Treatment of peritoneal carcinomatosis with intraperitoneal administration of Ad-hARF.

BACKGROUND: Peritoneal dissemination of cancer is a terminal condition with limited therapeutic options. Because the peritoneal cavity is a single enclosed space, regional treatment approaches for isolated peritoneal cancrinomatosis are appealing. There is a potential role for gene therapy in the management of peritoneal cancrinomatosis. MATERIALS AND METHODS: An adenoviral construct of the human p14ARF gene (a tumor suppressor) and a 22 amino acid sequence of the ARF gene product, which has cell membrane penetrating properties, were assayed for proapoptotic properties in a human colorectal cancer cell line (Clone A) cells in vitro. Peritoneal carcinomatosis derived from Clone A cells was also established in nude mice and then treated with intraperitoneal administration of an adenoviral construct of the human p14ARF gene. RESULTS: Treatment of ARF-negative Clone A cells with Ad-hARF in vitro reestablished ARF function. However, the cell penetrating ARF-related peptide did not restore ARF function in Clone A cells. Treatment of Clone A peritoneal xenografts with a single intraperitoneal dose of Ad-hARF (9 × 10(6) viral particles) suppressed the progression of peritoneal disease. Weekly (six times) administration of the Ad-hARF at a lower dose (3 × 10(6) viral particles) also suppressed tumor progression. CONCLUSIONS: Treatment of peritoneal carcinomatosis by intraperitoneal administration of adenoviral constructs of inactivated tumor suppressor genes may be a feasible clinical approach, and ARF may represent a suitable molecular target for tumors where the ARF gene is inactivated.

Targeting of C-terminal binding protein (CtBP) by ARF results in p53-independent apoptosis.

ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.

The alternative reading frame tumor suppressor antagonizes hypoxia-induced cancer cell migration via interaction with the COOH-terminal binding protein corepressor.

The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent activities critical to the prevention of cancer in mice and humans. Recent evidence from mouse models suggests that when p53 is absent, further loss of ARF can widen the tumor spectrum, and potentiate invasion and metastasis. A major target of the p53-independent activity of ARF is the COOH-terminal binding protein (CtBP) family of metabolically regulated transcriptional corepressors, which are degraded upon acute exposure to the ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, to repress epithelial and proapoptotic genes, and can mediate hypoxia-induced migration of cancer cells. The possibility that ARF could suppress tumor cell migration as part of its p53-independent activities was thus explored. Small-interfering RNA (siRNA)-mediated knockdown of ARF in human lung carcinoma cells led to increased cell migration, especially during hypoxia, and this effect was blocked by concomitant treatment with CtBP2 siRNA. Introduction of ARF into p53 and ARF-null human colon cancer cells inhibited hypoxia-induced migration. Furthermore, overexpression of CtBP2 in ARF-expressing cells enhanced cell migration, and an ARF mutant defective in CtBP-family binding was impaired in its ability to inhibit cell migration induced by CtBP2. ARF depletion or CtBP2 overexpression was associated with decreased PTEN expression and activation of the phosphatidylinositol 3-kinase pathway, and a phosphatidylinositol 3-kinase inhibitor blocked CtBP2-mediated cell migration. Thus, ARF can suppress cell migration by antagonizing CtBP2 and the phosphatidylinositol 3-kinase pathway, and these data may explain the increased aggressiveness of ARF-null tumors in mouse models.

CtBP2 Promotes Human Cancer Cell Migration by Transcriptional Activation of Tiam1.

The mammalian COOH-terminal binding proteins (CtBPs) CtBP1 and CtBP2 are metabolically regulated transcriptional co-repressors that are degraded upon acute exposure to the alternative reading frame (ARF) tumor suppressor. We reported previously that CtBP stimulates cell migration in certain contexts via repression of PTEN transcription and activation of the phosphatidylinositol 3-kinase (PI3K) pathway. We have now identified an additional and direct mechanism for CtBP stimulation of cell migration via regulation of T-cell lymphoma invasion and metastasis 1 (Tiam1) protein. Tiam1 is a guanine nucleotide exchange factor (GEF) for Rac GTPase that plays a critical role in regulating cell adhesion, invasion, and migration and has been directly implicated in the promotion of cancer progression and metastasis. We noted a strict positive correlation between CtBP2 and Tiam1 expression levels and that CtBP promotion of cell migration required CtBP-dependent transcriptional activation of Tiam1. RNA interference (RNAi)-mediated knockdown of CtBP2 in human colon or lung carcinoma cells led to decreased Tiam1 protein and mRNA expression, while overexpression of CtBP2 increased Tiam1 expression levels. RNAi and overexpression studies also demonstrated that Tiam1 is a key downstream mediator of CtBP2-mediated cell migration. An analysis of the Tiam1 promoter revealed binding sites for the CtBP-interacting Kruppel-like factor 8 (KLF8), and a Tiam1 promoter luciferase reporter was induced in the presence of both KLF8 and CtBP2, consistent with KLF8-dependent CtBP transactivation of Tiam1. Chromatin immunoprecipitation analyses demonstrated CtBP2 occupancy of the Tiam1 promoter that was dependent on the presence of KLF8. Our results indicate that Tiam1 is a transcriptional activation target of CtBP2 and that this interaction promotes the pro-oncogenic function of CtBP2 leading to cancer cell migration. Transcriptional activation thus plays a role in CtBP pro-oncogenic functions along with the previously characterized CtBP co-repressor function.

The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.

Therapeutic targeting of C-terminal binding protein in human cancer.

The CtBP transcriptional corepressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is antagonized by p14/p19(ARF) tumor suppressors. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells. MTOB induced apoptosis was p53-independent, correlated with the derepression of the proapoptotic CtBP repression target Bik, and was rescued by CtBP overexpression or Bik silencing. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy. In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer, the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP may represent a useful therapeutic strategy in human malignancies.

p19Arf inhibits the invasion of hepatocellular carcinoma cells by binding to C-terminal binding protein.

The INK4A/ARF tumor suppressor locus is frequently inactivated in hepatocellular carcinoma (HCC), yet the consequences of this remain unknown. We recently described a HCC mouse model in which loss of the Ink4a/Arf locus accelerates the development of metastasis and enhances tumor cell migration and invasion in cell culture assays. We show here that knockdown of p19Arf in an HCC cell line increases invasion in cell culture assays. Furthermore, reintroduction of p19(Arf) into HCC cell lines lacking Ink4a/Arf inhibits tumor cell invasion, without affecting cell proliferation, or cell transformation as measured by soft agar colony formation. Inhibition of cell invasion by p19(Arf) was dependent on its C-terminal binding protein (CtBP) interaction domain but independent of Mdm2 binding and nucleolar localization. Indeed, RNA interference-mediated knockdown of CtBP1 or CtBP2 decreased cell invasion, and ectopic expression of CtBP2 enhanced tumor cell migration and invasion. Thus, our data indicate a novel role for the Arf tumor suppressor protein in regulating phenotypes associated with tumor progression and metastasis in HCC cells.

P311 binds to the latency associated protein and downregulates the expression of TGF-beta1 and TGF-beta2.

P311 is an 8-kDa protein originally found in neurons and muscle. We recently showed that expression of P311 in NIH 3T3 cells induced a myofibroblast phenotype with low TGF-beta1 expression. Here we demonstrate that P311 downregulates not only TGF-beta1, but also TGF-beta2, expression, with no effect on TGF-beta3. In addition, P311 interacts with TGF-beta2 in a yeast two-hybrid system through a sequence encompassing part of the TGF-beta latent associated protein (LAP) and part of mature TGF-beta2. Coimmunoprecipitations demonstrated interaction between P311 and TGF-beta1 and 2, but not TGF-beta3. Additional coimmunoprecipitations after introducing LAP or mature TGF-beta1 into cells demonstrated P311 binding to LAP, but not to mature TGF-beta. P311 has a conserved PEST domain, which generally serves as a rapid degradation signal. Deletion of the PEST domain reversed the effect of P311 on TGF-beta isoforms. Finally, Smad3 activity was decreased in P311-expressing cells, but was corrected by exogenous TGF-beta1 treatment, which also elevated TGF-beta1 mRNA level. This suggested that P311 downregulates TGF-beta1 and 2 in part by blocking TGF-beta autoinduction.