in the cell while pro-apoptotic proteins are involved in the stimulation of apoptosis in the cell. 

The anti-apoptotic members of this family are usually mutated or dysregulated in the cancer cells. They provide a survival advantage to the cancerous cells and help them escape cell death. Moreover, the mutations in these proteins also make the tumor cells resistant to therapeutic strategies. Cells would not be able to respond to any treatment and will be proliferating indefinitely escaping apoptosis. 

Mcl-1 and Bcl-xL are members of the BCL-2 family that are anti-apoptotic and have such mutations that cause a gain of function in the tumor cells. They have increased expression in the tumor cells and are required for their survival. These proteins also help cancer cells to cope with oxidative stress in the cell and give them an advantage of repairing the damage caused by therapies given to kill the tumor cells. So, targeting these members of the BCL-2 family in tumors can also be effective to kill these cells (Eichhorn et al., 2014). 

Antisense Oligonucleotides (ASOs)

The most effective therapeutic strategies for neurological disorders and cancers is the use of antisense oligonucleotides that is in the clinical trials for different cancer types including breast cancer. These are short single-stranded sequences of DNA or RNA that are 12 to 25 nucleotides long. They have sequences complementary to the sequence of our gene of interest. They are unable to cross the plasma membrane as they have the chance of degradation by nucleases while crossing the cell membrane. To overcome this limitation, the ASOs are modified by using some chemicals in the laboratory, that help them in protection against nucleases and can cross the cell membrane easily. 

Mechanism of action of ASOs

Antisense oligonucleotides have different mechanisms of action based on their structure and sequence. The main aim of using ASOs is to decrease the expression of some specific protein in the cell that is involved in causing disorders like breast cancer. Some of the mechanisms of action are discussed below.

Degradation of mRNAmRNA can be degraded by miRNAs in the cell that are endogenous molecules. The process of degradation of mRNA was first studied in plants and later in C. elegans. This discovery gave the idea that if we need to decrease the expression of any specific protein in the cell, we can degrade the mRNA by using some exogenous compounds as well. ASOs are prepared in the laboratory, and they have sequences complementary to the sequence of mRNA that we want to degrade in the cell. When they are delivered in the cell by liposomes, they bind the specifically targeted mRNA and act like micro RNAs degrading that mRNA. 

The first ASO for the degradation of mRNA was prepared in the laboratory and approved by FDA in 1998, called Fomivirsen. It targets viral mRNA specifically. 

1. ASOs for modifying splicing events

Splicing is a modification that occurs in pre-mRNA. It is the removal of introns, and the exons are ligated together. Exons are a coding region of a gene that is then translated into the functional protein. This splicing event in the cell is regulated by many proteins like CPSF, CStF, etc. if there is any mutation in the splice donor site that is the last region of the intron and start of the exon or the mutation in the splice acceptor site that is the end of exon and start of next intron, then the splicing will not take place properly and there may be retention of an intron in the mRNA or there is a deletion of any exon that will result in the formation of abnormal protein. Splicing is usually dysregulated in the cell by slicing silencers that can be targeted to cure the disorder. ASOs against these splicing silencers are used in many diseases to make the splicing event normal in the cell. They will have a sequence complementary to the splicing silencer and will specifically bind it to inhibit its action in the dysregulating splicing process (Quemener et al., 2020). 

 

2. Release of sequestered protein

mRNA is always coding a protein. If there is any defect in mRNA this protein may have damaging effects on the cell that is formed from the mRNA. So, it should not be formed. In order to stop the formation of sequestered protein in the cell, mRNA coding it should be degraded. To degrade the mRNA, we can use antisense oligonucleotides having a complementary sequence that will target mRNA and degrade it (Quemener et al., 2020).

 

Antisense HER-2 and BCL-2 Oligonucleotides Treatment

A study was performed using Antisense Bcl-2 and HER-2 oligonucleotide whose results indicates that down-regulation of Bcl-2 and HER-2 increased drug-sensitivity by modulating drug-induced apoptotic pathways in breast cancer cells. The antitumor effects of a combination of antisense Oligonucleotides and anticancer drugs, including mitomycin C (MMC), adriamycin (ADM), paclitaxel (TXL), and docetaxel (TXT) was evaluated (Tanabe et al., 2003). 

 

As mentioned earlier in this article breast cancer cells have HER-2 receptors on their outer walls, and they are considered HER-2-positive breast cancer cells. HER-2 gives them surviving advantage and helps in their indefinite proliferation. So, targeting HER-2 for the cure of breast cancer in patients can be possible and may be an effective therapy.

Similarly, the BCL-2 family of proteins has a crucial role in apoptosis and if any of the proteins from this family is abnormal in the cell, the cell will get the survival advantage, escape apoptosis, and also will become resistant to cancer therapies. So, the breast cancer cells that have abnormal BCL-2, can be cured of tumors by targeting the abnormal protein. 

Antisense oligonucleotides are proven to be the best effective targeted therapy in cancers. It has passed many preclinical and clinical trials. HER-2 or BCL-2 can be targeted by using specific antisense oligonucleotides. 

A study was performed on the human breast cancer cell line BT474. These cells had overexpression of HER-2. They were treated with the ASOs for HER-2 and the effects were checked and compared with the control. It was observed that the number of HER-2 gene products was significantly reduced in these cells and the growth of breast carcinoma was also decreased as compared to the control cells. So, targeting HER-2 using antisense oligonucleotides will be an effective therapy for breast cancer patients that have multiple copies of HER-2 (Roh et al., 1998). 

Similarly, experiments were performed using antisense oligonucleotides for the BCL-2 protein that is proto-oncogene in the cell and any mutation in this gene will make the cells escape apoptosis and become cancerous. Targeting BCL-2 by oligonucleotides also showed significant outcomes. So, this can be an effective therapy for breast cancer patients that have mutations in the BCL-2 gene. 

 

References

Eichhorn, J. M., Alford, S. E., Sakurikar, N., & Chambers, T. C. (2014). Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival. Experimental Cell Research, 322(2), 415–424. https://doi.org/10.1016/j.yexcr.2014.02.010

Loibl, S., & Gianni, L. (2017). HER2-positive breast cancer. The Lancet, 389(10087), 2415–2429. https://doi.org/10.1016/S0140-6736(16)32417-5

Quemener, A. M., Bachelot, L., Forestier, A., Donnou-Fournet, E., Gilot, D., & Galibert, M. D. (2020). The powerful world of antisense oligonucleotides: From bench to bedside. Wiley Interdisciplinary Reviews: RNA, 11(5), 1–22. https://doi.org/10.1002/wrna.1594

Roh, H., Pippin, J., Boswell, C., & Drebin, J. A. (1998). Antisense oligonucleotides specific for the HER2/neu oncogene inhibit the growth of human breast carcinoma cells that overexpress HER/neu. Journal of Surgical Research, 77(1), 85–90. https://doi.org/10.1006/jsre.1998.5353