Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo.

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.

The tuberous sclerosis proteins regulate formation of the primary cilium via a rapamycin-insensitive and polycystin 1-independent pathway.

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome in which severe renal cystic disease can occur. Many renal cystic diseases, including autosomal dominant polycystic kidney disease (ADPKD), are associated with absence or dysfunction of the primary cilium. We report here that hamartin (TSC1) localizes to the basal body of the primary cilium, and that Tsc1(-/-) and Tsc2(-/-) mouse embryonic fibroblasts (MEFs) are significantly more likely to contain a primary cilium than wild-type controls. In addition, the cilia of Tsc1(-/-) and Tsc2(-/-) MEFs are 17-27% longer than cilia from wild-type MEFs. These data suggest a novel type of ciliary disruption in TSC, associated with enhanced cilia development. The TSC1 and TSC2 proteins function as a heterodimer to inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1). The enhanced ciliary formation in the Tsc1(-/-) and Tsc2(-/-) MEFs was not abrogated by rapamycin, which indicates a TORC1-independent mechanism. Polycystin 1 (PC1), the product of the PKD1 gene, has been found to interact with TSC2, but Pkd1(-/-) MEFs did not have enhanced ciliary formation. Furthermore, while activation of mTOR has been observed in renal cysts from ADPKD patients, Pkd1(-/-) MEFs did not have evidence of constitutive mTOR activation, thereby underscoring the independent functions of the TSC proteins and PC1 in regulation of primary cilia and mTOR. Our data link the TSC proteins with the primary cilium and reveal a novel phenotype of enhanced ciliary formation in a cyst-associated disease.

The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer.

The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G(1)/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.

Tumor suppressive functions of p53.

The majority of human cancers acquire mutations that abrogate the p53 tumor suppressor network and, as a consequence, p53 is one of the most extensively studied proteins in cancer research. Because of its potent tumor suppressive activity, it is widely assumed that a molecular understanding of p53 action will produce fundamental insights into natural processes that limit tumorigenesis and may identify key molecular targets for therapeutic intervention. p53 functions largely as a transcription factor, and can trigger a variety of antiproliferative programs by activating or repressing key effector genes. Despite a significant body of literature detailing the biochemical and biological functions of p53, much remains to be elucidated. Indeed, the p53 network is as complex and enigmatic as it is relevant. It is the goal of this article, written 30 years after the discovery of p53, to present a concise review of the tumor suppressor role of the p53 network and to highlight the context-dependent nature of p53 target-gene functions.

ARF induces autophagy by virtue of interaction with Bcl-xl.

The ARF tumor suppressor controls a well-described p53/Mdm2-dependent oncogenic stress checkpoint. In addition, ARF has recently been shown to localize to mitochondria, and to induce autophagy; however, this has never before been demonstrated for endogenous ARF, and the molecular basis for this activity of ARF has not been elucidated. Using an unbiased mass spectrometry-based approach, we show that mitochondrial ARF interacts with the Bcl2 family member Bcl-xl, which normally protects cells from autophagy by inhibiting the Beclin-1/Vps34 complex, which is essential for autophagy. We find that increased expression of ARF decreases Beclin-1/Bcl-xl complexes in cells, thereby providing a basis for ARF-induced autophagy. Our data also indicate that silencing p53 leads to high levels of ARF and increased autophagy, thereby providing a possible basis for the finding by others that p53 inhibits autophagy. The combined data support the premise that ARF induces autophagy in a p53-independent manner in part by virtue of its interaction with Bcl-xl.

The ARF tumor suppressor can promote the progression of some tumors.

p14/p19ARF (ARF) is a tumor suppressor gene that is frequently mutated in human cancer. ARF has multiple tumor suppressor functions, some of which are mediated by signaling to p53. Surprisingly, a significant fraction of human tumors retain persistently high levels of ARF, suggesting that ARF may possess a prosurvival function. We show that ARF protein is markedly up-regulated in cells exposed to nutrient starvation. Cells with silenced ARF show reduced autophagy and reduced viability when placed under conditions of starvation. We show for the first time that ARF silencing can limit the progression of some tumors, such as lymphoma, but not others, such as E1A/Ras-induced tumors. Specifically, myc-driven lymphomas with mutant p53 tend to overexpress ARF; we show that silencing ARF in these tumors greatly impedes their progression. These data are the first to show that ARF can act in a p53-independent manner to promote the progression of some tumors.

Inhibition of retinoblastoma tumor suppressor activity by RNA interference in lung cancer lines.

BACKGROUND: Inactivation of retinoblastoma (RB) tumor suppressor function occurs frequently in lung cancer. Short-hairpin RNA can be constructed to target specific sequences and efficiently knock down protein expression. We developed a short-hairpin RNA approach to specifically target Rb in lung cancer cells to determine the influence of RB knockdown on proliferation. METHODS: NCI-H520 human lung cancer cells (wild-type Rb) were transfected with pMSCVpuro-Rb3C, a plasmid containing a short-hairpin sequence targeted to human Rb. Transfectants harboring the construct were selected with puromycin. Loss of RB expression in selected cell populations was determined by immunoblotting. Proliferating cells were counted to establish growth rates. Retinoblastoma-proficient and RB-deficient tumor growth was monitored in nude mice. RESULTS: Transfection with pMSCVpuro-Rb3C dramatically diminished RB expression and led to aberrant expression of RB-regulated genes. Cells harboring pMSCVpuro-Rb3C grew at an increased rate compared with control cells: 480.6 +/- 37.7 versus 159.4 +/- 36.2 (relative cell count at 12 days). Tumor growth in nude mice also increased with RB knockdown compared with control mice: 135.2 +/- 73.6 mm3 versus 40.0 +/- 17.0 mm3 (tumor volume at 10 days). CONCLUSIONS: Inhibition of RB expression is efficiently achieved in lung cancer cells with short-hairpin RNA. Genetic targets of RB are deregulated with RB knockdown. Retinoblastoma depletion increases growth in vitro and in murine xenografts. These studies indicate that even in the context of an established tumor cell line, RB limits tumorigenic proliferation. Additionally, this model will serve as an ideal system to evaluate the role of RB activity on therapeutic response.

Slugging it out: fine tuning the p53-PUMA death connection.

In response to DNA damage, the tumor suppressor p53 elicits a complex cellular response. In this issue of Cell, Wu et al. (2005) show that the transcription factor SLUG is induced by p53 and protects hematopoietic progenitor cells from apoptosis triggered by DNA damage. SLUG exerts this protective role by repressing Puma, a proapoptotic target of p53. PUMA is also a key coordinator of apoptosis mediated by both nuclear and cytoplasmic functions of p53 (Chi-puk et al., 2005).

Transcriptional repression of the anti-apoptotic survivin gene by wild type p53.

Survivin is a member of the inhibitor of apoptosis family. This apoptosis inhibitor also has an evolutionarily conserved role as a mitotic spindle checkpoint protein. Previous studies on p53-repressed genes have implicated several genes involved in the G(2)/M transition of the cell cycle as targets of negative regulation by p53. However, few targets of p53 repression that are anti-apoptotic have been identified. This study identifies the anti-apoptotic survivin gene as a p53-repressed gene. Notably, Survivin repression by p53 is shown to be distinct from p53-dependent growth arrest. Chromatin immunoprecipitations indicate that p53 binds the survivin promoter in vivo; immunobinding studies indicate that this site overlaps with a binding site for E2F transcription factors and is subtly distinct from a canonical p53-transactivating element. The survivin-binding site contains a 3-nucleotide spacer between the two decamer "half-sites" of the p53 consensus element; deletion of this spacer is sufficient to convert the survivin site into a transactivating element. Finally, we show that overexpression of Survivin in cells sensitive to p53-dependent cell death markedly inhibits apoptosis induced by ultraviolet light. The identification of survivin as a p53 repressed gene should aid in the elucidation of the contribution of transcriptional repression to p53-dependent apoptosis.

Suppression of tumorigenesis by the p53 target PUMA.

The p53 tumor suppressor regulates diverse antiproliferative processes such that cells acquiring p53 mutations have impaired cell-cycle checkpoints, senescence, apoptosis, and genomic stability. Here, we use stable RNA interference to examine the role of PUMA, a p53 target gene and proapoptotic member of the Bcl2 family, in p53-mediated tumor suppression. PUMA short hairpin RNAs (shRNAs) efficiently suppressed PUMA expression and p53-dependent apoptosis but did not impair nonapoptotic functions of p53. Like p53 shRNAs, PUMA shRNAs promoted oncogenic transformation of primary murine fibroblasts by the E1A/ras oncogene combination and dramatically accelerated myc-induced lymphomagenesis without disrupting p53-dependent cell-cycle arrest. However, the ability of PUMA to execute p53 tumor suppressor functions was variable because, in contrast to p53 shRNAs, PUMA shRNAs were unable to cooperate with oncogenic ras in transformation. These results demonstrate that the p53 effector functions involved in tumor suppression are context dependent and, in some settings, depend heavily on the expression of a single proapoptotic effector. Additionally, they demonstrate the utility of RNA interference for evaluating putative tumor suppressor genes in vivo.