The incidence rates of renal cell carcinoma (RCC) have continued to rise with 58,000 new cases in the United States. RCC has notoriously been refractory to traditional chemotherapeutic including radiation and cytokine therapies. The advent of the use of molecularly targeted therapies for RCC has significantly improved the standard of care. Yet, there still remains room for improvement as many of the current therapies are limited by acquired resistance and dosing restrictions due to toxicity. In this review we discuss many potential therapeutic targets that have been suggested for development as advancements are made in discovering the underlying molecular biology of RCC. Among the targets that discussed are additional targets within the well-established VHL/HIF axis, the PI3K/AKT/mTOR pathway, independent targets, and those identified by synthetic lethality screens.

In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development and autophagy. Our CRISPR-generated, fluorescently tagged endogenous Caenorhabditis elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. LET-363/TOR and DAF-15/Raptor are required for development beyond the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs the decision of the animal to progress through development.

Background: Expression of Ror2 leads to increased tumorigenicity in RCC. Results: Ror2 expression stabilizes an increased pool of soluble ␤-catenin, which enhances the response to Wnt stimulation. Conclusion: Ror2 promotes basal and canonical Wnt3a ligand-enhanced ␤-catenin signaling in RCC. Significance: This work demonstrates a novel Ror2-dependent tiered state of canonical Wnt signaling, promoting basal signals and priming RCC cells for a heightened response to the ligand.

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.

The notoriety of the small GTPase Ras as the most mutated oncoprotein has led to a well-characterized signaling network largely conserved across metazoans. Yet the role of its close relative Rap1 (Ras Proximal), which shares 100% identity between their core effector binding sequences, remains unclear. A long-standing controversy in the field is whether Rap1 also functions to activate the canonical Ras effector, the S/T kinase Raf. We used the developmentally simpler Caenorhabditis elegans, which lacks the extensive paralog redundancy of vertebrates, to examine the role of RAP-1 in two distinct LET-60/Ras-dependent cell fate patterning events: induction of 1°vulval precursor cell (VPC) fate and of the excretory duct cell. Fluorescence-tagged endogenous RAP-1 is localized to plasma membranes and is expressed ubiquitously, with even expression levels across the VPCs. RAP-1 and its activating GEF PXF-1 function cell autonomously and are necessary for maximal induction of 1°VPCs. Critically, mutationally activated endogenous RAP-1 is sufficient both to induce ectopic 1°s and duplicate excretory duct cells. Like endogenous RAP-1, before induction GFP expression from the pxf-1 promoter is uniform across VPCs. However, unlike endogenous RAP-1, after induction GFP expression is increased in presumptive 1°s and decreased in presumptive 2°s. We conclude that RAP-1 is a positive regulator that promotes Ras-dependent inductive fate decisions. We hypothesize that PXF-1 activation of RAP-1 serves as a minor parallel input into the major LET-60/Ras signal through LIN-45/Raf.

The extracellular signal-regulated kinase (ERK) MAP kinase is utilized downstream of Ras>Raf>MEK signaling to control activation of a wide array of targets. Activation of ERK is elevated in Ras-driven tumors and RASopathies, and is thus a target for pharmacological inhibition. Regulatory mechanisms of ERK activation have been studied extensively in vitro and in cultured cells but little in living animals. We tagged the C. elegans ERK-encoding gene, mpk-1. MPK-1 is ubiquitously expressed with elevated expression in certain contexts. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During patterning of vulval precursor cells, MPK-1 is necessary and sufficient for the central cell, P6.p, to assume 1˚ fate. Yet MPK-1 translocates to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK-nKTR reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into regulation of MPK-1 activation within a complex intercellular signaling network.

The C. elegans vulva is an excellent model for the study of developmental biology and cell–cell signaling. The developmental induction of vulval precursor cells (VPCs) to assume the 3°-3°-2°-1°-2°-3° patterning of cell fates occurs with 99.8% accuracy. During C. elegans vulval development, an EGF signal from the anchor cell initiates the activation of RasLET-60 > RafLIN-45 > MEKMEK-2 > ERKMPK-1 signaling cascade to induce the 1° cell. The presumptive 1° cell signals its two neighboring cells via NotchLIN-12 to develop 2° cells. In addition, RasLET-60 switches effectors to RalGEFRGL-1 > RalRAL-1 to promote 2° fate. Shin et al. (2019) showed that RalGEFRGL-1 is a dual-function protein in VPCs fate patterning. RalGEFRGL-1 functions as a scaffold for PDKPDK-1 > AktAKT-1/2 modulatory signaling to promote 1° fate in addition to propagating the RasLET-60 modulatory signal through RalRAL-1 to promote 2° fate. The deletion of RalGEFRGL-1 increases the frequency of VPC patterning errors 15-fold compared to the wild-type control. We speculate that RalGEFRGL-1 represents an “insulated switch”, whereby the promotion of one signaling activity curtails the promotion of the opposing activity. This property might increase the impact of the switch on fidelity more than two separately encoded proteins could. Understanding how developmental fidelity is controlled will help us to better understand the origins of cancer and birth defects, which occur in part due to the misspecification of cell fates.

6 RAP-1 reinforces LET-60 signaling 7Abstract 16The notoriety of the small GTPase Ras as the most mutated oncoprotein has led to a well-17 characterized signaling network largely conserved across metazoans. Yet the role of its close 18 relative Rap1 (Ras Proximal), which shares 100% identity between their core effector binding 19 sequences, remains unclear. A long-standing controversy in the field is whether Rap1 also 20 functions to activate the canonical Ras effector, the S/T kinase Raf. We used the developmentally 21 simpler Caenorhabditis elegans, which lacks the extensive paralog redundancy of vertebrates, to 22 examine the role of RAP-1 in two distinct LET-60/Ras-dependent cell fate patterning events: 23 induction of 1˚ vulval precursor cell (VPC) fate and of the excretory duct cell. Fluorescently tagged 24 endogenous RAP-1 is localized to plasma membranes and is expressed ubiquitously, with even 25 expression levels across the VPCs. RAP-1 and its activating GEF PXF-1 function cell 26 autonomously and are necessary for maximal induction of 1˚ VPCs. Critically, mutationally 27 activated endogenous RAP-1 is sufficient both to induce ectopic 1˚s and duplicate excretory duct 28 cells. Like endogenous RAP-1, before induction GFP expression from the pxf-1 promoter is 29 uniform across VPCs. However, unlike endogenous RAP-1, after induction GFP expression is 30 increased in presumptive 1˚s and decreased in presumptive 2˚s. We conclude that RAP-1 is a 31 positive regulator that promotes Ras-dependent inductive fate decisions. We hypothesize that 32 PXF-1 activation of RAP-1 serves as a minor parallel input into the major LET-60/Ras signal 33 through LIN-45/Raf. 34 35Ras, the founder of the Ras superfamily of small GTPases, is the most mutated oncoprotein in 36 cancer (COSMIC v84; http://cancer.sanger.ac.uk/cosmic;(Papke and Der 2017). With diverse 37 functions throughout cell biology, members of the Ras superfamily are conserved across Metazoa. 38