Contribution

Our contribution is based upon the 2024 YiYe-WuHan iGEM project, we have focused on how our work can serve as a foundation for future iGEM teams by offering practical tools, strategies, and solutions to global problems like colorectal cancer (CRC). We consistently focused on developing tools and frameworks that other teams can build upon, both in terms of plasmid design and system modeling. By targeting TEAD4 in CRC through siRNA, our work offers promising methods that can be used as the basis for future innovations in cancer therapy. Our project leverages the Hippo signaling pathway, in which TEAD4 serves as a core transcription factor, promoting the growth and metastasis of CRC cells.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide(Baidoun et al., 2021; Dekker et al., 2019). According to the American Cancer Society, the general lifetime risk of developing CRC is approximately 1 in 23 for men and 1 in 25 for women. The mortality rate for CRC patients under the age of 55 has been increasing by approximately 1% per year since the mid-2000s. It is estimated that in 2024, CRC will cause around 53,010 deaths (American Cancer Society, 2024, www.cancer.org/cancer/types/colon-rectal-cancer/about/key-statistics.html). Our research focuses on exploring novel treatments for CRC by targeting transcriptional coactivators, specifically through the inhibition of the TEAD4 transcription factor in the Hippo signaling pathway.

One of our contributions to future iGEM teams lies in the development of new plasmids that downregulate TEAD4 using the siRNA mechanism. RNA interference (RNAi) technology, awarded the Nobel Prize in Physiology or Medicine in 2006, has revolutionized gene silencing approaches. However, the application of synthetic siRNA has encountered several stability challenges. To overcome these obstacles, researchers have introduced chemical modifications that significantly improve both the delivery efficiency and the stability of siRNA, particularly in cancer treatments (Hoogenboezem et al., 2024).

In our project, we are using plasmids that downregulate TEAD4, which plays a crucial role in CRC cell proliferation and metastasis. We have created both basic and improved parts, which will be made available for future teams. Our plasmids use siRNA to downregulate TEAD4 (Figure 1) in the Hippo signaling pathway, and we optimized our protocol by transfecting the SW480 (CRC cell line) with polyethyleneimine (PEI). The inhibition efficiency was found to be dose-dependent on the concentration of shRNA targeting TEAD4, and the results demonstrated the successful construction of the sh-TEAD4-1 and sh-TEAD4-2 plasmids. This work shows that TEAD4 is a viable therapeutic target for CRC, and future teams can build upon our findings by adapting these plasmids for other cancer types.

TEAD4 is a critical transcription factor in the Hippo signaling pathway, regulating cell proliferation, apoptosis, and organ size. When YAP/TAZ are dephosphorylated, they bind to TEAD4 in the nucleus, promoting the expression of genes that drive cell growth and survival. In this pathway, particularly hyperactivation of the YAP/TAZ-TEAD4 axis, leads to uncontrolled cell proliferation and tumorigenesis (Zanconato et al., 2018). Recent studies have shown that molecules capable of inhibiting the YAP/TAZ-TEAD4 complex could serve as potential cancer drugs (Baidoun et al., 2021; Liu et al., 2023; Sabnis, 2023).

In addition, we developed a plasmid that inhibits TEAD4 from entering the nucleus by deleting its nuclear localization sequence (NLS). This mutation allows the plasmid to act as a powerful competitor to wild-type TEAD4, providing another potential therapeutic strategy for CRC.

Our project also explored how energy stress, ER stress, and oxidative stress impact the Hippo pathway. Oxidative stress, triggered by reactive oxygen species (ROS) such as hydrogen peroxide, can stimulate cellular proliferation. We found that energy stress significantly affects the YAP/TAZ coactivators (Glorieux et al., 2024; Liu et al., 2024). Future iGEM teams could build on this work by investigating how environmental stresses could be modulated to alter TEAD4 activity, potentially offering new therapeutic strategies.

We utilized bioinformatics to model the relationship between the dosage of sh-TEAD4-1 and sh-TEAD4-2 and their effects on SW480 CRC cells. This provides future teams with a theoretical framework for designing TEAD4-targeted inhibition plasmids. In our statistical model of YiYe Wuhan, we offer a way to predict the efficiency of siRNA-based therapies by analyzing the impact of plasmid dosage on cell proliferation, migration, and ROS expression.

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Additionally, we developed a predictive model to assess how varying dosages of plasmids interact with tumor cells. This model provides a valuable tool for future iGEM teams by offering insights into the potential efficacy of different cell therapies. By offering this resource, we aim to support other teams in reducing experimental costs and optimizing their strategies for cell therapy research.

While our siRNA-based strategy shows promise, it also presents several challenges. These include off-target effects, delivery system efficiency, and the need for sustained siRNA delivery over time. Additionally, silencing TEAD4 completely in normal cells could cause harmful effects since TEAD4 is also involved in normal cellular functions. Future iGEM teams could explore alternative approaches, such as modulating upstream signals to selectively inhibit TEAD4 activity without disrupting normal cells(Papavassiliou et al., 2024; Pobbati and Hong, 2013).

One promising area of research focuses on blocking the nuclear entry of YAP/TAZ. Recent studies have shown that the protein SOX9 can bind to YAP and facilitate its nuclear translocation, suggesting that inhibiting SOX9-YAP interactions could effectively prevent YAP from entering the nucleus (Zheng and Pan, 2019). In our improved experiments, we constructed a plasmid with a deletion in the nuclear localization sequence (NLS) of TEAD4, aimed at colorectal cancer therapy. The results were consistent with our expectations, highlighting the potential efficacy of this approach. This strategy offers future iGEM teams a promising route to manipulate the Hippo pathway while minimizing harm to normal cells.

The tools and frameworks developed during the YiYe-WuHan iGEM 2024 project are designed to empower future iGEM teams in their research efforts. Our plasmids, bioinformatics models are valuable resources that can be adapted and expanded for other cancer research projects. TEAD4 has emerged as a promising target for CRC treatment(Sun et al., 2022), and our project lays the foundation for future teams to explore novel therapeutic strategies. By sharing our work, we aim to support other iGEM teams in developing innovative solutions for cancer and other diseases.

Baidoun, F., Elshiwy, K., Elkeraie, Y., Merjaneh, Z., Khoudari, G., Sarmini, M.T., Gad, M., Al-Husseini, M., and Saad, A. (2021). Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets 22, 998-1009. 10.2174/1389450121999201117115717.

Dekker, E., Tanis, P.J., Vleugels, J.L.A., Kasi, P.M., and Wallace, M.B. (2019). Colorectal cancer. Lancet 394, 1467-1480. 10.1016/S0140-6736(19)32319-0.

Glorieux, C., Liu, S., Trachootham, D., and Huang, P. (2024). Targeting ROS in cancer: rationale and strategies. Nat Rev Drug Discov 23, 583-606. 10.1038/s41573-024-00979-4. Hoogenboezem, E.N., Patel, S.S., Lo, J.H., Cavnar, A.B., Babb, L.M., Francini, N., Gbur, E.F., Patil, P., Colazo, J.M., Michell, D.L., et al. (2024). Structural optimization of siRNA conjugates for albumin binding achieves effective MCL1-directed cancer therapy. Nat Commun 15, 1581. 10.1038/s41467-024-45609-0.

Liu, Y., Yu, D., Ge, X., Huang, L., Pan, P.Y., Shen, H., Pettigrew, R.I., Chen, S.H., and Mai, J. (2024). Novel platinum therapeutics induce rapid cancer cell death through triggering intracellular ROS storm. Biomaterials 314, 122835. 10.1016/j.biomaterials.2024.122835.

Liu, Y., Zhang, B., Zhou, Y., Xing, Y., Wang, Y., Jia, Y., and Liu, D. (2023). Targeting Hippo pathway: A novel strategy for Helicobacter pylori-induced gastric cancer treatment. Biomed Pharmacother 161, 114549. 10.1016/j.biopha.2023.114549.

Papavassiliou, K.A., Gargalionis, A.N., and Papavassiliou, A.G. (2024). Direct YAP/TAZ-TEAD inhibitor paves the way toward realizing cancer mechanomedicine. Pharmacol Res 206, 107287. 10.1016/j.phrs.2024.107287.

Pobbati, A.V., and Hong, W. (2013). Emerging roles of TEAD transcription factors and its coactivators in cancers. Cancer Biol Ther 14, 390-398. 10.4161/cbt.23788.

Sabnis, R.W. (2023). Novel Compounds as TEAD Inhibitors for Treating Cancer. ACS Med Chem Lett 14, 1152-1153. 10.1021/acsmedchemlett.3c00346.

Sun, Y., Hu, L., Tao, Z., Jarugumilli, G.K., Erb, H., Singh, A., Li, Q., Cotton, J.L., Greninger, P., Egan, R.K., et al. (2022). Pharmacological blockade of TEAD-YAP reveals its therapeutic limitation in cancer cells. Nat Commun 13, 6744. 10.1038/s41467-022-34559-0.

Zanconato, F., Battilana, G., Forcato, M., Filippi, L., Azzolin, L., Manfrin, A., Quaranta, E., Di Biagio, D., Sigismondo, G., Guzzardo, V., et al. (2018). Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4. Nat Med 24, 1599-1610. 10.1038/s41591-018-0158-8.

Zheng, Y., and Pan, D. (2019). The Hippo Signaling Pathway in Development and Disease. Dev Cell 50, 264-282. 10.1016/j.devcel.2019.06.003.