Project Description

Abstract

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The World Health Organization reports that cancer is the leading cause of death worldwide. There is an urgent need to develop new targeted medicines to provide patients with more precise treatment options. The Hippo signaling pathway is a key regulator of organ size and tissue homeostasis in animals, and its dysfunction is one of the potential causes of cancer in many organs. In our project, we engineered E. coli to produce two key components (MST2 and STRN3) of the human hippo pathway, the interaction of which leads to dysfunctional hippo pathway signaling. We used this to develop a drug screening system to screen drug candidates that can restore normal Hippo signaling. This will contribute to the development of drugs targeting the Hippo pathway, improve the precision of oncology drugs, and alleviate the suffering of patients.

Inspiration

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As a group of high school students, cancer may seem distant, but in reality, it has the potential to affect those around us profoundly. Our knowledge about cancer stems from both educational lectures and personal encounters with individuals close to us. One such encounter involved my cousin and best friend, Leon, whose mother was diagnosed with bone cancer two years ago. This form of cancer has a five-year survival rate of only 60%. The news came unexpectedly, causing immense fear within our surroundings. Throughout her treatment, Leon's mother concealed her hair loss by wearing a hat. Although the situation eventually improved, we witnessed firsthand the arduousness of chemotherapy and the adverse physical and psychological effects caused by numerous medications. This experience prompted us to develop a deeper understanding of tumor treatment and drug advancement. We discovered that targeted anti-tumor drugs can concentrate at a higher level in specific localized areas, enhancing drug efficacy while mitigating toxic side effects and minimizing harm to normal tissues and cells. This revelation sparked our excitement and motivated us to embark on the development of a targeted drug screening system.

1.Current situation of cancer and cancer patients

Cancer arises from the unchecked growth of cancer cells that deplete the body's resources and disrupt the function of nearby organs. As a major contributor to global mortality, cancer accounted for almost one-sixth of all deaths in 2020. Its eradication is exceptionally challenging for several reasons:

• It is an endogenous disease. Even though cancer cells are bad human cells, they are still a type of human cell, which means that targeted drugs are currently poorly targeted and often eradicate normal cells in the body by mistake, causing serious damage to the body.
• It is not a single disease, it is a combination of thousands and thousands of diseases. Because of the diversity of cancers, pharmaceutical companies can only develop drugs for a small number of patients at a time, and the development cost of each new drug requires a large amount of manpower, time, and money, resulting in slow progress.
• Mutational resistance. Cancer cells have not only retained their basic ability to evolve, but they have become more powerful and are able to find ways to avoid the effects of drugs and develop resistance.

The following treatments are currently available on the market for cancer patients:

• Surgical treatment, including surgery, and minimally invasive surgical treatment, is the most effective treatment for most tumors.
• Radiotherapy
• Pharmacological treatments, including traditional chemotherapy, immunotherapy, and molecular targeted therapy.

Even after chemotherapy, patients still need to be assisted with anticancer drugs, because anticancer drugs can effectively inhibit the spread and metastasis of cancer cells and control the condition within a certain range. However, anti-cancer drugs at this stage have many side effects, such as allergic reactions, leading to a sharp decrease in the white blood cells and platelets in the blood cells, thus leading to the patient's body weakness and anemia, and will cause irreversible damage to the stomach and intestines. The symptoms are nausea, vomiting, abdominal pain and diarrhea. In addition, it can lead to hair loss, myocardial ischemia, palpitations, and numbness in the hands and feet. All this causes irreversible damage to the body, but anti-cancer drugs are still very necessary, so the development of new anti-cancer drugs is very necessary and important.

2. Interaction between MST2 and STRN3 in the hippo signaling pathway, a target for novel anti-cancer drug screening

2.1 Hippo signaling pathway

The Hippo pathway, initially discovered in Drosophila, controls tissue development, regulates organ sizes, and is critical in cellular proliferation, differentiation, growth, apoptosis, and tissue repair. In mammals, the hippo pathway is seen as an evolutionarily conserved pathway, which consists of a kinase cascade, MST1/2, and LATS1/2, as well as downstream effectors, transcriptional coactivators YAP, and YAZ. The hippo pathways in mammals include the STRN3-PP2A complex in the upstream which regulates MST1/2. MST1/2 and its scaffold protein SAV1 could phosphorylate LATS1/2 and its scaffold MOB1 with the help of WWC1–3. The phosphorylated MOB1 can also directly promote the activation of LATS1/2 by inducing the conformational change of LATS1/2. The activated LATS1/2 phosphorylated and inactivated YAP/TAZ, preventing it from translocating into the nucleus and binding to transcription factors TEAD1–4. Hippo pathway activity is frequently deregulated in different human cancers.

2.2 Reasons for using the Hippo signaling pathway as a target in screening anti-cancer drugs.

Signaling pathways refer to a collection of pathways responsible for the transmission of signals from extracellular spaces to intracellular spaces via receptors present on the cell membrane. These pathways play a pivotal role in regulating multiple aspects of cell behavior, such as cell differentiation, proliferation, and apoptosis. Disruption in signaling pathways can trigger aberrant cellular proliferation and ultimately lead to the formation of malignant tumors. The Hippo signaling pathway is a critical regulator of organ size across different species, and its malfunction contributes to the onset of diverse cancers, including liver, gastric, colon, and lung cancer. Therefore, the identification and implementation of targeted therapies against Hippo dysfunction could potentially increase the availability of precise and favorable therapeutic options for specific cancer types. Several drugs that target the Hippo signaling pathway (which is subdivided into different targets within the pathway) are currently being tested in laboratories or clinical trials. For example, serine/threonine protein kinases 3 and 4 (STK3 and STK4) are key components of the Hippo pathway. This regulates cell proliferation and death, making it a potential therapeutic target in acute myeloid leukemia (AML). Laboratory studies have shown that knocking down and pharmacologically inhibiting STK3 and STK4 can inhibit the proliferation of AML cells in vitro. Another class of compounds that targets the Hippo pathway is currently undergoing clinical development. Results from a phase I trial led by researchers at The University of Texas MD Anderson Cancer Center showed that the YAP/TEAD inhibitor VT3989 was well tolerated and produced durable anti-tumor responses in patients with advanced malignant mesothelioma and other NF2-mutated tumors. This is the first clinical trial to pharmacologically target the Hippo-YAP-TEAD pathway, and it demonstrates that targeting YAP can work in the clinic to provide real benefits for patients.

2.3 Potential of MST2-STRN3 interactions in the hippo signalling pathway as a target for anti-cancer drug screening

MST2 is a threonine protein kinase that inhibits cell proliferation and resists cell death by inhibiting the function of intranuclear YAP (Qin, Tian et al. 2013). STRN3 is an essential regulatory subunit of protein phosphatase 2A (PP2A), which leads to YAP activation and shutdown of Hippo signaling by recruiting and promoting dephosphorylation of MST1/2, resulting in oncogene overexpression and ultimately excessive proliferation of cancer cells (Tang, Fang et al. 2020). Therefore, we need to find small molecules that can disrupt the interaction between MST2 and STRN3 and restore the protein function of MST2 to turn on the Hippo signaling pathway and inhibit the further proliferation of cancer cells.

3.Reporting system---AlphaScreen

3.1 High-throughput screening

High-throughput screening (HTS) facilitates the discovery of potential compounds for the advancement of novel pharmaceuticals. HTS offers the benefits of enhanced sensitivity, precision, and efficiency, enabling rapid acquisition of significant data. Presently, various approaches exist for the identification of promising compounds, as depicted in the accompanying

Methods	Principles
FRNT	A non radiative energy transfer that allows observation of interaction between proteins in living cells.
NMR	The signals generated by hydrogen nuchleus in magnetic fields are reconstructed by a computer in process imagining so we can find out if drug works.
SPR	It's an optical reaction. We used it to find out signals between biomolecules by change of angles.
DSF	The amout of fluorescent dye bound to the structurally altered protein is measured during heating the sample. So we get data about protein stability.

The pharmaceutical substances utilized in high-throughput screening (HTS) have all been sanctioned by the United States Food and Drug Administration (FDA), thereby ensuring their efficacy and quality for human application. The rationale behind the Alpha screen's selection for this particular application can be attributed to its superior sensitivity, cost-effectiveness, a robust signal output, and the ability to robustly accommodate interactions spanning both low and high affinity.

3.2 AlphaScreen

AlphaScreen is a bead-based, non-radioactive Amplified Luminescent Proximity Homogeneous Assay. When a biological interaction brings the beads together, a cascade of chemical reactions acts to produce an amplified signal. On laser excitation, a photosensitizer in the "Donor" bead converts ambient oxygen to a more excited singlet state. The singlet state oxygen molecules diffuse across to react with a thioxene derivative in the Acceptor bead and further activates fluorophores contained in the same bead. The fluorophores subsequently emit light at 520-620 nm. In the absence of an Acceptor bead, singlet oxygen falls to ground state and no signal is produced.

3.3 Benefits of the AlphaScreen Report Screening System

The AlphaScreen Report-Screening System could be an ideal reason for screening anti-cancer drugs that "disrupt MST2 and STRN3 interactions in the hippo signaling pathway":

a: Very high sensitivity

The signal is a cascade reaction. Very small amounts of molecular interactions can be detected with sensitivity up to the nanomolar level.

b: High signal/Background ratios

Because of this combination of strong signal and low background, AlphaScreen S/B ratios tend to be outstanding.

c: True miniaturization

AlphaScreen assays can be easily miniaturized to 5 uL or less with no change in reagent concentration, no need for assay re-optimization, and no sacrifice in assay robustness.

d: Optimal versatility in assay design

If you can bind, label, or cleave, you can measure with AlphaScreen.

e: Low affinity to high-affinity interactions

AlphaScreen can measure virtually any strength of biological interaction. Unlike alternative technologies, AlphaScreen is suitable for low-affinity binding assays.

4.Overview

The research aims to screen candidate drugs that hinder the interaction between MST2 and STRN3 in the hippo signaling pathway to correct the abnormal hippo pathway, leading to a reduction of cancer cells and the treatment of cancer. In this investigation, E. coli was utilized as the chassis cells to express individual purified human MST2 and STRN3 proteins, respectively. These proteins were then assembled onto donor/acceptor magnetic beads within the AlphaScreen reporter-screening system through specific interactions. Subsequently, anticancer drugs were screened in a high-throughput manner by observing whether the system glowed upon addition of the drug candidates. The new drug candidates underwent testing on cancer cells in vitro and through Drosophila colorectal cancer models to validate their anti-cancer properties. This study could generate novel ideas for the treatment of colorectal cancer and prove to be an effective treatment strategy for improving the survival rate of patients.