Genetic inactivation of SKP2 has been shown to effectively prevent cancer initiation and block tumorigenesis. However, direct in vivo evidence for SKP2 on cancer initiation and prostatic microenvironment is still lacking and a SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting SKP2. We therefore have established a prostate-specific human SKP2 knock-in mouse model driven by an endogenous mouse probasin promoter. Overexpression of hSKP2 induces PIN and low-grade carcinoma. RNA-sequencing analysis revealed significant gene expression alterations in EMT, extracellular matrix, and interferon signaling. Single cell deconvolution showed an increase of fibroblast population and a decrease of CD8+ T cell and B cell populations. Consistently with these results from the SKP2 humanized mouse, SKP2 protein is overexpressed in human prostatic hyperplasia, PIN and prostate adenocarcinoma compared to normal prostate tissues. Overexpression of SKP2 markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decreased viability and altered the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.

Abstract Colorectal Cancer (CRC) remains the leading cause of cancer-related mortality worldwide. Inflammation is a key hallmark of many cancers, including CRC. Pro-inflammatory lipid mediators play a key role, while COX-2 inhibiting NSAIDs are promising, their chronic use is linked with unwanted side-effects. In this context, mechanistic studies suggest that targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) with natural products (NP) presents a valuable opportunity to intercept CRC and mitigate those side effects. Here we aimed to perform high-throughput screening (HTS) of NCI NPs library (∼500,000 semi-purified fractions) to identify potential inhibitors of 5-LOX and mPGES1; and further validate the purified compounds using secondary assays. To establish the enzyme activity inhibitory assays, first we developed a stable Human Embryonic Kidney (HEK) 293 cell lines with mPGES-1 and 5-LOX overexpression as well as 5-LOX overexpressing insect cells. Proteins expression was confirmed using western blotting. Lysates from human 5-LOX expressed in Sf9 insect cells and HEK-293 cells, were used to generate an assay format in 384-well microplates suitable for HTS. In this assay format, lysates are preincubated with inhibitors for 20 minutes, stimulated with arachidonic acid (AA) for 5 minutes. The production of free radicals, as a result of the conversion of AA to 5HPETE and LTA4 by 5-LOX activity, is detected upon the addition of 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA). The non-fluorescent H2DCFDA when oxidized by the free radicals, generates a highly fluorescent compound, which can be measured to quantify enzyme activity. The reaction was stopped after 15 minutes upon the addition of acetonitrile. Finally, enzyme activity was calculated by measuring “total relative fluorescence units (RFU) at 485-nm excitation and 530-nm emission spectra. The assay has been optimized using a final volume of 15ul and has a Z’ Factor score of 0.65 and a S/B of 3.5 in 384-well microplates. The 5-LOX activity was completely inhibited by 20uM NDGA, a known inhibitor of 5-LOX. Additionally, a pre-plated NCI library of semi-purified NP fractions (5mg/ml stock in DMSO) has been assessed to validate the assay and identify potential fractions that demonstrate 5-LOX inhibitory activity. The fractions with the most promising activity based on this evaluation will be presented at the meeting. We have also optimized the mPGES1 activity assay using HEK293 cell line overexpressing COX2 and mPGES-1. In this assay format, treatment with AA results in elevated levels of PGE2, as detected in a PGE2 HTRF assay. In summary, these optimized assays will be employed for large scale robotic HTS of NCI NP library to discover and develop safer inhibitors of proinflammatory targets mPGES1 and 5-LOX for intercepting inflammation associated cancers. (Funded by NCI-UG3CA290310-01). Citation Format: Krishnendu Goswami, Nataliya Smith, Ravi Manjhi, Gopal Pathuri, Brandon Somerville, Yurong Song, Venkateshwar Madka, Kajal Biswas, Altaf Mohammed, Robert H. Shoemaker, Matthew J. Hart, Chinthalapally V. Rao. Screening of natural products library against 5-lipoxygenase (5-LOX) and mPGES1 proinflammatory targets for CRC interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3653.

Abstract Background Natural products (NPs) represent a rich source of bioactive compounds with cancer prevention/interception potential. NPs offer unique chemical diversity and a history of safe human use, making them attractive candidates for long-term use. High-throughput screening and mechanistic studies are key to discovering promising leads and translating them into effective and safe cancer prevention and interception strategies. To support the Discovery and Development of Natural Products for Cancer Interception and Prevention (DDNP-CIP) Initiative, we aimed to validate potentially clinically relevant molecular targets (SKP2, TEAD2, 5-LOX and RUNX1) and establish and optimize assays to enable high-throughput NPs library screening for cancer prevention. Methods Target validation was performed using tissue microarrays. SKP2, a component of the SKP2-SCF E3 ligase complex, and TEAD2, a DNA-binding transcription factor, were evaluated for their expression levels in TMAs of prostate and liver cancer, respectively. In addition, cell-based platforms were developed or adapted to identify NPs exhibiting immune-modulating activity or targeting either 5-LOX, a lipid-peroxidizing enzyme, or RUNX1, a transcription factor critical for hematopoietic differentiation and frequently mutated in hematologic malignancies. These assays utilized stably expressing reporter cell lines. Results SKP2 was significantly over expressed in 68.6% of prostate hyperplasia, 97.6% of prostate intraepithelial neoplasia, and 81.9% of adenocarcinoma compared with normal tissue. TEAD2 was highly expressed in liver hyperplasia and significantly upregulated in hepatitis (p = 0.0041), hepatocellular carcinoma (p = 0.0012), and intrahepatic cholangiocarcinoma (p < 0.0001). To identify NPs with immune-modulating activity, an assay using the THP-1 ISRE FRET reporter cell line was adapted and optimized. Reference compounds and initial challenge plates were tested, with several samples eliciting positive responses. A cell line expressing 5-LOX was generated and validated. An inhibition assay using a positive compound nordihydroguaiaretic acid demonstrated dose-dependent inhibition (29% at 0.1uM to 92% at 1uM) upon arachidonic acid treatment without toxicity. To screen NPs for splicing modulating activity, nano-luciferase reporter cell lines with RUNX1 mutations identified in patients with familial platelet disorder with associated myeloid malignancy were generated. Several reference compounds showed positive responses in selected mutations. Summary Validated targets and assay platforms establish a foundation for high-throughput NPs screening. These efforts advance the DDNP-CIP’s mission to identify and develop NPs for cancer prevention and interception. Funded partly by the National Cancer Institute under Contract No. HHSN261201500003I Citation Format: Yurong Song, Brandon Somerville, Kajal Biswas, Karim Baktiar, Liankun Song, Sara Sanders, Tanja Grkovic, Ligia A. Pinto, Ana Catarina Menezes, Paul P. Liu, Matthew J. Hart, Chinthalapally V. Rao, Shugeng Cao, Xin Chen, Xu Wu, Xiaolin Zi, Mark J. Henderson, Barry R. O'Keefe, Altaf Mohammed, Robert H. Shoemaker. Target validation and high-throughput screening assay development for natural product discovery for cancer prevention and interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 942.

Drug repurposing is the process of reusing existing pharmaceuticals for novel clinical purposes, which offers advantages such as streamlined clinical trial access and reduced drug development costs. Clarithromycin (CAM), a member of the macrolide antibiotics family, is a promising candidate for repurposing in cancer therapy due to its known preclinical and clinical immunomodulatory and anticancer properties. In the current study, we investigated whether CAM could be repurposed as a preventive treatment for KRAS-mutant lung cancer, a subtype of lung adenocarcinoma that is strongly associated with heavy smoking. CCSPCre; LSL-KrasG12D mice at an early stage of tumor development were treated with different doses of CAM for 10 weeks. While exhibiting an excellent safety profile, CAM was able to prevent the development of premalignant and malignant lung lesions in a dose-dependent manner. In addition, CAM significantly reduced the infiltration of neutrophils/polymorphonuclear myeloid-derived suppressor cells and inhibited the mRNA expression of protumor inflammatory cytokines IL-6, TNFα, and IL-1β, as well as M2 macrophage markers Fizz1 and Arginase1 in the lung tumor microenvironment. Moreover, we investigated the effect of CAM in reshaping the intestinal and lung microbiome. Long-term CAM usage decreased intestinal microbiome diversity but, more notably, significantly increased the abundance of the probiotic genus Muribaculaceae while decreasing the abundance of Desulfovibrio, a genus associated with the promotion of various malignancies. Taken together, we conclude that CAM could provide promising cancer prevention efficacy in KRAS-mutant lung cancer due to its immunomodulatory properties on the tumor microenvironment and its regulatory effects on the microbiome.

Mono-allelic germline pathogenic variants (GPVs) in MMR genes (MLH1, MSH2, PMS2, and MSH6) predispose individuals to hereditary non-polyposis colorectal cancer (HNPCC), better known now as Lynch syndrome (LS). Bi-allelic GPVs in MMR genes lead to a severe phenotype, constitutional MMR deficiency (CMMRD), that is characterized by early onset of leukemia/lymphoma, colorectal/gastrointestinal tumors, brain tumors, and sarcomas. MMR deficient mouse models have provided many mechanistic and translational insights. Pms2-null (Pms2ko/ko) mice were found to be viable but showed an increase in microsatellite instability and developed lymphomas and sarcomas. One of the most frequently occurring human mutations in PMS2 is a founder mutation NM_000535.5:c.2002A>G (p.I668V), observed in the Canadian Inuit population (1 in 16 Inuits are carriers). This mutation acts as a de novo splice site that creates a 5 bp deletion resulting in a truncated protein (p.I668*). A mouse model with an equivalent germline mutation (Pms2c.1993A>G) results in a splicing defect like that observed in humans and exhibits a significant increase in microsatellite instability and intestinal adenomas on an Apc mutant (Apctm1Rak ) background. We are now further characterizing this model for intestinal tumor initiation and progression using time course experiments and comparing tumor development by histopathological examination with the Pms2ko/ko model. Moreover, we are also examining this model to identify neoantigens, that may lead to development of a new vaccine for PMS2 mutation carriers.This mouse model can be a valuable tool to test the efficacy of new strategies for cancer prevention in PMS2 mutation carriers. We have developed a luciferase based splicing reporter assay in HEK293 cells that can be used to screen splicing modulators to suppress aberrant splicing of this splice site mutation. The efficacy of these modulators to suppress aberrant splicing in the small intestine of mice can be further tested using this mouse model. Kajal Biswas, Yurong Song, Vaishnavi Peddibhotla, Brandon Somerville, Mary Albaugh, Dillon Dierman, Rameesha Mustafa, Altaf Mohammed, Shyam K. Sharan, Robert H. Shoemaker. A novel PMS2 mutant Lynch syndrome mouse model for cancer prevention studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6337.

Lynch syndrome (LS), also known as hereditary non-polyposis colorectal cancer (HNPCC), is caused by monoallelic germline mutations in mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2), which predispose individuals to an increased risk of developing various cancers (mainly colorectal cancer and endometrial cancer). The hallmark of LS-associated tumors is high microsatellite instability (MSI-H) characterized by increased mutations at coding mononucleotide repeats (cMNR), which usually lead to frameshift mutations (FSMs) in coding exons. These recurrent FSMs are thought to play a central role in the increased cancer risk. Vaccines targeting neoantigens produced by FSMs can elicit immune responses, which may prevent or suppress LS tumor formation and progression. Indeed, frameshift neoantigen-based vaccines have been developed and are being tested in LS or MSI-H patients in clinical trials (e.g., NOUS-209). To accelerate vaccine development and optimization, we have developed and characterized an intra-cecal tumor implantation model using tumor fragments derived from a LS mouse model (Msh2LoxP/LoxP;Villin-Cre; VCMsh2). The model was examined for its suitability for preclinical vaccine evaluation in prevention (vaccination followed by fragment implantation) and treatment (fragment implantation followed by vaccination) settings. Primary cecal tumors were adenocarcinoma mixed with mucinous feature. In contrast to subcutaneous tumor implant models that never develop metastasis, metastatic lesions were detected in the lung, liver, and lymph nodes within two months post intra-cecal tumor implantation. In both settings, vaccinated animals had intra-cecal tumors that were smaller in size and a lower rate of metastasis and lower number of metastatic nodules as compared to unvaccinated controls (2 vs. 13 nodules in preventive setting and 1 vs. 4 nodules in therapeutic setting, respectively). FSM analysis by targeted next generation sequencing of a panel of 50 genes revealed that some loci had lower mutation rates and variant allele frequencies, but not at all vaccine targets, in tumors from vaccinated groups compared to controls, indicating that tumor cells harboring certain FSMs targeted by the vaccine were not eliminated. Vaccine target composition and formulation need to be further optimized. Interestingly, plasma cell free DNA (cfDNA) showed lower FSM rates in vaccinated groups, which correlated with a lower metastatic rate. This is consistent with data from VCMsh2 model vaccinated with frameshift peptide vaccines. Additional studies are needed to confirm whether this is neoantigen vaccine specific. In summary, this intra-cecal implantation LS model appears suitable for preclinical neoantigen-based vaccine testing and may help elucidate aspects of tumor cell/DNA shedding and distant metastasis. Yurong Song, Brandon Somerville, Shaneen S. Baxter, Jason D. Marshall, Lei Wei, Chelsea Sanders, Qiang Hu, Song Liu, Alan Hutson, Baktiar Karim, Simone Difilippantonio, Ligia Pinto, Shizuko Sei, Robert H. Shoemaker. Frameshift neoantigen-based vaccine testing in an intra-cecal implantation mouse model of Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6343.

Abstract Monoallelic germline mutations in MMR genes (e.g., MLH1, MSH2, MSH6, and PMS2) predispose individuals to Lynch syndrome (LS) with microsatellite instability (MSI) throughout the genome. Mutations at coding mononucleotide repeats (MNR) usually result in frameshift mutations (FSMs). Recurrent FSMs are thought to play a central role in the increased risk of different types of cancer. Neoantigens produced by FSMs have been shown to elicit immune responses, which is the basis for frameshift neoantigen-based vaccines. To develop a prophylactic vaccine and prevention strategy for this high-risk population, we assessed FSMs during tumorigenesis from histologically normal mucosa and fecal DNA of a LS mouse model using targeted sequencing and a panel of FSMs in MNR regions. Msh2LoxP/LoxP;Villin-Cre mice (VCMsh2) started developing detectable tumors at 7-8 months and median survival was 11.5 months with 100% penetrance. Interestingly, FSMs were detectable not only in tumors and mucosa at 7-8 months, but also in young mice (1 month), embryos and pups although FSMs detected and variant allele frequency (VAF) were very low, indicating that FSMs accumulated over time, and FSMs alone may be not sufficient for tumorigenesis since tumors emerge at older age. To determine whether Msh2 was absent in embryos and pups, immunohistochemical (IHC) analysis was performed. Msh2 was lost in almost all intestine epithelial cells in 2-month-old mice but still present in young pups and embryos with very few cells absent of Msh2, indicating that emerging FSMs in these young pups may be due to decreased Msh2 expression because of delayed Cre function. To determine whether low Msh2 expression could lead to the emergence of FSMs, intestine mucosa from 8-, 10-, and 12-month-old Msh2LoxP/+;Villin-Cre heterozygous mice was sequenced. FSMs were detectable with low VAF, although they didn’t develop tumors. IHC staining revealed that Msh2 was absent in only a few intestine epithelial cells in these heterozygous mice, suggesting that loss of heterozygosity was a late event and rare at 12 months. To determine whether MSI emerged in young VCMsh2 pups and heterozygous mice due to haploinsufficiency of Msh2, MSI was assessed using seven markers and fragment size analysis. One marker (mBat67) showed instability in the intestinal mucosa of heterozygous mice and three showed instability in fecal DNA of one month old VCMsh2 mice. Interestingly, mucosal and fecal samples from a time course study in VCMsh2 mice showed progressive increase in MSI with a good correlation between MSI and FSMs during the tumorigenesis process. In summary, FSMs emerged at an early stage of tumorigenesis in VCMsh2 mice, which correlated with MSI status. Our data indicates that FSMs and MSI status can be used to monitor the tumor development of LS colorectal cancer. Funded by the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261201500003I Citation Format: Yurong Song, Lei Wei, Shaneen S. Baxter, Brandon Somerville, Holli Loomans-Kropp, Chelsea Sanders, Ryan N. Baugher, Stephanie D. Mellott, Todd B. Young, Heidi E. Lawhorn, Teri M. Plona, Qiang Hu, Song Liu, Alan Hutson, Simone Difilippantonio, Ligia Pinto, Steven M. Lipkin, Matthias Kloor, Shizuko Sei, Robert H. Shoemaker. Emergence of frameshift mutations during tumorigenesis in VCMsh2 mouse model of Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1445.

BACKGROUND:Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. METHODS:A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. RESULTS:Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. CONCLUSIONS:We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

PDF file - 325K, Nuclear p53 expression correlates positively with loss of wild type allele in serous epithelial ovarian carcinomas in p53m/+ mutant mice

DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, PMS2, and EPCAM) play an important role in maintaining genomic stability during DNA replication and recombination. Deficiency in MMR resulting from mutations in these genes leads to mutations in microsatellite regions throughout the genome (microsatellite instability; MSI) and in cancer driver oncogenes or tumor suppressor genes, which accumulate over time and eventually lead to cancer formation. Monoallelic germline mutation in MMR genes causes Lynch syndrome (LS). Among LS-related cancer types, the lifetime risk for colorectal cancer (CRC) is the highest (~80%). Frameshift mutations (FSMs) in coding microsatellites produce neoantigens, which have been shown to elicit immune responses. It was thus postulated that they can serve as vaccine targets. To develop a prophylactic vaccine and prevention strategy for this high-risk population, we characterized a LS mouse model (Msh2LoxP/LoxP;Villin-Cre) to determine whether these mice recapitulate the human LS oncogenic process. We found that tumor development was already notable at 7-8 months of age and median survival was 11.5 months. Histopathological analysis showed that tumors were adenoma or adenocarcinoma mixed with mucinous features. Using a targeted sequencing approach, a panel of FSMs in mononucleotide regions were identified in both tumors and histologically normal mucosa, suggesting that Msh2 deletion and FSMs were not sufficient for tumor development. In addition, Apc, Ctnnb, and Trp53 mutations were also observed with low frequency in organoids derived from these tumors, indicating that other driver mutations may be required for tumor initiation and progression, and most FSMs detected in tumors and mucosa were probably passenger mutations. To determine if fecal samples can be used to monitor the FSM load, fecal DNA from different time points was sequenced. We found that FSMs can be detected at 1month of age although the number of FSMs was relatively low compared to that from older mice, indicating that FSMs accumulate over time. MSI detection via fragment analysis confirmed that these tumors were MSI-H. Interestingly, mucosa and fecal samples from a time course study showed progressive increase in microsatellite instability, suggesting the possibility of using MSI score for disease monitoring. Our preliminary data indicates that combined fecal FSM status and MSI score can be potentially used as a biomarker to monitor the tumor development and disease progression for LS colorectal cancer. Funded by the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261201500003I Citation Format: Yurong Song, Shaneen Baxter, Lisheng Dai, Chelsea Sanders, Holli Loomans-Kropp, Brandon Somerville, Ryan N. Baugher, Stephanie D. Mellott, Todd B. Young, Heidi E. Lawhorn, Teri M. Plona, Bingfang Xu, Lei Wei, Qiang Hu, Song Liu, Alan Hutson, Baktiar Karim, Simone Difilippantonio, Ligia Pinto, Matthias Kloor, Steven M. Lipkin, Shizuko Sei, Robert H. Shoemaker. Time course genomic characterization reveals progressive accumulation of mutations during tumor development in a Lynch syndrome mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6518.

BackgroundGenome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed.MethodsTo better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization.ResultsThe organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node.ConclusionsThe established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.

PDF file - 267K, Widespread metabolomic alterations consistent with tumor development are found in blood of SEOC mice compared to wild type mice

PDF file - 161K, Analysis of recombination efficiency of targeted alleles in mutant mice

PDF file - 307K, lntrabursal injection of Adeno-Cre activates conditional alleles in OSE cells and induces histopathological changes of murine OSE post induction

PDF file - 253K, Gene expression analysis

PDF file - 53K, Tumor spectrum in Adeno-Cre injected mice

PDF file - 255K, Immunohistochemical staining for CA125, WT1 and calretinin

The Tripartite Motif Containing 44 (TRIM44) is highly expressed in a variety of tumours. However, the TRIM44's role in endometrial carcinoma (EC) progression remains unknown. To investigate the TRIM44's role in the development and metastasis of EC, we detected TRIM44 expression in EC cell lines and surgical specimens from patients with EC using immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and western blotting analysis. The biological functions of TRIM44 by loss-of-function analysis in RL95-2 and Ishikawa cells were studied. The effect of TRIM44 on the progression of EC in terms of cell proliferation, apoptosis, and invasion was examined and revealed its underlying mechanism in vitro using EC cell lines and in vivo using mouse xenograft models. The TRIM44's expression was positively correlated with EC progression and poor prognosis. The TRIM44 knockdown reduced the EC cell proliferation and invasion while promoting cell apoptosis. Mechanism experiments showed that the TRIM44 interacts with Fibroblast Growth Factor Receptor Substrate 2 (FRS2) and negatively regulates the expression of Bone Morphogenetic Protein 4(BMP4), β-catenin, and Transforming Growth Factor Beta Receptor 1(TGF-βR1). Moreover, the effect of TRIM44 overexpression on EC cell proliferation, invasion, and apoptosis is reversed by the FRS2 knockdown. Our study may provide a new perspective on targeting the TRIM44/FRS2 signaling pathway in treating EC, which deserves further investigation.

Malignant mesothelioma (MMe) is a rare malignancy originating from the linings of the pleural, peritoneal and pericardial cavities. The best-defined risk factor is exposure to carcinogenic mineral fibers (e.g., asbestos). Genomic studies have revealed that the most frequent genetic lesions in human MMe are mutations in tumor suppressor genes. Several genetically engineered mouse models have been generated by introducing the same genetic lesions found in human MMe. However, most of these models require specialized breeding facilities and long-term exposure of mice to asbestos for MMe development. Thus, an alternative model with high tumor penetrance without asbestos is urgently needed. We characterized an orthotopic model using MMe cells derived from Cdkn2a+/−;Nf2+/− mice chronically injected with asbestos. These MMe cells were tumorigenic upon intraperitoneal injection. Moreover, MMe cells showed mixed chromosome and microsatellite instability, supporting the notion that genomic instability is relevant in MMe pathogenesis. In addition, microsatellite markers were detectable in the plasma of tumor-bearing mice, indicating a potential use for early cancer detection and monitoring the effects of interventions. This orthotopic model with rapid development of MMe without asbestos exposure represents genomic instability and specific molecular targets for therapeutic or preventive interventions to enable preclinical proof of concept for the intervention in an immunocompetent setting.