Background and Inspiration
Cancer, characterized by its complexity and diversity, has emerged as one of the most formidable challenges confronting global health today. According to data from the Global Burden of Disease (GBD 2021), millions of individuals are diagnosed with cancer annually, a figure that continues to escalate. Cancer not only represents a significant threat to patients' physical well-being but also exerts profound effects on families, society, and the economy.
Figure 1.1. The number curve of incidence and death from GBD 2021 (1980-2021)
Meanwhile, WHO statistics reveal that in 2020 alone, there were approximately 19.2 million new cancer cases and 10 million deaths attributable to cancer worldwide, and cancer is in the top two causes of death in over half of the world's countries. Among these newly diagnosed cases, lung cancer, breast cancer, and colorectal cancer rank as the most prevalent types, underscoring the substantial impact of this disease on human health.
Figure 1.2. National Ranking of Cancer as a Cause of Death at Ages <70 Years in 2019. The numbers of countries represented in each ranking group are included in the legend.
Figure 1.3. Distribution of Cases and Deaths for the Top 10 Most Common Cancers in 2020.
Problem We Are Facing
Currently, chemotherapy and radiotherapy remain the predominant modalities for oncological treatment. By using alkylating agentss, antimetabolites, anti-tumor antibiotics and topoisomerase or mitotic inhibitors, chemotherapy targets rapidly proliferating cancer cells through pharmacological agents, while radiotherapy employs high-energy radiation to eradicate malignant cancer cells through X-ray, gamma-ray or high energy electron beam.
Figure 2.1. Different kinds of chemotherapy agents and their targets.
However, both therapeutic approaches are associated with considerable side effects that inflict additional suffering upon patients beyond their initial diagnosis, and whether the radiotherapy or chemotherapy, there is no doubt that the rapid development of drug resistance mechanisms in cancer cells in the face of the above two means of treatment has also left both doctors and patients helpless. At the same time, even nowadays the gradual development of small molecule targeted drugs have more and more types, like Gefitinib using EGFR as the anti-cancer target, in the T790M site mutation after the mutation has occurred in nearly 50 percent of the patients have a rapid drug-resistance, and the same situation is also very extensive in many other small molecule targeted anticancer drugs.
Figure 2.2. Molecular mechanisms of tumor chemo-resistance and radio-resistance./p>
Figure 2.3. Roles of genes in radioresistant cance cells.
Figure 2.4. The mutation gene on EGFR that cause the resistance of Gefitinib on NSCLC.
With ongoing advancements in science and technology, more precise targeted therapies and immunotherapies—such as CAR-T therapy—are gradually being integrated into clinical practice; however, the financial burden imposed by these innovative treatments often remains prohibitively high for many patients and its limited application on solid tumor is also a significant shortcome.
Figure 2.5. Prices of FDA-approved CAR T-cell products in the United States
Design
We refer to iGEM16_Slovenia and select the MscS (bacterial Mechanosensitive Channel ) (BBa_K1965000) and hTRPC1 (Transient Receptor Potential Channel 1) (BBa_K1965002) calcium ion channels that respond to physical stress on the membrane and can be activated by ultrasound stimulation. By introducing these channels into cancer cells, we aim to generate calcium overloading. Ultrasound stimulation will precisely control cell death by opening the channels and leading to calcium ion influx.
Figure 3.1. Structure of MscS from E.coli. MscS homoheptamer (left) and monomer (right) shown from the side based on the crystal structure.
hNOS2 (Nitric Oxide Synthase 2) is an enzyme that induces NO signaling by catalyzing the reaction: L-arginine + O2 + NADPH + H+ → NO + L-citrulline + NADP+ + H2O. Upregulation of the NO signal can induce the opening of the RyR calcium channel on the endoplasmic reticulum, dramatically increasing intracellular calcium levels.
Figure 3.2. Ca2+ sparks in failing human ventricular myocytes.
To deliver our target proteins to cancer cells, we propose using exosomes as the carrier vehicle (Salunkhe et al., 2020). We will utilize the exosomal transmembrane protein lamp2b, fused with our target proteins, to enhance the loading of MscS, hTRPC1, and hNOS2 into the exosomes. Indeed, Lamp2b has been widely used in the manufacture of engineered exosomes for targeted drug delivery.
Figure 3.3. The role of three LAMP2 isoforms in autophagy.
Our proposed system offers several advantages:
(1) using engineered exosomes ensures highly targeted delivery directly to cancer cells;
(2) specificity can be further improved and precision targeting can be achieved based on cancer properties through additional engineering of exosomes;
(3) precision-controlled cell death can be provided by ultrasound stimulation;
(4) exosome carriers have good biocompatibility and are immunologically safer compared to other nanoparticle carriers;
(5) calcium overload disrupts cell homeostasis, making it difficult for cancer cells to develop drug resistance. Hence, we believe this innovative system could provide specific, effective, and safe cancer therapies, offering new hope for patients who do not respond to existing treatments.
Project Description
Figure 4.1. The overall central design of our CavengerX project.
UM_Macau utilized synthetic biology to construct a controlled calcium overload system for killing cancer cells. We selected the MscS and hTPRC1 ion channels, which are sensitive to ultrasound, as the target proteins and introduced their gene sequences into different plasmids to express these ion channel proteins on the chassis cell HEK293. After successfully expressing the channel proteins, we used the lamp2b protein to package the them, also synthesized using synthetic biology, into vesicles (EVs) using exocytosis. We then allowed the vesicles to fuse with the target cell. Finally, we induced the calcium ion channels on the cancer cell membrane and the endoplasmic reticulum calcium release channel (RyR) in the cancer cells to open under the combined action of ultrasound and hNOS2, significantly increasing the intracellular calcium influx and releasing the calcium the endoplasmic reticulum. This triggered calcium overload in cancer cells, leading to cell death and achieving an anti-cancer effect.
Reference：
[1] GBD results. Institute for Health Metrics and Evaluation. (n.d.-b). https://gbd2019.healthdata.org/gbd-results/
[2] Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 71(3), 209–249. https://doi.org/10.3322/caac.21660
[3] How does chemo work?: Types of chemotherapy. How Does Chemo Work? | Types of Chemotherapy | American Cancer Society. (n.d.). https://www.cancer.org/cancer/managing-cancer/treatment-types/chemotherapy/how-chemotherapy-drugs-work.html
[4] Liu, Y., Zheng, C., Huang, Y., He, M., Xu, W. W., & Li, B. (2021). Molecular mechanisms of Chemo‐ and radiotherapy resistance and the potential implications for cancer treatment. MedComm, 2(3), 315–340. https://doi.org/10.1002/mco2.55
[5] Suda, K., Onozato, R., Yatabe, Y., & Mitsudomi, T. (2009). EGFR T790M mutation: A double role in lung cancer cell survival? Journal of Thoracic Oncology, 4(1), 1–4. https://doi.org/10.1097/jto.0b013e3181913c9f
[6] Cliff, E. R., Kelkar, A. H., Russler-Germain, D. A., Tessema, F. A., Raymakers, A. J. N., Feldman, W. B., & Kesselheim, A. S. (2023). High cost of chimeric antigen receptor T-cells: Challenges and solutions. American Society of Clinical Oncology Educational Book, (43). https://doi.org/10.1200/edbk_397912
[7] Hurst, A. C., Petrov, E., Kloda, A., Nguyen, T., Hool, L., & Martinac, B. (2008). MscS, the bacterial mechanosensitive channel of small conductance. The International Journal of Biochemistry & Cell Biology, 40(4), 581–585. https://doi.org/10.1016/j.biocel.2007.03.013
[8] Ziolo, M. T., Katoh, H., & Bers, D. M. (2001). Expression of inducible nitric oxide synthase depresses β-adrenergic–stimulated calcium release from the sarcoplasmic reticulum in intact ventricular myocytes. Circulation, 104(24), 2961–2966. https://doi.org/10.1161/hc4901.100379
[9] Qiao, L., Hu, J., Qiu, X., Wang, C., Peng, J., Zhang, C., Zhang, M., Lu, H., & Chen, W. (2023). Lamp2a, lamp2b and LAMP2C: Similar structures, Divergent Roles. Autophagy, 19(11), 2837–2852. https://doi.org/10.1080/15548627.2023.2235196
[10] Salunkhe, S., Dheeraj, Basak, M., Chitkara, D., & Mittal, A. (2020). Surface functionalization of exosomes for target-specific delivery and in vivo imaging & tracking: Strategies and significance. Journal of Controlled Release, 326, 599–614. https://doi.org/10.1016/j.jconrel.2020.07.042
