Authors: Pengze Yan, Ernesto Rojas Jimenez, Zheqi Li...

Aging is a pivotal risk factor for cancer, yet the underlying mechanisms remain poorly defined. Here, we explore age-related changes in the rat mammary gland by single-cell multiomics. Our findings include increased epithelial proliferation, loss of luminal identity, and decreased naive B and T cells with age. We discover a luminal progenitor population unique to old rats with profiles reflecting precancerous changes and identify midkine (Mdk) as a gene upregulated with age and a regulator of age-related luminal progenitors. Midkine treatment of young rats mimics age-related changes via activating PI3K-AKT-SREBF1 pathway and promotes nitroso-N-methylurea-induced mammary tumorigenesis. Midkine levels increase with age in human blood and mammary epithelium, and higher MDK in normal breast tissue is associated with higher breast cancer risk in younger women. Our findings reveal a link between aging and susceptibility to tumor initiation and identify midkine as a mediator of age-dependent increase in breast tumorigenesis.

Topics: Midkine; Animals; Female; Humans; Rats; Breast Neoplasms; Aging; Carcinogenesis; Mammary Glands, Animal; Cell Proliferation; Mammary Neoplasms, Experimental; Cell Transformation, Neoplastic; Signal Transduction; Methylnitrosourea

PubMed: 39366375
DOI: 10.1016/j.ccell.2024.09.002

Authors: Woosook Kim, Timothy H Chu, Henrik Nienhüser...

BACKGROUND & AIMS

Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1.

METHODS

Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage.

RESULTS

When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1β mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8 T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs.

CONCLUSIONS

In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8 T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.

Topics: Administration, Oral; Animals; Carcinogenesis; Gastric Mucosa; Gastrins; Helicobacter Infections; Helicobacter felis; Humans; Immune Checkpoint Inhibitors; Methylnitrosourea; Mice; Mice, Knockout; Myeloid-Derived Suppressor Cells; Neoplasms, Experimental; Programmed Cell Death 1 Receptor; Signal Transduction; Stomach Neoplasms; Tumor Microenvironment

PubMed: 33129844
DOI: 10.1053/j.gastro.2020.10.036

Authors: R B DuBridge, P Tang, H C Hsia...

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

Topics: Cell Line; Genetic Vectors; Herpesvirus 4, Human; Humans; Kidney; Methylnitrosourea; Mutation; Plasmids

PubMed: 3031469
DOI: 10.1128/mcb.7.1.379-387.1987

Authors: Masami Yamamoto, Sachiyo Nomura, Akihiro Hosoi...

Previously no mouse gastric cancer cell lines have been available for transplantation into C57BL/6 mice. However, a gastric cancer model in immunocompetent mice would be useful for analyzing putative therapies. N-Methyl-N-nitrosourea (MNU) was given in drinking water to C57BL/6 mice and p53 heterozygous knockout mice. Only 1 tumor from a p53 knockout mouse could be cultured and the cells s.c. transplanted into a C57BL/6 mouse. We cultured this s.c. tumor, and subcloned it. mRNA expression in the most aggressive YTN16 subline was compared to the less aggressive YTN2 subline by microarray analysis, and fibroblast growth factor receptor 4 (FGFR4) in YTN16 cells was knocked out with a CRISPR/Cas9 system and inhibited by an FGFR4 selective inhibitor, BLU9931. These transplanted cell lines formed s.c. tumors in C57BL/6 mice. Four cell lines (YTN2, YTN3, YTN5, YTN16) were subcloned and established. Their in vitro growth rates were similar. However, s.c. tumor establishment rates, metastatic rates, and peritoneal dissemination rates of YTN2 and YTN3 were lower than for YTN5 and YTN16. YTN16 established 8/8 s.c. tumors, 7/8 with lung metastases, 3/8 with lymph node metastases and 5/5 with peritoneal dissemination. FGFR4 expression by YTN16 was 121-fold higher than YTN2. FGFR4-deleted YTN16 cells failed to form s.c. tumors and showed lower rates of peritoneal dissemination. BLU9931 significantly inhibited the growth of peritoneal dissemination of YTN16. These studies present the first transplantable mouse gastric cancer lines. Our results further indicate that FGFR4 is an important growth signal receptor in gastric cancer cells with high FGFR4 expression.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Gene Knockout Techniques; Immunocompetence; Methylnitrosourea; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Metastasis; Neoplasm Transplantation; Receptor, Fibroblast Growth Factor, Type 4; Stomach Neoplasms; Tumor Suppressor Protein p53

PubMed: 29532565
DOI: 10.1111/cas.13569

Authors: Peng Song, Xiang Li, Shuai Chen...

YT521-B homology (YTH) domain family (YTHDF) proteins serve as readers that directly recognise m6A modifications. In this study, we aim to probe the role of YTHDF1 in environmental carcinogen-induced malignant transformation of gastric cells and gastric cancer (GC) carcinogenesis. We established a long-term low-dose MNU-induced malignant transformation model in gastric epithelial cells. In vivo and in vitro experiments were conducted to validate the malignant phenotype and characterise the roles of YTHDF1 and its downstream genes in malignant transformation cells. Additionally, we explored downstream m6A modification targets of YTHDF1 using RNA-sequencing, RNA immunoprecipitation, and proteomics analyses, and conducted validation experiments in cell experiments and clinical samples. Long-term low-dose exposure of MNU converted normal Gges-1 cells into malignant cells. YTHDF1 mRNA and protein expression are increased in MNU-induced malignant cells (p<0.001). Meanwhile, YTHDF1 knockdown inhibits the malignant potential of MNU-treated cells (p<0.01). YTHDF1 knockdown specifically suppresses HSPH1 protein, but not RNA levels. RIP-qPCR validates HSPH1 is the target of YTHDF1 (p<0.01). HSPH1 knockdown impairs the malignant potential of MNU-induced transformed cells. The increased expression of the key regulatory factor YTHDF1 in MNU-induced gastric carcinogenesis affects malignant transformation and tumorigenesis by regulating the translation of downstream HSPH1. These findings provide new potential targets for preventing and treating environmental chemical-induced gastric carcinogenesis.

Topics: Stomach Neoplasms; RNA-Binding Proteins; Humans; Animals; Methylnitrosourea; Mice; Carcinogenesis; Cell Transformation, Neoplastic; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Protein Biosynthesis; Male

PubMed: 38444279
DOI: 10.1111/cpr.13619

Authors: Wenchuan Zhou, Jincan He, Guiyan Shen...

Retinal degeneration is a collection of devastating conditions with progressive loss of vision which often lead to blindness. Research on retinal microglial cells offers great therapeutic potential in deterring the progression of degeneration. This study explored the mechanisms underlying the TREM2-mediated protective function of activated microglial cells during retinal degeneration. N-methyl-N-nitrosourea (MNU)-induced retinal degeneration was established in C57BL/6 J (WT) and Trem2 knockout (Trem2) mice. We discovered that MNU treatment led to the concurrent processes of photoreceptor apoptosis and microglia infiltration. A significant upregulation of disease-associated microglia signature genes was observed during photoreceptor degeneration. Following MNU treatment, Trem2 mice showed exacerbated photoreceptor cell death, decreased microglia migration and phagocytosis, reduced microglial PPARγ activation and CD36 expression. Pharmaceutical activation of PPARγ promoted microglial migration, ameliorated photoreceptor degeneration and restored CD36 expression in MNU-treated Trem2 mice. Inhibition of CD36 activity worsened photoreceptor degeneration in MNU-treated WT mice. Our findings suggested that the protective effect of microglia during retinal degeneration was dependent on Trem2 expression and carried out via the activation of PPARγ and the consequent upregulation of CD36 expression. Our study linked TREM2 signaling with PPARγ activation, and provided a potential therapeutic target for the management of retinal degeneration.

Topics: Animals; Mice; Apoptosis; CD36 Antigens; Cell Movement; Membrane Glycoproteins; Methylnitrosourea; Mice, Inbred C57BL; Mice, Knockout; Microglia; Phagocytosis; Photoreceptor Cells; PPAR gamma; Receptors, Immunologic; Retinal Degeneration

PubMed: 39187498
DOI: 10.1038/s41419-024-07002-z

Authors: Dong Guo, Yuntong Sun, Junqi Wu...

Photoreceptor loss is a primary pathological feature of retinal degeneration (RD) with limited treatment strategies. RNA interference (RNAi) has emerged as a promising method of gene therapy in regenerative medicine. However, the transfer of RNAi therapeutics to photoreceptors and the deficiency of effective therapeutic targets are still major challenges in the treatment of RD. In this study, photoreceptor-derived extracellular vesicles (PEVs) conjugated with photoreceptor-binding peptide MH42 (PEVs) were prepared using the anchoring peptide CP05. Transcriptome sequencing was applied to investigate the potential therapeutic target of RD. We then engineered PEVs with specific small-interfering RNAs (siRNAs) through electroporation and evaluated their therapeutic efficacy in N-methyl-N-nitrosourea (MNU)-induced RD mice and Pde6β mutant mice. PEVs were selectively accumulated in photoreceptors after intravitreal injection. Cullin-7 (Cul7) was identified as a novel therapeutic target of RD. Taking advantage of the established PEVs, siRNAs targeting Cul7 (siCul7) were efficiently delivered to photoreceptors and consequently blocked the expression of Cul7. Moreover, suppression of Cul7 effectively protected photoreceptors to alleviate RD both in MNU-induced mouse model and Pde6β mutant mouse model. Mechanistically, PEVs loaded with siCul7 (PEVs-siCul7)-induced Cul7 downregulation was responsible for preventing Cul7-mediated glutathione peroxidase 4 (Gpx4) ubiquitination and degradation, resulting in the inhibition of photoreceptor ferroptosis. In summary, PEVs-siCul7 attenuate photoreceptor ferroptosis to treat RD by inhibiting Cul7-induced ubiquitination of Gpx4. Our study develops a PEVs-based platform for photoreceptor-targeted delivery and highlights the potential of PEVs-siCul7 as effective therapeutics for RD.

Topics: Animals; Retinal Degeneration; Mice; Extracellular Vesicles; RNA, Small Interfering; Cullin Proteins; Disease Models, Animal; Mice, Inbred C57BL; Genetic Therapy; Photoreceptor Cells; Humans; Photoreceptor Cells, Vertebrate; Intravitreal Injections; Methylnitrosourea

PubMed: 39267786
DOI: 10.7150/thno.99484

Review

Authors: M D Reuber

Saccharin is carcinogenic for the urinary bladder in rats and mice, and most likely is carcinogenic in human beings. The neoplasms of the urinary bladder are malignant and invade and metastasize. Male rats are more susceptible to urinary bladder carcinogenesis than female rats. Rats exposed as fetuses develop neoplasms more readily than rats exposed as weanlings. The lesions in the urinary bladder go through the stages of hyperplasia, hyperplastic nodules, and later carcinomas. The male of the human species ingesting saccharin, as for rats, is more susceptible to carcinogenesis of the urinary bladder than the female. Neoplasms of the urinary bladder in rats were not caused by stones, parasites, sodium, or impurities. There is a cocarcinogenic effect between saccharin and methylnitrosurea for the urinary bladder. Even through carcinomas of the urinary bladder are present in rats given the higher doses of saccharin, one was observed in a female rat given 0.5%. Chronic renal disease develops in rats ingesting saccharin. The disease is more advanced at the lower doses than at the higher doses, suggesting that saccharin at the lower doses does not reach the urinary bladder. Early neoplasms are seen in the renal pelvis of rats given the higher doses of saccharin. The risk ratios for urinary bladder carcinomas in human beings increase with both frequency andduration of saccharin usage. Benign and malignant neoplasms at all sites are significantly increased in mice and rats ingesting the higher doses of saccharin. These neoplasms are present in the reproductive and hematopoietic systems, and to a lesser extent in the lungs, vascular system and squamous epithelium. Neoplasms in some organs develop with the lower doses of saccharin. Lymphosarcomas of the lung are significantly increased in rats given 0.01% saccharin. Chronic renal disease in rats given saccharin interferes with the health and life span and consequently with development of neoplasms. Saccharin initiates neoplasms of the skin when its application is followed by croton oil. Epidemiological studies have not been done for neoplasms other than the urinary bladder in human beings.

Topics: Animals; Carcinogens; Diet; Female; Lung Neoplasms; Male; Methylnitrosourea; Mice; Parasitic Diseases; Rats; Saccharin; Sex Factors; Species Specificity; Tosyl Compounds; United States; United States Food and Drug Administration; Urinary Bladder Calculi; Urinary Bladder Neoplasms; Urogenital Neoplasms

PubMed: 363408
DOI: 10.1289/ehp.7825173

Review

Authors: Ana I Faustino-Rocha, Rita Ferreira, Paula A Oliveira...

The administration of chemical carcinogens is one of the most commonly used methods to induce tumors in several organs in laboratory animals in order to study oncologic diseases of humans. The carcinogen agent N-methyl-N-nitrosourea (MNU) is the oldest member of the nitroso compounds that has the ability to alkylate DNA. MNU is classified as a complete, potent, and direct alkylating compound. Depending on the animals' species and strain, dose, route, and age at the administration, MNU may induce tumors' development in several organs. The aim of this manuscript was to review MNU as a carcinogenic agent, taking into account that this carcinogen agent has been frequently used in experimental protocols to study the carcinogenesis in several tissues, namely breast, ovary, uterus, prostate, liver, spleen, kidney, stomach, small intestine, colon, hematopoietic system, lung, skin, retina, and urinary bladder. In this paper, we also reviewed the experimental conditions to the chemical induction of tumors in different organs with this carcinogen agent, with a special emphasis in the mammary carcinogenesis.

Topics: Animals; Breast; Carcinogens; Cell Transformation, Neoplastic; DNA; Female; Humans; Mammary Neoplasms, Experimental; Methylnitrosourea

PubMed: 26386719
DOI: 10.1007/s13277-015-3973-2