Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomic analysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomic analysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds.

TRIB1 supports prostate tumorigenesis and tumor-propagating cell survival by regulation of endoplasmic reticulum chaperone expression.

Endocrine therapy is the standard treatment for advanced prostate cancer; however, relapse occurs in most patients with few treatment options available after recurrence. To overcome this therapeutic hurdle, the identification of new molecular targets is a critical issue. The capability to proliferate in three-dimensional (3D) conditions is a characteristic property of cancer cells. Therefore, factors that regulate 3D growth are considered rational targets for cancer therapy. Here, we applied a functional genomic approach to the 3D spheroid cell culture model and identified TRIB1, a member of the Trib family of serine/threonine kinase-like proteins, as an essential factor for prostate cancer cell growth and survival. RNAi-mediated silencing of TRIB1 suppressed prostate cancer cell growth selectively under the 3D conditions. This effect was rescued by ectopic expression of an RNAi-resistant TRIB1 exogene. Gene signature-based analysis revealed that TRIB1 was related to endoplasmic reticulum (ER) pathways in prostate cancer and was required for expression of the ER chaperone GRP78, which is critical for prostate tumorigenesis. Of note, GRP78 was expressed preferentially in a subpopulation of prostate cancer cells that possess tumor-propagating potential, and these tumor-propagating cells were highly sensitive to TRIB1 and GRP78 depletion. In a xenograft model of human prostate cancer, TRIB1 depletion strongly inhibited tumor formation. Supporting these observations, we documented frequent overexpression of TRIB1 in clinical specimens of prostate cancer. Overall, our results indicated that the TRIB1-ER chaperone axis drives prostate tumorigenesis and the survival of the tumor-propagating cells.

The lysosomal signaling anchor p18/LAMTOR1 controls epidermal development by regulating lysosome-mediated catabolic processes.

The lysosomal adaptor protein p18 is an essential anchor of a scaffolding complex for the mTORC1 and MAPK pathways, which play crucial roles in controlling cell growth and energy homeostasis. To elucidate the in vivo function of the p18-mediated pathway, we conditionally ablated p18 in the mouse epidermis. Mutant mice were born with severe defects in formation of the stratum corneum and died within 12 h after birth due to dehydration caused by loss of skin barrier function. Mutant epidermal cells can grow and differentiate into granular cells, but exhibit functional defects in corneocyte maturation. Electron microscopy identified abnormal immature cells, overlying the mutant granular cells, which accumulated autophagosomes, glycogen granules and dead nuclei. Cell culture analysis showed that loss of p18 attenuated lysosome function, resulting in accumulation of immature lysosomes and autophagosomes. Analyses of lysosome behavior revealed that p18 is required for functional interaction between lysosomes and target organelles including autophagosomes. These findings suggest that p18-mediated pathways control lysosome-mediated catabolic processes, which are crucial for the development of mouse epidermis.

The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes.

The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

Megalin is downregulated via LPS-TNF-α-ERK1/2 signaling pathway in proximal tubule cells.

Expression and function of megalin, an endocytic receptor in proximal tubule cells (PTCs), are reduced in diabetic nephropathy, involved in the development of proteinuria/albuminuria. Lipopolysaccharide (LPS) is chronically increased in diabetic sera, by the mechanism called metabolic endotoxemia. We investigated low-level LPS-mediated signaling that regulates megalin expression in immortalized rat PTCs (IRPTCs). Incubation of the cells with LPS (10 ng/ml) for 48 h suppressed megalin protein expression and its endocytic function. TNF-α mRNA expression was increased by LPS treatment, and knockdown of the mRNA with siRNA inhibited LPS-mediated downregulation of megalin mRNA expression at the 24-h time point. Incubation of IRPTCs with exogenous TNF-α also suppressed megalin mRNA and protein expression at the 24- and 48-h time points, respectively. MEK1 inhibitor PD98059 competed partially but significantly TNF-α-mediated downregulation of megalin mRNA expression. Collectively, low-level LPS-mediated TNF-α-ERK1/2 signaling pathway is involved in downregulation of megalin expression in IRPTCs.