# Loss of ING3 in the Prostate Leads to Activation of DNA Damage Repair Markers

## TL;DR

Removing ING3 in mouse prostate cells increases DNA damage markers but does not cause cancer, suggesting ING3 helps maintain genomic stability.

## Contribution

This study clarifies the role of ING3 in prostate cancer by showing its loss does not cause malignant transformation.

## Key findings

- Loss of ING3 in prostate cells increases DNA damage markers like γH2AX and 53BP1.
- ING3 deficiency does not lead to prostate cancer or PIN lesions in mice.
- ING3 is crucial for genomic stability but not a primary tumor suppressor in prostate cancer.

## Abstract

Prostate cancer is the most common cancer found in men, necessitating the study of genes implicated in the initiation and progression of this disease. The inhibitor of growth family member 3 (ING3) is an epigenetic regulator, whose role in prostate cancer is unknown. The aim of our study was to investigate the functional consequences of prostate-specific ablation of ING3 in mice. While we found normal prostate tissue histoarchitecture in the prostate-specific Ing3 knockout mice, increased expression of DNA-damage-associated markers suggests a role for ING3 in maintaining genomic stability. Altogether, our data show that loss of Ing3 does not lead to neoplastic transformation of the prostate.

Background/Objectives: The inhibitor of growth family member 3 (ING3) acts as an epigenetic reader through physical interactions with histone-modifying enzymes and subsequent chromatin remodelling processes. It is involved in various cellular functions, such as cell cycle control, cell growth, and apoptosis. Although ING3 was assigned tumour suppressor candidate status in some types of cancers, including prostate cancer, some studies suggest it acts to promote growth. To address these contradictory reports regarding its role in the initiation and progression of prostate cancer, we specifically addressed the question of whether ablation of ING3 in the mouse prostate is sufficient to initiate malignant transformation of the prostate and support its (candidate) tumour suppressor status. Methods: To generate the prostate-specific Ing3 knockout mouse, paternal inheritance of the PB-Cre4 transgene was used, while for the generation of a global knockout control, a female mouse harbouring the PB-Cre4 transgene was utilized. To determine the recombination efficiency of the Cre-LoxP system in the prostate at the Ing3 locus, a duplex probe-based digital PCR assay capable of counting undisrupted Ing3 copies was designed. The impact of DNA recombination on the protein level was investigated by immunohistochemical staining of prostate tissue samples. Results: In the prostate-specific knockout, digital PCR analysis revealed mosaic gene deletion. We found recombination efficiencies in the anterior, dorsolateral, and ventral prostate lobes ranging from approximately 15 to 30%. ING3 staining in the prostate was faint with no detectable differences in signal intensity between the knockout specimen and wild-type controls. This low ING3 expression in the prostate is consistent with observations of X-gal staining of an Ing3-LacZ reporter allele. Immunohistochemistry showed increased expression of DNA-damage-associated markers γH2AX and 53BP1. However, no gross anatomical abnormalities or prostate intraepithelial neoplasia (PIN) lesions in the prostate of tissue-specific knockout animals compared to wild-type controls were observed. Conclusions: Altogether, our data provide evidence that disruption of ING3 expression in prostate cells does not lead to malignant transformation and challenges the idea that ING3 acts primarily in a tumour-suppressive manner. Furthermore, this work supports the crucial role of ING3 in maintaining genomic stability, and we confirmed the embryonic lethal phenotype of homozygous Ing3 null mice that is rescued by ectopic expression of ING3.