ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response.

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) in multiple tumors is associated with a poor prognosis partly because of the metabolic diversion of cytosolic aspartate for pyrimidine synthesis, supporting proliferation and mutagenesis owing to nucleotide imbalance. Here, we find that prolonged loss of ASS1 promotes DNA damage in colon cancer cells and fibroblasts from subjects with citrullinemia type I. Following acute induction of DNA damage with doxorubicin, ASS1 expression is elevated in the cytosol and the nucleus with at least a partial dependency on p53; ASS1 metabolically restrains cell cycle progression in the cytosol by restricting nucleotide synthesis. In the nucleus, ASS1 and ASL generate fumarate for the succination of SMARCC1, destabilizing the chromatin-remodeling complex SMARCC1-SNF5 to decrease gene transcription, specifically in a subset of the p53-regulated cell cycle genes. Thus, following DNA damage, ASS1 is part of the p53 network that pauses cell cycle progression, enabling genome maintenance and survival. Loss of ASS1 contributes to DNA damage and promotes cell cycle progression, likely contributing to cancer mutagenesis and, hence, adaptability potential.

Myotubularin-related-protein-7 inhibits mutant (G12V) K-RAS by direct interaction.

Inhibition of K-RAS effectors like B-RAF or MEK1/2 is accompanied by treatment resistance in cancer patients via re-activation of PI3K and Wnt signaling. We hypothesized that myotubularin-related-protein-7 (MTMR7), which inhibits PI3K and ERK1/2 signaling downstream of RAS, directly targets RAS and thereby prevents resistance. Using cell and structural biology combined with animal studies, we show that MTMR7 binds and inhibits RAS at cellular membranes. Overexpression of MTMR7 reduced RAS GTPase activities and protein levels, ERK1/2 phosphorylation, c-FOS transcription and cancer cell proliferation in vitro. We located the RAS-inhibitory activity of MTMR7 to its charged coiled coil (CC) region and demonstrate direct interaction with the gastrointestinal cancer-relevant K-RASG12V mutant, favouring its GDP-bound state. In mouse models of gastric and intestinal cancer, a cell-permeable MTMR7-CC mimicry peptide decreased tumour growth, Ki67 proliferation index and ERK1/2 nuclear positivity. Thus, MTMR7 mimicry peptide(s) could provide a novel strategy for targeting mutant K-RAS in cancers.

Phosphorylation of PP2Ac by PKC is a key regulatory step in the PP2A-switch-dependent AKT dephosphorylation that leads to apoptosis.

BACKGROUND: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known. METHODS: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation. Other phosphorylations were monitored by commercial antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by a TUNEL assay as well as PARP1 cleavage using SDS-PAGE and Western blotting. RESULTS: We identified Ser24 as a phosphorylation site in PP2Ac. The phosphorylation is mediated mainly by classical PKCs (PKCα and PKCß) but not by novel PKCs (PKCδ and PKCε). By replacing the phosphorylated residue with either unphosphorylatable or phosphomimetic residues (S24A and S24E), we found that this phosphorylation event is necessary and sufficient to mediate the PP2A switch, which ultimately induces AKT inactivation, and a robust JNK-dependent apoptosis. CONCLUSION: Our results show that the PP2A switch is induced by PKC-mediated phosphorylation of Ser24-PP2Ac and that this phosphorylation leads to apoptosis upon GqPCR induction of various cells. We propose that this mechanism may provide an unexpected way to treat some cancer types or problems in the endocrine machinery.

Nuclear pore protein POM121 regulates subcellular localization and transcriptional activity of PPARγ.

Manipulation of the subcellular localization of transcription factors by preventing their shuttling via the nuclear pore complex (NPC) emerges as a novel therapeutic strategy against cancer. One transmembrane component of the NPC is POM121, encoded by a tandem gene locus POM121A/C on chromosome 7. Overexpression of POM121 is associated with metabolic diseases (e.g., diabetes) and unfavorable clinical outcome in patients with colorectal cancer (CRC). Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor with anti-diabetic and anti-tumoral efficacy. It is inhibited by export from the nucleus to the cytosol via the RAS-RAF-MEK1/2-ERK1/2 signaling pathway, a major oncogenic driver of CRC. We therefore hypothesized that POM121 participates in the transport of PPARγ across the NPC to regulate its transcriptional activity on genes involved in metabolic and tumor control. We found that POM121A/C mRNA was enriched and POM121 protein co-expressed with PPARγ in tissues from CRC patients conferring poor prognosis. Its interactome was predicted to include proteins responsible for tumor metabolism and immunity, and in-silico modeling provided insights into potential 3D structures of POM121. A peptide region downstream of the nuclear localization sequence (NLS) of POM121 was identified as a cytoplasmic interactor of PPARγ. POM121 positivity correlated with the cytoplasmic localization of PPARγ in patients with KRAS mutant CRC. In contrast, POM121A/C silencing by CRISPR/Cas9 sgRNA or siRNA enforced nuclear accumulation of PPARγ and activated PPARγ target genes promoting lipid metabolism and cell cycle arrest resulting in reduced proliferation of human CRC cells. Our data suggest the POM121-PPARγ axis as a potential drugable target in CRC.

Early Infiltration of Innate Immune Cells to the Liver Depletes HNF4α and Promotes Extrahepatic Carcinogenesis.

Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.

AKTs do not translocate to the nucleus upon stimulation but AKT3 can constitutively signal from the nuclear envelope.

AKT is a central signaling protein kinase that plays a role in the regulation of cellular survival metabolism and cell growth, as well as in pathologies such as diabetes and cancer. Human AKT consists of three isoforms (AKT1-3) that may fulfill different functions. Here, we report that distinct subcellular localization of the isoforms directly influences their activity and function. AKT1 is localized primarily in the cytoplasm, AKT2 in the nucleus, and AKT3 in the nucleus or nuclear envelope. None of the isoforms actively translocates into the nucleus upon stimulation. Interestingly, AKT3 at the nuclear envelope is constitutively phosphorylated, enabling a constant phosphorylation of TSC2 at this location. Knockdown of AKT3 induces moderate attenuation of cell proliferation of breast cancer cells. We suggest that in addition to the stimulation-induced activation of the lysosomal/cytoplasmic AKT1-TSC2 pathway, a subpopulation of TSC2 is constitutively inactivated by AKT3 at the nuclear envelope of transformed cells.

Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as ß-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-ß/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch.

BACKGROUND: G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. METHODS: Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. RESULTS: We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. CONCLUSION: Our results show a stimulated shift of PP2Ac from PI3K to AKT termed "PP2A switch" that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract.

C-Src is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells.

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gßγ and ß-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.

Nuclear P38: Roles in Physiological and Pathological Processes and Regulation of Nuclear Translocation.

The p38 mitogen-activated protein kinase (p38MAPK, termed here p38) cascade is a central signaling pathway that transmits stress and other signals to various intracellular targets in the cytoplasm and nucleus. More than 150 substrates of p38α/ß have been identified, and this number is likely to increase. The phosphorylation of these substrates initiates or regulates a large number of cellular processes including transcription, translation, RNA processing and cell cycle progression, as well as degradation and the nuclear translocation of various proteins. Being such a central signaling cascade, its dysregulation is associated with many pathologies, particularly inflammation and cancer. One of the hallmarks of p38α/ß signaling is its stimulated nuclear translocation, which occurs shortly after extracellular stimulation. Although p38α/ß do not contain nuclear localization or nuclear export signals, they rapidly and robustly translocate to the nucleus, and they are exported back to the cytoplasm within minutes to hours. Here, we describe the physiological and pathological roles of p38α/ß phosphorylation, concentrating mainly on the ill-reviewed regulation of p38α/ß substrate degradation and nuclear translocation. In addition, we provide information on the p38α/ß 's substrates, concentrating mainly on the nuclear targets and their role in p38α/b functions. Finally, we also provide information on the mechanisms of nuclear p38α/b translocation and its use as a therapeutic target for p38α/ß-dependent diseases.

Myotubularin-related protein 7 activates peroxisome proliferator-activated receptor-gamma.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor drugable by agonists approved for treatment of type 2 diabetes, but also inhibits carcinogenesis and cell proliferation in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene mitigate these beneficial effects by promoting a negative feedback-loop comprising extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated kinase kinase 1/2 (MEK1/2)-dependent inactivation of PPARγ. To overcome this inhibitory mechanism, we searched for novel post-translational regulators of PPARγ. Phosphoinositide phosphatase Myotubularin-Related-Protein-7 (MTMR7) was identified as cytosolic interaction partner of PPARγ. Synthetic peptides were designed resembling the regulatory coiled-coil (CC) domain of MTMR7, and their activities studied in human cancer cell lines and C57BL6/J mice. MTMR7 formed a complex with PPARγ and increased its transcriptional activity by inhibiting ERK1/2-dependent phosphorylation of PPARγ. MTMR7-CC peptides mimicked PPARγ-activation in vitro and in vivo due to LXXLL motifs in the CC domain. Molecular dynamics simulations and docking predicted that peptides interact with the steroid receptor coactivator 1 (SRC1)-binding site of PPARγ. Thus, MTMR7 is a positive regulator of PPARγ, and its mimicry by synthetic peptides overcomes inhibitory mechanisms active in cancer cells possibly contributing to the failure of clinical studies targeting PPARγ.

Beta-Like Importins Mediate the Nuclear Translocation of MAPKs.

BACKGROUND/AIMS: The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de-novo gene expression. However, the molecular mechanisms of this translocation is not well understood, although some studies suggest that much of this translocation may be mediated by beta-like importins (Imps). Here we undertook to study the stimulated nuclear shuttling of JNK and p38 MAPKs. METHODS: For this purpose, we used coimmunoprecipitation, proximity ligation assay, gel filtration and immunostaining to examine the mechanism of nuclear translocation of these proteins. RESULTS: We found that JNK and p38 MAPKs translocate into the nucleus in a Ran dependent, but NLS- or NTS-independent manner, unrelated to their catalytic activity. We show that this translocation involves three ß-like Imps, 3, 7 and 9. Knockdown of these Imps inhibits the nuclear translocation of the MAPKs, and thereby, phosphorylation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/ß bind to either Imp7 or Imp9 upon stimulated post-translational modifications of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope where Imp3 remains, while Imp7 or Imp9 escort the MAPKs into the nucleus. CONCLUSION: These results suggest that ß-like Imps are central mediators of stimulated nuclear translocation of signaling proteins, providing a central level of regulation of the induction of cellular processes such as transcription upon stimulation.

Author Correction: Combined inhibition of MEK and nuclear ERK translocation has synergistic antitumor activity in melanoma cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Nuclear ERK: Mechanism of Translocation, Substrates, and Role in Cancer.

The extracellular signal-regulated kinases 1/2 (ERK) are central signaling components that regulate stimulated cellular processes such as proliferation and differentiation. When dysregulated, these kinases participate in the induction and maintenance of various pathologies, primarily cancer. While ERK is localized in the cytoplasm of resting cells, many of its substrates are nuclear, and indeed, extracellular stimulation induces a rapid and robust nuclear translocation of ERK. Similarly to other signaling components that shuttle to the nucleus upon stimulation, ERK does not use the canonical importinmechanism of nuclear translocation. Rather, it has its own unique nuclear translocation signal (NTS) that interacts with importin7 to allow stimulated shuttling via the nuclear pores. Prevention of the nuclear translocation inhibits proliferation of B-Raf- and N/K-Ras-transformed cancers. This effect is distinct from the one achieved by catalytic Raf and MEK inhibitors used clinically, as cells treated with the translocation inhibitors develop resistance much more slowly. In this review, we describe the mechanism of ERK translocation, present all its nuclear substrates, discuss its role in cancer and compare its translocation to the translocation of other signaling components. We also present proof of principle data for the use of nuclear ERK translocation as an anti-cancer target. It is likely that the prevention of nuclear ERK translocation will eventually serve as a way to combat Ras and Raf transformed cancers with less side-effects than the currently used drugs.

The Nuclear Translocation of Mitogen-Activated Protein Kinases: Molecular Mechanisms and Use as Novel Therapeutic Target.

The mitogen-activated protein kinase (MAPK) cascades are central signaling pathways that play a central role in the regulation of most stimulated cellular processes including proliferation, differentiation, stress response and apoptosis. Currently 4 such cascades are known, each termed by its downstream MAPK components: the extracellular signal-regulated kinase 1/2 (ERK1/2), cJun-N-terminal kinase (JNK), p38 and ERK5. One of the hallmarks of these cascades is the stimulated nuclear translocation of their MAPK components using distinct mechanisms. ERK1/2 are shuttled into the nucleus by importin7, JNK and p38 by a dimer of importin3 with either importin9 or importin7, and ERK5 by importin-α/ß. Dysregulation of these cascades often results in diseases, including cancer and inflammation, as well as developmental and neurological disorders. Much effort has been invested over the years in developing inhibitors to the MAPK cascades to combat these diseases. Although some inhibitors are already in clinical use or clinical trials, their effects are hampered by development of resistance or adverse side-effects. Recently, our group developed 2 myristoylated peptides: EPE peptide, which inhibits the interaction of ERK1/2 with importin7, and PERY peptide, which prevents JNK/p38 interaction with either importin7 or importin9. These peptides block the nuclear translocation of their corresponding kinases, resulting in prevention of several cancers, while the PERY peptide also inhibits inflammation-induced diseases. These peptides provide a proof of concept for the use of the nuclear translocation of MAPKs as therapeutic targets for cancer and/or inflammation.

Gq-Induced Apoptosis is Mediated by AKT Inhibition That Leads to PKC-Induced JNK Activation.

BACKGROUND/AIMS: Gq protein-coupled receptors (GqPCRs) regulate various cellular processes including mainly proliferation and differentiation. In a previous study, we found that in prostate cancer cells, the GqPCR of GnRH induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Since it was thought that GqPCR induces mainly activation of AKT, we undertook to examine how general is this phenomenon and understand its signaling. METHODS: We used various cells to follow the phosphorylation of signaling components using western blotting. RESULTS: In a screen of 21 cell lines, we found that PKC activation results in the reduction of AKT activity, which correlates nicely to JNK activation and in some cases to apoptosis. To further understand the signaling pathways involved in this stimulation, we studied in detail the SVOG-4O and αT3-1 cells. We found that PGF2α and GnRH agonist (GnRH-a) indeed induce significant Gq- and PKC- dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists on c-Src activation of the JNK cascade and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3, to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. CONCLUSION: Our results present a general mechanism that mediates a GqPCR-induced, death receptors-independent, apoptosis in physiological, as well as cancer-related systems.

Intrinsically active MEK variants are differentially regulated by proteinases and phosphatases.

MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as cancers, arteriovenous malformation and RASopathies. Changes in their intrinsic activity do not seem to correlate with the severity of the diseases. Here we studied four MEK1/2 mutations using biochemical and molecular dynamic methods. Although the studied mutants elevated the activating phosphorylation of MEK they had no effect on the stimulated ERK1/2 phosphorylation. Studying the regulatory mechanism that may explain this lack of effect, we found that one type of mutation affects MEK stability and two types of mutations demonstrate a reduced sensitivity to PP2A. Together, our results indicate that some MEK mutations exert their function not only by their elevated intrinsic activity, but also by modulation of regulatory elements such as protein stability or dephosphorylation.

Pigment Epithelium-Derived Factor and its Phosphomimetic Mutant Induce JNK-Dependent Apoptosis and P38-Mediated Migration Arrest.

BACKGROUND/AIMS: Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis, and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated, and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity, and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (EC), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. METHODS: Here we used Western blotting, as well as in vitro binding, proliferation, apoptosis and migration assays to follow the signaling components responsible for the PEDF and EEE-PEDF effects. RESULTS: We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis, and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing stronger effect. The stronger activity of EEE-PEDF was correlated to a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found respectively regulated by differential activation of two distinct MAPK pathways, namely JNK and p38. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, while p38 leads to antimigratory effect in both EC and cancer cells. CONCLUSION: These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.

The nuclear translocation of the kinases p38 and JNK promotes inflammation-induced cancer.

The stimulated nuclear translocation of signaling proteins, such as MAPKs, is a necessity for the initiation and regulation of their physiological functions. Previously, we determined that nuclear translocation of the MAPKs p38 and JNK involves binding to heterodimers comprising importin 3 and either importin 7 or importin 9. Here, we identified the importin-binding region in p38 and JNK and developed a myristoylated peptide targeting this site that we called PERY. The PERY peptide specifically blocked the interaction of p38 and JNK with the importins, restricted their nuclear translocation, and inhibited phosphorylation of their nuclear (but not cytoplasmic) substrates. Through these effects, the PERY peptide reduced the proliferation of several (but not all) cancer cell lines in culture and inhibited the growth of a human breast cancer xenograft in mice. In addition, the PERY peptide substantially inhibited inflammation in mice, as manifested in models of colitis and colitis-associated colon cancer. The PERY peptide more effectively prevented colon cancer development than did a commercial p38 inhibitor. In vivo analysis further suggested that this effect was mediated by PERY peptide-induced prevention of the nuclear translocation of p38 in macrophages. Together, these results support the use of the nuclear translocation of p38 and JNK as a novel drug target to treat various cancers and inflammation-induced diseases.

Combined inhibition of MEK and nuclear ERK translocation has synergistic antitumor activity in melanoma cells.

Genetic alterations in BRAF, NRAS and NF1 that activate the ERK cascade, account for over 80% of metastatic melanomas. However, ERK cascade inhibitors have been proven beneficial almost exclusively for BRAF mutant melanomas. One of the hallmarks of the ERK cascade is the nuclear translocation of ERK1/2, which is important mainly for the induction of proliferation. This translocation can be inhibited by the NTS-derived peptide (EPE) that blocks the ERK1/2-importin7 interaction, inhibits the nuclear translocation of ERK1/2, and arrests active ERK1/2 in the cytoplasm. In this study, we found that the EPE peptide significantly reduced the viability of not only BRAF, but also several NRAS and NF1 mutant melanomas. Importantly, combination of the EPE peptide and trametinib showed synergy in reducing the viability of some NRAS mutant melanomas, an effect driven by the partial preservation of negative feedback loops. The same combination significantly reduced the viability of other melanoma cells, including those resistant to mono-treatment with EPE peptide and ERK cascade inhibitors. Our study indicates that targeting the nuclear translocation of ERK1/2, in combination with MEK inhibitors can be used for the treatment of different mutant melanomas.