No abstract

Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16 Ink4a) are frequently inactivated in these cancers. We tested whether transforming growth factor-b (TGF-b) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-b signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-b1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21Cip1 and p15 Ink4bwere up-regulated, and cells were arrested at G 1 . The expression of p16 Ink4a was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21Cip1 and p15Ink4b recapitulated TGF-b1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21 Cip1 and p15 Ink4b accumulation as well as the growth arrest in response to TGF-b. Human HCC tumors raised in immunodeficient mice also displayed TGF-b1-induced senescence. More importantly, peritumoral injection of TGFb1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-b receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. Conclusion: TGF-b induces p53-independent and p16 Ink4a -independent, but Nox4-dependent, p21 Cip1-dependent, p15 Ink4b-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-b-induced senescence in vivo is associated with a strong antitumor response against HCC. (HEPATOLOGY 2010;52:966-974)

Background: -catenin mutations that constitutively activate the canonical Wnt signaling have been observed in a subset of hepatocellular carcinomas (HCCs). These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling.

The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.

Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3′ splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-offunction mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner. reactive oxygen species | cancer A n evolutionarily conserved transcription factor, p53 has origins that can be traced back to the early metazoans, ∼700 Mya (1). This transcription factor plays a critical role in regulating many fundamental aspects of reversible and irreversible cellular stress responses, genome surveillance, and suppression of oncogenic transformation (1). In response to strong cellular stresses such as DNA damage or oncogenic signals, p53 regulates the expression of a large cohort of genes that affect cell cycle arrest, senescence, and apoptosis (1). Recent work has uncovered additional roles for p53 under basal physiological conditions, such as regulation of development, reproduction, metabolism, and self-renewal capacity (2-5). However, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation that involves alternative splicing of the TP53 gene. We found that the use of an alternative 3′ splice site in intron 6 generates a previously uncharacterized p53 isoform that we named p53Ψ. Interestingly, this isoform is highly expressed in cells characterized by a CD44 high /CD24 low immune type. At the molecular level, p53Ψ lacks major portions of the DNA-binding domain, the nuclear localization sequence, and the tetramerization domain, features that are normally present in full-length p53 (p53FL). Consequently, this isoform proved incapable of sequence-specific DNA binding and transactivation of canonical p53 target genes. However, expression of the p53Ψ isoform attenuated the expression of E-cadherin, induced expression of markers associated with epithelial-mesenchymal transition (EMT), and enhanced the motility and invasive capacity of normal and malignant cells. Consistent with a role of these features in enhancing the prometastatic capabilities of cells, we observed that in patients with early-stage nonsmall cell lung carcinoma (...

immortality ͉ liver cancer ͉ SIP1 ͉ telomerase ͉ p53

We have recently recapitulated metastasis of human PTEN/TP53-mutant PC in mouse using the RapidCaP system. Surprisingly, we found that this metastasis is driven by Myc-, and not Akt-activation. Here, we show that cell-cell communication by Il6 drives the Akt-Myc switch through activation of the Akt-suppressing phosphatase Phlpp2, when Pten and p53 are lost together, but not separately. Il6 then communicates a downstream program of Stat3-mediated Myc-activation, which drives cell proliferation. Similarly in tissues, peak proliferation in Pten/Trp53 mutant primary and metastatic PC does not correlate with activated Akt, but with Stat3/Myc activation instead. Mechanistically, Myc strongly activates the Akt phosphatase Phlpp2 in primary cells and PC metastasis. We show genetically that Phlpp2 is essential for dictating proliferation of Myc-mediated Akt-suppression. Collectively, our data reveal competition between two proto-oncogenes: Myc and Akt, which ensnarls the Phlpp2 gene to facilitate Myc-driven PC metastasis after loss of Pten and Trp53.

. Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment.