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

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

Ranking 3^rd in the most diagnosed cancer and 2^nd in the leading cause of cancer deaths globally, colorectal carcinoma (CRC) is characterized by the nuanced multi-step carcinogenesis in which it acquires the hallmarks of cancer. Metastatic CRC (mCRC), which majorly appears in an advanced stage, has only a 12% 5-year survival. Notably, immediate resection of mCRC is rare, and the lack of operability motivates the further uncovering of carcinogenic mechanisms to broaden our therapeutic options.
Our group has directed our focus on alternative splicing event which is frequently reported to be hijacked in cancer. By long-read sequencing in CRC organoids, we identified a novel isoform of citrate synthase (CS) with unprecedented exon-skipping (ΔCS) apart from the full-length isoform (CS-WT). Being the rate-limiting enzyme of the tricarboxylic acid cycle, differentially expressed ΔCS in CRC might be explanatory to metabolic reprogramming phenotype and may serve as a potential target of therapeutic intervention.

Aims & Methods

We validated the sequencing results in normal colonic (n=5), primary CRC (pCRC, n=10), and mCRC organoids (n=3), and in-house patient tumor and adjacent normal tissue pairs (n=97) by RT-qPCR. We performed survival analyses with progression-free survival, disease-free survival, and overall survival using all stages, late-stage, and early-stage stratification to explore the clinical relevance of ΔCS. We then proceeded into in vitro experimental validation in CRC cell lines, and in vivo tail-vein injection in NSG mice for investigating the biological phenomena. We also inspected the molecular mechanism to explain the oncogenic phenomena by focusing on the potential structural effects and enzymatic activity upon exon-skipping in ΔCS.

Results

An in-frame spliced CS transcript unreported in RefSeq and GENECODE was found upregulated in patient-derived pCRC organoids over normal organoids, and mCRC organoids have the highest ΔCS expression among the 3 groups. The clinical relevance of ΔCS was proven by 97 pairs of human CRC tissues, demonstrating a significant overexpression over non-tumoral colonic tissues (Paired student’s t-test, p<0.001). Tumor-to-adjacent normal (T/AN) ratio analysis indicated that 80.4% of patients showed increased expression of ΔCS in tumor tissues. A significant association was observed between high ΔCS expression and recurrence (Fisher’s exact test, p<0.01). Kaplan-Meier analyses, stratified by median expression, were performed and the high ΔCS expression group was found associated with lower survival rates when compared to the low ΔCS group in both PFS (Log-rank test, p<0.01) and DFS (p<0.005). We further separated groups by cancer stages and found that high ΔCS expression leads to worse survival outcomes in stage III & IV patients, indicating the metastatic feature associated with this isoform (OS: p<0.005; PFS: p<0.005).
Moreover, ΔCS silencing (shΔCS) demonstrated a more profound inhibition of cell proliferation compared with CS-WT knock-down (shCS-WT) and control. shΔCS groups also showed a significant increase in apoptosis cell counts. Overexpression of ΔCS showed enhanced migration ability in vitro and in vivo. The dimerization effect by ΔCS leads to enhanced CS activity, and metabolic shift to amino acids synthesis was found by GC-MS profiling.

Conclusion

Unannotated ΔCS isoform is explanatory to clinically important metastatic phenotype in CRC through metabolic alteration.

Introduction

Angiogenesis is a hallmark of cancer¹ and it plays a critical role in tumor development, especially in the proliferation and progression of colorectal cancer (CRC)^2,3.
Indeed, tumor vasculature prompts tumor growth and metastasis since it not only provides nutrients and oxygen for the rapid proliferation of cancer cells as well as a route for metastatization, but also affects the infiltration of immune cells to the tumor site. Thus, the targeting of angiogenesis has become an attractive strategy against various human malignancies, including CRC⁴_. In fact, tumor-associated vessels are leaky and poorly efficient due to the dismantlement of the basement membrane and the loss of pericytes, thus preventing proficient drug delivery to the tumor⁵_.
In this context, we demonstrated that Multimerin-2, an extracellular matrix glycoprotein deposited along the blood vessels in juxtaposition between endothelial cells and pericytes, mural cells fundamental in vessels stabilization, is key in the maintenance of vascular homoeostasis⁶_. However, the effects of this molecule on pericytes biology and its putative impact on the immune cells’ recruitment are still poorly understood.

Aims & Methods

Immunohistochemistry analysis on colorectal cancer patients’ biopsies revealed that colon cancer associated vessels display a downregulated expression of Multimerin-2. Thus, we aimed to verify the extent of pericyte recruitment by Multimerin-2-devoid vessels in a colon cancer model and to evaluate how Multimerin-2 loss affects the surrounding tumor microenvironment.
Human umbilical vein endothelial cells (HUVEC) and Human brain vascular pericytes (HBVP) were used as in vitro models for proliferation, functional assays, transcriptomic and proteomics analyses. Wild type and Multimerin-2^-/-C57BL/6 mice were subcutaneously injected with the syngeneic colon cancer MC-38 cells and the tumor sections were analysed by immunofluorescence analyses. Animals were then treated with cisplatin six hours prior sacrificing to evaluate the drug delivery.

Results

We demonstrated that Multimerin-2 affects pericytes migration, adhesion, and proliferation. MC-38 xenografts from Multimerin-2^-/-mice displayed lower number of pericytes along the vessels as well as increased hypoxia, likely due to vascular inefficiency. The delivery of drug to the tumors was impaired as assessed by the analysis of cisplatin DNA adducts, following the treatment with the chemotherapy drug.
Importantly, tumors from Multimerin-2^-/-mice also displayed an altered CD68⁺ cells infiltrate, suggesting that Multimerin-2 loss can affect macrophages recruitment to the tumor site. These results are in agreement with the finding that Multimerin-2 devoid endothelium was more permissive for the migration of the monocytic cell line THP-1. Finally, the loss of Multimerin-2 also associates with the expression of cytokines involved in macrophages recruitment.

Conclusion

In conclusion, loss of Multimerin-2 in CRC associates with poor pericyte recruitment, compromised delivery of drugs to the tumor, and an altered immune response. Our results suggest that Multimerin-2 expression could represent a promising marker to predict the efficacy of immunotherapy and the outcome of CRC patients.

References

1. Hanahan, D. & Weinberg, R. A. Hallmarks of Cancer: The Next Generation. Cell 144, 646–674 (2011).
2. Weis, S. M. & Cheresh, D. A. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med 17, 1359–1370 (2011).
3. Angiogenesis Factors Involved in the Pathogenesis of Colorectal Cancer. Current Health Sciences Journal 5–11 (2013).
4. Rivera, L. B. & Bergers, G. Tumor angiogenesis, from foe to friend. Science 349, 694–695 (2015).
5. Carmeliet, P. & Jain, R. K. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov 10, 417–427 (2011).
6. Pellicani, R. et al. Multimerin-2 maintains vascular stability and permeability. Matrix Biology 87, 11–25 (2020).

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

1. Wakabayashi Y, Kobayashi M, Akashi-Takamura S, et al. A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4. J Immunol. 2006;177(3):1772-9.
2.
3. Xiao L, Li XX, Chung HK, Kalakonda S, et al. RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3. Gastroenterology. 2019;157(3):731-743.
4. Ghait M, Husain RA, Duduskar SN, et al. The TLR-chaperone CNPY3 is a critical regulator of NLRP3-inflammasome activation. Eur J Immunol. 2022;52(6):907-923.
5. Zhou YQ, Bao TS, Xie JX, et al. The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells. Cell Oncol (Dordr). 2023;46(4):1049-1067.
6. Reddy BA, van der Knaap JA, Bot AG, et al. Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell. 2014;53(3):458-70.
7. Liu J, Zhang C, Xu D, et al. The ubiquitin ligase TRIM21 regulates mutant p53 accumulation and gain of function in cancer. J Clin Invest. 2023;133(6):e164354.