USP22 overexpression fails to augment tumor formation in MMTV-ERBB2 mice but loss of 1 function impacts MMTV promoter activity 2

35 The USP22 deubiquitinase, a component of the SAGA histone modifying complex, is 36 overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. 37 We reported previously that Usp22 loss in mice impacts execution of several signaling pathways 38 driven by growth factor receptors such as ERBB2. To determine whether changes in USP22 39 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or 40 deletion alleles of Usp22 into mice bearing the MMTV-NIC transgene, which drives both rat 41 ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in 42 mammary tumor formation. We found that USP22 overexpression in mammary glands did not 43 further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases 44 were observed. However, deletion of Usp22 significantly decreased tumor burden and increased 45 survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of 46 both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 47 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a 48 CMV-driven ERBB2 transgene or in HER2+ human SKBR3 and HCC1953 cells. Decreased 49 activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known 50 regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), 51 and the chromatin remodeling factor BRG1. Together our findings indicate that increased 52 expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene, but 53 impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected 54 effects on MMTV-driven Erbb2/Neu expression.

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