PIK3CA Cooperates with KRAS to Promote MYC Activity and Tumorigenesis via the Bromodomain Protein BRD9.

Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.

p53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb.

UNLABELLED: The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. IMPLICATIONS: This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype.

AMP-activated Protein Kinase (AMPK) Control of mTORC1 Is p53- and TSC2-independent in Pemetrexed-treated Carcinoma Cells.

The key sensor of energy status in mammalian cells, AMP-activated protein kinase (AMPK), can also be activated by the AMP analog aminoimidazolecarboxamide nucleoside monophosphate (ZMP) generated directly from aminoimidazolecarboxamide ribonucleoside (AICAR) or from inhibition of purine synthesis by the antifolate pemetrexed (PTX), a drug used extensively in the treatment of lung cancers. Despite this common mechanism, signaling downstream of AMPK activated by PTX or AICAR differed. AICAR-activated AMPK inhibited mTORC1 both directly by phosphorylation of the mTORC1 subunit Raptor and indirectly by phosphorylation of the regulator TSC2. In contrast, PTX-activated AMPK inhibited mTORC1 solely through Raptor phosphorylation. This dichotomy was due to p53 function. Transcription of p53 target genes, including TSC2, was activated by AICAR but not by PTX. Although both PTX and AICAR stabilized p53, only AICAR activated Chk2 phosphorylation, stimulating p53-dependent transcription. However, Raptor phosphorylation by AMPK was independent of p53 and was sufficient, after PTX treatment, to inhibit mTORC1. We concluded that PTX effects on mTORC1 were independent of TSC2 and p53 and that the activation of a p53 transcriptional response by AICAR was due to an activation of Chk2 that was not elicited by PTX.

Molecular characterization of the HIV type 1 subtype C accessory genes vif, vpr, and vpu.

HIV-1 Vif, Vpr, and Vpu proteins have a profound effect on efficient viral replication and pathogenesis. This study describes the genotypic characterisation of vif , vpr and vpu from 20 South African HIV-1 subtype C primary isolates, and extensive analysis and comparison of known motifs. All HIV-1 subtype C Vif, Vpr and Vpu proteins revealed the presence of highly conserved structural and functional motifs similar to other sub-types, for example, the Vif-APOBEC3G interaction domains. However, several differences were noted when these sequences were compared to subtype B, such as the presence of the LRLL motif which has been implicated in targeting subtype C Vpu predominantly to the cell surface, instead of the Golgi apparatus. A better understanding of the structure/function relationship of these proteins may lead to the development of new classes of antiviral drugs. These results indicate that antiviral drugs that target the conserved functional domains within Vif, Vpr or Vpu could be active against all circulating subtypes, including HIV-1 subtype C.