Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 (640PKLKRQ646) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence (S730AVACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in S730AVACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in S730AVACM-1/CUL5 sequences, is critical for their control of cell proliferation.

Establishment of genetically diverse patient-derived xenografts of colorectal cancer.

Preclinical compounds tested in animal models often show limited efficacy when transitioned into human clinical trials. As a result, many patients are stratified into treatment regimens that have little impact on their disease. In order to create preclinical models that can more accurately predict tumor responses, we established patient-derived xenograft (PDX) models of colorectal cancer (CRC). Surgically resected tumor specimens from colorectal cancer patients were implanted subcutaneously into athymic nude mice. Following successful establishment, fourteen models underwent further evaluation to determine whether these models exhibit heterogeneity, both at the cellular and genetic level. Histological review revealed properties not found in CRC cell lines, most notably in overall architecture (predominantly columnar epithelium with evidence of gland formation) and the presence of mucin-producing cells. Custom CRC gene panels identified somatic driver mutations in each model, and therapeutic efficacy studies in tumor-bearing mice were designed to determine how models with known mutations respond to PI3K, mTOR, or MAPK inhibitors. Interestingly, MAPK pathway inhibition drove tumor responses across most models tested. Noteworthy, the MAPK inhibitor PD0325901 alone did not significantly mediate tumor response in the context of a KRAS(G12D) model, and improved tumor responses resulted when combined with mTOR inhibition. As a result, these genetically diverse models represent a valuable resource for preclinical efficacy and drug discovery studies.

Persistent mitochondrial hyperfusion promotes G2/M accumulation and caspase-dependent cell death.

Cancer cells have several hallmarks that define their neoplastic behavior. One is their unabated replicative potential that allows cells to continually proliferate, and thereby contribute to increasing tumor burden. The progression of a cell through the cell cycle is regulated by a series of checkpoints that ensures successful transmission of genetic information, as well as various cellular components, including organelles and protein complexes to the two resulting daughter cells. The mitochondrial reticulum undergoes coordinated changes in shape to correspond with specific stages of the cell cycle, the most dramatic being complete mitochondrial fragmentation prior to cytokinesis. To determine whether mitochondrial fission is a required step to ensure proper mitochondrial segregation into two daughter cells, we investigated the importance of mitochondrial dynamics to cell cycle progression. We found that mitochondrial hyperfusion promotes a defect in cell cycle progression characterized by an inability for cells to exit G2/M. Additionally, extended periods of persistent mitochondrial fusion led to robust caspase-dependent cell death. The cell death signals were coordinated through activation and cleavage of caspase-8, promoting a potent death response. These results demonstrate the importance of mitochondrial dynamics in cell cycle progression, and that inhibiting mitochondrial fission regulators may provide a therapeutic strategy to target the replicative potential of cancer cells.