The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

mTORC1-Induced Bone Marrow-Derived Mesenchymal Stem Cell Exhaustion Contributes to the Bone Abnormalities in Klotho-Deficient Mice of Premature Aging.

Stem cell exhaustion is a hallmark of aging. Klotho-deficient mice (kl/kl mice) is a murine model that mimics human aging with significant bone abnormalities. The aim of this study is using kl/kl mice to investigate the functional change of bone marrow-derived mesenchymal stem cells (BMSCs) and explore the underlying mechanism. We found that klotho deficiency leads to bone abnormalities. In addition, kl/kl BMSCs manifested hyperactive proliferation but functionally declined both in vivo and in vitro. Mammalian target of rapamycin complex 1 (mTORC1) activity was higher in freshly isolated kl/kl BMSCs, and autophagy in kl/kl BMSCs was significantly decreased, possibly through mTORC1 activation. Conditional medium containing soluble Klotho protein (sKL) rescued hyperproliferation of kl/kl BMSCs by inhibiting mTORC1 activity and restoring autophagy. Finally, intraperitoneal injection of mTORC1 inhibitor rapamycin restored BMSC quiescence, ameliorated bone phenotype, and increased life span of kl/kl mice in vivo. This research highlights a therapeutic strategy to maintain the homeostasis of adult stem cell pool for healthy bone aging.

EZH2 engages TGFß signaling to promote breast cancer bone metastasis via integrin ß1-FAK activation.

Bone metastases occur in 50-70% of patients with late-stage breast cancers and effective therapies are needed. The expression of enhancer of zeste homolog 2 (EZH2) is correlated with breast cancer metastasis, but its function in bone metastasis hasn't been well-explored. Here we report that EZH2 promotes osteolytic metastasis of breast cancer through regulating transforming growth factor beta (TGFß) signaling. EZH2 induces cancer cell proliferation and osteoclast maturation, whereas EZH2 knockdown decreases bone metastasis incidence and outgrowth in vivo. Mechanistically, EZH2 transcriptionally increases ITGB1, which encodes for integrin ß1. Integrin ß1 activates focal adhesion kinase (FAK), which phosphorylates TGFß receptor type I (TGFßRI) at tyrosine 182 to enhance its binding to TGFß receptor type II (TGFßRII), thereby activating TGFß signaling. Clinically applicable FAK inhibitors but not EZH2 methyltransferase inhibitors effectively inhibit breast cancer bone metastasis in vivo. Overall, we find that the EZH2-integrin ß1-FAK axis cooperates with the TGFß signaling pathway to promote bone metastasis of breast cancer.

An inducible CRISPR/Cas9 screen identifies DTX2 as a transcriptional regulator of human telomerase.

Most tumor cells reactivate telomerase to ensure unlimited proliferation, whereas the expression of human telomerase reverse transcriptase (hTERT) is tightly regulated and rate-limiting for telomerase activity maintenance. Several general transcription factors (TFs) have been found in regulating hTERT transcription; however, a systematic study is lacking. Here we performed an inducible CRISPR/Cas9 KO screen using an hTERT core promoter-driven reporter. We identified numerous positive regulators including an E3 ligase DTX2. In telomerase-positive cancer cells, DTX2 depletion downregulated hTERT transcription and telomerase activity, contributing to progressive telomere shortening, growth arrest, and increased apoptosis. Utilizing BioID, we characterized multiple TFs as DTX2 proximal proteins, among which NFIC functioned corporately with DTX2 in promoting hTERT transcription. Further analysis demonstrated that DTX2 mediated K63-linked ubiquitination of NFIC, which facilitated NFIC binding to the hTERT promoter and enhanced hTERT expression. These findings highlight a new hTERT regulatory pathway that may be exploited for potential cancer therapeutics.

Blocking immunosuppressive neutrophils deters pY696-EZH2-driven brain metastases.

The functions of immune cells in brain metastases are unclear because the brain has traditionally been considered "immune privileged." However, we found that a subgroup of immunosuppressive neutrophils is recruited into the brain, enabling brain metastasis development. In brain metastatic cells, enhancer of zeste homolog 2 (EZH2) is highly expressed and phosphorylated at tyrosine-696 (pY696)-EZH2 by nuclear-localized Src tyrosine kinase. Phosphorylation of EZH2 at Y696 changes its binding preference from histone H3 to RNA polymerase II, which consequently switches EZH2's function from a methyltransferase to a transcription factor that increases c-JUN expression. c-Jun up-regulates protumorigenic inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), which recruits Arg1+- and PD-L1+ immunosuppressive neutrophils into the brain to drive metastasis outgrowth. G-CSF-blocking antibodies or immune checkpoint blockade therapies combined with Src inhibitors impeded brain metastasis in multiple mouse models. These findings indicate that pY696-EZH2 can function as a methyltransferase-independent transcription factor to facilitate the brain infiltration of immunosuppressive neutrophils, which could be clinically targeted for brain metastasis treatment.

mTORC1-Rps15 Axis Contributes to the Mechanisms Underlying Global Translation Reduction During Senescence of Mouse Embryonic Fibroblasts.

The reduction of protein translation is a common feature in senescent cells and aging organisms, yet the underlying mechanisms are not fully understood. Here we show that both global mRNA translation and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) kinase activity are declined in a senescent model of mouse embryonic fibroblasts (MEFs). Furthermore, RNA-seq analyses from polysomal versus total mRNA fractions identify TOP-like mRNA of Rps15 whose translation is regulated by mTORC1 during MEF senescence. Overexpression of Rps15 delays MEF senescence, possibly through regulating ribosome maturation. Together, these findings indicate that the activation of mTORC1-Rps15 axis ameliorate senescence by regulating ribosome biogenesis, which may provide further insights into aging research.

Oncogenic Kinase-Induced PKM2 Tyrosine 105 Phosphorylation Converts Nononcogenic PKM2 to a Tumor Promoter and Induces Cancer Stem-like Cells.

The role of pyruvate kinase M2 isoform (PKM2) in tumor progression has been controversial. Previous studies showed that PKM2 promoted tumor growth in xenograft models; however, depletion of PKM2 in the Brca1-loss-driven mammary tumor mouse model accelerates tumor formation. Because oncogenic kinases are frequently activated in tumors and PKM2 phosphorylation promotes tumor growth, we hypothesized that phosphorylation of PKM2 by activated kinases in tumor cells confers PKM2 oncogenic function, whereas nonphosphorylated PKM2 is nononcogenic. Indeed, PKM2 was phosphorylated at tyrosine 105 (Y105) and formed oncogenic dimers in MDA-MB-231 breast cancer cells, whereas PKM2 was largely unphosphorylated and formed nontumorigenic tetramers in nontransformed MCF10A cells. PKM2 knockdown did not affect MCF10A cell growth but significantly decreased proliferation of MDA-MB-231 breast cancer cells with tyrosine kinase activation. Multiple kinases that are frequently activated in different cancer types were identified to phosphorylate PKM2-Y105 in our tyrosine kinase screening. Introduction of the PKM2-Y105D phosphomimetic mutant into MCF10A cells induced colony formation and the CD44hi/CD24neg cancer stem-like cell population by increasing Yes-associated protein (YAP) nuclear localization. ErbB2, a strong inducer of PKM2-Y105 phosphorylation, boosted nuclear localization of YAP and enhanced the cancer stem-like cell population. Treatment with the ErbB2 kinase inhibitor lapatinib decreased PKM2-Y105 phosphorylation and cancer stem-like cells, impeding PKM2 tumor-promoting function. Taken together, phosphorylation of PKM2-Y105 by activated kinases exerts oncogenic functions in part via activation of YAP downstream signaling to increase cancer stem-like cell properties.Significance: These findings reveal PKM2 promotes tumorigenesis by inducing cancer stem-like cell properties and clarify the paradox of PKM2's dichotomous functions in tumor progression. Cancer Res; 78(9); 2248-61. ©2018 AACR.

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) promotes non-homologous end joining and inhibits homologous recombination repair upon DNA damage.

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage.