RSPOs (R-spondins) belong to a family of secreted proteins that was the leading cause of various cancers. In iGEM2022, NEU_CHINA team focused on developing a novel cancer targeted-therapy against all RSPOs. We originally designed and characterized a chimeric protein RTAC (RSPO-Targeting Anti-cancer Chimeric protein) through protein engineering. RTAC showed satisfactory anti-cancer effect through inhibition of RSPO-mediated Wnt/β-catenin signaling activation. For further improvement, a S.Cerevisiae-based probiotic was engineered, named as R-yeast, to deliver RTAC to RSPO-hyperactivated colorectal cancer locally. The mating pathway of R-yeast was reprogrammed to specifically sense the tumor environment-specific molecules, thus expressing RTAC in a self-tunable manner. Meanwhile, a unique copper-dependent kill switch was implanted to avoid R-yeast from causing potential environmental hazard. Collectively, we have presented the proof-of-concept of a novel anti-pan-RSPO protein and a probiotic with self-controllable machineries for cancer targeted treatment. Our work thus promises great value in synthetic biological research and translational medicine.
Cancer ranks as a leading cause of mortality and a frowning barrier to increasing life expectancy in every country of the world1. In 2020, there were 19.3 million of estimated new cancer cases and approximately 10.0 million of deaths caused by cancers1. As a genetic disease, the extraordinary diversity creates substantial difficulties for both basic biomedical research and clinical treatment. For decades, researchers have been seeking for personalized medical options to overcome the drawbacks of traditional therapies.
R-spondin (RSPO) encodes like-named secreted protein that participates in multiple critical biological processes including embryonic development, adult tissue homeostasis and oncogenesis2-11. In vertebrates, there are four conserved members in this family, going by the names RSPO1-4. Mechanistically, RSPO synergizes with Wnt ligand to potentiate Wnt/β-catenin signaling cascade, which critically governs cell proliferation, self-renewal and differentiation10. RSPO interacts to the extracellular domain of E3 ubiquitin ligase ZNRF3/RNF43 together with that of LGR4/5/6 receptor, thus inhibiting the E3 ligase by removing ZNRF3/RNF43 from the plasma membrane12,13. By preventing recycling and degradation, this process eventually enables the Wnt ligand (co-)receptors Frizzled and LRP5/6 to more easily contact ligands on the plasma membrane and trigger downstream signaling. As the final outcome of Wnt signaling activation, β-catenin accumulates in the cytosol and eventually translocates into the nucleus to initiate β-catenin-TCF/LEF target gene expression, which critically affects the cell proliferation and cell fate determination.
The prevalent traditional cancer therapies are surgery, chemotherapy and radiotherapy23. Surgery is often the first step in clinical treatment. Removal of the entire tumor tissue through surgery can strongly alleviate the cancer symptoms, however, pain and infection often occur post-surgically. Chemotherapy and radiotherapy, on the other hand, combat cancers in a logically-similar manner. The principle of these approaches is to kill fast-growing cells by chemicals or radiation, disabling the cell growth by breaking down the DNA contents or arresting cell cycles23. Due to the incapability of distinguishing cancer cells from healthy cells, these methods cause vast damage to normal tissues. The homeostasis and regeneration of blood, intestinal epithelium and hair follicle can be severely affected, and the patient can experience strong side effect including fatigue, nausea, hair loss and etc. Until novel therapies are fully developed to cure cancers, these traditional methods are indispensably exercised in cancer clinical treatment.
Cancer targeted therapy has emerged as a pivotal approach in precision medicine. It is based on the molecular dissection of a particular cancer type, and the specific oncogenic gene/protein is considered as the “target” for treatment24,25. As the greatest advantage, this treatment can focus on specifically treating tumor tissues while minimizing collateral damage to surrounding healthy tissues and distant organs. Multiple cancers have been treated or clinical trials conducted using discrete or combinatorial targeted therapy. Despite these progresses, therapeutic options are still largely lacking. Both fundamental biomedical research and translational medicine are particularly interested in the discovery of novel targets and the development of new methodologies.
Based on the previous literatures, we realized RSPO proteins play key roles in oncogenesis as reported in a variety of cancer types19. Despite the pathological importance, the anti-RSPO therapeutic approach is in great need. Inspired by the previous biochemical and structural biological clues12,13,26, we originally designed a novel anti-cancer chimeric secreted protein called RTAC by jointly combining several segments from different known proteins. We proposed that RTAC can effectively bind and neutralize RSPO extracellularly to antagonize RSPO hyperactivation-caused cancers.
First, we used the signaling peptide from Frizzled5 for optimized protein secretion. Then, we added the extracellular domains of ZNRF3 and LGR4 to specifically and potently bind RSPO, with a proper linker region in between to ensure the protein’s flexibility and solubility. At last, we added an IgG Fc region to the carboxyl terminal of the chimeric protein to provide further stability. This design ensured high secretion efficiency of RTAC, high pan-RSPO binding capacity and high protein stability.
After successful engineering, we employed in vitro pull-down assay to demonstrate that RTAC has strong pan-RSPO binding capacity. Following the binding studies, we evaluated RTAC's RSPO-inhibiting potential. We observed that in mammalian cells, the addition of RSPO-containing conditioned medium could augment Wnt signaling intensity by multiple folds from luciferase assay. And we visualized that RTAC treatment could effectively neutralize the Wnt/β-catenin activating effect of RSPO. Besides the luciferase assay, multiple experiments were exercised to confirm the anti-RSPO function of RTAC. Treatment of RTAC effectively nullified the accumulation of cytosolic β-catenin and Wnt target gene expressions caused by additional RSPO, shown by Western Blotting and quantitative RT-PCR assay, respectively.
Based on our successful engineering of RTAC, we further aim to incorporate synthetic biology
to expand the utilization of RTAC. Combining the facts that RSPO-hyperactivation causes 10% of all
CRC and microbiota essentially exists in the intestine, we propose to deliver RTAC via engineered microorganism to the tumor tissue. This microorganism is proposed to be beneficial or commensal in the intestinal tract, which can express RTAC in a self-controllable fashion. Therefore, it can be used as probiotic for cancer prevention and treatment, depending on the tumor (hyperplasia, adenoma or adenocarcinoma) stages.
For delivering the eukaryotic protein RTAC, the selected vessel demands the following three
characteristics: it can secrete fully active/functional RTAC proteins; it can harmlessly
colonize in the intestinal tract; it can self-regulate RTAC expression based on particular signals.
Based on these criteria, we consider the brewer’s yeast (Saccharomyces cerevisiae) as
the most suitable candidate, which is most commonly used in food production with high safety
and reliability. Therefore, we chose S. Cerevisiae as the ideal vector to deliver RTAC.
And based on the protein secretion efficiency, we further adapted the optimal signaling peptide sequence of RTAC particularly in yeast.
See more in "Design"
Tumor microenvironment (TME) has been proved to have special features compared to normal interstitium27. With the new techniques emerged in recent years, researchers discovered that extracellular ATP (eATP) specially exists in diseased tissues, in particular cancer tissues, the concentration of which at TME is 1,000-fold higher than that at the interstitium of the healthy tissue28,29. As the natural receptor of eATP, purinergic receptors can specifically sense eATP and promote tumor growth and progression via the GPCR (G-protein coupled receptor) activity27. We thus decided to employ eATP-purinergic receptors-G protein axis to sense the TME. Through gene editing, we introduced human P2Y2 purinergic receptor and chimeric Gα protein to the engineered probiotic S. Cerevisiae30. This is an important step of our treatment plan to achieve tumor targeting.
The mating pathway, also known as the pheromone pathway, is one of the most well-known signaling pathways in yeast31. It refers to the combination of the pheromone ligand and the GPCR to activate the associated G proteins, a process that amplifies the signal through the MAPK (mitogen-activated protein kinase) cascade and finally activates gene expression30,32. The original ligand of the pathway is pheromone α. In order to reprogram the pathway to sense the eATP at TME, we genetically removed ste2, the original GPCR that senses pheromone α, and embedded the human P2Y2 receptor and the chimeric yeast Gpa1-human Gαi3 protein to the engineered yeast30. And at the transcription level, we engineered RTAC downstream of the mating-responsive transcription factor, pFUS1, as the final outcome of this signaling cascade. The engineered yeast with reprogrammed mating pathway then specifically recognizes eATP from the TME of CRC, and triggers the expression of RTAC based on the amplitude of eATP.
See more in "Design".
As a proof of concept, we experimentally validated that engineered yeast can sense eATP and express substantial amount of RTAC. And co-culturing or the engineered yeast can significantly alleviate Wnt signaling in colon cancer cell and suppress the cancer cell growth.
During the project, our team paid much attention to biosafety and prevention of biological leakage. Thus, we developed a novel kill switch to prevent our engineered yeast from accidentally leaking and affecting the normal life activities of other cells or organisms in the environment. The trace element copper (Cu) is a cofactor for biochemical functions ranging from energy generation to iron (Fe) acquisition, angiogenesis, and free radical detoxification33. According to the high concentration of copper ions in the human intestinal microenvironment, we designed the kill switch based on sensing copper ions. For the kill switch, we replaced the original promoter of the HSP gene (an essential housekeeping gene in yeast that encodes HSF1 protein)34 to CUP1 promoter (a copper ion specific promotor)35,36. This design allows the engineered yeast to survive and grow only at the presence of adequate copper ions (such as in human intestine or culture medium with copper ion addition), while leading to the death of the engineered yeast in normal environment which lacks copper ion33.
See more in “Design”.
1. Pan-RSPO inhibitory effect: as a novel design, RTAC targets all four subtypes of RSPO family proteins with wide application potential in cancer targeted therapy.
2. “Dual targeting” and self-regulated release: while RTAC targets RSPO, the engineered yeast can target the colorectal cancer tissue by sensing TME-specific molecules. The TME-specific molecule triggers self-tunable expression of RTAC to specifically antagonize tumor growth.
3. An innovative kill switch in yeast：we originally designed and applied the copper ion-dependent switches to the engineered yeast to ensure the safety of our experiments and prevent potential biological hazard.
The future clinical application of RTAC will provide a new hope for cancer patients with RSPO hyperactivation by offering a choice with potentially low toxicity and satisfactory anti-cancer effects.
The RTAC-expressing engineered yeast promises a new probiotic approach for colorectal cancer treatment and prevention, which exceeds the usage of free RTAC by possible oral administration and intestinal specific colonization.
See more in "Results".
During the design and development of the RTAC, we conducted a thorough IHP process, going into hospitals and interviewing doctors and researchers to understand the real requirements. We talked with industry experts to optimize the design of the project and make it more feasible and effective.
See more in “Human Practices”.
We focus on safety and ethical issues to ensure patient privacy. We contact the FDA, pharmaceutical companies, and others to enhance drug safety.
See more in “Safety”.
We are engaged in a variety of new endeavors to disseminate synthetic biology and life education to various populations. We also focus on educational equity, reaching out to rural elementary schools and giving public lectures on their nutritional problems.
See more in “Communication”.
During the course of the project, we also exchanged ideas with many teams and gave constructive advice to each other.
See more in “Collaborations”.
We had a partnership with LZU-CHINA this year because of the similarity of projects. From April to October, we helped with each other in many aspects to improve our projects and cover difficulties. Plus, we raised many good ideas to do HP and education with frequent communication.
See more in “Partnership”.
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