Chemosensitization by Antisense Oligonucleotides Targeting MDM2 Expression

 

MDM2 gene

MDM2 oncogene in humans encodes Mouse double minute 2 homolog or MDM2 protein. This protein negatively regulates a tumor-suppressive gene in the cell called P53. It also acts as an E3-Ubiquitin ligase protein that binds the N-terminal of the p53 protein and inhibits the transcriptional activation of p53.

MDM2 plays a very important role in the differentiation of tissues and maintains homeostasis in cells. When any injury occurs in the tissue, it plays a role in wound healing. It acts as a transcription factor and activates NFK-B signaling pathway in the cell.

As MDM2 has a role in promoting differentiation in tissues, it can be a good target in different disorders like cancers. Inhibition of MDM2 has anti-inflammatory effects on cells and tissues (Sciot, 2021).

MDM2 is said to be a p53-responsive gene. Its transcription is activated only when the p53 gene is stabilized and acts as a tumor suppressor gene in the cell. When MDM2 levels increase in the cell, it will bind the transactivation domain TAD at the N-terminal of the P53 protein and inhibit its transcription generating a negative feedback loop. Thus, the cell will move towards increased proliferation and oncogenic phenotype.

MDM2 also acts as a ubiquitin ligase enzyme. It binds several lysine residues in the C-terminus of the p53 protein and causes its degradation by the proteasome. When p53 is degraded the cells will not have any checks and balances for proliferation. Moreover, MDM2 can also self-ubiquitinate along with p53.

Relationship between MDM2 and p53

MDM2 is an oncogene while p53 is a tumor suppressor gene. MDM2 is transcribed when the process is activated by p53 in the cell. When increased transcription of mDM2 occurs in the cell, and its level increases, it causes the inhibition or degradation of the P53 protein. So, a negative feedback loop mechanism exists between the two.

MDM2 may block the transcription of the p53 gene by binding the trans-activation domain at its N-terminal. Or it may cause degradation of p53 by acting as an E3 ubiquitin ligase and targeting the lysine residues in the C-terminal domain of the p53 protein.

P53 is a tumor suppressor gene that controls the activities of cells such as proliferation, differentiation, and growth. When it is degraded or its levels decrease in the cell, the control of the proliferation of the cell is lost. Cells will proliferate indefinitely and acquire the cancerous phenotype (Chène, 2003).

MDM2 overexpression is associated with chemotherapeutic resistance

MDM2 is found to be amplified in many human disorders particularly cancers like lung cancer and colon cancer. Its overexpression causes resistance against many therapeutic modalities in the tumor cells. This resistance is usually developed by MDM2-P53 loop-dependent and loop-independent mechanisms.

 

MDM2-P53 loop-dependent drug resistance

Recent literature has shown that MDM2 causes the inhibition of apoptosis in malignant cells that is caused by the chemotherapeutic drug Cisplatin in these cells. As a result, the cancer cells become resistant to cisplatin. Cisplatin phosphorylates p53 protein and activates it, this causes a reduction in resistance of cisplatin in cancer cells. But at the same time, p53 has the capability to cause activation of transcription of MDM2 in cells. Overexpression of MDM2 will cause the inhibition of p53 which will indirectly cause an increase in resistance against cisplatin in tumor cells. So, this is a loop-dependent mechanism of cisplatin resistance in tumor cells by MDM2.

Experiments performed on mice showed that the malignant cells of mice that were receiving doxorubicin as a drug, when MDM2 was amplified in these cells, they show an increased resistance against this drug. MDM2 downregulates the wtP53 in tumor cells and increases the resistance against chemotherapeutic drugs.

In vitro experiments have shown that when chemotherapeutic drugs like cisplatin and gemcitabine were given to the cancer cell lines. This causes DNA damage in these cell lines to kill the cancerous cells. As a result, both wild-type p53 and MDM2 expressions were increased in these cell lines. The expression of another protein Musashi-2 MSI was also increased in these cell lines that activate the E3 ubiquitin ligase activity of MDM2 and it causes degradation of p53. As a result, resistance to drugs developed in these cell lines (Hou et al., 2019).

MDM2-P53 loop-independent drug resistance

MDM2 can cause resistance to chemotherapeutic drugs in cancerous cells by increasing epithelial to mesenchymal transition EMT in these cells. Cells acquire characteristics like mesenchymal cells and have the ability to differentiate just like cells at the embryonic stage. This increased EMT will cause resistance against chemotherapeutic drugs. This mechanism does not involve the p53 gene, the so-called p53 loop-independent mechanism (Hou et al., 2019).

MDM2 overexpression and Radiotherapy resistance

MDM2 is also found to be associated with the resistance development to radiation therapy in the tumor cells. When tumor cells are treated with radiation, this causes breaks in their DNA to kill them. As a result, the expression of P53 and MDM2 increases in these cells. Experimental findings have suggested that the accumulation of MDM2 in tumor cells inhibits the transcriptional activation of p53 in these cells and also increases the EMT, which will repair the damages caused by radiation and will make the cells resistant to treatment.

If adenovirus-mediated gene therapy for p53 is given to these tumor cells, this will reduce the resistance and accumulation of MDM2 in the cells and they will respond to radiotherapy. Moreover, some MDM2 inhibitors are being used such as MI-219 that inhibit MDM2 and p53 expression causing a reduction in radiation therapy resistance in tumor cells (Carr et al., 2016).

 

MDM2 overexpression and TKIs resistance

Tyrosine kinase inhibitors TKIs are being used as a targeted therapy against many types of cancers. It is found that the overexpression of MDM2 in the cell causes resistance to TKIs. The overexpression of MDM2 in the tumor cell will stimulate the NF-kB signaling pathway in these cells. This is done through MDM2-P53 loop-dependent resistance. The NF-kB pathway is found to be associated with the development of resistance against tyrosine kinase inhibitors TKIs in tumor cells.

The accumulation of MDM2 increases the NF-kB signaling in tumor cells. That will in turn increase the proliferation of cells and will make them resistant to the therapy by TKIs. However, the exact mechanism of action of MDM2 against TKIs resistance in tumor cells is still unclear and needs further research (Trino et al., 2016).

It is found experimentally that if TKIs and MDM2 inhibitors are given simultaneously to cancer patients, they will have synergistic effects and will cause a more effective cure.

Chemo sensitization by antisense oligonucleotides targeting MDM2

Overexpression of MDM2 in tumor cells makes them resistant to different therapeutic modalities. So, targeting MDM2-p53 can be an effective therapy to reduce the resistance to other therapies such as chemotherapy, radiotherapy, and TKIs in cancer cells. For this purpose, different treatments are being researched that can target MDM2. MDM2 inhibitors and MDM2 antisense oligonucleotides have been proven effective and safe in in-vitro trials. They have shown anti-tumor activity in a significant way.  These MDM2 inhibitors and antisense oligonucleotides have also shown significant outcomes when tested on animal models in-vivo.

When MDM2 oligonucleotides were given, this activated p53 and p21 proteins that are tumor suppressors and ultimately inhibit the growth of tumor cells.

MDM2 inhibitors act as radiosensitizers and chemosensitizers that increase the sensitivity of tumor cells for radiation therapy and chemotherapy respectively. In the tumor cells that have mutations in the p53 gene, MDM2 inhibitors increase the sensitivity of chemotherapeutic drugs in them by both p53-dependent and p53-independent mechanisms.

MDM2 oligonucleotides act as a novel cancer therapeutic class. They have sequences complementary to human MDM2. When given to tumor cells, they will inhibit the translation of MDM2 by binding mRNA. And p53 will not be inhibited by MDM2 as occurs in tumor cells. P53 will act as a tumor suppressor gene and controls the proliferation of cells. It will also start the process of apoptosis or programmed cell death in tumor cells to kill them and reduce the progression of the tumor (Zhang et al., 2005).

Experiments on the use of MDM2 antisense oligonucleotides have been performed on human prostate cancer cells and have shown effective outcomes.

 

References

Carr, M. I., Roderick, J. E., Gannon, H. S., Kelliher, M. A., & Jones, S. N. (2016). Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis. Cell Reports, 16(10), 2618–2629. https://doi.org/10.1016/j.celrep.2016.08.014

Chène, P. (2003). Inhibiting the p53-MDM2 interaction: An important target for cancer therapy. Nature Reviews Cancer, 3(2), 102–109. https://doi.org/10.1038/nrc991

Hou, H., Sun, D., & Zhang, X. (2019). The role of MDM2 amplification and overexpression in therapeutic resistance of malignant tumors. Cancer Cell International, 19(1), 1–8. https://doi.org/10.1186/s12935-019-0937-4

Sciot, R. (2021). Mdm2 amplified sarcomas: A literature review. Diagnostics, 11(3). https://doi.org/10.3390/diagnostics11030496

Trino, S., De Luca, L., Laurenzana, I., Caivano, A., Del Vecchio, L., Martinelli, G., & Musto, P. (2016). P53-MDM2 pathway: Evidences for a new targeted therapeutic approach in B-acute lymphoblastic leukemia. Frontiers in Pharmacology, 7(DEC), 1–7. https://doi.org/10.3389/fphar.2016.00491

Zhang, R., Wang, H., & Agrawal, S. (2005). Novel Antisense Anti-MDM2 Mixed-Backbone Oligonucleotides: Proof of Principle, In Vitro and In Vivo Activities, and Mechanisms. Current Cancer Drug Targets, 5(1), 43–49. https://doi.org/10.2174/1568009053332663