Raw images of all western blots presented in S1 Fig.

Raw images of all western blots presented in S1 Fig.</p

Raw images of all western blots presented in the upper panel of Fig 2A.

Raw images of all western blots presented in the upper panel of Fig 2A.</p

Raw images of all western blots presented in Fig 4A.

Raw images of all western blots presented in Fig 4A.</p

qRT-PCR primers.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)—driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.</div

Decreased expression of SAGA components, ERBB2, and ERBB3 upon deletion of <i>Usp22</i> in mammary gland epithelial cells (MECs) collected from 100-day old NIC mice.

(A) Immunoblot analyses showed decreased levels of USP22, GCN5, ATXN7L3, ERBB2, phosphor-ERBB2 and ERBB3 proteins in MECs collected from Usp22FL/FL- NIC mice compared to Usp22+/+—NIC and Usp22FL/+—NIC mice. Despite the sharp decrease in the levels of the receptors, the levels of phosphorylated AKT and phosphorylated ERK show an increase. HSP70, HSP90AB1 and HSP90 protein levels were not affected when analyzed by western blotting in the same lysates. n = 3 for each group. (B, C) qRT-PCR analysis of total RNA collected from Usp22FL/FL- NIC MECs confirmed that mRNA levels of Usp22 and Erbb2 were drastically decreased compared to Usp22+/+- NIC. pUsp22 and Erbb2, as determined by two-tailed unpaired t test. n = 5 for each group. Each sample was measured in triplicate. (D) Immunoblot analyses using lysates from endpoint tumors from Usp22FL/FL- NIC mice showed that levels of USP22, ERBB2, ERBB3, phosphorylated ERK and H2Bub decreased relative to other genotypes, but levels of HSP70, HSP90, HSP90AB1, GCN5 and phosphorylated AKT did not change. n = 3 for each group.</p

Depletion or deletion of <i>USP22</i> in MCF10A cells that stably overexpress ERBB2 did not affect the levels of ERBB2.

(A) Efficient depletion of USP22 by lentiviral shRNA knock down in MCF10A-ERBB2 cells as verified by qRT-PCR analysis. (B) qRT-PCR analysis indicates levels of ERBB2 mRNA did not change upon USP22 shRNA knock down. ns: No statistical significance. (C) Immunoblot analysis of MCF10A-ERBB2 cell lysates showed that ERBB2 protein levels are not changed upon USP22 shRNA knock down. (D) Immunoblot analysis of MCF10A-ERBB2 cell lysates indicate that ERBB2 protein levels are not changed upon USP22 CRISPR/Cas9 knockout. (E). Immunoblot analysis of HER2-positive SKBR3 and HCC1954 cell lysates showed no difference in ERBB2 protein levels upon USP22 shRNA knock down.</p

Validation of high enrichment of epithelial cells vs fibroblasts in lysates from mammary epithelial cells (MECs) collected from 100-day old NIC mice.

Validation of high enrichment of epithelial cells vs fibroblasts in lysates from mammary epithelial cells (MECs) collected from 100-day old NIC mice.</p

USP22 lentiviral shRNA vectors.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)—driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.</div

Raw images of all western blots presented in Fig 6D.

Raw images of all western blots presented in Fig 6D.</p

Raw images of all western blots presented in the lower panel of Fig 2A.

Raw images of all western blots presented in the lower panel of Fig 2A.</p