Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells.

The histone methyltransferase G9a is overexpressed in a variety of cancer types, including pancreatic adenocarcinoma, and promotes tumor invasiveness and metastasis. We recently reported the discovery of BRD4770, a small-molecule inhibitor of G9a that induces senescence in PANC-1 cells. We observed that the cytotoxic effects of BRD4770 were dependent on genetic background, with cell lines lacking functional p53 being relatively resistant to compound treatment. To understand the mechanism of genetic selectivity, we used two complementary screening approaches to identify enhancers of BRD4770. The natural product and putative BH3 mimetic gossypol enhanced the cytotoxicity of BRD4770 in a synergistic manner in p53-mutant PANC-1 cells but not in immortalized non-tumorigenic pancreatic cells. The combination of gossypol and BRD4770 increased LC3-II levels and the autophagosome number in PANC-1 cells, and the compound combination appears to act in a BNIP3 (B-cell lymphoma 2 19-kDa interacting protein)-dependent manner, suggesting that these compounds act together to induce autophagy-related cell death in pancreatic cancer cells.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors.

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins.

BACKGROUND: In all organisms, nutrients are primary regulators of signaling pathways that control transcription. In Saccharomyces cerevisiae, the Tor proteins regulate the transcription of genes sensitive to the quality of available nitrogen and carbon sources. Formation of a ternary complex of the immunosuppressant rapamycin, its immunophilin receptor Fpr1p and Tor1p or Tor2p results in the nuclear import of several nutrient- and stress-responsive transcription factors. RESULTS: We show that treating yeast cells with rapamycin results in a broader modulation of functionally related gene sets than previously understood. Using chemical epistasis and vector-based global expression analyses, we partition the transcriptional program induced by rapamycin among five effectors (TAP42, MKS1, URE2, GLN3, GAT1) of the Tor proteins, and identify how the quality of carbon and nitrogen sources impinge upon components of the program. Biochemical data measuring Ure2p phosphorylation coupled with the partition analysis indicate that there are distinct signaling branches downstream of the Tor proteins. CONCLUSIONS: Whole-genome transcription profiling reveals a striking similarity between shifting to low-quality carbon or nitrogen sources and treatment with rapamycin. These data suggest that the Tor proteins are central sensors of the quality of carbon and nitrogen sources. Depending on which nutrient is limited in quality, the Tor proteins can modulate a given pathway differentially. Integrating the partition analysis of the transcriptional program of rapamycin with the biochemical data, we propose a novel architecture of Tor protein signaling and of the nutrient-response network, including the identification of carbon discrimination and nitrogen discrimination pathways.

Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.

The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.