Deletion of the POLR2J4 Gene in Breast Cancer Cells Promotes Bone Metastatic Tumor Growth

Background/Aim: Multiple genetic alterations accumulate during the progression of bone metastasis. Previously, we identified POLR2J4 as a causal gene associated with breast cancer bone metastasis. This study aimed to investigate the roles of POLR2J4 in breast cancer bone metastasis in a murine model.

Materials and Methods: We engineered and knocked out the POLR2J4 gene in luciferase-labeled MDA-MB-231 breast cancer cells using the CRISPR-spCas9 system. Parental or POLR2J4-knockout cells were injected into the systemic circulation of female nude mice. Development and growth of bone metastatic lesions were monitored by weekly bioluminescence imaging. After four weeks, mice were sacrificed for histological analysis of their bone lesions. In addition, transcriptomics analysis using RNA sequencing was performed in POLR2J4 knockout vs. parental cells to investigate gene expression changes. POLR2J4 knockout was confirmed by target sequencing and quantitative RT-PCR.

Results: The POLR2J4 knockout group had significantly increased incidence of bone metastasis, compared with the control group (p<0.01, Fisher’s exact test, n=28/group). However, no statistical significance was observed in the metastatic tumor size measured by bioluminescence signal intensities. Bioluminescence-positive metastatic lesions were confirmed for tumor cells microscopically. Among the genes differentially expressed between the knockout and control cells, CD74 was significantly up-regulated.

Conclusion: POLR2J4 is a negative regulator of breast cancer bone metastasis, and CD74 up-regulation in the POLR2J4 knockout cells contributes to the pro-metastatic phenotype. Overall, our data warrant further investigation on the role of POLR2J4 as a causal gene of breast cancer bone metastasis.