Protein synthesis control in cancer: selectivity and therapeutic targeting.

Translational control of mRNAs is a point of convergence for many oncogenic signals through which cancer cells tune protein expression in tumorigenesis. Cancer cells rely on translational control to appropriately adapt to limited resources while maintaining cell growth and survival, which creates a selective therapeutic window compared to non-transformed cells. In this review, we first discuss how cancer cells modulate the translational machinery to rapidly and selectively synthesize proteins in response to internal oncogenic demands and external factors in the tumor microenvironment. We highlight the clinical potential of compounds that target different translation factors as anti-cancer therapies. Next, we detail how RNA sequence and structural elements interface with the translational machinery and RNA-binding proteins to coordinate the translation of specific pro-survival and pro-growth programs. Finally, we provide an overview of the current and emerging technologies that can be used to illuminate the mechanisms of selective translational control in cancer cells as well as within the microenvironment.

Examining Myc-Dependent Translation Changes in Cellular Homeostasis and Cancer.

A central component of Myc's role as a master coordinator of energy metabolism and biomass accumulation is its ability to increase the rate of protein synthesis, driving cell cycle progression, and proliferation. Importantly, Myc-induced alterations in both global and specific mRNA translation is a key determinant of Myc's oncogenic function. Herein, we provide five assays to enable researchers to measure global protein synthesis changes, to identify the translatome uniquely regulated by Myc and to investigate the mechanisms generating the tailored Myc translation network. Metabolic labeling of cells with 35S-containing methionine and cysteine in culture and O-propargyl-puromycin (OP-Puro) incorporation in vivo are presented as methods to measure the overall rate of global protein synthesis. Isolation of polysome-associated mRNAs followed by quantitative real-time PCR (qRT-PCR) and the toeprint assay enable the detection of altered translation of specific mRNAs and isoforms, and visualization of differential ribosomal engagement at start codons uniquely mediated by Myc activation, respectively. Finally, the translation initiation reporter assay is utilized to uncover the molecular mechanism mediating altered translation initiation of a specific mRNA. Together, the protocols detailed in this chapter can be used to illuminate how and to what degree Myc-dependent regulation of translation influences homeostatic cellular functions as well as tumorigenesis.

KRAS regulation by small non-coding RNAs and SNARE proteins.

KRAS receives and relays signals at the plasma membrane (PM) where it transmits extracellular growth factor signals to downstream effectors. SNORD50A/B were recently found to bind KRAS and inhibit its tumorigenic action by unknown mechanisms. KRAS proximity protein labeling was therefore undertaken in SNORD50A/B wild-type and knockout cells, revealing that SNORD50A/B RNAs shape the composition of proteins proximal to KRAS, notably by inhibiting KRAS proximity to the SNARE vesicular transport proteins SNAP23, SNAP29, and VAMP3. To remain enriched on the PM, KRAS undergoes cycles of endocytosis, solubilization, and vesicular transport to the PM. Here we report that SNAREs are essential for the final step of this process, with KRAS localization to the PM facilitated by SNAREs but antagonized by SNORD50A/B. Antagonism between SNORD50A/B RNAs and specific SNARE proteins thus controls KRAS localization, signaling, and tumorigenesis, and disrupting SNARE-enabled KRAS function represents a potential therapeutic opportunity in KRAS-driven cancer.

Ras functional proximity proteomics establishes mTORC2 as new direct ras effector.

Although oncogenic mutations in the three major Ras isoforms, KRAS, HRAS and NRAS, are present in nearly a third of human cancers, therapeutic targeting of Ras remains a challenge due to its structure and complex regulation. However, an in-depth examination of the protein interactome of oncogenic Ras may provide new insights into key regulators, effectors and other mediators of its tumorigenic functions. Previous proteomic analyses have been limited by experimental tools that fail to capture the dynamic, transient nature of Ras cellular interactions. Therefore, in a recent study, we integrated proximity-dependent biotin labeling (BioID) proteomics with CRISPR screening of identified proteins to identify Ras proximal proteins required for Ras-dependent cancer cell growth. Oncogenic Ras was proximal to proteins involved in unexpected biological processes, such as vesicular trafficking and solute transport. Critically, we identified a direct, bona fide interaction between active Ras and the mTOR Complex 2 (mTORC2) that stimulated mTORC2 kinase activity. The oncogenic Ras-mTORC2 interaction resulted in a downstream pro-proliferative transcriptional program and promoted Ras-dependent tumor growth in vivo. Here we provide additional insight into the Ras isoform-specific protein interactomes, highlighting new opportunities for unique tumor-type therapies. Finally, we discuss the active Ras-mTORC2 interaction in detail, providing a more complete understanding of the direct relationship between Ras and mTORC2. Collectively, our findings support a model wherein Ras integrates an expanded array of pro-oncogenic signals to drive tumorigenic processes, including action on mTORC2 as a direct effector of Ras-driven proliferative signals.

The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.

Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-, K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycle transcriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia in vivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.

RNA-protein interaction detection in living cells.

RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.

Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence.

Neovascularization is a crucial component of tumor growth and ischemia. Although prior work primarily used disease models, delineation of neovascularization in the absence of disease can reveal intrinsic mechanisms of microvessel regulation amenable to manipulation in illness. We created a conditional model of epithelial HIF-1 induction in adult mice (TetON-HIF-1 mice). Longitudinal photoacoustic microscopy (L-PAM) was coincidentally developed for noninvasive, label-free serial imaging of red blood cell-perfused vasculature in the same mouse for weeks to months. TetON-HIF-1 mice evidenced 3 stages of neovascularization: development, maintenance, and transgene-dependent regression. Regression occurred despite extensive and tight pericyte coverage. L-PAM mapped microvascular architecture and quantified volumetric changes in neocapillary morphogenesis, arteriovenous remodeling, and microvessel regression. Developmental stage endothelial proliferation down-regulation was associated with a DNA damage checkpoint consisting of p53, p21, and endothelial γ-H2AX induction. The neovasculature was temporally responsive to VEGFR2 immuno-blockade, with the developmental stage sensitive, and the maintenance stage resistant, to DC101 treatment. L-PAM analysis also pinpointed microvessels ablated or resistant to VEGFR2 immuno-blockade. HIF-1-recruited myeloid cells did not mediate VEGFR2 inhibitor resistance. Thus, HIF-1 neovascularization in the absence of disease is self-regulated via cell autonomous endothelial checkpoints, and resistant to angiogenesis inhibitors independent of myeloid cells.