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Depletion of FZR1 leads to BRAF stabilization and subsequent ERK activation. Supplementary Figure S2. Depletion of FZR1 triggers the onset of premature senescence in primary melanocytes. Supplementary Figure S3. APCFZR1 promotes BRAF ubiquitination and subsequent degradation in a D-box dependent manner. Supplementary Figure S4. FZR1 disrupts the BRAF dimerization process. Supplementary Figure S5. Phosphorylation of FZR1 N-terminus by ERK and CYCLIN D1/CDK4 inhibits the APCFZR1 E3 ubiquitin ligase activity. Supplementary Figure S6. Pharmacologically inhibiting BRAF/ERK and CDK4 restores the APCFZR1 E3 ligase activity. Supplementary Figure S7. Depletion of FZR1 co-operates with PTEN deficiency to promote co-activation of BRAF/ERK and AKT oncogenic signaling both in vitro and in vivo. Supplementary Table S1. Mutation and deletion of FZR1 and 14 APC core complex subunits identified in melanoma patients from the TCGA melanoma dataset (cbioportal.org).

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following ɣ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. The oxidation of RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, is prevented by PRDX1. Alkylation of RAD51 Cys319 by the electrophilic fatty acid nitroalkene 10- nitro-octadec-9-enoic acid (OA-NO2) inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.

Peroxiredoxin 1 (PRDX1) is an oxidative stress protein sensor, which can coordinate cell signaling of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. Irradiation (IR) of MEFs proficient or deficient of PRDX1 with doses up to 10 Gy found Prdx1-/- MEFs were more sensitive to IR and exhibited reduced homologous recombination in DR-GFP reporter assays. Prdx1-/- MEFs also displayed enhanced DNA damage as measured by nuclear ɣH2AX phosphorylation and reduced RAD51 foci formation following IR. As PRDX1 has been previously identified to bind proteins under heightened oxidative insult, immunoprecipitation assays were conducted, which found PRDX1 directly bound RAD51 with increasing IR. Oxidation of cysteine residues to the sulfenic acid form was specifically probed with DAZ2 and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed C319 of RAD51 as a primary target of oxidation. Cells harboring overexpression of mutant RAD51 C319 revealed suppression of HR, IR-induced RAD51 foci formation, RAD51 enhanced malignancy in an anchorage-independent growth soft agar colony formation and proliferation. MDA-MB-231 breast cancer cells deficient of PRDX1 had decreased IR-induced RAD51 foci formation and were more sensitive to doxorubicin. In conclusion, PRDX1 binds RAD51 during heighted oxidative conditions and protects the functionally important RAD51 residue C319 from oxidation.

BRAF drives tumorigenesis by coordinating the activation of the RAS/RAF/MEK/ERK oncogenic signaling cascade. However, upstream pathways governing BRAF kinase activity and protein stability remain undefined. Here, we report that in primary cells with active APCFZR1, APCFZR1 earmarks BRAF for ubiquitination-mediated proteolysis, whereas in cancer cells with APC-free FZR1, FZR1 suppresses BRAF through disrupting BRAF dimerization. Moreover, we identified FZR1 as a direct target of ERK and CYCLIN D1/CDK4 kinases. Phosphorylation of FZR1 inhibits APCFZR1, leading to elevation of a cohort of oncogenic APCFZR1 substrates to facilitate melanomagenesis. Importantly, CDK4 and/or BRAF/MEK inhibitors restore APCFZR1 E3 ligase activity, which might be critical for their clinical effects. Furthermore, FZR1 depletion cooperates with AKT hyperactivation to transform primary melanocytes, whereas genetic ablation of Fzr1 synergizes with Pten loss, leading to aberrant coactivation of BRAF/ERK and AKT signaling in mice. Our findings therefore reveal a reciprocal suppression mechanism between FZR1 and BRAF in controlling tumorigenesis.Significance: FZR1 inhibits BRAF oncogenic functions via both APC-dependent proteolysis and APC-independent disruption of BRAF dimers, whereas hyperactivated ERK and CDK4 reciprocally suppress APCFZR1 E3 ligase activity. Aberrancies in this newly defined signaling network might account for BRAF hyperactivation in human cancers, suggesting that targeting CYCLIN D1/CDK4, alone or in combination with BRAF/MEK inhibition, can be an effective anti-melanoma therapy. Cancer Discov; 7(4); 424–41. ©2017 AACR.See related commentary by Zhang and Bollag, p. 356.This article is highlighted in the In This Issue feature, p. 339

The yeast succinic semi-aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi-aldehyde (Km = 15.48 ± 0.14 µm) and could use both NAD+ and NADP+ as co-factors, with Km values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD+ (Ki = 129.5 µm) but a non-competitive inhibitor with respect to succinate semi-aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with Ki = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD+ and mixed-competitive, non-competitive and non-competitive inhibition, respectively, with respect to succinate semi-aldehyde. The kinetic data suggest a 'ping-pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.

NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally-occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally-occurring PKCδ inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves PKCδ isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKCδ, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKCδ inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKCδ inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKCδ by novel small molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor-resistance.

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following γ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. PRDX1 was found to directly bind RAD51 with increasing irradiation and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, as a primary target of oxidation. Pharmacological treatment with the thiol reactive electrophilic fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) targeted the C319 residue of RAD51. Alkylation of RAD51 Cys319 OA-NO2 inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation, enhanced H2AX phosphorylation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.

GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α-ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild-type and single mutant strains. Although wild-type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non-lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild-type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild-type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N-tert-butyl-α-phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild-type. Measurement of intracellular GABA, glutamate and α-ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α-ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α-ketoglutarate to succinate during heat stress.

PAX3 (paired box 3) is known to have an important role in melanocyte development through modulation of microphthalmia-associated transcription factor transcription. Here we found that PAX3 transcriptional activity could be regulated through FGF2 (basic fibroblast growth factor)-STAT3 (signal transducer and activator of transcription 3) signaling in the pigment cells. To study its function in vivo, we have generated a transgenic mouse model expressing PAX3 driven by tyrosinase promoter in a tissue-specific fashion. These animals exhibit hyperpigmentation in the epidermis, evident in the skin color of their ears and tails. We showed that the darker skin color results from both increased melanocyte numbers and melanin synthesis. Together, our study delineated a novel pathway in the melanocyte lineage, linking FGF2-STAT3 signaling to increased PAX3 transcription. Moreover, our results suggest that this pathway might contribute to the regulation of melanocyte numbers and melanin levels, and thereby provide an alternative strategy to induce pigmentation.

It is widely accepted that reactive oxygen species (ROS) promote tumorigenesis. However, the exact mechanisms are still unclear. As mice lacking the peroxidase peroxiredoxin1 (Prdx1) produce more cellular ROS and die prematurely of cancer, they offer an ideal model system to study ROS-induced tumorigenesis. Prdx1 ablation increased the susceptibility to Ras-induced breast cancer. We, therefore, investigated the role of Prdx1 in regulating oncogenic Ras effector pathways. We found Akt hyperactive in fibroblasts and mammary epithelial cells lacking Prdx1. Investigating the nature of such elevated Akt activation established a novel role for Prdx1 as a safeguard for the lipid phosphatase activity of PTEN, which is essential for its tumour suppressive function. We found binding of the peroxidase Prdx1 to PTEN essential for protecting PTEN from oxidation-induced inactivation. Along those lines, Prdx1 tumour suppression of Ras- or ErbB-2-induced transformation was mediated mainly via PTEN.

The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor-suppressor gene syndrome caused by inactivating mutations in either TSC1 or TSC2, and the TSC protein complex is an essential regulator of mTOR complex 1 (mTORC1). Patients with TSC develop hypomelanotic macules (white spots), but the molecular mechanisms underlying their formation are not fully characterized. Using human primary melanocytes and a highly pigmented melanoma cell line, we demonstrate that reduced expression of either TSC1 or TSC2 causes reduced pigmentation through mTORC1 activation, which results in hyperactivation of glycogen synthase kinase 3β (GSK3β), followed by phosphorylation of and loss of β-catenin from the nucleus, thereby reducing expression of microphthalmia-associated transcription factor (MITF), and subsequent reductions in tyrosinase and other genes required for melanogenesis. Genetic suppression or pharmacological inhibition of this signaling cascade at multiple levels restored pigmentation. Importantly, primary melanocytes isolated from hypomelanotic macules from 6 patients with TSC all exhibited reduced TSC2 protein expression, and 1 culture showed biallelic mutation in TSC2, one of which was germline and the second acquired in the melanocytes of the hypomelanotic macule. These findings indicate that the TSC/mTORC1/AKT/GSK3β/β-catenin/MITF axis plays a central role in regulating melanogenesis. Interventions that enhance or diminish mTORC1 activity or other nodes in this pathway in melanocytes could potentially modulate pigment production.

Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored poly-ubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma.

The yeast succinic semi-aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS-PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µM NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi-aldehyde (Km = 15.48 ± 0.14 µM) and could use both NAD(+) and NADP(+) as co-factors, with Km values of 579.06 ± 30.1 µM and 1.017 ± 0.46 mM, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD(+) (Ki = 129.5 µM) but a non-competitive inhibitor with respect to succinate semi-aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with Ki = 1.13-10.2 mM, and showed competitive inhibition with respect to NAD(+) and mixed-competitive, non-competitive and non-competitive inhibition, respectively, with respect to succinate semi-aldehyde. The kinetic data suggest a 'ping-pong' mechanism.

GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces.

The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.

The paired box homeotic gene 3 (PAX3) is a crucial regulator for the maintenance of melanocytic progenitor cells and has a poorly defined role in melanoma. To understand how PAX3 affects melanocyte and melanoma proliferation, we identified potential PAX3 downstream targets through gene expression profiling. Here, we identify T-box 2 (TBX2), a key developmental regulator of cell identity and an antisenescence factor in melanoma, as a directly regulated PAX3 target. We also found that TBX2 is involved in the survival of melanoma cells and is overexpressed in some melanoma specimens. The identification of TBX2 as a target for PAX3 provides a key insight into how PAX3 may contribute to melanoma evolution and may provide opportunities for prosenescence therapeutic intervention aimed at disrupting the ability of PAX3 to regulate TBX2. © 2012 John Wiley and Sons A/S.

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α-ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild-type and single mutant strains. Although wild-type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non-lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild-type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild-type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N-tert-butyl-α-phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild-type. Measurement of intracellular GABA, glutamate and α-ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α-ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α-ketoglutarate to succinate during heat stress.

Peroxiredoxins (Prdxs) are a family of small (22–27 kDa) nonseleno peroxidases currently known to possess six mammalian isoforms. Although their individual roles in cellular redox regulation and antioxidant protection are quite distinct, they all catalyze peroxide reduction of H(2)O(2), organic hydroperoxides and peroxynitrite.(1,2) They are found to be expressed ubiquitously and in high levels,(3) suggesting that they are both an ancient and important enzyme family. Prdxs can be divided into three major subclasses: typical 2-cysteine (2-Cys) Prdxs (Prdx1–4), atypical 2-Cys Prdx (Prdx 5) and 1-Cys Prdx (Prdx 6). Recent evidence suggests that 2-Cys peroxiredoxins are more than “just simple peroxidases”. This hypothesis has been discussed elegantly in recent review articles, considering “over”-oxidation of the protonated thiolate peroxidatic cysteine and post-translational modification of Prdxs as processes initiating a mechanistic switch from peroxidase to chaperon function.(4–6) The process of over-oxidation of the peroxidatic cysteine (C(P)) occurs during catalysis in the presence of thioredoxin (Trx), thus rendering the sulfenic moiety to sulfinic acid,(7) which can be reduced by sulfiredoxin (Srx).(8,9) However, further oxidation to sulfonic acid is believed to promote Prdx degradation or, as recently shown, the formation of oligomeric peroxidase-inactive chaperones(10) with questionable H(2)O(2)-scavenging capacity. In the light of this and given that Prdx1 has recently been shown by us(11) and by others(12–17) to interact directly with signaling molecules, we will explore the possibility that H(2)O(2) regulates signaling in the cell in a temporal and spatial fashion via oxidizing Prdx1. Therefore, this review will focus on H(2)O(2) modulating cell signaling via Prdxs by discussing: (1) the activity of Prdxs towards H(2)O(2); (2) sub cellular localization and availability of other peroxidases, such as catalase or glutathione peroxidases; (3) the availability of Prdxs reducing systems, such as thioredoxin and sulfiredoxin and lastly, (4) Prdx1 interacting signaling molecules.

Melanoma is an aggressive skin cancer characterized by gene mutations, high metastatic potential and few clinically effective systemic therapies. BRAF, MEK1 kinase inhibitors and CTLA4 antagonist have improved patient survival, but only a subset of patients respond or derived long-lasting clinical benefit, illustrating the need for discovery of additional targets and pathways that may be inhibited with small molecules. Given the clinical impact of inhibitors of the ubiquitin-proteasome system in other cancers, we explored the potential for aberrant deubiquitinase (DUB) activity to play a role in the tumorigenic properties of melanoma. We also assessed whether specific DUBs play a role in apoptotic responsiveness and resistance to kinase inhibitors. Initially, we noted that the activity of several DUBs, including Usp9x and Usp5, was elevated in melanoma compared to normal melanocytes. Further, Usp9x and Usp5 expression and activity were up-regulated by expression of BRAFV600E in 293T cells and kinase inhibition partially suppressed both Usp9x and Usp5 activity in vemurafenib sensitive, but not resistant cells. Usp9x knockdown (KD) in BRAF mutant or wild-type melanoma did not affect 2-dimensional cell growth or survival but significantly amplified the apoptotic activity of BRAF or MEK1 inhibitors, respectively. Usp5 KD in melanoma suppressed cell growth by reinforced the S/G2-M checkpoint, enhanced extrinsic caspase activation through modulation of p53 and FAS levels and amplified the apoptotic activity of kinase inhibitors (and other chemotherapeutic agents) and overcame vemurafenib resistance in A375R cells. To further assess the clinical relevance of activated DUBs in melanoma, primary human melanoma explants characterized for their metastatic efficiency in patients and NSG mice demonstrated that Usp9x and Usp5 expression and activity were elevated in efficient metastasizers, but only a subset of inefficient metastasizers, suggesting a potential role for DUBs in the regulation of metastatic potential. To investigate that possibly, control and Usp9x KD cells were grown in Matrigel or suspension culture and colony growth and survival, as well as Usp9x activity were examined. Usp9x KD significantly suppressed melanoma colony growth and fully reduced survival of highly metastatic SK-Mel147 cells in 3D culture. Growth in 3D conditions resulted in activation of Usp9x, but also increased their sensitivity (IC50 Citation Format: Harish Potu, Anupama Pal, Hanshi Sun, Luke Peterson, Moshe Talpaz, Monique Verhaegen, Juxiang Cao, Ugur Eskiocak, Sean Morrison, Nicholas J. Donato. Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2014-4435