ABSTRACT
The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53, is required and sufficient for tumorigenesis. While RB1-mutant cells fail to arrest at G1-S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1-deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1-deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1, inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment.
Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Lung Neoplasms/drug therapy , Retinoblastoma Protein/deficiency , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Small Cell Lung Carcinoma/drug therapy , Animals , CDC2-CDC28 Kinases/genetics , CDC2-CDC28 Kinases/metabolism , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Mice , Mice, Knockout , Molecular Targeted Therapy , Retinoblastoma Binding Proteins/deficiency , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV- (dipeptidyl peptidase-4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)-deficient Gunn rats (model of Crigler-Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti-hYAP-ERT2 (mutated estrogen receptor ligand-binding domain 2)-transduced hepatocytes (YAP-Hc). Yap (yes-associated protein) nuclear translocation/function in YAP-Hc was regulated by tamoxifen. Repopulating YAP-Hc and host hepatocytes were fluorescence-activated cell sorting-purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP-Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte-specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up-regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP-Hc. In Gunn rats transplanted with YAP-Hc+tamoxifen, there was a 65%-81% decline in serum bilirubin over 6 months versus 8%-20% with control-Hc, representing a 3-4-fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1-positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen-regulated nuclear translocation/function of hYAP-ERT2 enabled long-term repopulation of DPPIV-/- and Gunn rat livers by hYAP-ERT2-transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.
