Abstract 4434: Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor

Abstract

Abstract Deubiquitinase Usp9x has been shown to promote tumor cell survival and resistance to chemo- and radiotherapy through effects on the Mcl-1 pro-survival protein. It is predicted that a small molecule Usp9x inhibitor could have a major impact in cancer therapy. However, recent data has raised caution about therapeutic targeting of Usp9X in cancer. In a study to identify genes that reduce the latency of pancreatic tumor formation in a mutant Kras model, Pérez-Mancera et al. 2012 reported that the Usp9x locus was frequently inactivated in pancreatic cancer cells, resulting in protection from anoikis and enhanced transformation. In order to clarify the role of Usp9X in pancreatic cancer, we used a newly created cell line 4668 established from mouse pancreatic tumors with doxycycline (DOX) inducible expression of KrasG12D and Tp53R172H. Stable Usp9x knockdown (KD) in 4668 cells had no effect on their proliferation or phenotype in 2D cultures. However, withdrawal of DOX from DOX-treated Usp9x KD 4668 cells resulted in 4-fold increase in colony formation, unlike DOX depleted control KD 4668 cells which undergo apoptosis resulting in colony disintegration. DOX treated control KD and Usp9x KD 4668 cells did not show any difference in basal 3D colony formation. These results suggest that Usp9x loss in the absence of continuous mutant Kras/p53 expression is sufficient to sustain 3D cancer cell growth, confirming a tumor suppressor role for Usp9x in a Kras mouse model of pancreatic cancer. To determine if Usp9x also plays a tumor suppressor role in human pancreatic tumors, we did Usp9x KD in three human pancreatic cancer cell lines with varied Kras mutational status; BxPC3 (wild type), PANC1 (heterozygous) and MIA-PACA2 (homozygous) which expressed high basal levels of Usp9x. MIA-PACA2-Usp9x KD cells undergo apoptosis and caspase activation. In contrast, BxPC3-Usp9x KD cells showed no effect on growth in 2D or in 3D culture. However, PANC1-Usp9x KD cells showed more rapid (38% p=0.004) 2D growth, but 3D colony formation was consistently reduced by >50%, (p=0.03). These results suggest that the effect of Usp9x loss in established human pancreatic tumor cells is distinct from that detected in a murine pancreatic tumor model. Distinctions may be partially dependent on their Kras mutational status or Usp9x substrate utilization in human vs. murine cells. To further assess the clinical relevance of our observations, Usp9x KD was evaluated in four recently established, low passage number human pancreatic patient tumor cell lines (UM2, UM6, UM16, UM76). Usp9x KD did not affect 2D growth but fully suppressed 3D colony growth. This activity was phenocopied by our small molecule Usp9x inhibitor, EOAI3402143 (G9), with nM efficacy. Together, these results suggest that possible species distinctions exist in the tumorigenic functions of Usp9x and necessitate further assessment of its role in pancreatic tumor development and treatment. Citation Format: Anupama Pal, Marina Pasca di Magliano, Moshe Talpaz, Michelle Dziubinski, Diane Simeone, Nicholas J. Donato. Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2014-4434