Team:BNUZH-CHINA/Description
Inspiration
Many of our team members have witnessed their loved ones or individuals close to them suffering from cancer. Unfortunately, despite the multitude of therapies employed, cancer continues to prevail and ultimately claim their lives. Cancer not only inflicts physical pain upon patients, but also brings immense sadness and shock to those who care for them. Additionally, their family was severely affected by the burden of cancer treatment. Having witnessed such tragedies, our team is resolute in developing a new solution to improve this dire situation.
The five-year survival rate of many types of cancer (e.g., lung cancer, liver cancer, pancreatic cancer) is still quite low. While certain types of cancer can be managed, they are seldom curable. Is it truly impossible for humans to overcome cancer? Fortunately, we realized the potential of clinical application of ferroptosis. Tumor cells tend to accumulate intracellular iron, rendering them susceptible to ferroptosis. The challenge lies in inducing ferroptosis in cancer cells. Through literature reviewing, we found that VNP20009, a strain of attenuated Salmonella typhimurium, can be used as the microbial chassis that selectively targets tumors and inhibits their growth. Based on this, we devised a plan to utilize genetically engineered bacteria, specifically VNP20009, to induce ferroptosis in tumor cells.
The five-year survival rate of many types of cancer (e.g., lung cancer, liver cancer, pancreatic cancer) is still quite low. While certain types of cancer can be managed, they are seldom curable. Is it truly impossible for humans to overcome cancer? Fortunately, we realized the potential of clinical application of ferroptosis. Tumor cells tend to accumulate intracellular iron, rendering them susceptible to ferroptosis. The challenge lies in inducing ferroptosis in cancer cells. Through literature reviewing, we found that VNP20009, a strain of attenuated Salmonella typhimurium, can be used as the microbial chassis that selectively targets tumors and inhibits their growth. Based on this, we devised a plan to utilize genetically engineered bacteria, specifically VNP20009, to induce ferroptosis in tumor cells.
Background
Cancer is an encompassing term for a diverse cluster of illnesses that have the potential to impact any region of the human body. One critical characteristic of cancer is the swift proliferation of irregular cells surpassing their normal limitations. Consequently, these cells have the ability to infiltrate neighboring body parts and disseminate to other organs. This phenomenon is recognized as metastasis and is predominantly accountable for fatalities resulting from cancer.
According to the latest global Cancer burden data in 2020 released by the International Agency for Research on Cancer (IARC), the number of new cancer cases worldwide in 2020 was nearly 20 million, with almost 10 million reported death cases. The global burden of cancer is of significant concern1. In this context, China accounted for 4.569 million new cancer cases and 3.003 million deaths, ranking first in the world2.
As the aging population continues to grow, industrialization and urbanization processes accelerate, and factors like unhealthy lifestyles accumulate, the number of cancer incidence and death cases in China is still on the rise3.
Due to the characteristics of cancer, such as its typically lengthy course, complex diagnostic and therapeutic techniques, and significant treatment challenges, the associated high treatment costs impose a heavy economic burden on patients' families and society as a whole4,5. The substantial economic burden brought about by cancer treatment and the physical and mental health issues it entails severely impact the patients' quality of life and societal participation6. Although these issues have been recognized, they remain inadequately addressed, placing immense burdens on patients, families, and society. To fundamentally tackle these problems, there is an urgent need to identify a more precise and safe cancer treatment method.
Currently, the mainstream methods for cancer treatment primarily consist of physical therapies, exemplified by surgery and radiation therapy; chemical treatments, represented by chemotherapy and hormone therapy; and biological therapies, represented by immunotherapy and cancer vaccines.
However, these methods all carry certain disadvantages. For instance, surgical operations may prove insufficient in managing the local spread or potential metastasis of cancer cells. The surgical trauma is substantial, and in some anatomical locations, surgery is intricate and carries significant risks7. Radiotherapy has a considerable degree of toxic side effects on the body, and long-term toxic reactions and dose-limiting toxicity must be thoroughly estimated prior to the therapy8. The utilization of chemotherapy drugs can readily induce adverse reactions within the patient's digestive system and other areas, such as the heart, liver, and urinary system, thereby impacting the physical recovery of patients9.
In the context of immunotherapy, immune checkpoint inhibitors enhance the immune response, thereby assuming an anti-tumor role. However, they can disrupt the immune homeostasis mediated by the PD-1 pathway and destroy the immune tolerance of T cells to autoantigens, leading to a high incidence of immune-related adverse reactions among patients receiving antibody therapy10. Additionally, due to the long-term duration of immune checkpoint inhibitor treatment compared to chemotherapy, the occurrence of adverse immune reactions is delayed11. Cancer vaccines are faced with the dilemma of solving the inherent low response rate of cancer to immunotherapy, overcoming the immune escape of tumor cells, and how to carry out cancer immunotherapy strategies for tumors with low mutation load.
Bacterial therapy for cancer relies on the virulence, autonomous locomotion ability, and tendency and invasiveness of bacteria in nature, enabling them to regulate the tumor immune microenvironment and induce anti-tumor immune responses12. The inhibition of immune monitoring within the tumor, the increase of nutrients in the necrotic tumor core, and the unique hypoxic microenvironment of the tumor work together to enable microrganisms to selectively grow in the tumor without causing adverse effects on other healthy tissues of the host. Therefore, bacterial therapy is expected to become a more suitable cancer treatment method.
We have learnt that ferroptosis, a form of iron-dependent regulated cell death (RCD) discovered in recent years, exhibits key biochemical characteristics, including an intracellular redox homeostasis imbalance and the generation of lipid peroxidation13. These factors result in the accumulation of iron-dependent lipid peroxides within cells. This buildup heightens lipid ROS levels in the cytoplasm, causing plasma membrane blistering, mitochondrial contraction, reduced or absent ridges, and increased membrane density13. In comparison to normal cells, tumor cells possess a higher iron content and greater sensitivity to ferroptosis due to their high metabolic activity. Therefore, inducing ferroptosis in tumor cells as a means of cancer treatment has emerged as a prominent research focus14.
The molecular mechanism of ferroptosis is related to iron toxicity, lipid peroxidation, and antioxidant system. Compared to normal cells, tumor cells exhibit a greater reliance on iron within the microenvironment for their growth. When the dynamic equilibrium of intracellular iron is disrupted, an excess of Fe2+ can generate oxygen radicals and reactive oxygen species (ROS) through iron-dependent Fenton reactions. This activation subsequently triggers iron-dependent enzymes like lipoxygenases, leading to cellular oxidative stress and lipid peroxidation, ultimately inducing a process known as ferroptosis15. Ferroptosis in cells is usually accompanied by an imbalance between the antioxidant defense system and lipid peroxidation. The intracellular antioxidant defense system is unable to limit the degree of lipid peroxidation and maintain intracellular stability, ultimately resulting in cell death. So, cells that undergo ferroptosis experience unchecked lipid peroxidation, which ruptures the cell membrane and leads to cell death.
Research indicates that the accumulation of iron within cells, the occurrence of excessive lipid peroxidation, and the suppression of the antioxidant defense system can all culminate in ferroptosis in tumor cells. Furthermore, with the unveiling of several pathways associated with ferroptosis, numerous approaches have emerged in tumor treatment that leverage cell ferroptosis. These include exploiting the susceptibility of ferroptosis-sensitive tumors to induce the demise of these cells, inhibiting ferroptosis defense mechanism to make ferroptosis resistant tumor cells sensitive to ferroptosis again, combining ferroptosis inducer and traditional tumor treatment scheme to synergistically inhibit tumor growth. However, due to the problems of effectiveness, targeting and safety, there are still many challenges in the development of drugs targeting ferroptosis.
This year, the BNUZH-China 2023 iGEM team aims to employ engineered bacteria to induce ferroptosis in tumor cells. The objective is to enhance the targeting and safety of cancer therapy, overcome current limitations in treatment, and offer a fresh approach to cancer treatment.
According to the latest global Cancer burden data in 2020 released by the International Agency for Research on Cancer (IARC), the number of new cancer cases worldwide in 2020 was nearly 20 million, with almost 10 million reported death cases. The global burden of cancer is of significant concern1. In this context, China accounted for 4.569 million new cancer cases and 3.003 million deaths, ranking first in the world2.
As the aging population continues to grow, industrialization and urbanization processes accelerate, and factors like unhealthy lifestyles accumulate, the number of cancer incidence and death cases in China is still on the rise3.
Due to the characteristics of cancer, such as its typically lengthy course, complex diagnostic and therapeutic techniques, and significant treatment challenges, the associated high treatment costs impose a heavy economic burden on patients' families and society as a whole4,5. The substantial economic burden brought about by cancer treatment and the physical and mental health issues it entails severely impact the patients' quality of life and societal participation6. Although these issues have been recognized, they remain inadequately addressed, placing immense burdens on patients, families, and society. To fundamentally tackle these problems, there is an urgent need to identify a more precise and safe cancer treatment method.
Currently, the mainstream methods for cancer treatment primarily consist of physical therapies, exemplified by surgery and radiation therapy; chemical treatments, represented by chemotherapy and hormone therapy; and biological therapies, represented by immunotherapy and cancer vaccines.
However, these methods all carry certain disadvantages. For instance, surgical operations may prove insufficient in managing the local spread or potential metastasis of cancer cells. The surgical trauma is substantial, and in some anatomical locations, surgery is intricate and carries significant risks7. Radiotherapy has a considerable degree of toxic side effects on the body, and long-term toxic reactions and dose-limiting toxicity must be thoroughly estimated prior to the therapy8. The utilization of chemotherapy drugs can readily induce adverse reactions within the patient's digestive system and other areas, such as the heart, liver, and urinary system, thereby impacting the physical recovery of patients9.
In the context of immunotherapy, immune checkpoint inhibitors enhance the immune response, thereby assuming an anti-tumor role. However, they can disrupt the immune homeostasis mediated by the PD-1 pathway and destroy the immune tolerance of T cells to autoantigens, leading to a high incidence of immune-related adverse reactions among patients receiving antibody therapy10. Additionally, due to the long-term duration of immune checkpoint inhibitor treatment compared to chemotherapy, the occurrence of adverse immune reactions is delayed11. Cancer vaccines are faced with the dilemma of solving the inherent low response rate of cancer to immunotherapy, overcoming the immune escape of tumor cells, and how to carry out cancer immunotherapy strategies for tumors with low mutation load.
Bacterial therapy for cancer relies on the virulence, autonomous locomotion ability, and tendency and invasiveness of bacteria in nature, enabling them to regulate the tumor immune microenvironment and induce anti-tumor immune responses12. The inhibition of immune monitoring within the tumor, the increase of nutrients in the necrotic tumor core, and the unique hypoxic microenvironment of the tumor work together to enable microrganisms to selectively grow in the tumor without causing adverse effects on other healthy tissues of the host. Therefore, bacterial therapy is expected to become a more suitable cancer treatment method.
We have learnt that ferroptosis, a form of iron-dependent regulated cell death (RCD) discovered in recent years, exhibits key biochemical characteristics, including an intracellular redox homeostasis imbalance and the generation of lipid peroxidation13. These factors result in the accumulation of iron-dependent lipid peroxides within cells. This buildup heightens lipid ROS levels in the cytoplasm, causing plasma membrane blistering, mitochondrial contraction, reduced or absent ridges, and increased membrane density13. In comparison to normal cells, tumor cells possess a higher iron content and greater sensitivity to ferroptosis due to their high metabolic activity. Therefore, inducing ferroptosis in tumor cells as a means of cancer treatment has emerged as a prominent research focus14.
The molecular mechanism of ferroptosis is related to iron toxicity, lipid peroxidation, and antioxidant system. Compared to normal cells, tumor cells exhibit a greater reliance on iron within the microenvironment for their growth. When the dynamic equilibrium of intracellular iron is disrupted, an excess of Fe2+ can generate oxygen radicals and reactive oxygen species (ROS) through iron-dependent Fenton reactions. This activation subsequently triggers iron-dependent enzymes like lipoxygenases, leading to cellular oxidative stress and lipid peroxidation, ultimately inducing a process known as ferroptosis15. Ferroptosis in cells is usually accompanied by an imbalance between the antioxidant defense system and lipid peroxidation. The intracellular antioxidant defense system is unable to limit the degree of lipid peroxidation and maintain intracellular stability, ultimately resulting in cell death. So, cells that undergo ferroptosis experience unchecked lipid peroxidation, which ruptures the cell membrane and leads to cell death.
Research indicates that the accumulation of iron within cells, the occurrence of excessive lipid peroxidation, and the suppression of the antioxidant defense system can all culminate in ferroptosis in tumor cells. Furthermore, with the unveiling of several pathways associated with ferroptosis, numerous approaches have emerged in tumor treatment that leverage cell ferroptosis. These include exploiting the susceptibility of ferroptosis-sensitive tumors to induce the demise of these cells, inhibiting ferroptosis defense mechanism to make ferroptosis resistant tumor cells sensitive to ferroptosis again, combining ferroptosis inducer and traditional tumor treatment scheme to synergistically inhibit tumor growth. However, due to the problems of effectiveness, targeting and safety, there are still many challenges in the development of drugs targeting ferroptosis.
This year, the BNUZH-China 2023 iGEM team aims to employ engineered bacteria to induce ferroptosis in tumor cells. The objective is to enhance the targeting and safety of cancer therapy, overcome current limitations in treatment, and offer a fresh approach to cancer treatment.
Brief Introduction
Due to the hypoxic living microenvironment and the suppression of immune monitoring of tumor tissue,
bacteria can selectively grow in tumor tissue. Abnormal iron and redox metabolism during tumor growth and
proliferation lead to increasing levels of iron ions and reactive oxygen species within tumor cells,
making them highly sensitive to ferroptosis. Building on this understanding, we design a novel cancer
treatment that utilizes engineered bacteria to mediate ferroptosis in tumor cells.
Our project aims to use attenuated S. typhimurium as chassis and will introduce custom-built plasmids, which can induce ferroptosis in tumor cells. To further enhance the bacteria's tumor-specific targeting ability, we will engineer S. typhimurium to express carcinoembryonic (CEA) antigen-specific single chain antibody fragments (scFv) on the outer membrane. Once the engineered bacteria reach the tumor tissue, they will respond to the microenvironment by activating anaerobic promoter and secrete glucose oxidase into the tumor cells through the type III secretion system, thereby producing H2O2 and hydroxyl radicals (·OH) to facilitate lipid peroxidation. In addition, we design to engineer S. typhimurium to mediate the silencing of SLC7A11 gene after it invades tumor cells. This process will lead to a decrease of glutathione (GSH) and inactivation of GPX4, further accelerating the occurrence of ferroptosis in tumor cells.
In our safety module, a toxin-antitoxin system is designed to prevent the loss of two functional plasmids. And engineered bacteria are able to self-destruct by taking doxycycline.
The fight against cancer has a powerful new ally in our project, which utilizes genetically-engineered bacteria to provide a safe and precise cancer therapy alternative. Boasting a sophisticated targeting mechanism and bolstered efficacy, our approach is new and effective strategies in cancer treatment.
Our project aims to use attenuated S. typhimurium as chassis and will introduce custom-built plasmids, which can induce ferroptosis in tumor cells. To further enhance the bacteria's tumor-specific targeting ability, we will engineer S. typhimurium to express carcinoembryonic (CEA) antigen-specific single chain antibody fragments (scFv) on the outer membrane. Once the engineered bacteria reach the tumor tissue, they will respond to the microenvironment by activating anaerobic promoter and secrete glucose oxidase into the tumor cells through the type III secretion system, thereby producing H2O2 and hydroxyl radicals (·OH) to facilitate lipid peroxidation. In addition, we design to engineer S. typhimurium to mediate the silencing of SLC7A11 gene after it invades tumor cells. This process will lead to a decrease of glutathione (GSH) and inactivation of GPX4, further accelerating the occurrence of ferroptosis in tumor cells.
In our safety module, a toxin-antitoxin system is designed to prevent the loss of two functional plasmids. And engineered bacteria are able to self-destruct by taking doxycycline.
The fight against cancer has a powerful new ally in our project, which utilizes genetically-engineered bacteria to provide a safe and precise cancer therapy alternative. Boasting a sophisticated targeting mechanism and bolstered efficacy, our approach is new and effective strategies in cancer treatment.
References
1 Deo, S. V. S., Sharma, J. & Kumar, S. GLOBOCAN 2020 Report on Global Cancer Burden: Challenges and
Opportunities for Surgical Oncologists. Ann Surg Oncol 29, 6497-6500, doi:10.1245/s10434-022-12151-6
(2022).
2 McGuire, S. World Cancer Report 2014. Geneva, Switzerland: World Health Organization, International Agency for Research on Cancer, WHO Press, 2015. Advances in Nutrition 7, 418-419, doi:10.3945/an.116.012211 (2016).
3 Chen, W. et al. Cancer statistics in China, 2015. CA Cancer J Clin 66, 115-132, doi:10.3322/caac.21338 (2016).
4 Garaszczuk, R., Yong, J. H. E., Sun, Z. & de Oliveira, C. The Economic Burden of Cancer in Canada from a Societal Perspective. Curr Oncol 29, 2735-2748, doi:10.3390/curroncol29040223 (2022).
5 Guha, A. et al. Socio-Economic Burden of Myocardial Infarction Among Cancer Patients. Am J Cardiol 141, 16-22, doi:10.1016/j.amjcard.2020.11.005 (2021).
6 Pham, H., Torres, H. & Sharma, P. Mental health implications in bladder cancer patients: A review. Urol Oncol 37, 97-107, doi:10.1016/j.urolonc.2018.12.006 (2019).
7 Chen, Z. et al. Surgical stress and cancer progression: the twisted tango. Mol Cancer 18, 132, doi:10.1186/s12943-019-1058-3 (2019).
8 Wang, K. & Tepper, J. E. Radiation therapy-associated toxicity: Etiology, management, and prevention. CA Cancer J Clin 71, 437-454, doi:10.3322/caac.21689 (2021).
9 Perez-Herrero, E. & Fernandez-Medarde, A. Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. Eur J Pharm Biopharm 93, 52-79, doi:10.1016/j.ejpb.2015.03.018 (2015).
10 Khan, S. & Gerber, D. E. Autoimmunity, checkpoint inhibitor therapy and immune-related adverse events: A review. Semin Cancer Biol 64, 93-101, doi:10.1016/j.semcancer.2019.06.012 (2020).
11 Wu, Y. L. et al. A consensus on immunotherapy from the 2017 Chinese Lung Cancer Summit expert panel. Transl Lung Cancer Res 7, 428-436, doi:10.21037/tlcr.2018.04.15 (2018).
12 Raman, V., Van Dessel, N., O'Connor, O. M. & Forbes, N. S. The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella. J Immunother Cancer 7, 44, doi:10.1186/s40425-018-0490-z (2019).
13 Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060-1072, doi:10.1016/j.cell.2012.03.042 (2012).
14 Salnikow, K. Role of iron in cancer. Semin Cancer Biol 76, 189-194, doi:10.1016/j.semcancer.2021.04.001 (2021).
15 Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A 113, E4966-4975, doi:10.1073/pnas.1603244113 (2016).
2 McGuire, S. World Cancer Report 2014. Geneva, Switzerland: World Health Organization, International Agency for Research on Cancer, WHO Press, 2015. Advances in Nutrition 7, 418-419, doi:10.3945/an.116.012211 (2016).
3 Chen, W. et al. Cancer statistics in China, 2015. CA Cancer J Clin 66, 115-132, doi:10.3322/caac.21338 (2016).
4 Garaszczuk, R., Yong, J. H. E., Sun, Z. & de Oliveira, C. The Economic Burden of Cancer in Canada from a Societal Perspective. Curr Oncol 29, 2735-2748, doi:10.3390/curroncol29040223 (2022).
5 Guha, A. et al. Socio-Economic Burden of Myocardial Infarction Among Cancer Patients. Am J Cardiol 141, 16-22, doi:10.1016/j.amjcard.2020.11.005 (2021).
6 Pham, H., Torres, H. & Sharma, P. Mental health implications in bladder cancer patients: A review. Urol Oncol 37, 97-107, doi:10.1016/j.urolonc.2018.12.006 (2019).
7 Chen, Z. et al. Surgical stress and cancer progression: the twisted tango. Mol Cancer 18, 132, doi:10.1186/s12943-019-1058-3 (2019).
8 Wang, K. & Tepper, J. E. Radiation therapy-associated toxicity: Etiology, management, and prevention. CA Cancer J Clin 71, 437-454, doi:10.3322/caac.21689 (2021).
9 Perez-Herrero, E. & Fernandez-Medarde, A. Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. Eur J Pharm Biopharm 93, 52-79, doi:10.1016/j.ejpb.2015.03.018 (2015).
10 Khan, S. & Gerber, D. E. Autoimmunity, checkpoint inhibitor therapy and immune-related adverse events: A review. Semin Cancer Biol 64, 93-101, doi:10.1016/j.semcancer.2019.06.012 (2020).
11 Wu, Y. L. et al. A consensus on immunotherapy from the 2017 Chinese Lung Cancer Summit expert panel. Transl Lung Cancer Res 7, 428-436, doi:10.21037/tlcr.2018.04.15 (2018).
12 Raman, V., Van Dessel, N., O'Connor, O. M. & Forbes, N. S. The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella. J Immunother Cancer 7, 44, doi:10.1186/s40425-018-0490-z (2019).
13 Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060-1072, doi:10.1016/j.cell.2012.03.042 (2012).
14 Salnikow, K. Role of iron in cancer. Semin Cancer Biol 76, 189-194, doi:10.1016/j.semcancer.2021.04.001 (2021).
15 Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A 113, E4966-4975, doi:10.1073/pnas.1603244113 (2016).
