Differential effects on ARF stability by normal versus oncogenic levels of c-Myc expression.

ARF suppresses aberrant cell growth upon c-Myc overexpression by activating p53 responses. Nevertheless, the precise mechanism by which ARF specifically restrains the oncogenic potential of c-Myc without affecting its normal physiological function is not well understood. Here, we show that low levels of c-Myc expression stimulate cell proliferation, whereas high levels inhibit by activating the ARF/p53 response. Although the mRNA levels of ARF are induced in both scenarios, the accumulation of ARF protein occurs only when ULF-mediated degradation of ARF is inhibited by c-Myc overexpression. Moreover, the levels of ARF are reduced through ULF-mediated ubiquitination upon DNA damage. Blocking ARF degradation by c-Myc overexpression dramatically stimulates the apoptotic responses. Our study reveals that ARF stability control is crucial for differentiating normal (low) versus oncogenic (high) levels of c-Myc expression and suggests that differential effects on ULF- mediated ARF ubiquitination by c-Myc levels act as a barrier in oncogene-induced stress responses.

Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer.

Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.

Prostate Cancer-associated SPOP mutations enhance cancer cell survival and docetaxel resistance by upregulating Caprin1-dependent stress granule assembly.

BACKGROUND: The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in primary prostate cancer, but how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. Stress granules (SG) assembly is an evolutionarily conserved strategy for survival of cells under stress, and often upregulated in human cancers. We investigated the role of SPOP mutations in aberrant activation of the SG in prostate cancer and explored the relevanve of the mechanism in therapy resistance. METHODS: We identified SG nucleating protein Caprin1 as a SPOP interactor by using the yeast two hybrid methods. A series of functional analyses in cell lines, patient samples, and xenograft models were performed to investigate the biological significance and clinical relevance of SPOP regulation of SG signaling in prostate cancer. RESULTS: The cytoplasmic form of wild-type (WT) SPOP recognizes and triggers ubiquitin-dependent degradation of Caprin1. Caprin1 abundance is elevated in SPOP-mutant expressing prostate cancer cell lines and patient specimens. SPOP WT suppresses SG assembly, while the prostate cancer-associated mutants enhance SG assembly in a Caprin1-dependent manner. Knockout of SPOP or expression of prostate cancer-associated SPOP mutants conferred resistance to death caused by SG inducers (e.g. docetaxel, sodium arsenite and H2O2) in prostate cancer cells. CONCLUSIONS: SG assembly is aberrantly elevated in SPOP-mutated prostate cancer. SPOP mutations cause resistance to cellular stress induced by chemtherapeutic drug such as docetaxel in prostate cancer.

Overexpression of MTA1 inhibits the metastatic ability of ZR-75-30 cells in vitro by promoting MTA2 degradation.

BACKGROUND: As the first member of the metastasis-associated protein (MTA) family, MTA1 and another MTA family member, MTA2, have both been reported to promote breast cancer progression and metastasis. However, the difference and relationship between MTA1 and MTA2 have not been fully elucidated. METHODS: Transwell assays were used to assess the roles of MTA1 and MTA2 in the metastasis of ZR-75-30 luminal B breast cancer cells in vitro. Immunoblotting and qRT-PCR were used to evaluate the effect of MTA1 overexpression on MTA2. Proteases that cleave MTA2 were predicted using an online web server. The role of neutrophil elastase (NE) in MTA1 overexpression-induced MTA2 downregulation was confirmed by specific inhibitor treatment, knockdown, overexpression and immunocytochemistry, and NE cleavage sites in MTA2 were confirmed by MTA2 truncation and mutation. The effect of MTA1 overexpression on the intrinsic inhibitor of NE, elafin, was detected by qRT-PCR, immunoblotting and treatment with inhibitors. RESULTS: MTA1 overexpression inhibited, while MTA2 promoted the metastasis of ZR-75-30 cells in vitro. MTA1 overexpression downregulated MTA2 expression at the protein level rather than the mRNA level. NE was predicted to cleave MTA2 and was responsible for MTA1 overexpression-induced MTA2 degradation. NE was found to cleave MTA2 in the C-terminus at the 486, 497, 542, 583 and 621 sites. MTA1 overexpression activated NE by downregulating elafin in a histone deacetylase- and DNA methyltransferase-dependent manner. CONCLUSIONS: MTA1 and MTA2 play opposing roles in the metastasis of ZR-75-30 luminal B breast cancer cells in vitro. MTA1 downregulates MTA2 at the protein level by epigenetically repressing the expression of elafin and releasing the inhibition of neutrophil elastase, which cleaves MTA2 in the C-terminus at multiple specific sites.

Front-signal-dependent accumulation of the RHOA inhibitor FAM65B at leading edges polarizes neutrophils.

A hallmark of neutrophil polarization is the back localization of active RHOA and phosphorylated myosin light chain (pMLC, also known as MYL2). However, the mechanism for the polarization is not entirely clear. Here, we show that FAM65B, a newly identified RHOA inhibitor, is important for the polarization. When FAM65B is phosphorylated, it binds to 14-3-3 family proteins and becomes more stable. In neutrophils, chemoattractants stimulate FAM65B phosphorylation largely depending on the signals from the front of the cells that include those mediated by phospholipase Cß (PLCß) and phosphoinositide 3-kinase γ (PI3Kγ), leading to FAM65B accumulation at the leading edge. Concordantly, FAM65B deficiency in neutrophils resulted in an increase in RHOA activity and localization of pMLC to the front of cells, as well as defects in chemotaxis directionality and adhesion to endothelial cells under flow. These data together elucidate a mechanism for RHOA and pMLC polarization in stimulated neutrophils through direct inhibition of RHOA by FAM65B at the leading edge.

The tumor suppressor proteins ASPP1 and ASPP2 interact with C-Nap1 and regulate centrosome linker reassembly.

Centrosome linker tethers interphase centrosomes together allowing them to function as a single microtubule organization center. The centrosome linker is disrupted at the onset of mitosis to ensure timely centrosome disjunction and bipolar spindle formation and is reassembled at the end of mitosis. While the mechanism controlling centrosome linker disassembly at early mitosis has been well explored, little is known about how the linker is subsequently reassembled before mitotic exit. Here we report that ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are involved in centrosome linker reassembly. We showed that ASPP1/2 interacted with centrosome linker protein C-Nap1. Co-depletion of ASPP1 and ASPP2 inhibited re-association of C-Nap1 with centrosome at the end of mitosis. Moreover, ASPP1/2 facilitated the interaction between C-Nap1 and PP1α, and this interaction was significantly reduced by co-depletion of ASPP1/2. ASPP1/2 antagonized the NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner. Co-depletion of ASPP1 and ASPP2 inhibited dephosphorylation of C-Nap1 (Ser2417/2421) at the end of mitosis. Based on these findings, we propose that ASPP1/2 act as PP1-targeting subunits to facilitate C-Nap1 dephosphorylation and centrosome linker reassembly at the end of mitosis.

HDGF-related protein-2 (HRP-2) acts as an oncogene to promote cell growth in hepatocellular carcinoma.

HDGFRP2 (HRP-2) belongs to the Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) family, which are characterized by a conserved HATH/PWWP domain at a well-conserved region of the N-terminus. However, the cellular function of HRP-2 remains unknown. In this study, we showed for the first time that HRP-2 is frequently overexpressed in human HCC tissues at mRNA and protein levels. We further showed that HRP-2 can promote HCC cells growth in vitro and xenograft tumors in vivo. Using protein affinity purification methods, we searched for functional partners of HRP-2, and found that HRP-2 interacts with various proteins known to be involved in transcription elongation and processing. Furthermore, we demonstrate HRP-2 interacts and co-localizes with RNA processing regulator IWS1, and positively regulated the mRNA level of Cyclin D1. Together, our study suggests HRP-2 may act as an mRNA processing co-factor to promote cells growth by regulating the mRNA of key oncogenes, which can be explored further for cancer treatment.

Destruction of DDIT3/CHOP protein by wild-type SPOP but not prostate cancer-associated mutants.

Characterization of the exome and genome of prostate cancers by next-generation sequencing has identified numerous genetic alternations. SPOP (speckle-type POZ protein) was identified as one of the most frequently affected genes by somatic point mutations in prostate cancer, suggesting SPOP is potentially a key driver for prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer remains to be elucidated. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex and selectively recruits substrates for their ubiquitination and subsequent degradation. DDIT3 is an endoplasmic reticulum (ER) stress-responsive transcription factor playing an essential role in apoptotic execution pathways triggered by ER stress. Here, we identified DDIT3/CHOP as a bona fide substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP recognizes a Ser/Thr-rich degron in the transactivation domain of DDIT3 and triggers DDIT3 degradation via the ubiquitin-proteasome pathway. Strikingly, prostate cancer-associated mutants of SPOP are defective in promoting DDIT3 degradation. This study reveals novel molecular events underlying the regulation of DDIT3 protein homeostasis and provides insight in understanding the relationship between SPOP mutations and ER stress dysregulation in prostate cancer.

Polycomb group protein PHF1 regulates p53-dependent cell growth arrest and apoptosis.

Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression.

HDGF-related protein-3 is required for anchorage-independent survival and chemoresistance in hepatocellular carcinomas.

OBJECTIVE: Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of six members and are characterised by a conserved HATH domain. Among the family members, HDGF was the first to be identified as a mitogenic factor and shown to play an important role in hepatocellular carcinoma pathogenesis. The aim of the present study is to examine the relevance of HDGF-related protein-3 (HRP-3), another member of the HRP family in hepatocellular carcinoma (HCC). DESIGN: HRP-3 expression in HCC tissues was measured by quantitative reverse transcriptase PCR, western blot and immunohistochemistry analysis. The biological consequences of overexpression and knockdown of HRP-3 in HCC cell lines were studied in vitro and in vivo. RESULTS: Expression of HRP-3 mRNA and protein was shown to be highly upregulated in HCC tissues. While knockdown of HRP-3 by small interference RNAs failed to affect anchorage-dependent growth of HCC cells, it inhibited anchorage-independent growth of HCC cells in vitro and xenograft tumour growth in vivo. Further, knockdown of HRP-3 was shown to sensitise HCC cells to anoikis. Moreover, HRP-3 specifically activated the extracellular-signal-regulated kinase (ERK) pathway without affecting c-Jun N-terminal kinase (JNK), p38, AKT and signal transducer and activator of transcription 3 (STAT3). Importantly, inhibition of the ERK pathway diminished HRP-3-mediated protection of HCC cells from anoikis. Finally, knockdown of HRP-3 was shown to enhance apoptosis of HCC cells induced by multiple chemotherapeutic drugs. CONCLUSION: These findings indicate that HRP-3 plays an essential role in HCC pathogenesis and suggest that it may serve as a novel prognostic marker and molecular target for development of drugs for treatment of HCC.

The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation.

AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.

An audit of autopsy-confirmed diagnostic errors in perinatal deaths: What are the most common major missed diagnoses.

Perinatal autopsies are essential to establish the cause of stillbirth or neonatal death and improve clinical practice. Limited studies have provided detailed major missed diagnoses of perinatal deaths in current clinical practice. In this retrospective audit of 177 perinatal autopsies including 99 stillbirths and 78 neonatal deaths with complete pathologic evaluation, 66 cases (21 Class I and 45 Class II diagnostic errors) were revealed as major discrepancies (37.3%), with complete agreements in 80 cases (45.2%). The difference in major discrepancies between stillbirth and neonatal death groups was significant (P < 0.001), with neonatal deaths being more prone to Class I errors. Various respiratory diseases (25/66, 37.9%) and congenital malformations (16/66, 24.2%) accounted for the majority of missed diagnoses (41/66, 62.1%). More importantly, neonatal respiratory distress syndrome (NRDS) was the most common type I missed diagnosis (7/8, 87.5%), markedly higher than the average 11.9% of all Class I errors. Our findings suggest that there are high disparities between clinical diagnoses and autopsy findings in perinatal deaths, and that various respiratory diseases are mostly inclined to cause major diagnostic errors. We first demonstrated that NRDS is the most common type I missed diagnosis in perinatal deaths, which clinicians should pay special attention to in practice.

Disruption of the Keap1-mTORC2 axis by cancer-derived Keap1/mLST8 mutations leads to oncogenic mTORC2-AKT activation.

The mechanistic target of the rapamycin (mTOR) pathway, which participates in the regulation of cellular growth and metabolism, is aberrantly regulated in various cancer types. The mTOR complex 2 (mTORC2), which consists of the core components mTOR, Rictor, mSin1, and mLST8, primarily responds to growth signals. However, the coordination between mTORC2 assembly and activity remains poorly understood. Keap1, a major sensor of oxidative stress in cells, functions as a substrate adaptor for Cullin 3-RING E3 ubiquitin ligase (CRL3) to promote proteasomal degradation of NF-E2-related factor 2 (NRF2), which is a transcription factor that protects cells against oxidative and electrophilic stress. In the present study, we demonstrate that Keap1 binds to mLST8 via a conserved ETGE motif. The CRL3Keap1 ubiquitin ligase complex promotes non-degradative ubiquitination of mLST8, thus reducing mTORC2 complex integrity and mTORC2-AKT activation. However, this effect can be prevented by oxidative/electrophilic stresses and growth factor signaling-induced reactive oxygen species (ROS) burst. Cancer-derived Keap1 or mLST8 mutations disrupt the Keap1-mLST8 interaction and allow mLST8 to evade Keap1-mediated ubiquitination, thereby enhancing mTORC2-AKT activation and promoting cell malignancy and remodeling cell metabolism. Our findings provide new insights into the molecular mechanisms of Keap1/mLST8 mutation-driven tumorigenesis by promoting mTORC2-AKT activation, which is independent of the canonical NRF2 pathway.

Metallothionein MT1M is a tumor suppressor of human hepatocellular carcinomas.

Members of the metallothionein (MT) family are short, cysteine-rich proteins involved in metal metabolism and detoxification, suggesting that MT proteins protect cells from damage caused by electrophilic carcinogens and thereby constitute a critical surveillance system against carcinogenesis. However, the roles of MT proteins in human hepatocellular carcinoma (HCC) are not fully understood. We identified a member of the MT family, termed MT1M. MT1M is expressed in various normal tissues with the highest level in the liver. MT1M expression can be induced by heavy metals and protect Escherichia coli from heavy metal toxicity. However, MT1M expression markedly decreased in human HCC specimens. A methylation profiling analysis indicated that the MT1M promoter is methylated in the majority of HCC tumors examined. Moreover, restored expression of MT1M in the HCC cell line Hep3B, which lacks endogenous MT1M expression, suppressed cell growth in vitro and in vivo and augmented apoptosis induced by tumor necrosis factor α. Furthermore, stable expression of MT1M in Hep3B cells blocked tumor necrosis factor α-induced degradation of IκBα and transactivation of NF-κB. We conclude that MT1M is a novel member of the MT family. Frequent downregulation of MT1M in human HCC may contribute to liver tumorigenesis by increasing cellular NF-κB activity.

KCTD7 mutations impair the trafficking of lysosomal enzymes through CLN5 accumulation to cause neuronal ceroid lipofuscinoses.

Lysosomes are central organelles for cellular degradation and energy metabolism. Neuronal ceroid lipofuscinoses (NCLs) are a group of the most common neurodegenerative lysosomal storage disorders characterized by intracellular accumulation of ceroid in neurons. Mutations in KCTD7, a gene encoding an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, are categorized as a unique NCL subtype. However, the underlying mechanisms remain elusive. Here, we report various lysosomal and autophagic defects in KCTD7-deficient cells. Mechanistically, the CRL3-KCTD7 complex degrades CLN5, whereas patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive accumulation of CLN5. The accumulated CLN5 disrupts the interaction between CLN6/8 and lysosomal enzymes at the endoplasmic reticulum (ER), subsequently impairing ER-to-Golgi trafficking of lysosomal enzymes. Our findings reveal previously unrecognized roles of KCTD7-mediated CLN5 proteolysis in lysosomal homeostasis and demonstrate that KCTD7 and CLN5 are biochemically linked and function in a common neurodegenerative pathway.

SPOP inhibits BRAF-dependent tumorigenesis through promoting non-degradative ubiquitination of BRAF.

BACKGROUND: The gene encoding the E3 ubiquitin ligase substrate-binding adapter Speckle-type BTB/POZ protein (SPOP) is frequently mutated in prostate cancer (PCa) and endometrial cancer (EC); however, the molecular mechanisms underlying the contribution of SPOP mutations to tumorigenesis remain poorly understood. METHODS: BRAF harbors a potential SPOP-binding consensus motif (SBC) motif. Co-immunoprecipitation assays demonstrated that BRAF interacts with SPOP. A series of functional analyses in cell lines were performed to investigate the biological significance of MAPK/ERK activation caused by SPOP mutations. RESULTS: Cytoplasmic SPOP binds to and induces non-degradative ubiquitination of BRAF, thereby reducing the interaction between BRAF and other core components of the MAPK/ERK pathway. SPOP ablation increased MAPK/ERK activation. EC- or PCa-associated SPOP mutants showed a reduced capacity to bind and ubiquitinate BRAF. Moreover, cancer-associated BRAF mutations disrupted the BRAF-SPOP interaction and allowed BRAF to evade SPOP-mediated ubiquitination, thereby upregulating MAPK/ERK signaling and enhancing the neoplastic phenotypes of cancer cells. CONCLUSIONS: Our findings provide new insights into the molecular link between SPOP mutation-driven tumorigenesis and aberrant BRAF-dependent activation of the MAPK/ERK pathway.

DCAF12 promotes apoptosis and inhibits NF-κB activation by acting as an endogenous antagonist of IAPs.

Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival and appear to neutralize the cell death machinery by binding pro-apoptotic caspases. dcaf12 was recently identified as an apoptosis regulator in Drosophila. However, the underlying molecular mechanisms are unknown. Here we revealed that human DCAF12 homolog binds multiple IAPs, including XIAP, cIAP1, cIAP2, and BRUCE, through recognition of BIR domains in IAPs. The pro-apoptotic function of DCAF12 is dependent on its capacity to bind IAPs. In response to apoptotic stimuli, DCAF12 translocates from the nucleus to the cytoplasm, where it blocks the interaction between XIAP and pro-apoptotic caspases to facilitate caspase activation and apoptosis execution. Similarly, DCAF12 suppresses NF-κB activation in an IAP binding-dependent manner. Moreover, DCAF12 acts as a tumor suppressor to restrict the malignant phenotypes of cancer cells. Together, our results suggest that DCAF12 is an evolutionarily conserved IAP antagonist.

SPOP mutations promote p62/SQSTM1-dependent autophagy and Nrf2 activation in prostate cancer.

p62/SQSTM1 is a selective autophagy receptor that drives ubiquitinated cargos towards autophagic degradation. This receptor is also a stress-induced scaffold protein that helps cells to cope with oxidative stress through activation of the Nrf2 pathway. Functional disorders of p62 are closely associated with multiple neurodegenerative diseases and cancers. The gene encoding the E3 ubiquitin ligase substrate-binding adapter SPOP is frequently mutated in prostate cancer (PCa), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis remain poorly understood. Here, we report that cytoplasmic SPOP binds and induces the non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This protein modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing p62-dependent autophagy. Moreover, we show that SPOP relieves p62-mediated Keap1 sequestration, which ultimately decreases Nrf2-mediated transcriptional activation of antioxidant genes. We further show that PCa-associated SPOP mutants lose the capacity to ubiquitinate p62 and instead promote autophagy and the redox response in a dominant-negative manner. Thus, our findings indicate oncogenic roles of autophagy and Nrf2 activation in the tumorigenesis of SPOP-mutated PCa.

CRL2-KLHDC3 E3 ubiquitin ligase complex suppresses ferroptosis through promoting p14ARF degradation.

The cystine/glutamate antiporter SLC7A11 (commonly known as xCT) functions to import cystine for glutathione biosynthesis, thereby protecting cells from oxidative stress and ferroptosis, a regulated form of non-apoptotic cell death driven by the accumulation of lipid-based reactive oxygen species (ROS). p14ARF, a well-established tumor suppressor, promotes ferroptosis by inhibiting NRF2-mediated SLC7A11 transcription. Here, we demonstrate the crucial role of Cullin 2 RING E3 ligase (CRL2)-KLHDC3 E3 ubiquitin ligase complex in regulating p14ARF protein stability. KLHDC3 acts as a CRL2 adaptor that specifically recognizes a C-terminal degron in p14ARF and triggers p14ARF for ubiquitin-proteasomal degradation. This regulation mode is absent in the murine p14ARF homolog, p19arf which lacks the C-terminal degron. We also show that KLHDC3 suppresses ferroptosis in vitro and supports tumor growth in vivo by relieving p14ARF-mediated suppression of SLC7A11 transcription. Overall, these findings reveal that the protein stability and pro-ferroptotic function of p14ARF are controlled by a CRL2 E3 ubiquitin ligase complex, and suggest that suppression of the p14ARF-NRF2-SLC7A11 regulatory pathway by KLHDC3 overexpression likely contributes to cancer progression.

ZNF191 alters DNA methylation and activates the PI3K-AKT pathway in hepatoma cells via transcriptional regulation of DNMT1.

BACKGROUND: Alteration of DNA methylation is an important event in pathogenesis and progression of hepatocellular carcinoma (HCC). DNA methyltransferase (DNMT) 1, the foremost contributor in DNA methylation machinery, was revealed elevated in HCC and significantly correlates with poor prognosis. However, the transcriptional regulation of DNMT1 in HCC remains unknown. METHODS: Real-time PCR and immunohistochemistry were performed to detect DNMT1 and zinc finger transcription factor 191 (ZNF191) expressions in HCCs. Transcription activity of DNMT1promoter was analyzed with Luciferase reporter activity assay. The binding capacity of ZNF191 protein to DNMT1 promoter was examined with chromatin immunoprecipitation-qPCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA). DNA methylation level of hepatoma cells was detected with Methylation array. RESULTS: ZNF191 can regulate DNMT1 mRNA and protein expression positively, and increase the transcription activity of the DNMT1 promoter. ChIP-qPCR and EMSA revealed that ZNF191 protein directly binds to the DNMT1 promoter at nt-240 AT(TCAT)3 TC. Moreover, DNMT1 and ZNF191 expression correlate positively in human HCCs. With methylation array, DNA methylation alteration was observed in hepatoma cells with ZNF191 knockdown, and the differential methylation sites are enriched in the PI3K-AKT pathway. Furthermore, we proved DNMT1 contributes the effect of ZNF191 on hepatoma cell growth via the PI3K-AKT pathway. CONCLUSION: ZNF191 is a novel transcription regulator for DNMT1, and the pro-proliferation effect of ZNF191/DNMT1/p-AKT axis in hepatoma cells implies that ZNF191 status in HCCs may affect the therapeutic effect of DNMTs inhibitors and PI3K inhibitors for precise treatment of the disease.