Yongmei Feng

Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-Inducing Chemotherapies

Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5(hi)/SLC1A5/38A2(lo) expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.

Degradation of Newly Synthesized Polypeptides by Ribosome-Associated RACK1/c-Jun N-Terminal Kinase/Eukaryotic Elongation Factor 1A2 Complex

ABSTRACT Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.

Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of Braf V600E ::Pten –/– melanoma

Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3′-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten–/– or BrafV600E::Cdkn2a–/–::Pten–/– mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten–/– mice. Pdk1–/– melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1–/– melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

PDK1 and SGK3 contribute to the growth of BRAF mutant melanomas and are potential therapeutic targets

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutant melanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. The PDK1 substrate SGK3 was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment.

Structure-based design of covalent siah inhibitors.

The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.

Downregulation of the Ubiquitin Ligase RNF125 Underlies Resistance of Melanoma Cells to BRAF Inhibitors via JAK1 Deregulation

SUMMARY Despite the remarkable clinical response of melanoma harboring BRAF mutations to BRAF inhibitors (BRAFi), most tumors become resistant. Here, we identified the downregulation of the ubiquitin ligase RNF125 in BRAFi-resistant melanomas and demonstrated its role in intrinsic and adaptive resistance to BRAFi in cultures as well as its association with resistance in tumor specimens. Sox10/MITF expression correlated with and contributed to RNF125 transcription. Reduced RNF125 was associated with elevated expression of receptor tyrosine kinases (RTKs), including EGFR. Notably, RNF125 altered RTK expression through JAK1, which we identified as an RNF125 substrate. RNF125 bound to and ubiquitinated JAK1, prompting its degradation and suppressing RTK expression. Inhibition of JAK1 and EGFR signaling overcame BRAFi resistance in melanoma with reduced RNF125 expression, as shown in culture and in in vivo xenografts. Our findings suggest that combination therapies targeting both JAK1 and EGFR could be effective against BRAFi-resistant tumors with de novo low RNF125 expression.

Effective inhibition of melanoma by BI-69A11 is mediated by dual targeting of the AKT and NF-κB pathways

In melanoma, the activation of pro‐survival signaling pathways, such as the AKT and NF‐κB pathways, is critical for tumor growth. We have recently reported that the AKT inhibitor BI‐69A11 causes efficient inhibition of melanoma growth. Here, we show that in addition to its AKT inhibitory activity, BI‐69A11 also targets the NF‐κB pathway. In melanoma cell lines, BI‐69A11 inhibited TNF‐α‐stimulated IKKα/β and IκB phosphorylation as well as NF‐κB reporter gene expression. Furthermore, the effective inhibition of melanoma growth by BI‐69A11 was attenuated upon NF‐κB activation. Mechanistically, reduced NF‐κB signaling by BI‐69‐A11 is mediated by the inhibition of sphingosine kinase 1, identified in a screen of 315 kinases. Significantly, we demonstrate that BI‐69A11 is well tolerated and orally active against UACC 903 and SW1 melanoma xenografts. Our results demonstrate that BI‐69A11 inhibits both the AKT and the NF‐κB pathways and that the dual targeting of these pathways may be efficacious as a therapeutic strategy in melanoma.

The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation

Suppressing ATF2 or supplementing the diet with fucose impairs growth and metastasis of melanoma in mice. Stopping melanoma metastasis with fucose Metastatic melanoma is particularly challenging to treat. Lau et al. found that activation of the transcription factor ATF2 by the kinase PKCε, which was more prevalent in advanced-stage melanomas than in primary melanocytes or early-stage tumors, promoted the metastatic behavior of melanoma cells in culture and in mice. ATF2 repressed the expression of the gene encoding fucokinase (FUK), an enzyme that promotes global protein fucosylation. Supplementing drinking water with dietary fucose suppressed the growth and metastasis of melanoma in mice, likely by promoting protein fucosylation, which enhanced cell adhesion and reduced cell migration. Thus, inhibiting PKCε or ATF2 activity or increasing protein fucosylation in tumor cells may be therapeutic for melanoma patients. Melanoma is one of the most lethal skin cancers worldwide, primarily because of its propensity to metastasize. Thus, the elucidation of mechanisms that govern metastatic propensity is urgently needed. We found that protein kinase Cε (PKCε)–mediated activation of activating transcription factor 2 (ATF2) controls the migratory and invasive behaviors of melanoma cells. PKCε-dependent phosphorylation of ATF2 promoted its transcriptional repression of the gene encoding fucokinase (FUK), which mediates the fucose salvage pathway and thus global cellular protein fucosylation. In primary melanocytes and cell lines representing early-stage melanoma, the abundance of PKCε-phosphorylated ATF2 was low, thereby enabling the expression of FUK and cellular protein fucosylation, which promoted cellular adhesion and reduced motility. In contrast, increased expression of the gene encoding PKCε and abundance of phosphorylated, transcriptionally active ATF2 were observed in advanced-stage melanomas and correlated with decreased FUK expression, decreased cellular protein fucosylation, attenuated cell adhesion, and increased cell motility. Restoring fucosylation in mice either by dietary fucose supplementation or by genetic manipulation of murine Fuk expression attenuated primary melanoma growth, increased the number of intratumoral natural killer cells, and decreased distal metastasis in murine isograft models. Tumor microarray analysis of human melanoma specimens confirmed reduced fucosylation in metastatic tumors and a better prognosis for primary melanomas that had high abundance of fucosylation. Thus, inhibiting PKCε or ATF2 or increasing protein fucosylation in tumor cells may improve clinical outcome in melanoma patients.

Inhibition of melanoma development in the Nras((Q61K)) ::Ink4a(-/-) mouse model by the small molecule BI-69A11.

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras(Q61K)::Ink4a−/−). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras(Q61K)::Ink4a−/− tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.

Inhibition of Melanoma Growth by Small Molecules That Promote the Mitochondrial Localization of ATF2

Purpose: Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKCϵ in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization. Therefore, promoting the nuclear export of ATF2, which sensitizes melanoma cells to apoptosis, represents a novel therapeutic modality. Experimental Design: We conducted a pilot high-throughput screen of 3,800 compounds to identify small molecules that promote melanoma cell death by inducing the cytoplasmic localization of ATF2. The imaging-based ATF2 translocation assay was conducted using UACC903 melanoma cells that stably express doxycycline-inducible GFP-ATF2. Results: We identified two compounds (SBI-0089410 and SBI-0087702) that promoted the cytoplasmic localization of ATF2, reduced cell viability, inhibited colony formation, cell motility, and anchorage-free growth, and increased mitochondrial membrane permeability. SBI-0089410 inhibited the 12-O-tetradecanoylphorbol-l3-acetate (TPA)–induced membrane translocation of protein kinase C (PKC) isoforms, whereas both compounds decreased ATF2 phosphorylation by PKCϵ and ATF2 transcriptional activity. Overexpression of either constitutively active PKCϵ or phosphomimic mutant ATF2T52E attenuated the cellular effects of the compounds. Conclusion: The imaging-based high-throughput screen provides a proof-of-concept for the identification of small molecules that block the oncogenic addiction to PKCϵ signaling by promoting ATF2 nuclear export, resulting in mitochondrial membrane leakage and melanoma cell death. Clin Cancer Res; 19(10); 2710–22. ©2013 AACR.