The translational landscape of mTOR signalling steers cancer initiation and metastasis.

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.

Cell type-specific abundance of 4EBP1 primes prostate cancer sensitivity or resistance to PI3K pathway inhibitors.

Pharmacological inhibitors against the PI3K-AKT-mTOR (phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin) pathway, a frequently deregulated signaling pathway in cancer, are clinically promising, but the development of drug resistance is a major limitation. We found that 4EBP1, the central inhibitor of cap-dependent translation, was a critical regulator of both prostate cancer initiation and maintenance downstream of mTOR signaling in a genetic mouse model. 4EBP1 abundance was distinctly different between the epithelial cell types of the normal prostate. Of tumor-prone prostate epithelial cell types, luminal epithelial cells exhibited the highest transcript and protein abundance of 4EBP1 and the lowest protein synthesis rates, which mediated resistance to both pharmacologic and genetic inhibition of the PI3K-AKT-mTOR signaling pathway. Decreasing total 4EBP1 abundance reversed resistance in drug-insensitive cells. Increased 4EBP1 abundance was a common feature in prostate cancer patients who had been treated with the PI3K pathway inhibitor BKM120; thus, 4EBP1 may be associated with drug resistance in human tumors. Our findings reveal a molecular program controlling cell type-specific 4EBP1 abundance coupled to the regulation of global protein synthesis rates that renders each epithelial cell type of the prostate uniquely sensitive or resistant to inhibitors of the PI3K-AKT-mTOR signaling pathway.

PI3K-AKT-mTOR signaling in prostate cancer progression and androgen deprivation therapy resistance.

Prostate cancer (PCa) is the second most common malignancy among men in the world. Castration-resistant prostate cancer (CRPC) is the lethal form of the disease, which develops upon resistance to first line androgen deprivation therapy (ADT). Emerging evidence demonstrates a key role for the PI3K-AKT-mTOR signaling axis in the development and maintenance of CRPC. This pathway, which is deregulated in the majority of advanced PCas, serves as a critical nexus for the integration of growth signals with downstream cellular processes such as protein synthesis, proliferation, survival, metabolism and differentiation, thus providing mechanisms for cancer cells to overcome the stress associated with androgen deprivation. Furthermore, preclinical studies have elucidated a direct connection between the PI3K-AKT-mTOR and androgen receptor (AR) signaling axes, revealing a dynamic interplay between these pathways during the development of ADT resistance. Thus, there is a clear rationale for the continued clinical development of a number of novel inhibitors of the PI3K pathway, which offer the potential of blocking CRPC growth and survival. In this review, we will explore the relevance of the PI3K-AKT-mTOR pathway in PCa progression and castration resistance in order to inform the clinical development of specific pathway inhibitors in advanced PCa. In addition, we will highlight current deficiencies in our clinical knowledge, most notably the need for biomarkers that can accurately predict for response to PI3K pathway inhibitors.

The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium.

Agrobacterium tumefaciens is capable of transferring and integrating an oncogenic T-DNA (transferred DNA) from its tumor-inducing (Ti) plasmid into dicotyledonous plants. This transfer requires that the virulence genes (vir regulon) be induced by plant signals such as acetosyringone in an acidic environment. Salicylic acid (SA) is a key signal molecule in regulating plant defense against pathogens. However, how SA influences Agrobacterium and its interactions with plants is poorly understood. Here we show that SA can directly shut down the expression of the vir regulon. SA specifically inhibited the expression of the Agrobacterium virA/G two-component regulatory system that tightly controls the expression of the vir regulon including the repABC operon on the Ti plasmid. We provide evidence suggesting that SA attenuates the function of the VirA kinase domain. Independent of its effect on the vir regulon, SA up-regulated the attKLM operon, which functions in degrading the bacterial quormone N-acylhomoserine lactone. Plants defective in SA accumulation were more susceptible to Agrobacterium infection, whereas plants overproducing SA were relatively recalcitrant to tumor formation. Our results illustrate that SA, besides its well known function in regulating plant defense, can also interfere directly with several aspects of the Agrobacterium infection process.

Effects of cetylpyridinium chloride resistance and treatment on fluconazole activity versus Candida albicans.

Mouthwash antiseptic cetylpyridinium chloride (CPC) has potent activity against Candida albicans; however, two of five azole-resistant strains showed reduced CPC susceptibility. To further examine the potential for cross-resistance, CPC-resistant mutants were selected in vitro and their fluconazole susceptibility was tested. MICs were unchanged, and trailing growth generally decreased. With CPC-fluconazole combinations, both antagonism and synergism were observed, which can be explained, in part, by CDR1-CDR2 multidrug transporter upregulation.

Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast.

PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5' ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent < or =2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes (SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicans LEM3 and RHO1, specificity was 79-95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity (slt2Delta and RHO1/rho1Delta), cycloheximide hypersensitivity (pdr1Delta), or miltefosine resistance (lem3Delta and lem3Delta/lem3Delta). PRODIGE application to the S. cerevisiae PDR5 gene in strains with and without the Pdr1-Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter-URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond.