Introduction

p53 is frequently mutated in cancer which leads to attenuated DNA-binding or complete loss of p53. Further assuming, that p53 primarily acts as a transcription factor, it has therefore long been considered as undruggable. The presence of multiple protein isoforms and their respective cellular ratios often determine therapeutic success and therefore provide novel targets for drug treatment of p53. Those mainly include FLp53, Δ40p53, Δ133p53, and Δ160p53. FLp53 is responsible for the proliferative and metabolic regulation. Δ40p53, an N-terminally truncated isoform lacking TAD I (1 – 40 aa), is transcriptionally inactive. Together with FLp53, Δ40p53 can address other tumor-suppressive genes. Higher Δ40p53:FLp53 ratios are linked to increased apoptotic and decreased tumor recurrence rates. The Δ133p53 and Δ160p53 isoforms are known for their tumor-progressive abilities and are considered as negative regulators of tumor suppressive p53. We introduced an exon‑resident and intein-luciferase-based reporter system (Truong, Tümen et al., 2021, Nat. Cell Biol.) that first successfully quantifies FLp53, Δ40p53, Δ133p53, and Δ160p53 non-invasively and in real-time. Its access to high-throughput screenings allowed us to monitor the differential p53 isoform expression for 4,863 anti-cancer compounds and identified two potential drug candidates to provide new p53-tailored therapeutic options for the effective treatment of colorectal cancer (CRC).

Aims & Methods

We introduced three exon-specific intein luciferase reporter systems (EXSISERS) into tumor suppressor p53 (TP53) coding exon 2, 4, and 7 of HCT116 cells. Knock-in of the reporter systems was conducted with CRISPR/Cas9. We demonstrated spatial separation and integrity of both, p53 and the intein-luciferase reporters by Western blot and immunofluorescence. The luminescent read-out of EXSISERS provides the quantity of cellular p53 isoforms in real-time. We screened the p53 isoform expression via EXSISERS for over 4,863 anti-cancer compounds (MCE, HY-L025) and measured the luminescent signal alterations with a Multiplate Reader during the drug treatment.

Results

With the implementation of EXSISERS, we demonstrated the advantageous application over conventional protein detection methods. Immunoblot and immunoprecipitation, for instance, failed to quantify the isoform diversity of p53. Moreover, absolute p53-peptide quantification via mass spectrometry showed low FLp53-peptide sensitivity, which exclusively allowed distinction between tumor suppressive vs. oncogenic p53. EXSISERS, however, was able to non-invasively distinguish FLp53, Δ40p53, Δ133p53, and Δ160p53. The screen of 4,863 small molecules led to the identification of specific drug candidates upregulating pro-apoptotic p53 isoform members. We validated the efficacy of these drugs on different colon cancer cell lines. The drugs were applicable in nanomolar concentrations and resulted in efficient induction of cell death (60-90%). Drug treatment of primary colonic organoids has shown to predominantly induce cell death in malignant cells, but not in healthy colonic organoids.

Conclusion

In summary, our study demonstrated the integration of EXSISERS into TP53 and allowed us to rapidly quantify essential p53 isoforms to provide new approaches to treat CRC via p53. By screening various anti-cancer compounds, we identified potential drug candidates efficiently upregulating tumor suppressive p53 and consequently inducing cell death in CRC. Here, we underline the particular importance of our new universally applicable method to expand the CRC treatment options by drugging p53.

References

Truong, D.-J.J.; Phlairaharn, T.; Eßwein, B.; Gruber, C.; Tümen, D.; Baligács, E.; Armbrust, N.; Vaccaro, F.L.; Lederer, E.-M.; Beck, E.M.; et al. Non-Invasive and High-Throughput Interrogation of Exon-Specific Isoform Expression. Nat. Cell Biol. 2021, doi:10.1038/s41556-021-00678-x .

Disclosure

Nothing to disclose

Introduction

Tumor-related peripheral tolerance is responsible for the limited ability of the immune system to control tumor growth. In addition to the known functions of p53 as a tumor suppressor, there is increasing evidence that p53 also regulates innate and adaptive immune responses. Wild-type p53 affects the innate immune system by modulating macrophage function via the induction of cytokine expression, thereby sharpening the immune system to suppress tumorigenesis. When p53 is mutated or deleted, as is the case in many tumors, macrophages are not attracted to the tumor and immune evasion occurs. In addition to soluble factors that activate innate immunity, the glycerophospholipid phosphatidylserine also plays a key role in peripheral tolerance. This lipid is localized on the inner side of the plasma membrane facing the cell matrix. The exposure of phosphatidylserine to the outside of the cell, e.g. during apoptosis, causes the induction of peripheral tolerance and helps to suppress inflammation. The localization of phosphatidylserine is regulated by the flippase complex. Here, we demonstrate for the first time that p53 exerts a direct influence on the composition of the plasma membrane and can control immune responses by regulation of flippase protein expression. When p53 is deleted or mutated, phosphatidylserine is externalized and tolerance towards the tumor is induced.

Aims & Methods

We generated stable HCT116 cell lines using CRISPR/Cas9 that have either a complete knock-out of p53 or knock-out of the DNA-binding domain of p53. We assessed exposure of phosphatidylserine and induction of cell death by flow cytometry and determined the metabolic activity of the cells by measuring NAD(P)H and ATP. In addition, the expression of flippase subunits was analyzed by Western blot and quantitative PCR. Comparative transcriptome analysis of wild-type and p53 knock-out cells was performed by next-generation sequencing to further investigate the regulation of phosphatidylserine exposure.

Results

Following the knock-out of p53 or deletion of the p53 DNA-binding domain, we detected a translocation of phosphatidylserine to the outer side of the plasma membrane. Of importance, cells with knock-out of the p53 DNA-binding domain, as well as cells with complete p53 knock-out, remained metabolically active. Proliferation was comparable to wild-type cells. Thus, cell death can be excluded as the underlying mechanism of phosphatidylserine externalization. Subsequently, the expression of essential flippase subunits was examined. We observed a downregulation of the protein expression of ATP11A and ATP11C subunits in p53 knock-out cells. This explains why there is an increased phosphatidylserine exposure in these cells and how tumors with p53 deletions may evade the immune system. Moreover, transcriptome analysis revealed that genes upregulated in p53 knock-out cells are mainly induced by interferons, demonstrating involvement of the immune system.

Conclusion

Here we report a novel function of p53 as an immunomodulator. In addition to its tumor suppressor properties, p53 regulates plasma membrane composition. Deletion of p53 or its DNA-binding domain, as frequently observed in malignant tumors, leads to the downregulation of essential flippase subunits and thus, to the externalization of phosphatidylserine. In this way, peripheral tolerance can be induced and the malignant tumor can escape the immune system. Thus, we describe a novel function of p53 in mediating peripheral tolerance in malignant tumors.

Introduction

Colorectal cancer is one of the most frequent malignant tumors worldwide. With increasing use of prognostic biomarkers, molecularly stratified treatment of colorectal cancer is improving in clinical practice. However, identifying stable and easily detectable biomarkers for clinical use remains a challenge. Among these biomarkers, epigenetic changes, especially DNA methylation, are the most promising candidates for clinical practice. Prominent examples of such methylation markers are SEPTIN9, the Secretin Receptor (SCTR), and Engrailed Homeobox 1 (EN1). The methylation preferentially affects CpG islands within promoter regions of these genes. Aberrant methylation of CpG islands in the promoter region can be associated with the silencing of tumor suppressor genes and promotes tumorigenesis and tumor growth.

Aims & Methods

Our goal is to develop an innovative diagnostic tool for the reliable detection of malignant colon tissue based on the recognition of specific methylation patterns. This reporter system will detect hypermethylated genetic regions in vivo using luminescence. Suitable biomarkers for this approach will be identified by methylation profiling of colorectal carcinoma cell lines as well as organoids from patients with colorectal cancer.
Our reporter system consists of two constructs. The first construct, the key element of which is the methyl-binding domain of Methyl-CpG-binding domain protein 1 (MBD1), recognizes hypermethylated CpG islands that are frequently located within the promoter region. The second construct can bind with high specificity to the genomic region of interest via the sgRNA of a deadCas9. When the two components combine in close proximity, a SplitTEV protease induces the newly developed flipNluc luciferase. The emitted luminescence signal then detects the tumor in a highly specific manner and can be quantified. In addition, whole-genome bisulfite sequencing on colon carcinoma cell lines HCT116 and CaCo2 and organoids from colon biopsies were used to identify additional specific hypermethylated regions. The raw data was analyzed bioinformatically using Bismark and the R package DSS.

Results

We verified the functionality of our new assay by assessing the induction of the newly developed flipNLuc luciferase by the TEV protease. We detected a significant increase in luminescence, confirming the functionality of the luciferase. Thereafter, we validated the reporter system by analyzing the methylation marker genes EN1 and SCTR, which are typically hypermethylated in colorectal carcinoma. In particular, significantly increased luminescence was measured compared to the unmethylated housekeeping genes GAPDH and GPI. To optimize the sensitivity and specificity of our new detection system, additional genes with differentially methylated regions were identified during bioinformatic analysis of the sequencing data. Currently, we are adapting our reporter system to these newly identified genes to validate their potential role as new specific biomarkers.

Conclusion

Reliable DNA methylation biomarkers for clinical practice in the diagnosis of colorectal cancer are still limited. The developed method allows the highly specific identification of malignant tissue based on the detection of hypermethylated CpG islands. Furthermore, our method can be used for targeted screening for new methylation markers. By replacing the sgRNAs of deadCas9, our newly developed method is not limited to the detection of colon tumors and can be adapted to the diagnosis of other tumor entities.

Introduction

Canopy FGF signaling regulator 3 (CNPY3) is a protein predominantly localized in the endoplasmic reticulum. Its expression is increased in several cancers, including colorectal cancer (CRC). However, how CNPY3 affects CRC progression and the function of CNPY3 translocation to the nucleus in CRC is not clear.

Aims & Methods

This study aimed to investigate the role of CNPY3 in the development of CRC. In vitro, the impacts of CNPY3 on CRC growth were evaluated by 5-Ethynyl-2′-deoxyuridine staining, colony formation, wound healing, transwell migration, and apoptosis assay. Tumor xenograft mouse models and orthotopic mouse models of colorectal cancer metastasis were used to assess in vivo. Furthermore, the underlying mechanism of CNPY3 in CRC was revealed by RT-qPCR, western blot, LC-MS/MS analysis, immunofluorescence staining, coimmunoprecipitation, GST-pulldown, confocal microscopy, ubiquitination assays, and rescue experiments.

Results

CNPY3 was significantly upregulated in CRC tissue and cell lines, correlating with their poor prognosis. Functional experiments demonstrated that CNPY3 enhances CRC proliferation, migration, invasion, and tumor growth. Notably, our investigation reveals a nucleus translocation of cnpy3 by activation of nuclear localization sequences through protein conformational changes. Meanwhile, nuclear localization of CNPY3 promotes the binding of TRIM21 and p53 and forms a CNPY3-TRIM21-p53 complex, thereby promoting the ubiquitination-proteasomal degradation of wild-type p53 protein, inhibiting the related genes of the TP53 signaling pathway, and promoting apoptosis of tumor cells. In vivo and in vitro, inhibiting the degradation of p53 was able to suppress CNPY3 in cancer-promoting tumors, whereas the knockdown of CNPY3 in p53^-/- HCT116 cells did not significantly inhibit CRC.

Conclusion

CNPY3, after nuclear translocated, binds to TRIM21 and p53 and stabilizes the TRIM21–p53 interaction. This CNPY3–TRIM21–p53 complex facilitates TRIM21‐mediated p53 ubiquitinoylation and causes p53 proteasome degradation. Collectively, these findings support that CNPY3, an oncogene with CRC progression and poor prognosis, serves as a biomarker and therapeutic target correlated to suppress p53 function.

References

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Introduction

Colonoscopy is the "gold-standard" technique for early diagnosis and resection of colorectal cancer precursor lesions. However, this technique has limitations, and can result in missed adenomas and polyps.
In recent years, artificial intelligence (AI) systems have been developed for the automatic detection of polyps. This study aims to investigate the impact of an AI system applied in real time on the detection rate, compared with conventional detection by the endoscopist.

Aims & Methods

A prospective observational study was carried out by seven gastroenterologists in the Digestive Endoscopy Unit of a tertiary hospital. Colonoscopies were performed on consecutive patients for polyp screening or surveillance. High-definition colonoscopes were used for the examinations and an AI system was applied in real time.
Each procedure consisted of two phases: (1) an initial assessment was performed by the endoscopist who withdrew the endoscope after reaching the cecum, identifying all visualized lesions without resecting them, and (2) the colonoscope was reintroduced up to the cecal pole with an AI device. The automatic detection of the AI device was assessed by means of a light and sound signal.
The primary objective was to compare the detection rate of adenomas and polyps. Secondary objectives were to compare the total number of polyps and serrated lesions detected, as well as detection of polyps by size, location and morphology, with and without the device.

Results

The sixty-six patients included in the analysis were examined simultaneously by colonoscopy, and the AI device was applied. The AI system identified a greater mean number of adenomas (2.85 vs 2.41, p<0.05) and polyps (3.68 vs 2.92, p<0.05), as well as a greater number of serrated lesions (0.83 vs 0.52, p <0.05), compared with the endoscopist. No significant differences were found in the adenoma detection rate (78.8% vs 75.7%; p > 0.05).
AI significantly increased the detection of small lesions (178 vs 135, p<0.001), and those of Paris type IIa flat morphology (165 vs 123, p<0.001). There were no differences in the detection of lesions larger than 5 mm or with other morphologies.

Conclusion

In a high-risk population, the application of an AI system allowed a significantly higher number of polyps and adenomatous lesions to be detected, at the expense of small lesions and those with flat morphology, compared with conventional evaluation by the endoscopist. There were no differences in the adenoma detection rate.